@article {pmid32461256, year = {2020}, author = {Geng, X and Zhang, S and He, J and Ma, A and Li, Y and Li, M and Zhou, H and Chen, G and Yang, B}, title = {The urea transporter UT-A1 plays a predominant role in a urea-dependent urine-concentrating mechanism.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1074/jbc.RA120.013628}, pmid = {32461256}, issn = {1083-351X}, abstract = {Urea transporters are a family of urea-selective channel proteins expressed in multiple tissues and play an important role in the urine-concentrating mechanism of the mammalian kidney. Previous studies have shown that knockout of urea transporter-B (UT-B), UT-A1/A3, or all-UT lead to urea-selective diuresis, indicating that urea transporters have important roles in urine concentration. Here, we sought to determine the role of UT-A1 in the urine-concentrating mechanism in a newly developed UT-A1-knockout mouse model. Phenotypically, daily urine output in UT-A1-knockout mice was nearly 3-fold that of wild-type mice and 82% of all-UT-knockout mice, and the UT-A1-knockout mice had significantly lower urine osmolality than wild-type mice. After 24 h water restriction, acute urea loading, or high-protein (40%) intake, UT-A1 knockout mice were unable to increase urine-concentrating ability. Compared with all-UT-knockout mice, the UT-A1-null mice exhibited similarly elevated daily urine output and decreased urine osmolality, indicating impaired urea-selective urine concentration. Our experimental findings reveal that UT-A1 has a predominant role in urea-dependent urine-concentrating mechanisms, suggesting that UT-A1 represents a promising diuretic target.}, }
@article {pmid32270050, year = {2019}, author = {Evanoff, M and Komor, AC}, title = {Base Editors: Modular Tools for the Introduction of Point Mutations in Living Cells.}, journal = {Emerging topics in life sciences}, volume = {3}, number = {5}, pages = {483-491}, pmid = {32270050}, issn = {2397-8554}, support = {T32 GM007240/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; Cell Line ; Cytidine Deaminase/chemistry/genetics ; DNA, Single-Stranded/chemistry/metabolism ; Endonucleases/chemistry/*genetics ; Gene Editing/*methods ; Genetic Enhancement ; *Point Mutation ; RNA, Guide/chemistry/metabolism ; Recombination, Genetic/genetics ; Synthetic Biology ; Transcription, Genetic ; }, abstract = {Base editors are a new family of programmable genome editing tools that fuse ssDNA (single stranded DNA) modifying enzymes to catalytically inactive CRISPR-associated (Cas) endonucleases to induce highly efficient single base changes. With dozens of base editors now reported, it is apparent that these tools are highly modular; many combinations of ssDNA modifying enzymes and Cas proteins have resulted in a variety of base editors, each with its own unique properties and potential uses. In this perspective, we describe currently available base editors, highlighting their modular nature and describing the various options available for each component. Furthermore, we briefly discuss applications in synthetic biology and genome engineering where base editors have presented unique advantages over alternative techniques.}, }
@article {pmid31659913, year = {2019}, author = {Zoppo, M and Luca, MD and Villarreal, SN and Poma, N and Barrasa, MI and Bottai, D and Vyas, VK and Tavanti, A}, title = {A CRISPR/Cas9-based strategy to simultaneously inactivate the entire ALS gene family in Candida orthopsilosis.}, journal = {Future microbiology}, volume = {14}, number = {}, pages = {1383-1396}, doi = {10.2217/fmb-2019-0168}, pmid = {31659913}, issn = {1746-0921}, mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Candida parapsilosis/*genetics/growth & development ; Candidiasis/microbiology ; Cell Adhesion ; Cells, Cultured ; Epithelial Cells/microbiology ; Gene Editing/*methods ; *Genes, Fungal ; Humans ; Hyphae/growth & development ; Mouth/cytology ; Multigene Family ; RNA, Guide/genetics ; }, abstract = {Aim: In this study, the CRISPR gene-editing approach was used to simultaneously inactivate all three members of the ALS gene family in the opportunistic pathogen Candida orthopsilosis. Materials & methods: Using a single gRNA and repair template, CRISPR-edited clones were successfully generated in a one-step process in both C. orthopsilosis reference and clinical strains. Results: The phenotypic characterization of the ALS triple-edited strains revealed no impact on growth in liquid or solid media. However, pseudohyphal formation and the ability to adhere to human buccal epithelial cells were significantly decreased in triple-edited clones. Conclusion: Our CRISPR/Cas9 system is a powerful tool for simultaneous editing of fungal gene families, which greatly accelerates the generation of multiple gene-edited Candida strains. Data deposition: Nucleotide sequence data are available in the GenBank databases under the accession numbers MK875971, MK875972, MK875973, MK875974, MK875975, MK875976, MK875977.}, }
@article {pmid31626775, year = {2019}, author = {Feldman, D and Singh, A and Schmid-Burgk, JL and Carlson, RJ and Mezger, A and Garrity, AJ and Zhang, F and Blainey, PC}, title = {Optical Pooled Screens in Human Cells.}, journal = {Cell}, volume = {179}, number = {3}, pages = {787-799.e17}, doi = {10.1016/j.cell.2019.09.016}, pmid = {31626775}, issn = {1097-4172}, support = {R01 HG009283/HG/NHGRI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; P50 HG006193/HG/NHGRI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Nucleus/genetics/metabolism ; *Genetic Testing ; *Genomics ; Humans ; Mediator Complex/genetics ; NF-kappa B/*genetics ; RNA, Guide/genetics ; Transcription Factor RelA/*genetics ; }, abstract = {Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.}, }
@article {pmid30996093, year = {2019}, author = {Luteijn, RD and van Diemen, F and Blomen, VA and Boer, IGJ and Manikam Sadasivam, S and van Kuppevelt, TH and Drexler, I and Brummelkamp, TR and Lebbink, RJ and Wiertz, EJ}, title = {A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection.}, journal = {Journal of virology}, volume = {93}, number = {13}, pages = {}, pmid = {30996093}, issn = {1098-5514}, mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cowpox virus/genetics ; DNA Viruses ; Gene Knockout Techniques ; Genetic Testing ; Golgi Apparatus ; HEK293 Cells ; *Haploidy ; HeLa Cells ; Heparitin Sulfate/*genetics/*isolation & purification/metabolism ; Host Specificity ; Host-Pathogen Interactions ; Humans ; Membrane Proteins ; Monkeypox virus/genetics ; N-Acetylglucosaminyltransferases ; Phosphatidylserines/metabolism ; Pinocytosis/*physiology ; Poxviridae/genetics ; Vaccinia/*virology ; Vaccinia virus/*genetics/*metabolism ; Vesicular Transport Proteins/*metabolism ; Virus Attachment ; }, abstract = {Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.}, }
@article {pmid30996089, year = {2019}, author = {Zhang, W and Yang, F and Zhu, Z and Yang, Y and Wang, Z and Cao, W and Dang, W and Li, L and Mao, R and Liu, Y and Tian, H and Zhang, K and Liu, X and Ma, J and Zheng, H}, title = {Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway.}, journal = {Journal of virology}, volume = {93}, number = {13}, pages = {}, pmid = {30996089}, issn = {1098-5514}, mesh = {Animals ; Antiviral Agents/metabolism/pharmacology ; CRISPR-Cas Systems ; Capsid Proteins/*metabolism ; Cell Line ; Foot-and-Mouth Disease Virus/*drug effects ; Gene Knockout Techniques ; HEK293 Cells ; HSP40 Heat-Shock Proteins/*antagonists & inhibitors/chemistry/genetics/*metabolism ; Host-Pathogen Interactions ; Humans ; Interferon Regulatory Factor-3 ; Interferon-beta/*metabolism ; Lysosomes/*metabolism ; Phosphorylation ; Proteasome Endopeptidase Complex ; Protein Interaction Domains and Motifs ; Signal Transduction/*drug effects ; Viral Proteins/metabolism ; Virus Replication/*drug effects ; }, abstract = {DnaJ heat shock protein family (Hsp40) member A3 (DNAJA3) plays an important role in viral infections. However, the role of DNAJA3 in replication of foot-and-mouth-disease virus (FMDV) remains unknown. In this study, DNAJA3, a novel binding partner of VP1, was identified using yeast two-hybrid screening. The DNAJA3-VP1 interaction was further confirmed by coimmunoprecipitation and colocalization in FMDV-infected cells. The J domain of DNAJA3 (amino acids 1 to 168) and the lysine at position 208 (K208) of VP1 were shown to be critical for the DNAJA3-VP1 interaction. Overexpression of DNAJA3 dramatically dampened FMDV replication, whereas loss of function of DNAJA3 elicited opposing effects against FMDV replication. Mechanistical study demonstrated that K208 of VP1 was critical for reducing virus titer caused by DNAJA3 using K208A mutant virus. DNAJA3 induced lysosomal degradation of VP1 by interacting with LC3 to enhance the activation of lysosomal pathway. Meanwhile, we discovered that VP1 suppressed the beta interferon (IFN-β) signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. This inhibitory effect was considerably boosted in DNAJA3-knockout cells. In contrast, overexpression of DNAJA3 markedly attenuated VP1-mediated suppression on the IFN-β signaling pathway. Poly(I⋅C)-induced phosphorylation of IRF3 was also decreased in DNAJA3-knockout cells compared to that in the DNAJA3-WT cells. In conclusion, our study described a novel role for DNAJA3 in the host's antiviral response by inducing the lysosomal degradation of VP1 and attenuating the VP1-induced suppressive effect on the IFN-β signaling pathway.IMPORTANCE This study pioneeringly determined the antiviral role of DNAJA3 in FMDV. DNAJA3 was found to interact with FMDV VP1 and trigger its degradation via the lysosomal pathway. In addition, this study is also the first to clarify the mechanism by which VP1 suppressed IFN-β signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. Moreover, DNAJA3 significantly abrogated VP1-induced inhibitive effect on the IFN-β signaling pathway. These data suggested that DNAJA3 plays an important antiviral role against FMDV by both degrading VP1 and restoring of IFN-β signaling pathway.}, }
@article {pmid32459325, year = {2020}, author = {Wang, J and Dai, W and Li, J and Xie, R and Dunstan, RA and Stubenrauch, C and Zhang, Y and Lithgow, T}, title = {PaCRISPR: a server for predicting and visualizing anti-CRISPR proteins.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkaa432}, pmid = {32459325}, issn = {1362-4962}, abstract = {Anti-CRISPRs are widespread amongst bacteriophage and promote bacteriophage infection by inactivating the bacterial host's CRISPR-Cas defence system. Identifying and characterizing anti-CRISPR proteins opens an avenue to explore and control CRISPR-Cas machineries for the development of new CRISPR-Cas based biotechnological and therapeutic tools. Past studies have identified anti-CRISPRs in several model phage genomes, but a challenge exists to comprehensively screen for anti-CRISPRs accurately and efficiently from genome and metagenome sequence data. Here, we have developed an ensemble learning based predictor, PaCRISPR, to accurately identify anti-CRISPRs from protein datasets derived from genome and metagenome sequencing projects. PaCRISPR employs different types of feature recognition united within an ensemble framework. Extensive cross-validation and independent tests show that PaCRISPR achieves a significantly more accurate performance compared with homology-based baseline predictors and an existing toolkit. The performance of PaCRISPR was further validated in discovering anti-CRISPRs that were not part of the training for PaCRISPR, but which were recently demonstrated to function as anti-CRISPRs for phage infections. Data visualization on anti-CRISPR relationships, highlighting sequence similarity and phylogenetic considerations, is part of the output from the PaCRISPR toolkit, which is freely available at http://pacrispr.erc.monash.edu/.}, }
@article {pmid32455882, year = {2020}, author = {Sledzinski, P and Nowaczyk, M and Olejniczak, M}, title = {Computational Tools and Resources Supporting CRISPR-Cas Experiments.}, journal = {Cells}, volume = {9}, number = {5}, pages = {}, doi = {10.3390/cells9051288}, pmid = {32455882}, issn = {2073-4409}, support = {2018/29/B/NZ1/00293//National Science Center, Poland/ ; }, abstract = {The CRISPR-Cas system has become a cutting-edge technology that revolutionized genome engineering. The use of Cas9 nuclease is currently the method of choice in most tasks requiring a specific DNA modification. The rapid development in the field of CRISPR-Cas is reflected by the constantly expanding ecosystem of computational tools aimed at facilitating experimental design and result analysis. The first group of CRISPR-Cas-related tools that we review is dedicated to aid in guide RNA design by prediction of their efficiency and specificity. The second, relatively new group of tools exploits the observed biases in repair outcomes to predict the results of CRISPR-Cas edits. The third class of tools is developed to assist in the evaluation of the editing outcomes by analysis of the sequencing data. These utilities are accompanied by relevant repositories and databases. Here we present a comprehensive and updated overview of the currently available CRISPR-Cas-related tools, from the perspective of a user who needs a convenient and reliable means to facilitate genome editing experiments at every step, from the guide RNA design to analysis of editing outcomes. Moreover, we discuss the current limitations and challenges that the field must overcome for further improvement in the CRISPR-Cas endeavor.}, }
@article {pmid31894108, year = {2019}, author = {Chen, Z and Song, Z and Yang, J and Huang, J and Jiang, H}, title = {Sp7/osterix positively regulates dlx2b and bglap to affect tooth development and bone mineralization in zebrafish larvae.}, journal = {Journal of biosciences}, volume = {44}, number = {6}, pages = {}, pmid = {31894108}, issn = {0973-7138}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Calcification, Physiologic/genetics ; Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; Homeodomain Proteins/genetics ; Humans ; Larva/genetics/growth & development ; Osteocalcin/genetics ; Osteogenesis/*genetics ; Sp7 Transcription Factor/*genetics ; Tooth/growth & development ; Transcription Factors/genetics ; Zebrafish/*genetics/growth & development ; Zebrafish Proteins/*genetics ; }, abstract = {Osterix (or Sp7) is an important transcription factor that promotes osteoblast differentiation by modulating the expression of a range of target genes. Although many studies have focused on Osterix/Sp7 regulatory mechanisms, the detailed functions have not been fully elucidated. Toward this end, in this study, we used CRISPR/Cas9 technology to knock out the zebrafish sp7 gene, and then analyzed its phenotype and biological function. Two knockout sp7 mutant lines were successfully obtained. The bone mineralization level was significantly reduced in the zebrafish sp7-/- homozygote, resulting in abnormal tooth development in the larvae. Quantitative real-time polymerase chain reaction showed that loss of sp7 led to down-regulated expression of the dlx2b and bglap genes related to tooth development and bone mineralization, respectively. Moreover, cell transfection experiments demonstrated that Sp7 directly regulates the expression of dlx2b and bglap through Sp7-binding sites on the promoter regions of these two genes. Overall, this study provides new insight into the role of Sp7 in bone mineralization and tooth development.}, }
@article {pmid31761532, year = {2019}, author = {Morton, AR and Dogan-Artun, N and Faber, ZJ and MacLeod, G and Bartels, CF and Piazza, MS and Allan, KC and Mack, SC and Wang, X and Gimple, RC and Wu, Q and Rubin, BP and Shetty, S and Angers, S and Dirks, PB and Sallari, RC and Lupien, M and Rich, JN and Scacheri, PC}, title = {Functional Enhancers Shape Extrachromosomal Oncogene Amplifications.}, journal = {Cell}, volume = {179}, number = {6}, pages = {1330-1341.e13}, pmid = {31761532}, issn = {1097-4172}, support = {R01 CA169117/CA/NCI NIH HHS/United States ; R01 CA204279/CA/NCI NIH HHS/United States ; R01 CA160356/CA/NCI NIH HHS/United States ; TL1 TR000441/TR/NCATS NIH HHS/United States ; R01 DA043980/DA/NIDA NIH HHS/United States ; R01 CA143237/CA/NCI NIH HHS/United States ; R01 NS087913/NS/NINDS NIH HHS/United States ; R01 NS103434/NS/NINDS NIH HHS/United States ; R01 CA193677/CA/NCI NIH HHS/United States ; R01 CA171652/CA/NCI NIH HHS/United States ; R01 NS089272/NS/NINDS NIH HHS/United States ; F30 CA236313/CA/NCI NIH HHS/United States ; R35 CA197718/CA/NCI NIH HHS/United States ; T32 GM007250/GM/NIGMS NIH HHS/United States ; R01 CA154130/CA/NCI NIH HHS/United States ; }, mesh = {Acetylation ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/genetics ; Chromatin/metabolism ; Chromosomes, Human/*genetics ; DNA, Neoplasm/genetics ; *Enhancer Elements, Genetic ; ErbB Receptors/genetics/metabolism ; *Gene Amplification ; Genes, Neoplasm ; Genetic Loci ; Glioblastoma/genetics/pathology ; Histones/metabolism ; Humans ; Neuroglia/metabolism ; *Oncogenes ; }, abstract = {Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.}, }
@article {pmid31703769, year = {2019}, author = {Gentner, B and Naldini, L}, title = {In Vivo Selection for Gene-Corrected HSPCs Advances Gene Therapy for a Rare Stem Cell Disease.}, journal = {Cell stem cell}, volume = {25}, number = {5}, pages = {592-593}, doi = {10.1016/j.stem.2019.10.004}, pmid = {31703769}, issn = {1875-9777}, mesh = {CRISPR-Cas Systems ; DNA Breaks ; *Fanconi Anemia ; Genetic Therapy ; Genetic Vectors ; *Hematopoietic Stem Cell Transplantation ; Humans ; Lentivirus/genetics ; Mutation ; }, abstract = {Two recent papers (one by Román-Rodríguez et al., 2019 in this issue of Cell Stem Cell) highlight how the power of biological selection on hematopoietic stem cell fitness can facilitate gene therapies for Fanconi Anemia. A clinical trial using lentiviral gene replacement and a proof-of-concept targeted genome editing study show robust engraftment and expansion of gene-corrected cells at levels reaching therapeutic relevance.}, }
@article {pmid31662324, year = {2019}, author = {Chen, G and Park, D and Magis, AT and Behera, M and Ramalingam, SS and Owonikoko, TK and Sica, GL and Ye, K and Zhang, C and Chen, Z and Curran, WJ and Deng, X}, title = {Mcl-1 Interacts with Akt to Promote Lung Cancer Progression.}, journal = {Cancer research}, volume = {79}, number = {24}, pages = {6126-6138}, pmid = {31662324}, issn = {1538-7445}, support = {R01 CA136534/CA/NCI NIH HHS/United States ; R01 CA193828/CA/NCI NIH HHS/United States ; R01 CA200905/CA/NCI NIH HHS/United States ; P50 CA217691/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Carcinoma, Non-Small-Cell Lung/drug therapy/mortality/*pathology ; Cell Line, Tumor ; Disease Progression ; Follow-Up Studies ; Gene Knockout Techniques ; Humans ; Kaplan-Meier Estimate ; Lung/pathology ; Lung Neoplasms/drug therapy/mortality/*pathology ; Male ; Mice ; Molecular Docking Simulation ; Myeloid Cell Leukemia Sequence 1 Protein/genetics/*metabolism ; Prognosis ; Protein Binding/drug effects ; Protein Domains/drug effects ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors/*metabolism ; Signal Transduction/drug effects ; Tissue Array Analysis ; Xenograft Model Antitumor Assays ; }, abstract = {Mcl-1 is a unique antiapoptotic Bcl2 family protein that functions as a gatekeeper in manipulating apoptosis and survival in cancer cells. Akt is an oncogenic kinase that regulates multiple cellular functions and its activity is significantly elevated in human cancers. Here we discovered a cross-talk between Mcl-1 and Akt in promoting lung cancer cell growth. Depletion of endogenous Mcl-1 from human lung cancer cells using CRISPR/Cas9 or Mcl-1 shRNA significantly decreased Akt activity, leading to suppression of lung cancer cell growth in vitro and in xenografts. Mechanistically, Mcl-1 directly interacted via its PEST domain with Akt at the pleckstrin homology (PH) domain. It is known that the interactions between the PH domain and kinase domain (KD) are important for maintaining Akt in an inactive state. The binding of Mcl-1/PH domain disrupted intramolecular PH/KD interactions to activate Akt. Intriguingly, Mcl-1 expression correlated with Akt activity in tumor tissues from patients with non-small cell lung cancer. Using the Mcl-1-binding PH domain of Akt as a docking site, we identified a novel small molecule, PH-687, that directly targets the PH domain and disrupts Mcl-1/Akt binding, leading to suppression of Akt activity and growth inhibition of lung cancer in vitro and in vivo. By targeting the Mcl-1/Akt interaction, this mechanism-driven agent provides a highly attractive strategy for the treatment of lung cancer. SIGNIFICANCE: These findings indicate that targeting Mcl-1/Akt interaction by employing small molecules such as PH-687 represents a potentially new and effective strategy for cancer treatment.}, }
@article {pmid31201381, year = {2019}, author = {Memczak, S and Shao, Y and Izpisua Belmonte, JC}, title = {Towards precise, safe genome editing.}, journal = {Cell research}, volume = {29}, number = {9}, pages = {687-689}, pmid = {31201381}, issn = {1748-7838}, mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *Gene Editing ; RNA Editing ; Transcriptome ; }, }
@article {pmid31006600, year = {2019}, author = {Bryant, JM and Baumgarten, S and Glover, L and Hutchinson, S and Rachidi, N}, title = {CRISPR in Parasitology: Not Exactly Cut and Dried!.}, journal = {Trends in parasitology}, volume = {35}, number = {6}, pages = {409-422}, doi = {10.1016/j.pt.2019.03.004}, pmid = {31006600}, issn = {1471-5007}, mesh = {Animals ; *CRISPR-Cas Systems ; Congresses as Topic ; Humans ; Parasites/genetics ; Parasitology/*trends ; Research/trends ; }, abstract = {CRISPR/Cas9 technology has been developing rapidly in the field of parasitology, allowing for the dissection of molecular processes with unprecedented efficiency. Optimization and implementation of a new technology like CRISPR, especially in nonmodel organisms, requires communication and collaboration throughout the field. Recently, a 'CRISPR in Parasitology' symposium was held at the Institut Pasteur Paris, bringing together scientists studying Leishmania, Plasmodium, Trypanosoma, and Anopheles. Here we share technological advances and challenges in using CRISPR/Cas9 in the parasite and vector systems that were discussed. As CRISPR/Cas9 continues to be applied to diverse parasite systems, the community should now focus on improvement and standardization of the technique as well as expanding the CRISPR toolkit to include Cas9 alternatives/derivatives for more advanced applications like genome-wide functional screens.}, }
@article {pmid30648968, year = {2019}, author = {Voss, JE and Gonzalez-Martin, A and Andrabi, R and Fuller, RP and Murrell, B and McCoy, LE and Porter, K and Huang, D and Li, W and Sok, D and Le, K and Briney, B and Chateau, M and Rogers, G and Hangartner, L and Feeney, AJ and Nemazee, D and Cannon, P and Burton, DR}, title = {Reprogramming the antigen specificity of B cells using genome-editing technologies.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {30648968}, issn = {2050-084X}, support = {5R01DE025167-05/NH/NIH HHS/United States ; UM1 AI100663/AI/NIAID NIH HHS/United States ; MR/R008698/1/MRC_/Medical Research Council/United Kingdom ; UL1 TR001114/TR/NCATS NIH HHS/United States ; R01 AI073148/AI/NIAID NIH HHS/United States ; R01 DE025167/DE/NIDCR NIH HHS/United States ; FP7-PEOPLE-2013-IOF//FP7 People: Marie-Curie Actions/International ; Ramón y Cajal Merit Award RYC-2016-21155//Ministerio de Ciencia, Innovacion y Universidades/International ; R37 AI059714/AI/NIAID NIH HHS/United States ; RYC-2016-21155//Ramón y Cajal Merit Award, Ministerio de Ciencia, Innovacion y Universidades/International ; FP7-PEOPLE-2013-IOF//Marie-Curie Fellowship/International ; OPP1183956//Bill and Melinda Gates Foundation/International ; }, mesh = {Antibodies, Neutralizing/immunology ; Antibody Specificity ; Antigen-Antibody Reactions/*genetics ; B-Lymphocytes/*immunology ; CRISPR-Cas Systems ; Cell Line ; Cytidine Deaminase/metabolism ; Gene Editing/*methods ; HIV Antibodies/immunology ; Humans ; Immunoglobulin Heavy Chains/genetics ; Receptors, Antigen, B-Cell/genetics/immunology ; }, abstract = {We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.}, }
@article {pmid32453626, year = {2020}, author = {Reimann, V and Ziemann, M and Li, H and Zhu, T and Behler, J and Lu, X and Hess, WR}, title = {Specificities and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups.}, journal = {RNA biology}, volume = {}, number = {}, pages = {}, doi = {10.1080/15476286.2020.1774197}, pmid = {32453626}, issn = {1555-8584}, abstract = {Many bacteria and archaea possess an RNA-guided adaptive and inheritable immune system that consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. In most CRISPR-Cas systems, the maturation of CRISPR-derived small RNAs (crRNAs) is essential for functionality. Cas6 endonucleases function as the most frequent CRISPR RNA maturation enzymes. In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR loci are present, but only two cas gene cassettes plus a Tn7-associated eleventh array. In this study, we deleted the two cas6 genes alr1482 (Type III-D) or alr1566 (Type I-D) and tested the specificities of the two corresponding enzymes in the resulting mutant strains, as recombinant proteins and in a cell-free transcription-translation system. The results assign the direct repeats (DRs) to three different groups. While Alr1566 is specific for one group, Alr1482 has a higher preference for the DRs of the second group but can also cleave those of the first group. We found that this cross-recognition limits crRNA accumulation for the Type I-D system in vivo. We also show that the DR of the cas gene-free CRISPR array of cyanophage N-1 is processed by these enzymes, suggesting that it is fully competent in association with host-encoded Cas proteins. The data support the functionality of CRISPR arrays that frequently appear fragmented to multiple genomic loci in multicellular cyanobacteria and disfavor other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our results show the functional coordination of Cas6 endonucleases with both neighboring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria.}, }
@article {pmid32450802, year = {2020}, author = {Wada, N and Ueta, R and Osakabe, Y and Osakabe, K}, title = {Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering.}, journal = {BMC plant biology}, volume = {20}, number = {1}, pages = {234}, doi = {10.1186/s12870-020-02385-5}, pmid = {32450802}, issn = {1471-2229}, support = {JP19H02932//Japan Society for the Promotion of Science/ ; }, abstract = {Traditionally, generation of new plants with improved or desirable features has relied on laborious and time-consuming breeding techniques. Genome-editing technologies have led to a new era of genome engineering, enabling an effective, precise, and rapid engineering of the plant genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a new genome-editing tool, extensively applied in various organisms, including plants. The use of CRISPR/Cas9 allows generating transgene-free genome-edited plants ("null segregants") in a short period of time. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9 derived technologies for inducing mutations at target sites in the genome and controlling the expression of target genes. We highlight the major breakthroughs in applying CRISPR/Cas9 to plant engineering, and challenges toward the production of null segregants. We also provide an update on the efforts of engineering Cas9 proteins, newly discovered Cas9 variants, and novel CRISPR/Cas systems for use in plants. The application of CRISPR/Cas9 and related technologies in plant engineering will not only facilitate molecular breeding of crop plants but also accelerate progress in basic research.}, }
@article {pmid32132707, year = {2020}, author = {Guo, X and Aviles, G and Liu, Y and Tian, R and Unger, BA and Lin, YT and Wiita, AP and Xu, K and Correia, MA and Kampmann, M}, title = {Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway.}, journal = {Nature}, volume = {579}, number = {7799}, pages = {427-432}, doi = {10.1038/s41586-020-2078-2}, pmid = {32132707}, issn = {1476-4687}, support = {1S10OD010786-01/NH/NIH HHS/United States ; R01 GM044037/GM/NIGMS NIH HHS/United States ; R01 DK026506/DK/NIDDK NIH HHS/United States ; DP2 GM123500/GM/NIGMS NIH HHS/United States ; DK26506/NH/NIH HHS/United States ; OD022552/NH/NIH HHS/United States ; GM44037/NH/NIH HHS/United States ; DP2 GM119139/GM/NIGMS NIH HHS/United States ; GM119139/NH/NIH HHS/United States ; }, mesh = {Activating Transcription Factor 4/biosynthesis/metabolism ; CRISPR-Cas Systems ; Cell Line ; Cytosol/enzymology/*metabolism ; Enzyme Activation ; Eukaryotic Initiation Factor-2/metabolism ; Humans ; Male ; Metalloendopeptidases/*metabolism ; Mitochondria/*metabolism/*pathology ; Mitochondrial Proteins/chemistry/*metabolism ; Molecular Chaperones/metabolism ; Phosphorylation ; Protein Binding ; *Stress, Physiological ; eIF-2 Kinase/*metabolism ; }, abstract = {In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF41-3. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.}, }
@article {pmid32111843, year = {2020}, author = {de Jong, OG and Murphy, DE and Mäger, I and Willms, E and Garcia-Guerra, A and Gitz-Francois, JJ and Lefferts, J and Gupta, D and Steenbeek, SC and van Rheenen, J and El Andaloussi, S and Schiffelers, RM and Wood, MJA and Vader, P}, title = {A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {1113}, doi = {10.1038/s41467-020-14977-8}, pmid = {32111843}, issn = {2041-1723}, support = {BB/M024393/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Biological Transport ; *CRISPR-Cas Systems ; Cell Communication ; Cell Line ; Endocytosis/genetics ; Extracellular Vesicles/genetics/*metabolism ; Fluorescence ; Genes, Reporter/genetics ; HEK293 Cells ; Humans ; RNA, Guide/genetics/metabolism ; RNA, Small Untranslated/genetics/*metabolism ; }, abstract = {Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.}, }
@article {pmid31749445, year = {2019}, author = {Harding, HP and Ordonez, A and Allen, F and Parts, L and Inglis, AJ and Williams, RL and Ron, D}, title = {The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {31749445}, issn = {2050-084X}, support = {C14801/A21211/CRUK_/Cancer Research UK/United Kingdom ; Wellcome 100140//Wellcome/International ; Wellcome 200848/Z/16/Z//Wellcome/International ; }, mesh = {Amino Acids/*metabolism ; Animals ; CHO Cells ; CRISPR-Cas Systems ; Cricetulus ; Endoplasmic Reticulum/metabolism ; Gene Expression Regulation, Enzymologic ; HeLa Cells ; Humans ; Kinetics ; Ligands ; Mice ; Models, Molecular ; Mutagenesis ; Phosphorylation ; Protein Binding ; Protein Conformation ; Protein Unfolding ; Protein-Serine-Threonine Kinases/chemistry/genetics/*metabolism ; RNA, Transfer/metabolism ; Ribosomes/chemistry/*metabolism ; Signal Transduction ; Starvation/*metabolism ; Transcriptome ; eIF-2 Kinase/genetics/metabolism ; }, abstract = {The eukaryotic translation initiation factor 2α (eIF2α) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2α kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2α phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation.}, }
@article {pmid31741433, year = {2019}, author = {Zeng, H and Castillo-Cabrera, J and Manser, M and Lu, B and Yang, Z and Strande, V and Begue, D and Zamponi, R and Qiu, S and Sigoillot, F and Wang, Q and Lindeman, A and Reece-Hoyes, JS and Russ, C and Bonenfant, D and Jiang, X and Wang, Y and Cong, F}, title = {Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {31741433}, issn = {2050-084X}, mesh = {A549 Cells ; Adaptor Proteins, Signal Transducing/metabolism ; Animals ; *CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*genetics ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cullin Proteins ; ErbB Receptors/genetics ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Guanine Nucleotide Exchange Factors/genetics ; HEK293 Cells ; Humans ; Methionyl Aminopeptidases/metabolism ; Mice ; Mice, Nude ; Receptors, Lysophosphatidic Acid/metabolism ; Signal Transduction ; Transcription Factors/metabolism ; Transcriptome ; Ubiquitin-Protein Ligases/genetics ; rhoA GTP-Binding Protein/metabolism ; }, abstract = {EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses.}, }
@article {pmid31636095, year = {2019}, author = {Kang, Y and Chu, C and Wang, F and Niu, Y}, title = {CRISPR/Cas9-mediated genome editing in nonhuman primates.}, journal = {Disease models & mechanisms}, volume = {12}, number = {10}, pages = {}, pmid = {31636095}, issn = {1754-8411}, mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Humans ; Mosaicism ; Mutation/genetics ; Primates/*genetics ; }, abstract = {Owing to their high similarity to humans, non-human primates (NHPs) provide an exceedingly suitable model for the study of human disease. In this Review, we summarize the history of transgenic NHP models and the progress of CRISPR/Cas9-mediated genome editing in NHPs, from the first proof-of-principle green fluorescent protein-expressing monkeys to sophisticated NHP models of human neurodegenerative disease that accurately phenocopy several complex disease features. We discuss not only the breakthroughs and advantages, but also the potential shortcomings of the application of the CRISPR/Cas9 system to NHPs that have emerged from the expanded understanding of this technology in recent years. Although off-target and mosaic mutations are the main concerns in CRISPR/Cas9-mediated NHP modeling, recent progress in genome editing techniques make it likely that these technical limitations will be overcome soon, bringing excellent prospects to human disease studies.}, }
@article {pmid31494407, year = {2019}, author = {Mansourian, S and Fandino, RA and Riabinina, O}, title = {Progress in the use of genetic methods to study insect behavior outside Drosophila.}, journal = {Current opinion in insect science}, volume = {36}, number = {}, pages = {45-56}, doi = {10.1016/j.cois.2019.08.001}, pmid = {31494407}, issn = {2214-5753}, mesh = {Animals ; Appetitive Behavior ; *Behavior, Animal ; CRISPR-Cas Systems ; Insecta/*genetics/*physiology ; Mutagenesis, Site-Directed ; Oviposition ; RNA Interference ; Social Behavior ; }, abstract = {In the span of a decade we have seen a rapid progress in the application of genetic tools and genome editing approaches in 'non-model' insects. It is now possible to target sensory receptor genes and neurons, explore their functional roles and manipulate behavioral responses in these insects. In this review, we focus on the latest examples from Diptera, Lepidoptera and Hymenoptera of how applications of genetic tools advanced our understanding of diverse behavioral phenomena. We further discuss genetic methods that could be applied to study insect behavior in the future.}, }
@article {pmid31391075, year = {2019}, author = {Pacini, V and Petit, F and Querat, B and Laverriere, JN and Cohen-Tannoudji, J and L'hôte, D}, title = {Identification of a pituitary ERα-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor.}, journal = {Epigenetics &amp; chromatin}, volume = {12}, number = {1}, pages = {48}, pmid = {31391075}, issn = {1756-8935}, support = {Allocation de recherche 2018//Société Française d'Endocrinologie/International ; }, mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Chromatin/metabolism ; Chromatin Assembly and Disassembly ; DNA-Directed RNA Polymerases/metabolism ; Enhancer Elements, Genetic ; Estrogen Receptor alpha/*metabolism ; Gonadotrophs/cytology/metabolism ; Histones/metabolism ; Humans ; Mice ; Pituitary Gland/growth &amp; development/metabolism ; Promoter Regions, Genetic ; Sequence Alignment ; Steroidogenic Factor 1/genetics/*metabolism ; Transcription, Genetic ; }, abstract = {BACKGROUND: Gonadotrope lineage differentiation is a stepwise process taking place during pituitary development. The early step of gonadotrope lineage specification is characterized by the expression of the Nr5a1 transcription factor, a crucial factor for gonadotrope cell fate determination. Abnormalities affecting Nr5a1 expression lead to hypogonadotropic hypogonadism and infertility. Although significant knowledge has been gained on the signaling and transcriptional events controlling gonadotrope differentiation, epigenetic mechanisms regulating Nr5a1 expression during early gonadotrope lineage specification are still poorly understood.<br><br>RESULTS: Using ATAC chromatin accessibility analyses on three cell lines recapitulating gradual stages of gonadotrope differentiation and in vivo on developing pituitaries, we demonstrate that a yet undescribed enhancer is transiently recruited during gonadotrope specification. Using CRISPR/Cas9, we show that this enhancer is mandatory for the emergence of Nr5a1 during gonadotrope specification. Furthermore, we identify a highly conserved estrogen-binding element and demonstrate that the enhancer activation is dependent upon estrogen acting through ERα. Lastly, we provide evidence that binding of ERα is crucial for chromatin remodeling of Nr5a1 enhancer and promoter, leading to RNA polymerase recruitment and transcription.<br><br>CONCLUSION: This study identifies the earliest regulatory sequence involved in gonadotrope lineage specification and highlights the key epigenetic role played by ERα in this differentiation process.}, }
@article {pmid31266541, year = {2019}, author = {Schneider, D and Chua, RL and Molitor, N and Hamdan, FH and Rettenmeier, EM and Prokakis, E and Mishra, VK and Kari, V and Wegwitz, F and Johnsen, SA and Kosinsky, RL}, title = {The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells.}, journal = {Clinical epigenetics}, volume = {11}, number = {1}, pages = {98}, pmid = {31266541}, issn = {1868-7083}, mesh = {Apoptosis ; CRISPR-Cas Systems ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms/*genetics ; Epigenesis, Genetic ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Neoplastic ; *Gene Silencing ; HCT116 Cells ; HT29 Cells ; Histones/metabolism ; Humans ; Ubiquitin-Protein Ligases/*genetics ; Ubiquitination ; }, abstract = {BACKGROUND: Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The loss of monoubiquitinated histone H2B (H2Bub1) was correlated with poor prognosis of CRC patients and, accordingly, H2Bub1 was suggested as a tumor-suppressive mark. Surprisingly, our previous work revealed that the H2B ubiquitin ligase RING finger protein 40 (RNF40) might exert tumor-promoting functions. Here, we investigated the effect of RNF40 loss on tumorigenic features of CRC cells and their survival in vitro.<br><br>METHODS: We evaluated the effects of RNF40 depletion in several human CRC cell lines in vitro. To evaluate cell cycle progression, cells were stained with propidium iodide and analyzed by flow cytometry. In addition, to assess apoptosis rates, caspase 3/7 activity was assessed in a Celigo® S-based measurement and, additionally, an Annexin V assay was performed. Genomic occupancy of H2Bub1, H3K79me3, and H3K27ac was determined by chromatin immunoprecipitation. Transcriptome-wide effects of RNF40 loss were evaluated based on mRNA-seq results, qRT-PCR, and Western blot. To rescue apoptosis-related effects, cells were treated with Z-VAD-FMK.<br><br>RESULTS: Human CRC cell lines displayed decreased cell numbers in vitro after RNF40 depletion. While the differences in confluence were not mediated by changes in cell cycle progression, we discovered highly increased apoptosis rates after RNF40 knockdown due to elevated caspase 3/7 activity. This effect can be explained by reduced mRNA levels of anti-apoptotic and upregulation of pro-apoptotic BCL2 family members. Moreover, the direct occupancy of the RNF40-mediated H2B monoubiquitination was observed in the transcribed region of anti-apoptotic genes. Caspase inhibition by Z-VAD-FMK treatment rescued apoptosis in RNF40-depleted cells. However, knockdown cells still displayed decreased tumorigenic features despite the absence of apoptosis.<br><br>CONCLUSIONS: Our findings reveal that RNF40 is essential for maintaining tumorigenic features of CRC cells in vitro by controlling the expression of genes encoding central apoptotic regulators.}, }
@article {pmid31217031, year = {2019}, author = {Sen, M and Wang, X and Hamdan, FH and Rapp, J and Eggert, J and Kosinsky, RL and Wegwitz, F and Kutschat, AP and Younesi, FS and Gaedcke, J and Grade, M and Hessmann, E and Papantonis, A and Strӧbel, P and Johnsen, SA}, title = {ARID1A facilitates KRAS signaling-regulated enhancer activity in an AP1-dependent manner in colorectal cancer cells.}, journal = {Clinical epigenetics}, volume = {11}, number = {1}, pages = {92}, pmid = {31217031}, issn = {1868-7083}, mesh = {CRISPR-Cas Systems ; Cell Proliferation ; Colorectal Neoplasms/*genetics/metabolism ; *DNA Methylation ; DNA-Binding Proteins/*genetics/*metabolism ; Enhancer Elements, Genetic ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Genome-Wide Association Study ; HCT116 Cells ; HT29 Cells ; Humans ; MAP Kinase Signaling System ; Mutation ; Proto-Oncogene Proteins p21(ras)/*genetics ; Signal Transduction ; Transcription Factor AP-1/*genetics ; Transcription Factors/*genetics/*metabolism ; }, abstract = {BACKGROUND: ARID1A (AT-rich interactive domain-containing protein 1A) is a subunit of the BAF chromatin remodeling complex and plays roles in transcriptional regulation and DNA damage response. Mutations in ARID1A that lead to inactivation or loss of expression are frequent and widespread across many cancer types including colorectal cancer (CRC). A tumor suppressor role of ARID1A has been established in a number of tumor types including CRC where the genetic inactivation of Arid1a alone led to the formation of invasive colorectal adenocarcinomas in mice. Mechanistically, ARID1A has been described to largely function through the regulation of enhancer activity.<br><br>METHODS: To mimic ARID1A-deficient colorectal cancer, we used CRISPR/Cas9-mediated gene editing to inactivate the ARID1A gene in established colorectal cancer cell lines. We integrated gene expression analyses with genome-wide ARID1A occupancy and epigenomic mapping data to decipher ARID1A-dependent transcriptional regulatory mechanisms.<br><br>RESULTS: Interestingly, we found that CRC cell lines harboring KRAS mutations are critically dependent on ARID1A function. In the absence of ARID1A, proliferation of these cell lines is severely impaired, suggesting an essential role for ARID1A in this context. Mechanistically, we showed that ARID1A acts as a co-factor at enhancers occupied by AP1 transcription factors acting downstream of the MEK/ERK pathway. Consistently, loss of ARID1A led to a disruption of KRAS/AP1-dependent enhancer activity, accompanied by a downregulation of expression of the associated target genes.<br><br>CONCLUSIONS: We identify a previously unknown context-dependent tumor-supporting function of ARID1A in CRC downstream of KRAS signaling. Upon the loss of ARID1A in KRAS-mutated cells, enhancers that are co-occupied by ARID1A and the AP1 transcription factors become inactive, thereby leading to decreased target gene expression. Thus, targeting of the BAF complex in KRAS-mutated CRC may offer a unique, previously unknown, context-dependent therapeutic option in CRC.}, }
@article {pmid31161803, year = {2019}, author = {Garcia-Leon, JA and Vitorica, J and Gutierrez, A}, title = {Use of human pluripotent stem cell-derived cells for neurodegenerative disease modeling and drug screening platform.}, journal = {Future medicinal chemistry}, volume = {11}, number = {11}, pages = {1305-1322}, doi = {10.4155/fmc-2018-0520}, pmid = {31161803}, issn = {1756-8927}, mesh = {Animals ; CRISPR-Cas Systems ; Drug Evaluation, Preclinical/*methods ; Gene Editing/methods ; Humans ; Neurodegenerative Diseases/*drug therapy/genetics/pathology ; Neurogenesis/drug effects ; Neurons/*cytology/drug effects/metabolism/pathology ; Pluripotent Stem Cells/*cytology/metabolism/pathology ; }, abstract = {Most neurodegenerative diseases are characterized by a complex and mostly still unresolved pathology. This fact, together with the lack of reliable disease models, has precluded the development of effective therapies counteracting the disease progression. The advent of human pluripotent stem cells has revolutionized the field allowing the generation of disease-relevant neural cell types that can be used for disease modeling, drug screening and, possibly, cell transplantation purposes. In this Review, we discuss the applications of human pluripotent stem cells, the development of efficient protocols for the derivation of the different neural cells and their applicability for robust in vitro disease modeling and drug screening platforms for most common neurodegenerative conditions.}, }
@article {pmid31128273, year = {2019}, author = {Ballard, E and Weber, J and Melchers, WJG and Tammireddy, S and Whitfield, PD and Brakhage, AA and Brown, AJP and Verweij, PE and Warris, A}, title = {Recreation of in-host acquired single nucleotide polymorphisms by CRISPR-Cas9 reveals an uncharacterised gene playing a role in Aspergillus fumigatus azole resistance via a non-cyp51A mediated resistance mechanism.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {130}, number = {}, pages = {98-106}, pmid = {31128273}, issn = {1096-0937}, support = {MR/M026663/1/MRC_/Medical Research Council/United Kingdom ; MR/N006364/1/MRC_/Medical Research Council/United Kingdom ; BB/M010996/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Antifungal Agents/pharmacology ; Aspergillosis/microbiology ; Aspergillus fumigatus/*drug effects/*genetics/growth &amp; development/isolation &amp; purification ; Azoles/*pharmacology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Multiple, Fungal/*genetics ; Ergosterol ; Fungal Proteins/genetics ; Genotype ; Host-Pathogen Interactions ; Humans ; Itraconazole/pharmacology ; Microbial Sensitivity Tests ; Mycelium/drug effects/growth &amp; development ; Phenotype ; *Polymorphism, Single Nucleotide ; }, abstract = {The human host comprises a range of specific niche environments. In order to successfully persist, pathogens such as Aspergillus fumigatus must adapt to these environments. One key example of in-host adaptation is the development of resistance to azole antifungals. Azole resistance in A. fumigatus is increasingly reported worldwide and the most commonly reported mechanisms are cyp51A mediated. Using a unique series of A. fumigatus isolates, obtained from a patient suffering from persistent and recurrent invasive aspergillosis over 2 years, this study aimed to gain insight into the genetic basis of in-host adaptation. Single nucleotide polymorphisms (SNPs) unique to a single isolate in this series, which had developed multi-azole resistance in-host, were identified. Two nonsense SNPs were recreated using CRISPR-Cas9; these were 213* in svf1 and 167* in uncharacterised gene AFUA_7G01960. Phenotypic analyses including antifungal susceptibility testing, mycelial growth rate assessment, lipidomics analysis and statin susceptibility testing were performed to associate genotypes to phenotypes. This revealed a role for svf1 in A. fumigatus oxidative stress sensitivity. In contrast, recapitulation of 167* in AFUA_7G01960 resulted in increased itraconazole resistance. Comprehensive lipidomics analysis revealed decreased ergosterol levels in strains containing this SNP, providing insight to the observed itraconazole resistance. Decreases in ergosterol levels were reflected in increased resistance to lovastatin and nystatin. Importantly, this study has identified a SNP in an uncharacterised gene playing a role in azole resistance via a non-cyp51A mediated resistance mechanism. This mechanism is of clinical importance, as this SNP was identified in a clinical isolate, which acquired azole resistance in-host.}, }
@article {pmid31048007, year = {2019}, author = {Schuster, M and Kahmann, R}, title = {CRISPR-Cas9 genome editing approaches in filamentous fungi and oomycetes.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {130}, number = {}, pages = {43-53}, doi = {10.1016/j.fgb.2019.04.016}, pmid = {31048007}, issn = {1096-0937}, mesh = {*CRISPR-Cas Systems ; Fungi/*genetics ; *Gene Editing ; Genome, Fungal ; Mutation ; Oomycetes/*genetics ; }, abstract = {Due to their biotechnological relevance as well as their importance as disease agents, filamentous fungi and oomycetes have been prime candidates for genetic selection and in vitro manipulation for decades. With the advent of new genome editing technologies such manipulations have reached a new level of speed and sophistication. The CRISPR-Cas9 genome editing technology in particular has revolutionized the ways how desired mutations can be introduced. To date, the CRISPR-Cas9 genome editing system has been established in more than 40 different species of filamentous fungi and oomycetes. In this review we describe the various approaches taken to assure expression of the components necessary for editing and describe the varying strategies used to achieve gene disruptions, gene replacements and precise editing. We discuss potential problems faced when establishing the system, propose ways to circumvent them and suggest future approaches not yet realized in filamentous fungi or oomycetes.}, }
@article {pmid31034868, year = {2019}, author = {Darma, R and Lutz, A and Elliott, CE and Idnurm, A}, title = {Identification of a gene cluster for the synthesis of the plant hormone abscisic acid in the plant pathogen Leptosphaeria maculans.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {130}, number = {}, pages = {62-71}, doi = {10.1016/j.fgb.2019.04.015}, pmid = {31034868}, issn = {1096-0937}, mesh = {Abscisic Acid/*metabolism ; Ascomycota/*genetics/*metabolism/pathogenicity ; Brassica napus/microbiology ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; Gene Expression Regulation, Fungal ; Genes, Fungal/*genetics ; Multigene Family/*genetics ; Plant Diseases/microbiology ; Plant Growth Regulators/*biosynthesis/*genetics ; Polyketide Synthases/genetics ; Secondary Metabolism ; Transcription Factors/genetics ; Up-Regulation ; Virulence/genetics ; }, abstract = {Leptosphaeria maculans is an ascomycetous fungus that causes the disease blackleg on Brassica napus (canola). In spite of the importance of the disease worldwide, the mechanisms of disease development are poorly understood. Secondary metabolites, which are one of the common virulence factors of pathogenic fungi, have not been extensively explored from this fungus. An RNA-seq dataset was examined to find genes responsible for secondary metabolite synthesis by this fungus during infection. One polyketide synthase gene, pks5, was found to be upregulated during the early biotrophic stage of development. In addition to pks5, six other genes adjacent to the pks5 gene, including one encoding a Zn(II)2Cys6 transcription factor abscisic acid-like 7 gene (abl7), were also upregulated during that time. A striking feature of the L. maculans genome is that it contains large AT-rich regions that are gene-poor and large GC-rich regions that are gene rich. This set of seven co-regulated genes is embedded within and separated by two such AT-rich regions. Three of the genes in the cluster have similarities to those known to be involved in the synthesis of abscisic acid (ABA) in other fungi. When L. maculans is grown in axenic culture the genes in this cluster are not expressed and ABA is not produced. Overexpressing abl7, encoding the putative transcription factor, resulted in the transcription of the six adjacent genes in axenic culture and in the production of ABA, as detected by liquid chromatography quadrupole-time-of-flight mass spectrometry analysis. Mutation of two genes of the cluster using CRISPR/Cas9 did not affect pathogenicity on canola cotyledons. The characterization of the ABA gene cluster has led to the discovery of the co-regulation of genes within an AT-rich region by a transcription factor, and the first report of the plant hormone abscisic acid being produced by L. maculans.}, }
@article {pmid31026589, year = {2019}, author = {Kunitake, E and Tanaka, T and Ueda, H and Endo, A and Yarimizu, T and Katoh, E and Kitamoto, H}, title = {CRISPR/Cas9-mediated gene replacement in the basidiomycetous yeast Pseudozyma antarctica.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {130}, number = {}, pages = {82-90}, doi = {10.1016/j.fgb.2019.04.012}, pmid = {31026589}, issn = {1096-0937}, mesh = {Base Sequence ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Fungal/genetics ; Fungal Proteins/genetics ; Gene Editing/*methods ; Gene Targeting/*methods ; Genes, Fungal/genetics ; Genetic Loci ; Homologous Recombination ; Ribonucleoproteins/genetics ; Transformation, Genetic ; Ustilaginales/*genetics ; }, abstract = {The basidiomycetous yeast, Pseudozyma antarctica, has the ability to express industrially beneficial biodegradable plastic-degrading enzyme (PaE) and glycolipids. In this study, we developed a highly efficient gene-targeting method in P. antarctica using a CRISPR/Cas9 gene-editing approach. Transformation of protoplast cells was achieved by incubation with a ribonucleoprotein (RNP) complex prepared by mixing the Cas9 protein with a single-guide RNA together with donor DNA (dDNA) containing a selectable marker in vitro. The PaE gene was selected as the targeted locus for gene disruption and gene-disrupted colonies were readily detected by their ability to degrade polybutylene succinate-co-adipate on solid media. The accuracy of the gene conversion event was confirmed by colony PCR. An increase in the RNP mix increased both transformation and gene disruption efficiencies. Examining the effect of the homology arm length of the dDNA revealed that dDNA with homology arms longer than 0.1 kb induced efficient homologous recombination in our system. Furthermore, this system was successful in another targeted locus, PaADE2. Following the creation of RNP-induced double-strand break of the chromosomal DNA, dDNA could be inserted into the target locus even in the absence of homology arms.}, }
@article {pmid30844313, year = {2019}, author = {Shi, Z and Xin, H and Tian, D and Lian, J and Wang, J and Liu, G and Ran, R and Shi, S and Zhang, Z and Shi, Y and Deng, Y and Hou, C and Chen, Y}, title = {Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {33}, number = {6}, pages = {6962-6968}, doi = {10.1096/fj.201802661R}, pmid = {30844313}, issn = {1530-6860}, mesh = {Abnormalities, Multiple/*genetics ; Albinism, Oculocutaneous/*genetics ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Female ; Genotype ; Heart Defects, Congenital/*genetics ; Heart Septal Defects, Atrial/*genetics ; Humans ; Lower Extremity Deformities, Congenital/*genetics ; Male ; *Point Mutation ; Upper Extremity Deformities, Congenital/*genetics ; Xenopus/*embryology ; }, abstract = {Precise single-base editing in Xenopus tropicalis would greatly expand the utility of this true diploid frog for modeling human genetic diseases caused by point mutations. Here, we report the efficient conversion of C-to-T or G-to-A in X. tropicalis using the rat apolipoprotein B mRNA editing enzyme catalytic subunit 1-XTEN-clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9) nickase-uracil DNA glycosylase inhibitor-nuclear localization sequence base editor [base editor 3 (BE3)]. Coinjection of guide RNA and the Cas9 mutant complex mRNA into 1-cell stage X. tropicalis embryos caused precise C-to-T or G-to-A substitution in 14 out of 19 tested sites with efficiencies of 5-75%, which allowed for easy establishment of stable lines. Targeting the conserved T-box 5 R237 and Tyr C28 residues in X. tropicalis with the BE3 system mimicked human Holt-Oram syndrome and oculocutaneous albinism type 1A, respectively. Our data indicate that BE3 is an easy and efficient tool for precise base editing in X. tropicalis.-Shi, Z., Xin, H., Tian, D., Lian, J., Wang, J., Liu, G., Ran, R., Shi, S., Zhang, Z., Shi, Y., Deng, Y., Hou, C., Chen, Y. Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.}, }
@article {pmid30844312, year = {2019}, author = {Ko, T and Li, S}, title = {Genome-wide screening identifies novel genes and biological processes implicated in cisplatin resistance.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {33}, number = {6}, pages = {7143-7154}, doi = {10.1096/fj.201801534RR}, pmid = {30844312}, issn = {1530-6860}, mesh = {Antineoplastic Agents/*pharmacology ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism ; Cell Line, Tumor ; Cisplatin/*pharmacology ; Drug Resistance, Neoplasm ; Eukaryotic Initiation Factor-4E/genetics/metabolism ; Gene Deletion ; Gene Expression Regulation, Neoplastic/*drug effects ; Genome-Wide Association Study ; Humans ; Melanocytes/drug effects ; Melanoma/*drug therapy ; Neurofibromin 2/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; Up-Regulation ; Verteporfin/pharmacology ; beta Catenin/genetics/metabolism ; }, abstract = {Cisplatin-based chemotherapeutic regimens are frequently used for treatments of solid tumors. However, tumor cells may have inherent or acquired cisplatin resistance, and the underlying mechanisms are largely unknown. We performed genome-wide screening of genes implicated in cisplatin resistance in A375 human melanoma cells. A substantial fraction of genes whose disruptions cause cisplatin sensitivity or resistance overlap with those whose disruptions lead to increased or decreased cell growth, respectively. Protein translation, mitochondrial respiratory chain complex assembly, signal recognition particle-dependent cotranslational protein targeting to membrane, and mRNA catabolic processes are the top biologic processes responsible for cisplatin sensitivity. In contrast, proteasome-mediated ubiquitin-dependent protein catabolic process, negative regulations of cellular catabolic process, and regulation of cellular protein localization are the top biologic processes responsible for cisplatin resistance. ZNRF3, a ubiquitin ligase known to be a target and negative feedback regulator of Wnt-β-catenin signaling, enhances cisplatin resistance in normal and melanoma cells independently of β-catenin. Ariadne-1 homolog (ARIH1), another ubiquitin ligase, also enhances cisplatin resistance in normal and melanoma cells. By regulating ARIH1, neurofibromin 2, a tumor suppressor, enhances cisplatin resistance in melanoma but not normal cells. Our results shed new lights on cisplatin resistance mechanisms and may be useful for development of cisplatin-related treatment strategies.-Ko, T., Li, S. Genome-wide screening identifies novel genes and biological processes implicated in cisplatin resistance.}, }
@article {pmid31877894, year = {2019}, author = {Bae, T and Kim, H and Kim, JH and Kim, YJ and Lee, SH and Ham, BJ and Hur, JK}, title = {Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences.}, journal = {Molecules (Basel, Switzerland)}, volume = {25}, number = {1}, pages = {}, pmid = {31877894}, issn = {1420-3049}, support = {NRF-2017R1E1A1A01074529//national research foundation of Korea/ ; }, mesh = {CRISPR-Cas Systems ; DNA, Complementary/genetics ; DNA, Intergenic/genetics ; ErbB Receptors/genetics ; Gene Editing/*methods ; Humans ; *Point Mutation ; }, abstract = {In CRISPR genome editing, CRISPR proteins form ribonucleoprotein complexes with guide RNAs to bind and cleave the target DNAs with complete sequence complementarity. CRISPR genome editing has a high potential for use in precision gene therapy for various diseases, including cancer and genetic disorders, which are caused by DNA mutations within the genome. However, several studies have shown that targeting the DNA via sequence complementarity is imperfect and subject to unintended genome editing of other genomic loci with similar sequences. These off-target problems pose critical safety issues in the therapeutic applications of CRISPR technology, with particular concerns in terms of the genome editing of pathogenic point mutations, where non-mutant alleles can become an off-target with only a one-base difference. In this study, we sought to assess a novel CRISPR genome editing technique that has been proposed to achieve a high specificity by positioning the mismatches within the protospacer adjacent motif (PAM) sequence. To this end, we compared the genome editing specificities of the PAM-based and conventional methods on an oncogenic single-base mutation in the endothelial growth factor receptor (EGFR). The results indicated that the PAM-based method provided a significantly increased genome editing specificity for pathogenic mutant alleles with single-base precision.}, }
@article {pmid30664728, year = {2019}, author = {Guo, M and Ren, K and Zhu, Y and Tang, Z and Wang, Y and Zhang, B and Huang, Z}, title = {Structural insights into a high fidelity variant of SpCas9.}, journal = {Cell research}, volume = {29}, number = {3}, pages = {183-192}, pmid = {30664728}, issn = {1748-7838}, mesh = {CRISPR-Associated Protein 9/*chemistry ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Crystallography, X-Ray ; DNA, Bacterial/*chemistry ; Gene Editing/methods ; Molecular Conformation ; RNA, Guide/*chemistry ; Streptococcus pyogenes/genetics ; }, abstract = {The RNA-guided endonucleases of the CRISPR-Cas9 system, including the most widely used Cas9 from Streptococcus pyogenes (SpCas9), are becoming a robust genome editing tool in model organisms and hold immense promise for therapeutic applications. Many strategies have been employed to overcome the limitations caused by SpCas9's off-target effects and its stringent requirement for the protospacer adjacent motif (PAM) sequence. However, the structural mechanisms underlying these strategies remain undefined. Here, we present crystal structure of a SpCas9 variant, xCas9 3.7 that has broad PAM compatibility and high DNA targeting specificity, in complex with a single-guide RNA and its double-stranded DNA targets. Structural comparison revealed that salt bridge-stabilized R1335 is critical for the stringent selection of PAM sequence by SpCas9. Unrestricted rotamerization of this residue by the E1219V mutation in xCas9 3.7 lessens the stringency for PAM recognition and allows SpCas9 to recognize multiple PAM sequences as further supported by biochemical data. Compared to those in wild-type (WT) SpCas9, REC2 and REC3 domains in xCas9 3.7 undergo striking conformational changes, leading to reduced contact with DNA substrate. SpCas9 mutants engineered to display less interaction with DNA and have conformationally more flexible REC2 and REC3 domains display enhanced specificity for DNA substrates in both biochemical and cellular assays. Taken together, our findings reveal the structural mechanisms underlying the broadened PAM compatibility and high DNA fidelity of xCas9 3.7, which can assist rational engineering of more efficient SpCas9 variants and probably other Cas9 orthologs.}, }
@article {pmid32439162, year = {2020}, author = {Kaneko, T and Tanaka, S}, title = {Improvement of genome editing by electroporation using embryos artificially removed cumulus cells in the oviducts.}, journal = {Biochemical and biophysical research communications}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.bbrc.2020.05.034}, pmid = {32439162}, issn = {1090-2104}, abstract = {Many genome-edited animals have been produced using the CRISPR/Cas system. Genome-edited strains were produced by introducing nucleases into pronuclear stage embryos. Recently, a new electroporation technique (TAKE: Technique for Animal Knockout system by Electroporation) was developed for the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. Furthermore, this method, which can introduce nucleases into intact embryos, enables genome editing of mouse embryos in the oviducts. However, the present protocol required improvements for low litter size and restriction of operation time. In this study, the influence on the development and genome editing of mouse embryos in the oviducts by electroporation and operation time was examined. The genome-editing rate was higher in the embryos electroporated at 16:00-17:00 (PM) (54%) on the following day of natural mating compared to that of embryos at 10:00-11:00 (AM) (27%). The embryos at AM formed a complex with cumulus cells, and cumulus cells were freed from embryos by treatment with hyalronidase before electroporation. The results showed that the genome-editing rate was significantly increased in the embryos treated with hyalronidase at AM, because the cumulus cells surrounding the embryos interfered with the introduction of nucleases into embryos. This study demonstrated that it was possible to adjust the operation time for the introduction of nucleases into embryos in the oviducts by treatment with hyalronidase before electroporation. However, litter size and development of embryos after electroporation was quite low in all experiments (5-7) compared with the control without operation (11).}, }
@article {pmid32439069, year = {2020}, author = {Hahn, E and Hiemenz, M}, title = {Therapeutic Gene Editing with CRISPR: A Laboratory Medicine Perspective.}, journal = {Clinics in laboratory medicine}, volume = {40}, number = {2}, pages = {205-219}, doi = {10.1016/j.cll.2020.02.008}, pmid = {32439069}, issn = {1557-9832}, abstract = {Therapeutic gene editing with the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system offers significant improvements in specificity and programmability compared with previous methods. CRISPR editing strategies can be used ex vivo and in vivo with many theoretic disease applications. Off-target effects of CRISPR-mediated gene editing are an important outcome to be aware of, minimize, and detect. The current methods of regulatory approval for personalized therapies are complex and may be proved inefficient as these therapies are implemented more widely. The role of pathologists and laboratory medicine practitioners is vital to the clinical implementation of therapeutic gene editing.}, }
@article {pmid31855178, year = {2019}, author = {Palit, SA and Vis, D and Stelloo, S and Lieftink, C and Prekovic, S and Bekers, E and Hofland, I and Šuštić, T and Wolters, L and Beijersbergen, R and Bergman, AM and Győrffy, B and Wessels, LF and Zwart, W and van der Heijden, MS}, title = {TLE3 loss confers AR inhibitor resistance by facilitating GR-mediated human prostate cancer cell growth.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {31855178}, issn = {2050-084X}, support = {NKI2014-7080//KWF Kankerbestrijding/International ; Alpe d'HuZes Bas Mulder Award//KWF Kankerbestrijding/International ; VIDI grant//NWO/International ; }, mesh = {Androgen Receptor Antagonists/pharmacology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Co-Repressor Proteins/*genetics ; Drug Resistance, Neoplasm/genetics ; Gene Expression Regulation, Neoplastic/drug effects ; HEK293 Cells ; Hepatocyte Nuclear Factor 3-alpha/*genetics ; Humans ; Male ; Phenylthiohydantoin/analogs &amp; derivatives/pharmacology ; Prostate/metabolism ; Prostatic Neoplasms/drug therapy/*genetics/pathology ; Receptors, Androgen/*genetics ; Receptors, Glucocorticoid/genetics ; Transcriptional Activation/drug effects ; }, abstract = {Androgen receptor (AR) inhibitors represent the mainstay of prostate cancer treatment. In a genome-wide CRISPR-Cas9 screen using LNCaP prostate cancer cells, loss of co-repressor TLE3 conferred resistance to AR antagonists apalutamide and enzalutamide. Genes differentially expressed upon TLE3 loss share AR as the top transcriptional regulator, and TLE3 loss rescued the expression of a subset of androgen-responsive genes upon enzalutamide treatment. GR expression was strongly upregulated upon AR inhibition in a TLE3-negative background. This was consistent with binding of TLE3 and AR at the GR locus. Furthermore, GR binding was observed proximal to TLE3/AR-shared genes. GR inhibition resensitized TLE3KO cells to enzalutamide. Analyses of patient samples revealed an association between TLE3 and GR levels that reflected our findings in LNCaP cells, of which the clinical relevance is yet to be determined. Together, our findings reveal a mechanistic link between TLE3 and GR-mediated resistance to AR inhibitors in human prostate cancer.}, }
@article {pmid30802454, year = {2019}, author = {Hayashida, G and Shioi, S and Hidaka, K and Fujikane, R and Hidaka, M and Tsurimoto, T and Tsuzuki, T and Oda, S and Nakatsu, Y}, title = {Differential genomic destabilisation in human cells with pathogenic MSH2 mutations introduced by genome editing.}, journal = {Experimental cell research}, volume = {377}, number = {1-2}, pages = {24-35}, doi = {10.1016/j.yexcr.2019.02.020}, pmid = {30802454}, issn = {1090-2422}, mesh = {*CRISPR-Cas Systems ; Colorectal Neoplasms, Hereditary Nonpolyposis/genetics/*pathology ; *Gene Editing ; Genomics/*methods ; HeLa Cells ; Humans ; *Microsatellite Instability ; MutS Homolog 2 Protein/*genetics ; *Mutation ; Phenotype ; }, abstract = {Repeat destabilisation is variously associated with human disease. In neoplastic diseases, microsatellite instability (MSI) has been regarded as simply reflecting DNA mismatch repair (MMR) deficiency. However, several discrepancies have been pointed out. Firstly, the MSI+ phenotype is not uniform in human neoplasms. Established classification utilises the frequency of microsatellite changes, i.e. MSI-H (high) and -L (low), the former regarded as an authentic MMR-defective phenotype. In addition, we have observed the qualitatively distinct modes of MSI, i.e. Type A and Type B. One discrepancy we previously pointed out is that tumours occurring in MMR gene knockout mice exhibited not drastic microsatellite changes typical in MSI-H tumours (i.e. Type B mode) but minor and more subtle alterations (i.e. Type A mode). In the present study, MSH2 mutations reported in Lynch syndrome (LS) kindred have been introduced into HeLa cells using the CRISPR/Cas9 system. The established mutant clones clearly exhibited MMR-defective phenotypes with alkylating agent-tolerance and elevated mutation frequencies. Nevertheless, microsatellites were not markedly destabilised as in MSI-H tumours occurring in LS patients, and all the observed alterations were uniformly Type A, which confirms the results in mice. Our findings suggest added complexities to the molecular mechanisms underlying repeat destabilisation in human genome.}, }
@article {pmid31742594, year = {2019}, author = {Pettingill, P and Weir, GA and Wei, T and Wu, Y and Flower, G and Lalic, T and Handel, A and Duggal, G and Chintawar, S and Cheung, J and Arunasalam, K and Couper, E and Haupt, LM and Griffiths, LR and Bassett, A and Cowley, SA and Cader, MZ}, title = {A causal role for TRESK loss of function in migraine mechanisms.}, journal = {Brain : a journal of neurology}, volume = {142}, number = {12}, pages = {3852-3867}, pmid = {31742594}, issn = {1460-2156}, support = {J-0901/PUK_/Parkinson's UK/United Kingdom ; MR/L023784/1/MRC_/Medical Research Council/United Kingdom ; MR/L023784/2/MRC_/Medical Research Council/United Kingdom ; MR/M024962/1/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; }, mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Humans ; Induced Pluripotent Stem Cells/metabolism ; *Loss of Function Mutation ; Mice ; Migraine Disorders/chemically induced/*genetics/metabolism ; Nitroglycerin ; Nociception/*physiology ; Nociceptors/*metabolism ; Pain Measurement ; Patch-Clamp Techniques ; Potassium Channels/*genetics/metabolism ; }, abstract = {The two-pore potassium channel, TRESK has been implicated in nociception and pain disorders. We have for the first time investigated TRESK function in human nociceptive neurons using induced pluripotent stem cell-based models. Nociceptors from migraine patients with the F139WfsX2 mutation show loss of functional TRESK at the membrane, with a corresponding significant increase in neuronal excitability. Furthermore, using CRISPR-Cas9 engineering to correct the F139WfsX2 mutation, we show a reversal of the heightened neuronal excitability, linking the phenotype to the mutation. In contrast we find no change in excitability in induced pluripotent stem cell derived nociceptors with the C110R mutation and preserved TRESK current; thereby confirming that only the frameshift mutation is associated with loss of function and a migraine relevant cellular phenotype. We then demonstrate the importance of TRESK to pain states by showing that the TRESK activator, cloxyquin, can reduce the spontaneous firing of nociceptors in an in vitro human pain model. Using the chronic nitroglycerine rodent migraine model, we demonstrate that mice lacking TRESK develop exaggerated nitroglycerine-induced mechanical and thermal hyperalgesia, and furthermore, show that cloxyquin conversely is able to prevent sensitization. Collectively, our findings provide evidence for a role of TRESK in migraine pathogenesis and its suitability as a therapeutic target.}, }
@article {pmid31692906, year = {2019}, author = {Jo, DH and Song, DW and Cho, CS and Kim, UG and Lee, KJ and Lee, K and Park, SW and Kim, D and Kim, JH and Kim, JS and Kim, S and Kim, JH and Lee, JM}, title = {CRISPR-Cas9-mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis.}, journal = {Science advances}, volume = {5}, number = {10}, pages = {eaax1210}, pmid = {31692906}, issn = {2375-2548}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Dependovirus/metabolism ; Disease Models, Animal ; *Gene Editing ; Genome ; Leber Congenital Amaurosis/*genetics/*therapy ; Male ; Mice ; Mice, Inbred C57BL ; Mutation/genetics ; Phenotype ; Recombinational DNA Repair ; Retina/pathology/physiopathology ; cis-trans-Isomerases/*genetics/metabolism ; }, abstract = {Leber congenital amaurosis (LCA), one of the leading causes of childhood-onset blindness, is caused by autosomal recessive mutations in several genes including RPE65. In this study, we performed CRISPR-Cas9-mediated therapeutic correction of a disease-associated nonsense mutation in Rpe65 in rd12 mice, a model of human LCA. Subretinal injection of adeno-associated virus carrying CRISPR-Cas9 and donor DNA resulted in >1% homology-directed repair and ~1.6% deletion of the pathogenic stop codon in Rpe65 in retinal pigment epithelial tissues of rd12 mice. The a- and b-waves of electroretinograms were recovered to levels up to 21.2 ± 4.1% and 39.8 ± 3.2% of their wild-type mice counterparts upon bright stimuli after dark adaptation 7 months after injection. There was no definite evidence of histologic perturbation or tumorigenesis during 7 months of observation. Collectively, we present the first therapeutic correction of an Rpe65 nonsense mutation using CRISPR-Cas9, providing new insight for developing therapeutics for LCA.}, }
@article {pmid30991115, year = {2019}, author = {Nagy, G and Vaz, AG and Szebenyi, C and Takó, M and Tóth, EJ and Csernetics, Á and Bencsik, O and Szekeres, A and Homa, M and Ayaydin, F and Galgóczy, L and Vágvölgyi, C and Papp, T}, title = {CRISPR-Cas9-mediated disruption of the HMG-CoA reductase genes of Mucor circinelloides and subcellular localization of the encoded enzymes.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {129}, number = {}, pages = {30-39}, doi = {10.1016/j.fgb.2019.04.008}, pmid = {30991115}, issn = {1096-0937}, mesh = {*CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Endoplasmic Reticulum/*enzymology ; Gene Deletion ; Hydroxymethylglutaryl CoA Reductases/*genetics ; Mevalonic Acid/metabolism ; Microscopy, Fluorescence ; Mucor/*enzymology/*genetics ; Mutation ; }, abstract = {Terpenoid compounds, such as sterols, carotenoids or the prenyl groups of various proteins are synthesized via the mevalonate pathway. A rate-limiting step of this pathway is the conversion of 3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid catalyzed by the HMG-CoA reductase. Activity of this enzyme may affect several biological processes, from the synthesis of terpenoid metabolites to the adaptation to various environmental conditions. In this study, the three HMG-CoA reductase genes (i.e. hmgR1, hmgR2 and hmgR3) of the β-carotene producing filamentous fungus, Mucor circinelloides were disrupted individually and simultaneously by a recently developed in vitro plasmid-free CRISPR-Cas9 method. Examination of the mutants revealed that the function of hmgR2 and hmgR3 are partially overlapping and involved in the general terpenoid biosynthesis. Moreover, hmgR2 seemed to have a special role in the ergosterol biosynthesis. Disruption of all three genes affected the germination ability of the spores and the sensitivity to hydrogen peroxide. Disruption of the hmgR1 gene had no effect on the ergosterol production and the sensitivity to statins but caused a reduced growth at lower temperatures. By confocal fluorescence microscopy using strains expressing GFP-tagged HmgR proteins, all three HMG-CoA reductases were localized in the endoplasmic reticulum.}, }
@article {pmid30808210, year = {2019}, author = {Li, X and Yu, X and Chen, X and Liu, Z and Wang, G and Li, C and Wong, EYM and Sham, MH and Tang, J and He, J and Xiong, W and Liu, Z and Huang, P}, title = {Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {33}, number = {6}, pages = {6838-6851}, doi = {10.1096/fj.201802155RR}, pmid = {30808210}, issn = {1530-6860}, mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems ; *Gene Expression Regulation, Developmental ; Hair Cells, Auditory/*metabolism ; Hair Cells, Auditory, Inner/*metabolism ; Mechanotransduction, Cellular ; Membrane Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Mice ; Mice, Knockout ; }, abstract = {The channel that governs mechanotransduction (MT) by hair cells in the inner ear has been investigated intensively for 4 decades, but its precise molecular composition remains enigmatic. Transmembrane channel-like protein 1 (TMC1) was recently identified as a component of the MT channel, and lipoma HMGIC fusion partner-like 5 (LHFPL5) is considered to be part of the MT complex and may functionally couple the tip link to the MT channel. As components of the MT complex, TMC1 and LHFPL5 are expected to localize at the lower end of the tip link in hair cells, a notion generally supported by previous studies on neonatal mice. However, the localization of these 2 proteins, particularly in the hair cells of adult mice, remains incompletely elucidated. Because determination of TMC1 and LHFPL5 localization at distinct developmental stages is essential for understanding their function and regulation, we used several approaches to examine the localization of these proteins in neonatal and adult hair cells in the mouse. We report several notable findings: 1) TMC1 and LHFPL5 predominantly localize at the tip of the shorter rows of stereocilia in neonatal hair cells, which largely verifies the previously published findings in neonatal hair cells; 2) LHFPL5 persists in the hair bundle of hair cells after postnatal day (P)7, which clarifies the previously reported unexpected absence of LHFPL5 after P7 and supports the view that LHFPL5 is a permanent component in the MT complex; and 3) TMC1 and LHFPL5 remain at the tip of the shorter rows of stereocilia in adult outer hair cells, but in adult inner hair cells, TMC1 is uniformly distributed in both the tallest row and the shorter rows of stereocilia, whereas LHFPL5 is uniformly distributed in the shorter rows of stereocilia. These findings raise intriguing questions regarding the turnover rate, regulation, additional functions, and functional interaction of TMC1 and LHFPL5. Our study confirms the previous findings in neonatal hair cells and reveals several previously unidentified aspects of TMC1 and LHFPL5 localization in more mature hair cells.-Li, X., Yu, X., Chen, X., Liu, Z., Wang, G., Li, C., Wong, E. Y. M., Sham, M. H., Tang, J., He, J., Xiong, W., Liu, Z., Huang, P. Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.}, }
@article {pmid32433763, year = {2020}, author = {Roberts, A and Barrangou, R}, title = {Applications of CRISPR-Cas systems in Lactic Acid Bacteria.}, journal = {FEMS microbiology reviews}, volume = {}, number = {}, pages = {}, doi = {10.1093/femsre/fuaa016}, pmid = {32433763}, issn = {1574-6976}, abstract = {As a phenotypically and phylogenetically diverse group, lactic acid bacteria are found in a variety of natural environments and occupy important roles in medicine, biotechnology, food and agriculture. The widespread use of lactic acid bacteria across these industries fuels the need for new and functionally diverse strains that may be utilized as starter cultures or probiotics. Originally characterized in lactic acid bacteria, CRISPR-Cas systems and derived molecular machines can be used natively or exogenously to engineer new strains with enhanced functional attributes. Research on CRISPR-Cas biology and its applications has exploded over the past decade with studies spanning from the initial characterization of CRISPR-Cas immunity in Streptococcus thermophilus to the use of CRISPR-Cas for clinical gene therapies. Here, we discuss CRISPR-Cas classification, overview CRISPR biology and mechanism of action, and discuss current and future applications in lactic acid bacteria, opening new avenues for their industrial exploitation and manipulation of microbiomes.}, }
@article {pmid32426802, year = {2020}, author = {Singhal, J and Chikara, S and Horne, D and Awasthi, S and Salgia, R and Singhal, SS}, title = {Targeting RLIP with CRISPR/Cas9 controls tumor growth.}, journal = {Carcinogenesis}, volume = {}, number = {}, pages = {}, doi = {10.1093/carcin/bgaa048}, pmid = {32426802}, issn = {1460-2180}, abstract = {Breast cancer (BC) remains one of the major causes of cancer deaths in women. Over half of all BCs carry genetic defects in the gene encoding p53, a powerful tumor suppressor. P53 is known as the &quot;guardian of the genome&quot; because it is essential for regulating cell division and preventing tumor formation. Ral-interacting protein (RLIP) is a modular protein capable of participating in many cellular functions. Blocking this stress-responsive protein, which is overexpressed during malignancy, enables BC cells to overcome the deleterious effects of p53 loss more effectively. In the CRISPR/Cas9 system, a single-guide RNA (sgRNA) recognizes a specific DNA sequence and directs the endonuclease Cas9 to make a double-strand break, which enables editing of targeted genes. Here, we harnessed CRISPR/Cas9 technology to target the RLIP gene in BC cells. We screened sgRNAs using a reporter system and lentivirally delivered them, along with Cas9, to BC cells for validation. We then assessed the survival, proliferation, and tumorigenicity of BC cells in vitro and the growth of tumors in vivo after CRISPR-mediated knockdown of RLIP. Doxycycline-inducible expression of Cas9 in BC cells transduced with lentiviral vectors encoding the sgRNAs disrupted the RLIP gene, leading to inhibition of BC cell proliferation both in vitro and in vivo, with resected tumors showing reduced levels of the survival and proliferation markers Ki67, RLIP, pAkt, and survivin, the cell cycle protein CDK4, and the mesenchymal marker vimentin, as well as elevated levels of the differentiation protein E-cadherin and pro-apoptotic protein Bim. Inducible Cas9/sgRNA-transduced BC cells without doxycycline treatment did not exhibit altered cell survival or proliferation in vitro or in vivo. Our study provides proof-of-concept that the CRISPR/Cas9 system can be utilized to target RLIP in vitro and in vivo.}, }
@article {pmid32353252, year = {2020}, author = {Abbott, TR and Dhamdhere, G and Liu, Y and Lin, X and Goudy, L and Zeng, L and Chemparathy, A and Chmura, S and Heaton, NS and Debs, R and Pande, T and Endy, D and La Russa, MF and Lewis, DB and Qi, LS}, title = {Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza.}, journal = {Cell}, volume = {181}, number = {4}, pages = {865-876.e12}, doi = {10.1016/j.cell.2020.04.020}, pmid = {32353252}, issn = {1097-4172}, mesh = {A549 Cells ; Antibiotic Prophylaxis/methods ; Antiviral Agents/*pharmacology ; Base Sequence ; Betacoronavirus/*drug effects/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computer Simulation ; Conserved Sequence ; Coronavirus/drug effects/genetics/growth &amp; development ; Coronavirus Infections/drug therapy ; Epithelial Cells/virology ; Humans ; Influenza A Virus, H1N1 Subtype/*drug effects/genetics/growth &amp; development ; Lung/pathology/virology ; Nucleocapsid Proteins/genetics ; Pandemics ; Phylogeny ; Pneumonia, Viral/drug therapy ; RNA Replicase/genetics ; RNA, Viral/*antagonists &amp; inhibitors ; Viral Nonstructural Proteins/genetics ; }, abstract = {The coronavirus disease 2019 (COVID-19) pandemic, caused by the SARS-CoV-2 virus, has highlighted the need for antiviral approaches that can target emerging viruses with no effective vaccines or pharmaceuticals. Here, we demonstrate a CRISPR-Cas13-based strategy, PAC-MAN (prophylactic antiviral CRISPR in human cells), for viral inhibition that can effectively degrade RNA from SARS-CoV-2 sequences and live influenza A virus (IAV) in human lung epithelial cells. We designed and screened CRISPR RNAs (crRNAs) targeting conserved viral regions and identified functional crRNAs targeting SARS-CoV-2. This approach effectively reduced H1N1 IAV load in respiratory epithelial cells. Our bioinformatic analysis showed that a group of only six crRNAs can target more than 90% of all coronaviruses. With the development of a safe and effective system for respiratory tract delivery, PAC-MAN has the potential to become an important pan-coronavirus inhibition strategy.}, }
@article {pmid31980824, year = {2020}, author = {Szentes, S and Zsibrita, N and Koncz, M and Zsigmond, E and Salamon, P and Pletl, Z and Kiss, A}, title = {I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins.}, journal = {Nucleic acids research}, volume = {48}, number = {5}, pages = {e28}, pmid = {31980824}, issn = {1362-4962}, mesh = {Base Sequence ; Binding Sites ; Binding, Competitive ; *Biological Assay ; CRISPR-Cas Systems ; DNA, Bacterial/*genetics/metabolism ; DNA-Directed RNA Polymerases/*genetics/metabolism ; Deoxyribonucleases, Type II Site-Specific/genetics/metabolism ; Escherichia coli/*genetics/metabolism ; Escherichia coli Proteins/genetics ; Lac Repressors/deficiency/genetics ; Plasmids/chemistry/metabolism ; Promoter Regions, Genetic ; Protein Binding ; *Transcription, Genetic ; beta-Galactosidase/genetics/metabolism ; }, abstract = {We have developed a simple method called I-Block assay, which can detect sequence-specific binding of proteins to DNA in Escherichia coli. The method works by detecting competition between the protein of interest and RNA polymerase for binding to overlapping target sites in a plasmid-borne lacI promoter variant. The assay utilizes two plasmids and an E. coli host strain, from which the gene of the Lac repressor (lacI) has been deleted. One of the plasmids carries the lacI gene with a unique NheI restriction site created in the lacI promoter. The potential recognition sequences of the tested protein are inserted into the NheI site. Introduction of the plasmids into the E. coliΔlacI host represses the constitutive β-galactosidase synthesis of the host bacterium. If the studied protein expressed from a compatible plasmid binds to its target site in the lacI promoter, it will interfere with lacI transcription and lead to increased β-galactosidase activity. The method was tested with two zinc finger proteins, with the lambda phage cI857 repressor, and with CRISPR-dCas9 targeted to the lacI promoter. The I-Block assay was shown to work with standard liquid cultures, with cultures grown in microplate and with colonies on X-gal indicator plates.}, }
@article {pmid31956898, year = {2020}, author = {Tian, B and Minero, GAS and Fock, J and Dufva, M and Hansen, MF}, title = {CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification.}, journal = {Nucleic acids research}, volume = {48}, number = {5}, pages = {e30}, pmid = {31956898}, issn = {1362-4962}, mesh = {Bacterial Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded/*genetics/metabolism ; Endonucleases/*genetics/metabolism ; Gene Editing/methods ; Nucleic Acid Amplification Techniques/*standards ; RNA, Guide/genetics/metabolism ; Reference Standards ; }, abstract = {False-positive results cause a major problem in nucleic acid amplification, and require external blank/negative controls for every test. However, external controls usually have a simpler and lower background compared to the test sample, resulting in underestimation of false-positive risks. Internal negative controls, performed simultaneously with amplification to monitor the background level in real-time, are therefore appealing in both research and clinic. Herein, we describe a nonspecific product-activated single-stranded DNA-cutting approach based on CRISPR (clustered regularly interspaced short palindromic repeats) Cas12a (Cpf1) nuclease. The proposed approach, termed Cas12a-based internal referential indicator (CIRI), can indicate the onset of nonspecific amplification in an exponential rolling circle amplification strategy here combined with an optomagnetic readout. The capability of CIRI as an internal negative control can potentially be extended to other amplification strategies and sensors, improving the performance of nucleic acid amplification-based methodologies.}, }
@article {pmid31943080, year = {2020}, author = {Yu, J and Xiang, X and Huang, J and Liang, X and Pan, X and Dong, Z and Petersen, TS and Qu, K and Yang, L and Zhao, X and Li, S and Zheng, T and Xu, Z and Liu, C and Han, P and Xu, F and Yang, H and Liu, X and Zhang, X and Bolund, L and Luo, Y and Lin, L}, title = {Haplotyping by CRISPR-mediated DNA circularization (CRISPR-hapC) broadens allele-specific gene editing.}, journal = {Nucleic acids research}, volume = {48}, number = {5}, pages = {e25}, pmid = {31943080}, issn = {1362-4962}, mesh = {Alleles ; Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Circular/*genetics/metabolism ; Gene Editing/methods ; HEK293 Cells ; Haplotypes ; Hep G2 Cells ; Humans ; Plasmids/chemistry/metabolism ; RNA, Guide/*genetics/metabolism ; }, abstract = {Allele-specific protospacer adjacent motif (asPAM)-positioning SNPs and CRISPRs are valuable resources for gene therapy of dominant disorders. However, one technical hurdle is to identify the haplotype comprising the disease-causing allele and the distal asPAM SNPs. Here, we describe a novel CRISPR-based method (CRISPR-hapC) for haplotyping. Based on the generation (with a pair of CRISPRs) of extrachromosomal circular DNA in cells, the CRISPR-hapC can map haplotypes from a few hundred bases to over 200 Mb. To streamline and demonstrate the applicability of the CRISPR-hapC and asPAM CRISPR for allele-specific gene editing, we reanalyzed the 1000 human pan-genome and generated a high frequency asPAM SNP and CRISPR database (www.crispratlas.com/knockout) for four CRISPR systems (SaCas9, SpCas9, xCas9 and Cas12a). Using the huntingtin (HTT) CAG expansion and transthyretin (TTR) exon 2 mutation as examples, we showed that the asPAM CRISPRs can specifically discriminate active and dead PAMs for all 23 loci tested. Combination of the CRISPR-hapC and asPAM CRISPRs further demonstrated the capability for achieving highly accurate and haplotype-specific deletion of the HTT CAG expansion allele and TTR exon 2 mutation in human cells. Taken together, our study provides a new approach and an important resource for genome research and allele-specific (haplotype-specific) gene therapy.}, }
@article {pmid31872378, year = {2020}, author = {Li, J and Li, HY and Gu, SY and Zi, HX and Jiang, L and Du, JL}, title = {One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting.}, journal = {Science China. Life sciences}, volume = {63}, number = {1}, pages = {59-67}, doi = {10.1007/s11427-019-1607-9}, pmid = {31872378}, issn = {1869-1889}, mesh = {Alleles ; Animals ; Animals, Genetically Modified ; Base Sequence ; Basic Helix-Loop-Helix Transcription Factors/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Expression ; Gene Knock-In Techniques/methods ; Gene Knockout Techniques/methods ; Gene Targeting/*methods ; Genetic Engineering ; Genotype ; Green Fluorescent Proteins/genetics ; Homologous Recombination/*genetics ; Introns/*genetics ; Zebrafish ; Zebrafish Proteins/*genetics ; }, abstract = {The zebrafish has become a popular vertebrate animal model in biomedical research. However, it is still challenging to make conditional gene knockout (CKO) models in zebrafish due to the low efficiency of homologous recombination (HR). Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin (KI) switch (zCKOIS) coupled with dual-color fluorescent reporters. Using this strategy, we generated hey2zKOIS which served as a hey2 KI reporter with EGFP expression. Upon Cre induction in targeted cells, the hey2zCKOIS was switched to a non-functional CKO allele hey2zCKOIS-invassociated with TagRFP expression, enabling visualization of the CKO alleles. Thus, simplification of the design, and the visibility and combination of both CKO and KI alleles make our zCKOIS strategy an applicable CKO approach for zebrafish.}, }
@article {pmid31756263, year = {2020}, author = {Wang, X and Ma, Y and Wang, F and Yang, Y and Wu, S and Wu, Y}, title = {Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella.}, journal = {Pest management science}, volume = {76}, number = {5}, pages = {1618-1625}, doi = {10.1002/ps.5689}, pmid = {31756263}, issn = {1526-4998}, support = {KYT201803//Fundamental Research Funds for the Central Universities of China/ ; 2016YFD0200500//National Key Research and Development Program of China/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Insecticide Resistance ; Insecticides ; Moths ; Receptors, Nicotinic ; }, abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system provides some advantages over other reverse genetic techniques to investigate the causal relationship between insecticide resistance phenotype and candidate gene. Several studies published to date point to the nicotinic acetylcholine receptor (nAChR) α6 subunit strongly associated with spinosyns resistance in insects, including Plutella xylostella. However, reverse genetic verification of the P. xylostella nAChRα6 has not yet been achieved via an in vivo approach.<br><br>RESULTS: Here, we successfully constructed a homozygous strain (Pxα6-KO) with a 2-nt deletion mutation of nAChRα6 by CRISPR/Cas9 coupled with non-homologous end joining approach in P. xylostella. The manipulated mutation results in a frame shift in the open reading frame of transcripts, which produces a predicted protein truncated in the TM3-TM4 loop region. When compared to the background strain IPP-S, the knockout strain Pxα6-KO exhibited 229- and 1462-fold resistance to spinosad and spinetoram, respectively, but no or limited (resistance ratios <3-fold) effects on the toxicities of imidacloprid, abamectin, β-cypermethrin, indoxacarb, metaflumizone and chlorantraniliprole. Furthermore, the mode of inheritance of the acquired spinetoram resistance was autosomal recessive and significantly linked with the 2-nt deletion mutation of nAChRα6 in the Pxα6-KO strain.<br><br>CONCLUSION: In vivo functional investigation demonstrates the causality of the Pxα6 truncating mutation with high levels of resistance to spinosyns in P. xylostella. Our results suggest the Pxα6-KO strain underlies an autosomal, recessive mode of inheritance for spinetoram resistance, and reinforces the association of this gene to the mode of action of spinosyns. © 2019 Society of Chemical Industry.}, }
@article {pmid31701407, year = {2019}, author = {Hong, W and Huang, M and Wei, Y and Wei, X}, title = {A new and promising application of gene editing: CRISPR-controlled smart materials for tissue engineering, bioelectronics, and diagnostics.}, journal = {Science China. Life sciences}, volume = {62}, number = {11}, pages = {1547-1549}, doi = {10.1007/s11427-019-1576-0}, pmid = {31701407}, issn = {1869-1889}, mesh = {Base Sequence ; Biocatalysis ; Biosensing Techniques ; CRISPR-Cas Systems/*genetics ; Diagnostic Techniques and Procedures ; Electronics ; Gene Editing/*methods ; Gene Expression ; Smart Materials/*metabolism ; Tissue Engineering ; Transfection ; }, }
@article {pmid31686319, year = {2019}, author = {Li, G and Liu, YG and Chen, Y}, title = {Genome-editing technologies: the gap between application and policy.}, journal = {Science China. Life sciences}, volume = {62}, number = {11}, pages = {1534-1538}, doi = {10.1007/s11427-019-1566-1}, pmid = {31686319}, issn = {1869-1889}, mesh = {Animals ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Gene Editing/*methods ; Gene Knockdown Techniques ; Genetic Therapy/methods ; Humans ; Patient Safety ; Social Control, Formal ; }, }
@article {pmid31620779, year = {2020}, author = {Zhang, Y and Zhang, Z and Zhang, H and Zhao, Y and Zhang, Z and Xiao, J}, title = {PADS Arsenal: a database of prokaryotic defense systems related genes.}, journal = {Nucleic acids research}, volume = {48}, number = {D1}, pages = {D590-D598}, pmid = {31620779}, issn = {1362-4962}, mesh = {Archaea/*genetics/virology ; Archaeal Viruses/pathogenicity ; Bacteria/*genetics/virology ; Bacteriophages/pathogenicity ; CRISPR-Cas Systems ; DNA Restriction-Modification Enzymes ; *Databases, Genetic ; *Host-Pathogen Interactions ; *Software ; }, abstract = {Defense systems are vital weapons for prokaryotes to resist heterologous DNA and survive from the constant invasion of viruses, and they are widely used in biochemistry investigation and antimicrobial drug research. So far, numerous types of defense systems have been discovered, but there is no comprehensive defense systems database to organize prokaryotic defense gene datasets. To fill this gap, we unveil the prokaryotic antiviral defense system (PADS) Arsenal (https://bigd.big.ac.cn/padsarsenal), a public database dedicated to gathering, storing, analyzing and visualizing prokaryotic defense gene datasets. The initial version of PADS Arsenal integrates 18 distinctive categories of defense system with the annotation of 6 600 264 genes retrieved from 63,701 genomes across 33 390 species of archaea and bacteria. PADS Arsenal provides various ways to retrieve defense systems related genes information and visualize them with multifarious function modes. Moreover, an online analysis pipeline is integrated into PADS Arsenal to facilitate annotation and evolutionary analysis of defense genes. PADS Arsenal can also visualize the dynamic variation information of defense genes from pan-genome analysis. Overall, PADS Arsenal is a state-of-the-art open comprehensive resource to accelerate the research of prokaryotic defense systems.}, }
@article {pmid31556487, year = {2019}, author = {Dandage, R and Landry, CR}, title = {Paralog dependency indirectly affects the robustness of human cells.}, journal = {Molecular systems biology}, volume = {15}, number = {9}, pages = {e8871}, pmid = {31556487}, issn = {1744-4292}, support = {RN348479-387697//Canadian Institute of Health Research (CIHR)/International ; }, mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Cell Line, Tumor ; Computational Biology ; Gene Dosage/*genetics ; Gene Duplication/*genetics ; Genetic Fitness/genetics ; Humans ; Loss of Function Mutation/genetics ; Protein Interaction Maps/*genetics ; }, abstract = {The protective redundancy of paralogous genes partly relies on the fact that they carry their functions independently. However, a significant fraction of paralogous proteins may form functionally dependent pairs, for instance, through heteromerization. As a consequence, one could expect these heteromeric paralogs to be less protective against deleterious mutations. To test this hypothesis, we examined the robustness landscape of gene loss-of-function by CRISPR-Cas9 in more than 450 human cell lines. This landscape shows regions of greater deleteriousness to gene inactivation as a function of key paralog properties. Heteromeric paralogs are more likely to occupy such regions owing to their high expression and large number of protein-protein interaction partners. Further investigation revealed that heteromers may also be under stricter dosage balance, which may also contribute to the higher deleteriousness upon gene inactivation. Finally, we suggest that physical dependency may contribute to the deleteriousness upon loss-of-function as revealed by the correlation between the strength of interactions between paralogs and their higher deleteriousness upon loss of function.}, }
@article {pmid31355781, year = {2019}, author = {Lou, WP and Wang, L and Long, C and Liu, L and Fei, JF}, title = {Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {149}, pages = {}, doi = {10.3791/59850}, pmid = {31355781}, issn = {1940-087X}, mesh = {Ambystoma mexicanum ; Animals ; CRISPR-Associated Protein 9/*genetics/*metabolism ; CRISPR-Cas Systems/genetics ; *Electroporation ; Gene Knockout Techniques/*methods ; Neural Stem Cells/*metabolism ; RNA, Guide/*genetics/metabolism ; Regeneration/genetics ; Spinal Cord/cytology/physiology ; }, abstract = {The axolotl has the unique ability to fully regenerate its spinal cord. This is largely due to the ependymal cells remaining as neural stem cells (NSCs) throughout life, which proliferate to reform the ependymal tube and differentiate into lost neurons after spinal cord injury. Deciphering how these NSCs retain pluripotency post-development and proliferate upon spinal cord injury to reform the exact pre-injury structure can provide valuable insight into how mammalian spinal cords may regenerate as well as potential treatment options. Performing gene knock-outs in specific subsets of NSCs within a restricted time period will allow study of the molecular mechanisms behind these regenerative processes, without being confounded by development perturbing effects. Described here is a method to perform gene knock-out in axolotl spinal cord NSCs using the CRISPR-Cas9 system. By injecting the CAS9-gRNA complex into the spinal cord central canal followed by electroporation, target genes are knocked out in NSCs within specific regions of the spinal cord at a desired timepoint, allowing for molecular studies of spinal cord NSCs during regeneration.}, }
@article {pmid31228045, year = {2019}, author = {Liu, X and Zheng, G and Wang, G and Jiang, W and Li, L and Lu, Y}, title = {Overexpression of the diguanylate cyclase CdgD blocks developmental transitions and antibiotic biosynthesis in Streptomyces coelicolor.}, journal = {Science China. Life sciences}, volume = {62}, number = {11}, pages = {1492-1505}, doi = {10.1007/s11427-019-9549-8}, pmid = {31228045}, issn = {1869-1889}, mesh = {Anthraquinones/metabolism ; Anti-Bacterial Agents/biosynthesis/*metabolism ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems ; Cyclic GMP/*genetics/metabolism ; DNA-Binding Proteins ; Escherichia coli Proteins/*genetics/metabolism ; Fermentation ; Gene Editing ; Gene Expression Regulation, Bacterial ; Mutation ; Phosphorus-Oxygen Lyases/*genetics/metabolism ; Prodigiosin/analogs &amp; derivatives/metabolism ; Streptomyces coelicolor/*metabolism ; Transfection ; }, abstract = {Cyclic dimeric GMP (c-di-GMP) has emerged as the nucleotide second messenger regulating both development and antibiotic production in high-GC, Gram-positive streptomycetes. Here, a diguanylate cyclase (DGC), CdgD, encoded by SCO5345 from the model strain Streptomyces coelicolor, was functionally identified and characterized to be involved in c-di-GMP synthesis through genetic and biochemical analysis. cdgD overexpression resulted in significantly reduced production of actinorhodin and undecylprodigiosin, as well as completely blocked sporulation or aerial mycelium formation on two different solid media. In the cdgD-overexpression strain, intracellular c-di-GMP levels were 13-27-fold higher than those in the wild-type strain. In vitro enzymatic assay demonstrated that CdgD acts as a DGC, which could efficiently catalyze the synthesis of c-di-GMP from two GTP molecules. Heterologous overproduction of cdgD in two industrial Streptomyces strains could similarly impair developmental transitions as well as antibiotic biosynthesis. Collectively, our results combined with previously reported data clearly demonstrated that c-di-GMP-mediated signalling pathway plays a central and universal role in the life cycle as well as secondary metabolism in streptomycetes.}, }
@article {pmid30951374, year = {2019}, author = {Lee, HJ and Yoon, JW and Jung, KM and Kim, YM and Park, JS and Lee, KY and Park, KJ and Hwang, YS and Park, YH and Rengaraj, D and Han, JY}, title = {Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {33}, number = {7}, pages = {8519-8529}, doi = {10.1096/fj.201802671R}, pmid = {30951374}, issn = {1530-6860}, mesh = {Animals ; Animals, Genetically Modified/genetics ; CRISPR-Cas Systems/genetics ; Chickens/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genome/genetics ; Germ Cells/*physiology ; Green Fluorescent Proteins/genetics ; Mutagenesis, Insertional/methods ; RNA, Guide/genetics ; Sex Chromosomes/*genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) have facilitated the production of genome-edited animals for use as models. Because of their unique developmental system, avian species offer many advantages as model vertebrates. Here, we report the development of novel chicken models using the CRISPR/Cas9-mediated nonhomologous end joining repair pathway in chicken primordial germ cells (PGCs). Through the introduction of a donor plasmid containing short guide RNA recognition sequences and CRISPR/Cas9 plasmids into chicken PGCs, exogenous genes of donor plasmids were precisely inserted into target loci, and production of transgenic chickens was accomplished through subsequent transplantation of the Z chromosome-targeted PGCs. Using this method, we successfully accomplished the targeted gene insertion to the chicken sex Z chromosome without detected off-target effects. The genome-modified chickens robustly expressed green fluorescent protein from the Z chromosome, which could then be used for easy sex identification during embryogenesis. Our results suggest that this powerful genome-editing method could be used to develop many chicken models and should significantly expand the application of genome-modified avians.-Lee, H. J., Yoon, J. W., Jung, K. M., Kim, Y. M., Park, J. S., Lee, K. Y., Park, K. J., Hwang, Y. S., Park, Y. H., Rengaraj, D., Han, J. Y. Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.}, }
@article {pmid30923099, year = {2019}, author = {González-Romero, E and Martínez-Valiente, C and García-Ruiz, C and Vázquez-Manrique, RP and Cervera, J and Sanjuan-Pla, A}, title = {CRISPR to fix bad blood: a new tool in basic and clinical hematology.}, journal = {Haematologica}, volume = {104}, number = {5}, pages = {881-893}, pmid = {30923099}, issn = {1592-8721}, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genetic Engineering ; *Genetic Therapy ; Hematologic Diseases/genetics/*therapy ; Humans ; Neoplasm Proteins/antagonists &amp; inhibitors/*genetics ; }, abstract = {Advances in genome engineering in the last decade, particularly in the development of programmable nucleases, have made it possible to edit the genomes of most cell types precisely and efficiently. Chief among these advances, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a novel, versatile and easy-to-use tool to edit genomes irrespective of their complexity, with multiple and broad applications in biomedicine. In this review, we focus on the use of CRISPR/Cas9 genome editing in the context of hematologic diseases and appraise the major achievements and challenges in this rapidly moving field to gain a clearer perspective on the potential of this technology to move from the laboratory to the clinic. Accordingly, we discuss data from studies editing hematopoietic cells to understand and model blood diseases, and to develop novel therapies for hematologic malignancies. We provide an overview of the applications of gene editing in experimental, preclinical and clinical hematology including interrogation of gene function, target identification and drug discovery and chimeric antigen receptor T-cell engineering. We also highlight current limitations of CRISPR/Cas9 and the possible strategies to overcome them. Finally, we consider what advances in CRISPR/Cas9 are needed to move the hematology field forward.}, }
@article {pmid30600536, year = {2019}, author = {Foss, DV and Hochstrasser, ML and Wilson, RC}, title = {Clinical applications of CRISPR-based genome editing and diagnostics.}, journal = {Transfusion}, volume = {59}, number = {4}, pages = {1389-1399}, doi = {10.1111/trf.15126}, pmid = {30600536}, issn = {1537-2995}, mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing ; Humans ; Neoplasms/*diagnosis/*genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-driven genome editing has rapidly transformed preclinical biomedical research by eliminating the underlying genetic basis of many diseases in model systems and facilitating the study of disease etiology. Translation to the clinic is under way, with announced or impending clinical trials utilizing ex vivo strategies for anticancer immunotherapy or correction of hemoglobinopathies. These exciting applications represent just a fraction of what is theoretically possible for this emerging technology, but many technical hurdles must be overcome before CRISPR-based genome editing technology can reach its full potential. One exciting recent development is the use of CRISPR systems for diagnostic detection of genetic sequences associated with pathogens or cancer. We review the biologic origins and functional mechanism of CRISPR systems and highlight several current and future clinical applications of genome editing.}, }
@article {pmid32421986, year = {2020}, author = {Hashmat, R and Yousaf, MZ and Rahman, Z and Anjum, KM and Yaqoob, A and Imran, M}, title = {CRISPR-CAS Replacing Antiviral Drugs against HIV: An Update.}, journal = {Critical reviews in eukaryotic gene expression}, volume = {30}, number = {1}, pages = {77-83}, doi = {10.1615/CritRevEukaryotGeneExpr.2020028233}, pmid = {32421986}, issn = {1045-4403}, abstract = {Various antiretroviral drugs do not kill or cure the human immunodeficiency virus (HIV) but do prevent the replication of the virus. The combination of antiretroviral drugs is known as highly active antiretroviral therapy (HAART). Current drug therapies effectively suppress HIV-1 replication but do not inactivate the provirus that persists in latent reservoirs. Guide RNA (gRNA)-directed CRISPR/Cas9 system can be used for sequence-specific attacks on this proviral DNA. The biggest achievement might be the complete elimination of HIV from infected cells. A study revealed that the tail injection, in transgenic mice and rats having HIV-1 genome, of an adenoassociated virus (AAV) vector expressing a short version of the Cas9 endonuclease (saCas9) and the gRNAs resulted in the cleavage of integrated HIV-1 DNA and excision of a DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, kidney, and circulating lymphocytes. HIV-1 has capacity to escape the attack on its genome from most of inhibitors. Thus, to achieve successful antiretroviral treatment, combinations of several antiviral therapies have been applied that are based on two important facts. The first is that multiple drugs lead to synergistic or additive inhibition, and the second is that the combinational therapy increases chances of drug resistance. The success that has been achieved with the help of the genetically engineered tool CRISPR is that dCas9 protein alone can efficiently silence viral gene expression in bacteria with sgRNA. All the reported investigations have indicated that CRISPR/Cas9 can be used as immune machinery into human cells in the form of novel antivirus tools.}, }
@article {pmid32421984, year = {2020}, author = {Ashfaq, UA and Khalid, H}, title = {CRISPR/CAS9-Mediated Antiviral Activity: A Tool to Combat Viral Infection.}, journal = {Critical reviews in eukaryotic gene expression}, volume = {30}, number = {1}, pages = {45-56}, doi = {10.1615/CritRevEukaryotGeneExpr.2020028207}, pmid = {32421984}, issn = {1045-4403}, abstract = {Viruses hijack host cellular receptors and functions for replication, thereby posing a complication in identifying therapeutic targets. The CRISPR/Cas 9 platform has revolutionized gene-editing modalities in a wide range of cells and organisms with high potential in therapeutics. Recently, it has been put to work targeting human pathogenic viruses that interrupt receptors and functions with viral replication. This review encompasses major discoveries in CRISPR/Cas as an antiviral strategy. Additionally, challenges that demand consideration prior to its use in the clinic as part of the antiviral armamentarium are briefly addressed.}, }
@article {pmid32421777, year = {2020}, author = {Garrett, S and Shiimori, M and Watts, EA and Clark, L and Graveley, BR and Terns, MP}, title = {Primed CRISPR DNA uptake in Pyrococcus furiosus.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkaa381}, pmid = {32421777}, issn = {1362-4962}, abstract = {CRISPR-Cas adaptive immune systems are used by prokaryotes to defend against invaders like viruses and other mobile genetic elements. Immune memories are stored in the form of 'spacers' which are short DNA sequences that are captured from invaders and added to the CRISPR array during a process called 'adaptation'. Spacers are transcribed and the resulting CRISPR (cr)RNAs assemble with different Cas proteins to form effector complexes that recognize matching nucleic acid and destroy it ('interference'). Adaptation can be 'naïve', i.e. independent of any existing spacer matches, or it can be 'primed', i.e. spurred by the crRNA-mediated detection of a complete or partial match to an invader sequence. Here we show that primed adaptation occurs in Pyrococcus furiosus. Although P. furiosus has three distinct CRISPR-Cas interference systems (I-B, I-A and III-B), only the I-B system and Cas3 were necessary for priming. Cas4, which is important for selection and processing of new spacers in naïve adaptation, was also essential for priming. Loss of either the I-B effector proteins or Cas3 reduced naïve adaptation. However, when Cas3 and all crRNP genes were deleted, uptake of correctly processed spacers was observed, indicating that none of these interference proteins are necessary for naïve adaptation.}, }
@article {pmid32421209, year = {2020}, author = {Bouchaut, B and Asveld, L}, title = {Safe-by-Design: Stakeholders' Perceptions and Expectations of How to Deal with Uncertain Risks of Emerging Biotechnologies in the Netherlands.}, journal = {Risk analysis : an official publication of the Society for Risk Analysis}, volume = {}, number = {}, pages = {}, doi = {10.1111/risa.13501}, pmid = {32421209}, issn = {1539-6924}, support = {15809//Stichting voor de Technische Wetenschappen/ ; }, abstract = {Advanced gene editing techniques such as Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas have increased the pace of developments in the field of industrial biotechnology. Such techniques imply new possibilities when working with living organisms, possibly leading to uncertain risks. In the Netherlands, current policy fails to address these uncertain risks because risk classification is determined process-wise (i.e., genetically modified organism [GMO] and non-GMO), there is a strong focus on quantifiable risks, and the linearity within current governance (science-policy-society) hinders iterative communication between stakeholders, leaving limited room to anticipate uncertainties at an early stage of development. A suggested concept to overcome these shortcomings is the Safe-by-Design (SbD) approach, which, theoretically, allows stakeholders to iteratively incorporate safety measures throughout a technology's development process, creating a dynamic environment for the anticipation of uncertain risks. Although this concept originates from chemical engineering and is already widely applied in nanotechnology, for the field of biotechnology, there is no agreed upon definition yet. To explore the possibilities of SbD for future governance of biotechnology, we should gain insight in how various stakeholders perceive notions of risk, safety, and inherent safety, and what this implies for the applicability of SbD for risk governance concerning industrial biotechnology. Our empirical research reveals three main themes: (1) diverging expectations with regard to safety and risks, and to establish an acceptable level of risk; (2) different applications of SbD and inherent safety, namely, product- and process-wise; and (3) unclarity in allocating responsibilities to stakeholders in the development process of a biotechnology and within society.}, }
@article {pmid32124178, year = {2020}, author = {Ding, N and Qin, Q and Wu, X and Miller, R and Zaitlin, D and Li, D and Yang, S}, title = {Antagonistic regulation of axillary bud outgrowth by the BRANCHED genes in tobacco.}, journal = {Plant molecular biology}, volume = {103}, number = {1-2}, pages = {185-196}, pmid = {32124178}, issn = {1573-5028}, support = {Gift funding//R.J. Reynolds Tobacco Company/ ; Grant//Kentucky Tobacco Research and Development Center/ ; Gift funding//Council for Burley Tobacco/ ; }, mesh = {CRISPR-Cas Systems ; Down-Regulation ; Gene Expression Regulation, Plant ; *Genes, Plant ; Phylogeny ; Plant Development/*genetics ; RNA Interference ; Signal Transduction ; Tobacco/genetics/*growth &amp; development ; Transcriptome ; Up-Regulation ; }, abstract = {As a key integrator of shoot branching, BRANCHED 1 (BRC1) coordinates and is orchestrated by endogenous and environmental signals involved in the regulation of axillary bud outgrowth. In the present study, we characterized the regulatory roles of five BRC gene members in tobacco (Nicotiana tabacum L.) using CRISPR site-directed mutagenesis and overexpression assays. It was shown that lateral branching was negatively regulated by NtBRC1A-1, 1B-1, and 1B-2, but was unexpectedly promoted by NtBRC2A. Suppression of bud growth may be attained by direct binding of NtBRCs to the Tassels Replace Upper Ears 1 (TRU1) genes. It was speculated that NtBRC2A probably confers a dominant negative effect by interfering with the branching-inhibitory BRC1 genes. Our results suggested that highly homologous gene family members may function antagonistically in the same signaling pathway. However, the molecular mechanism underlying NtBRC2A-mediated outgrowth of axillary buds needs to be further addressed. KEY MESSAGE: Axillary bud outgrowth in general is negatively regulated by the BRANCHED gene. Here we show that the BRANCHED genes play opposing regulatory roles in tobacco lateral branching.}, }
@article {pmid32040758, year = {2020}, author = {Shockey, J}, title = {Gene editing in plants: assessing the variables through a simplified case study.}, journal = {Plant molecular biology}, volume = {103}, number = {1-2}, pages = {75-89}, pmid = {32040758}, issn = {1573-5028}, support = {6435-41000-083-00D//USDA-Agricultural Research Service (US)/ ; USM-GR05635-01//National Science Foundation/ ; }, mesh = {Arabidopsis/*genetics ; CRISPR-Cas Systems ; Cytosine/metabolism ; *Gene Editing ; Gene Expression Regulation, Plant ; Genome, Plant ; Guanosine/metabolism ; Mutation ; }, abstract = {KEY MESSAGE: Multiple variables that control the relative levels of successful heritable plant genome editing were addressed using simple case studies in Arabidopsis thaliana. The recent advent of genome editing technologies (especially CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats) has revolutionized various fields of scientific research. The process is much more specific than previous mutagenic processes and allows for targeting of nearly any gene of interest for the creation of loss-of-function mutations and many other types of editing, including gene-replacement and gene activation. However, not all CRISPR construct designs are successful, due to several factors, including differences in the strength and cell- or tissue-type specificity of the regulatory elements used to express the Cas9 (CRISPR Associated protein 9) DNA nuclease and single guide RNA components, and differences in the relative editing efficiency at different target areas within a given gene. Here we compare the levels of editing created in Arabidopsis thaliana by CRISPR constructs containing either different promoters, or altered target sites with varied levels of guanine-cytosine base content. Additionally, nuclease activity at sites targeted by imperfectly matched single guide RNAs was observed, suggesting that while the primary goal of most CRISPR construct designs is to achieve rapid, robust, heritable gene editing, the formation of unintended mutations at other genomic loci must be carefully monitored.}, }
@article {pmid31789494, year = {2019}, author = {Echave, MC and Domingues, RMA and Gómez-Florit, M and Pedraz, JL and Reis, RL and Orive, G and Gomes, ME}, title = {Biphasic Hydrogels Integrating Mineralized and Anisotropic Features for Interfacial Tissue Engineering.}, journal = {ACS applied materials &amp; interfaces}, volume = {11}, number = {51}, pages = {47771-47784}, doi = {10.1021/acsami.9b17826}, pmid = {31789494}, issn = {1944-8252}, mesh = {Animals ; CRISPR-Cas Systems/genetics/physiology ; Cellulose/chemistry ; Gelatin/chemistry ; Gene Editing/*methods ; Humans ; Hydrogels/chemistry ; Microscopy ; Nanoparticles/chemistry ; Swine ; Tendons/cytology ; Tissue Engineering/*methods ; Transglutaminases/metabolism ; }, abstract = {The innate graded structural and compositional profile of musculoskeletal tissue interfaces is disrupted and replaced by fibrotic tissue in the context of disease and degeneration. Tissue engineering strategies focused on the restoration of the transitional complexity found in those junctions present special relevance for regenerative medicine. Herein, we developed a gelatin-based multiphasic hydrogel system, where sections with distinct composition and microstructure were integrated in a single unit. In each phase, hydroxyapatite particles or cellulose nanocrystals (CNC) were incorporated into an enzymatically cross-linked gelatin network to mimic bone or tendon tissue, respectively. Stiffer hydrogels were produced with the incorporation of mineralized particles, and magnetic alignment of CNC resulted in anisotropic structure formation. The evaluation of the biological commitment with human adipose-derived stem cells toward the tendon-to-bone interface revealed an aligned cell growth and higher synthesis and deposition of tenascin in the anisotropic phase, while the activity of the secreted alkaline phosphatase and the expression of osteopontin were induced in the mineralized phase. These results highlight the potential versatility offered by gelatin-transglutaminase enzyme tandem for the development of strategies that mimic the graded, composite, and complex intersections of the connective tissues.}, }
@article {pmid31773942, year = {2019}, author = {Li, S and Song, Z and Liu, C and Chen, XL and Han, H}, title = {Biomimetic Mineralization-Based CRISPR/Cas9 Ribonucleoprotein Nanoparticles for Gene Editing.}, journal = {ACS applied materials &amp; interfaces}, volume = {11}, number = {51}, pages = {47762-47770}, doi = {10.1021/acsami.9b17598}, pmid = {31773942}, issn = {1944-8252}, mesh = {Biomimetics/methods ; Biotechnology/methods ; CRISPR-Cas Systems/genetics/*physiology ; Gene Editing/methods ; Ribonucleoproteins/genetics/*metabolism ; }, abstract = {Delivery of the CRISPR/Cas9 ribonucleoprotein (RNP) complex has provided an alternative strategy for the regulation of CRISPR functions, offering a transient and DNA-free means for genomic editing. Chemical methods of delivering the RNPs via nanocomplexes have the potential to address these delivery problems for efficiency, safety, and packaging capacity. Here, we developed a biomimetic mineralization-mediated strategy for efficient DNA-free genome editing by using CRISPR/Cas9 RNPs. We found that the RNPs have the ability to form the biomimetic mineralized RNP nanoparticles (Bm-RNP NPs) quickly in situ and can be effectively delivered into the fungal protoplast cells. Biomimetic mineralization can maintain the natural function of Cas9 protein and protect the sgRNA activity. At the same time, the encapsulated RNPs can be effectively released into the cytoplasm, and the Sytalone dehydratase (SDH) gene can be edited in a targeted manner. Except for phenotypic defects, molecular detections indicated that the delivery of Bm-RNP NPs achieved 20% genomic editing for Magnaporthe oryzae compared to RNPs alone. Moreover, the Bm-RNP NP-mediated editing of the SDH gene significantly affects the appressorium-mediated penetration and invasive growth in M. oryzae. Our system has the advantages of being cheap, fast, and effective, without the traditional transformation process, suggesting the potential application of this DNA-free gene-editing strategy in different organisms.}, }
@article {pmid31450674, year = {2019}, author = {Brandt, ZJ and North, PN and Link, BA}, title = {Somatic Mutations of lats2 Cause Peripheral Nerve Sheath Tumors in Zebrafish.}, journal = {Cells}, volume = {8}, number = {9}, pages = {}, pmid = {31450674}, issn = {2073-4409}, support = {R01 EY029267/EY/NEI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cell Proliferation ; Gene Editing ; *Mutation ; Neoplasms, Experimental/genetics/metabolism/*pathology ; Nerve Sheath Neoplasms/genetics/metabolism/*pathology ; Protein-Serine-Threonine Kinases/*genetics/metabolism ; Signal Transduction ; Zebrafish/genetics/growth &amp; development ; Zebrafish Proteins/metabolism ; }, abstract = {The cellular signaling pathways underlying peripheral nerve sheath tumor (PNST) formation are poorly understood. Hippo signaling has been recently implicated in the biology of various cancers, and is thought to function downstream of mutations in the known PNST driver, NF2. Utilizing CRISPR-Cas9 gene editing, we targeted the canonical Hippo signaling kinase Lats2. We show that, while germline deletion leads to early lethality, targeted somatic mutations of zebrafish lats2 leads to peripheral nerve sheath tumor formation. These peripheral nerve sheath tumors exhibit high levels of Hippo effectors Yap and Taz, suggesting that dysregulation of these transcriptional co-factors drives PNST formation in this model. These data indicate that somatic lats2 deletion in zebrafish can serve as a powerful experimental platform to probe the mechanisms of PNST formation and progression.}, }
@article {pmid31378590, year = {2019}, author = {Towers, CG and Fitzwalter, BE and Regan, D and Goodspeed, A and Morgan, MJ and Liu, CW and Gustafson, DL and Thorburn, A}, title = {Cancer Cells Upregulate NRF2 Signaling to Adapt to Autophagy Inhibition.}, journal = {Developmental cell}, volume = {50}, number = {6}, pages = {690-703.e6}, pmid = {31378590}, issn = {1878-1551}, support = {K01 OD022982/OD/NIH HHS/United States ; L30 TR002126/TR/NCATS NIH HHS/United States ; L30 TR002126/TR/NCATS NIH HHS/United States ; K01 OD022982/CD/ODCDC CDC HHS/United States ; R01 CA150925/CA/NCI NIH HHS/United States ; R01 CA190170/CA/NCI NIH HHS/United States ; T32 CA190216/CA/NCI NIH HHS/United States ; P30 CA046934/CA/NCI NIH HHS/United States ; }, mesh = {*Adaptation, Physiological/drug effects ; *Autophagy/drug effects ; Autophagy-Related Protein 7/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/drug effects ; Clone Cells ; Gene Knockout Techniques ; Genes, Essential ; Humans ; NF-E2-Related Factor 2/*metabolism ; Neoplasms/*metabolism/*pathology ; Proteasome Endopeptidase Complex/metabolism ; Proteasome Inhibitors/pharmacology ; Ribonucleoproteins/metabolism ; *Signal Transduction/drug effects ; *Up-Regulation/drug effects ; }, abstract = {While autophagy is thought to be an essential process in some cancer cells, it is unknown if or how such cancer cells can circumvent autophagy inhibition. To address this, we developed a CRISPR/Cas9 assay with dynamic live-cell imaging to measure acute effects of knockout (KO) of autophagy genes compared to known essential and non-essential genes. In some cancer cells, autophagy is as essential for cancer cell growth as mRNA transcription or translation or DNA replication. However, even these highly autophagy-dependent cancer cells evolve to circumvent loss of autophagy by upregulating NRF2, which is necessary and sufficient for autophagy-dependent cells to circumvent ATG7 KO and maintain protein homeostasis. Importantly, however, this adaptation increases susceptibly to proteasome inhibitors. These studies identify a common mechanism of acquired resistance to autophagy inhibition and show that selection to avoid tumor cell dependency on autophagy creates new, potentially actionable cancer cell susceptibilities.}, }
@article {pmid31297692, year = {2019}, author = {Ouyang, Y}, title = {Understanding and breaking down the reproductive barrier between Asian and African cultivated rice: a new start for hybrid rice breeding.}, journal = {Science China. Life sciences}, volume = {62}, number = {8}, pages = {1114-1116}, doi = {10.1007/s11427-019-9592-6}, pmid = {31297692}, issn = {1869-1889}, mesh = {Africa ; Alleles ; Asia ; CRISPR-Cas Systems/genetics ; Genetic Variation ; Hybridization, Genetic ; Mutation ; Oryza/*genetics/*growth &amp; development ; Phylogeny ; Plant Breeding/*methods ; Reproduction ; }, }
@article {pmid31124706, year = {2019}, author = {Fan, J and Xia, Y and Wang, GL}, title = {An improved heteroduplex analysis for rapid genotyping of SNPs and single base pair indels.}, journal = {BioTechniques}, volume = {67}, number = {1}, pages = {6-10}, doi = {10.2144/btn-2019-0012}, pmid = {31124706}, issn = {1940-9818}, mesh = {Base Pairing ; CRISPR-Cas Systems ; DNA, Plant/genetics ; Genotyping Techniques/economics/methods ; Heteroduplex Analysis/economics/*methods ; *INDEL Mutation ; Oryza/genetics ; *Polymorphism, Single Nucleotide ; Time Factors ; }, abstract = {SNPs and single base pair (SBP) insertion/deletions (indels) are not only the most abundant genetic markers for genetic mapping and breeding selection, but also always occur in the mutants generated from chemical mutagenesis or CRISPR/Cas9-mediated genome editing. Most of the current SNP and SBP indel genotyping methods are time-consuming and/or require special equipment or reagents. Here, we describe an improved heteroduplex analysis method, named iHDA, that can readily discriminate SNP and SBP indel alleles with specially designed DNA probes that harbor a couple of nucleotides adjacent to the SNP site. By hybridizing with the same probe, SNP and SBP indel alleles form different heteroduplexes, differing in bulge size, which show different mobility on a polyacrylamide gel. Therefore, iHDA is an easy, fast and inexpensive method for SNP and SBP indel genotyping.}, }
@article {pmid31124015, year = {2019}, author = {Wang, Z and Yang, L and Qu, S and Zhang, C}, title = {CRISPR-mediated gene editing to rescue haploinsufficient obesity syndrome.}, journal = {Protein &amp; cell}, volume = {10}, number = {10}, pages = {705-708}, pmid = {31124015}, issn = {1674-8018}, mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; Obesity ; }, }
@article {pmid30988149, year = {2019}, author = {Sun, Q and Wang, Y and Dong, N and Shen, L and Zhou, H and Hu, Y and Gu, D and Chen, S and Zhang, R and Ji, Q}, title = {Application of CRISPR/Cas9-Based Genome Editing in Studying the Mechanism of Pandrug Resistance in Klebsiella pneumoniae.}, journal = {Antimicrobial agents and chemotherapy}, volume = {63}, number = {7}, pages = {}, pmid = {30988149}, issn = {1098-6596}, mesh = {CRISPR-Cas Systems/genetics ; Colistin/pharmacology ; Gene Editing/*methods ; Klebsiella pneumoniae/*drug effects/genetics ; Tigecycline/pharmacology ; }, abstract = {In this study, a CRISPR/Cas9-mediated genome editing method was used to study the functions of the mgrB, tetA, and ramR genes in mediating colistin and tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP). Inactivation of the tetA or ramR gene or the mgrB gene by CRISPR/Cas9 affected bacterial susceptibility to tigecycline or colistin, respectively. This study proved that the CRISPR/Cas9-based genome editing method could be effectively applied to K. pneumoniae and should be further utilized for genetic characterization.}, }
@article {pmid30978509, year = {2019}, author = {Chin, JS and Chooi, WH and Wang, H and Ong, W and Leong, KW and Chew, SY}, title = {Scaffold-mediated non-viral delivery platform for CRISPR/Cas9-based genome editing.}, journal = {Acta biomaterialia}, volume = {90}, number = {}, pages = {60-70}, doi = {10.1016/j.actbio.2019.04.020}, pmid = {30978509}, issn = {1878-7568}, mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Editing ; *Gene Transfer Techniques ; Humans ; Indoles/chemistry/pharmacology ; Nanofibers/chemistry ; Polymers/chemistry/pharmacology ; }, abstract = {Genome editing, especially via the simple and versatile type II CRISPR/Cas9 system, offers an effective avenue to precisely control cell fate, an important aspect of tissue regeneration. Unfortunately, most CRISPR/Cas9 non-viral delivery strategies only utilise micro-/nano-particle delivery methods. While these approaches provide reasonable genomic editing efficiencies, their systemic delivery may lead to undesirable off-target effects. For in vivo applications, a more localized and sustained delivery approach may be useful, particularly in the context of tissue regeneration. Here, we developed a scaffold that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and non-viral manner. Specifically, using mussel-inspired bioadhesive coating, polyDOPA-melanin (pDOPA), we adsorbed Cas9:sgRNA lipofectamine complexes onto bio-mimicking fiber scaffolds. To evaluate the genome-editing efficiency of this platform, U2OS.EGFP cells were used as the model cell type. pDOPA coating was essential in allowing Cas9:sgRNA lipofectamine complexes to adhere onto the scaffolds with a higher loading efficiency, while laminin coating was necessary for maintaining cell viability and proliferation on the pDOPA-coated fibers for effective gene editing (21.5% editing efficiency, p < 0.001). Importantly, U2OS.EGFP cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds via reverse transfection. Overall, we demonstrate the efficacy of such fiber scaffolds in providing localized, sustained and non-viral delivery of Cas9:sgRNA complexes. Such genome editing scaffolds may find useful applications in tissue regeneration. STATEMENT OF SIGNIFICANCE: Currently, there is a lack of effective non-viral means to deliver CRISPR/Cas9 components for genome editing. Most existing approaches only utilize micro-/nano-particles by injection or systemic delivery, which may lead to undesirable off-target effects. Here, we report a platform that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and sustained manner. We used mussel-inspired bioadhesive coating to functionalize the bio-mimicking fiber scaffolds with Cas9:sgRNA lipofectamine complexes, to allow effective gene editing for the cells seeded on the scaffolds. Importantly, the cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds. Such genome editing scaffolds may find useful applications in tissue regeneration.}, }
@article {pmid30947515, year = {2019}, author = {Dunbar, GL and Koneru, S and Kolli, N and Sandstrom, M and Maiti, P and Rossignol, J}, title = {Silencing of the Mutant Huntingtin Gene through CRISPR-Cas9 Improves the Mitochondrial Biomarkers in an In Vitro Model of Huntington's Disease.}, journal = {Cell transplantation}, volume = {28}, number = {4}, pages = {460-463}, pmid = {30947515}, issn = {1555-3892}, mesh = {Biomarkers/*metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Huntington Disease/*genetics ; Mitochondria/*metabolism ; }, abstract = {During the 25-year history of the American Society for Neural Therapy and Repair (ASNTR) there have been several breakthroughs in the area of neurotherapeutics, which was the case during the 2014-2105 year when one of us (GLD) had the privilege of serving as its president. During that year, the use of a newly developed gene-editing tool, the CRISPR-Cas9 system, started to skyrocket. Although scientists unraveled the use of &quot;clustered regularly interspaced short palindromic repeats&quot; (CRISPR) and its associated genes from the Cas family as an evolved mechanism of some bacterial and archaeal genomes to protect themselves from being hijacked by invasive viral genes, its use as a therapeutic tool was not fully appreciated until further research revealed how this system operated and how it might be developed technologically to manipulate genes of any species. By 2015, this technology had exploded to the point that close to 2,000 papers that used this technology were published during that year alone.}, }
@article {pmid30926729, year = {2019}, author = {Tapscott, T and Guarnieri, MT and Henard, CA}, title = {Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing.}, journal = {Applied and environmental microbiology}, volume = {85}, number = {11}, pages = {}, pmid = {30926729}, issn = {1098-5336}, mesh = {Bacterial Proteins/*genetics ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Codon, Terminator ; Deoxyribonuclease I/metabolism ; Gene Editing/*methods ; Gene Expression Regulation, Bacterial ; Genome, Bacterial ; Methylococcus capsulatus/*genetics ; Oxygenases/genetics ; Plasmids/genetics ; }, abstract = {Methanotrophic bacteria play a crucial role in the Earth's biogeochemical cycle and have the potential to be employed in industrial biomanufacturing processes due to their capacity to use natural gas- and biogas-derived methane as a sole carbon and energy source. Advanced gene-editing systems have the potential to enable rapid, high-throughput methanotrophic genetics and biocatalyst development. To this end, we employed a series of broad-host-range expression plasmids to construct a conjugatable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene-editing system in Methylococcus capsulatus (Bath). Heterologous coexpression of the Streptococcus pyogenes Cas9 endonuclease and a synthetic single guide RNA (gRNA) showed efficient Cas9 DNA targeting and double-stranded DNA (dsDNA) cleavage that resulted in cell death. We demonstrated effective in vivo editing of plasmid DNA using both Cas9 and Cas9D10A nickase to convert green fluorescent protein (GFP)- to blue fluorescent protein (BFP)-expressing cells with 71% efficiency. Further, we successfully introduced a premature stop codon into the soluble methane monooxygenase (sMMO) hydroxylase component-encoding mmoX gene with the Cas9D10A nickase, disrupting sMMO function. These data provide proof of concept for CRISPR/Cas9-mediated gene editing in M. capsulatus Given the broad-host-range replicons and conjugation capability of these CRISPR/Cas9 tools, they have potential utility in other methanotrophs and a wide array of Gram-negative microorganisms.IMPORTANCE In this study, we targeted the development and evaluation of broad-host-range CRISPR/Cas9 gene-editing tools in order to enhance the genetic-engineering capabilities of an industrially relevant methanotrophic biocatalyst. The CRISPR/Cas9 system developed in this study expands the genetic tools available to define molecular mechanisms in methanotrophic bacteria and has the potential to foster advances in the generation of novel biocatalysts to produce biofuels, platform chemicals, and high-value products from natural gas- and biogas-derived methane. Further, due to the broad-host-range applicability, these genetic tools may also enable innovative approaches to overcome the barriers associated with genetically engineering diverse, industrially promising nonmodel microorganisms.}, }
@article {pmid30853558, year = {2019}, author = {Xu, H and Wang, B and Ono, M and Kagita, A and Fujii, K and Sasakawa, N and Ueda, T and Gee, P and Nishikawa, M and Nomura, M and Kitaoka, F and Takahashi, T and Okita, K and Yoshida, Y and Kaneko, S and Hotta, A}, title = {Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility.}, journal = {Cell stem cell}, volume = {24}, number = {4}, pages = {566-578.e7}, doi = {10.1016/j.stem.2019.02.005}, pmid = {30853558}, issn = {1875-9777}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Female ; *Gene Editing ; HLA Antigens/*genetics/immunology ; Histocompatibility/*immunology ; Humans ; Induced Pluripotent Stem Cells/*immunology/*metabolism ; Male ; Mice ; Mice, Inbred NOD ; }, abstract = {Induced pluripotent stem cells (iPSCs) have strong potential in regenerative medicine applications; however, immune rejection caused by HLA mismatching is a concern. B2M gene knockout and HLA-homozygous iPSC stocks can address this issue, but the former approach may induce NK cell activity and fail to present antigens, and it is challenging to recruit rare donors for the latter method. Here, we show two genome-editing strategies for making immunocompatible donor iPSCs. First, we generated HLA pseudo-homozygous iPSCs with allele-specific editing of HLA heterozygous iPSCs. Second, we generated HLA-C-retained iPSCs by disrupting both HLA-A and -B alleles to suppress the NK cell response while maintaining antigen presentation. HLA-C-retained iPSCs could evade T cells and NK cells in vitro and in vivo. We estimated that 12 lines of HLA-C-retained iPSCs combined with HLA-class II knockout are immunologically compatible with >90% of the world's population, greatly facilitating iPSC-based regenerative medicine applications.}, }
@article {pmid30586281, year = {2019}, author = {Chang, J and Chen, X and Glass, Z and Gao, F and Mao, L and Wang, M and Xu, Q}, title = {Integrating Combinatorial Lipid Nanoparticle and Chemically Modified Protein for Intracellular Delivery and Genome Editing.}, journal = {Accounts of chemical research}, volume = {52}, number = {3}, pages = {665-675}, pmid = {30586281}, issn = {1520-4898}, support = {R01 EB027170/EB/NIBIB NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Drug Carriers/*chemistry ; Gene Editing/methods ; Gene Transfer Techniques ; Humans ; Lipids/*chemistry ; Mice ; Nanoparticles/*chemistry ; Proteins/*metabolism ; Rats ; }, abstract = {The use of protein to precisely manipulate cell signaling is an effective approach for controlling cell fate and developing precision medicine. More recently, programmable nucleases, such as CRISPR/Cas9, have shown extremely high potency for editing genetic flow of mammalian cells, and for treating genetic disorders. The therapeutic potential of proteins with an intracellular target, however, is mostly challenged by their low cell impermeability. Therefore, a developing delivery system to transport protein to the site of action in a spatiotemporal controlled manner is of great importance to expand the therapeutic index of the protein. In this Account, we first summarize our most recent advances in designing combinatorial lipid nanoparticles with diverse chemical structures for intracellular protein delivery. By designing parallel Michael addition or ring-opening reaction of aliphatic amines, we have generated a combinatorial library of cationic lipids, and identified several leading nanoparticle formulations for intracellular protein delivery both in vitro and in vivo. Moreover, we optimized the chemical structure of lipids to control lipid degradation and protein release inside cells for CRISPR/Cas9 genome-editing protein delivery. In the second part of this Account, we survey our recent endeavor in developing a chemical approach to modify protein, in particular, coupled with the nanoparticle delivery platform, to improve protein delivery for targeted diseases treatment and genome editing. Chemical modification of protein is a useful tool to modulate protein function and to improve the therapeutic index of protein drugs. Herein, we mostly summarize our recent advances on designing chemical approaches to modify protein with following unique findings: (1) chemically modified protein shows selective turn-on activity based on the specific intracellular microenvironment, with which we were able to protein-based targeted cancer therapy; (2) the conjugation of hyaluronic acid (HA) to protein allows cancer cell surface receptor-targeted delivery of protein; (3) the introduction of nonpeptidic boronic acid into protein enabled cell nucleus targeted delivery; this is the first report that a nonpeptidic signal can direct protein to subcellular compartment; and (4) the fusion of protein with negatively supercharged green fluorescent protein (GFP) facilitates the self-assembly of protein with lipid nanoparticle for genome-editing protein delivery. At the end of the Account, we give a perspective of expanding the chemistry that could be integrated to design biocompatible lipid nanocarriers for protein delivery and genome editing in vitro and in vivo, as well as the chemical approaches that we can harness to modulate protein activity in live cells for targeted diseases treatment.}, }
@article {pmid32416935, year = {2020}, author = {Levesque, S and Agudelo, D and Doyon, Y}, title = {Rewired Cas9s with Minimal Sequence Constraints.}, journal = {Trends in pharmacological sciences}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tips.2020.04.009}, pmid = {32416935}, issn = {1873-3735}, abstract = {The genome editing toolkit is ever expanding. Although CRISPR-Cas systems can target virtually any gene, single-nucleotide resolution is yet to be achieved. Walton and colleagues engineered nucleases and base editors compatible with every protospacer adjacent motif (PAM) to achieve high-precision targeting. Their findings revealed the striking plasticity of Cas9.}, }
@article {pmid32416111, year = {2020}, author = {Wan, T and Ping, Y}, title = {Delivery of genome-editing biomacromolecules for treatment of lung genetic disorders.}, journal = {Advanced drug delivery reviews}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.addr.2020.05.002}, pmid = {32416111}, issn = {1872-8294}, abstract = {Genome-editing systems based on clustered, regularly interspaced, short palindromic repeat (CRISPR)/associated protein (CRISPR/Cas), are emerging as a revolutionary technology for the treatment of various genetic diseases. To date, the delivery of genome-editing biomacromolecules by viral or non-viral have been proposed as new therapeutic options for lung genetic disorders, such as cystic fibrosis (CF) and α-1 antitrypsin deficiency (AATD), and it has been accepted that these delivery vectors can introduce CRISPR/Cas9 machineries into target cells or tissues in vitro, ex vivo and in vivo. However, the efficient local or systemic delivery of CRISPR/Cas9 elements to the lung, enabled by either viral or by non-viral carriers, still remains elusive. Herein, we first introduce lung genetic disorders and their current treatment options, and then summarize CRISPR/Cas9-based strategies for the therapeutic genome editing of these disorders. We further summarize the pros and cons of different routes of administration for lung genetic disorders. In particular, the potentials of aerosol delivery for therapeutic CRISPR/Cas9 biomacromolecules for lung genome editing are discussed highlighted. Finally, current challenges and future outlooks in this emerging area are briefly discussed.}, }
@article {pmid32413572, year = {2020}, author = {Liu, D and Zhao, X and Tang, A and Xu, X and Liu, S and Zha, L and Ma, W and Zheng, J and Shi, M}, title = {Potential of CRISPR screen in target discovery for cancer immunotherapy.}, journal = {Biochimica et biophysica acta. Reviews on cancer}, volume = {}, number = {}, pages = {188378}, doi = {10.1016/j.bbcan.2020.188378}, pmid = {32413572}, issn = {1879-2561}, abstract = {CRISPR/Cas-based genetic perturbation screens have emerged as powerful tools for large-scale identification of new targets for cancer immunotherapy. Various strategies of CRISPR screen have been used for immune-oncology (IO) target discovery. The genomic sequences targeted by CRISPR/Cas system range from coding sequences to non-coding RNA/DNA, including miRNAs, LncRNAs, circRNAs, promoters, and enhancers, which may be potential targets for future pharmacological and therapeutic interventions. Rapid progresses have been witnessed in finding novel targets for enhancing tumor antigen presentation, sensitizing of tumor cells to immune-mediated cytotoxicity, and reinvigorating tumor-specific T cells by using CRISPR technologies. In combination with other strategies, the detailed characteristics of the targets for immunotherapy have been obtained by CRISPR screen. In this review, we present an overview of recent progresses in the development of CRISPR-based screens for IO target identification and discuss the challenges and possible solutions in this rapidly growing field.}, }
@article {pmid32413257, year = {2020}, author = {Boonekamp, FJ and Dashko, S and Duiker, D and Gehrmann, T and van den Broek, M and den Ridder, M and Pabst, M and Robert, V and Abeel, T and Postma, ED and Daran, JM and Daran-Lapujade, P}, title = {Design and experimental evaluation of a minimal, innocuous watermarking strategy to distinguish near-identical DNA and RNA sequences.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.0c00045}, pmid = {32413257}, issn = {2161-5063}, abstract = {The construction of powerful cell factories requires intensive and extensive remodelling of microbial genomes. Considering the rapidly increasing number of these synthetic biology endeavours, there is an increasing need for DNA watermarking strategies that enable the discrimination between synthetic and native gene copies. While it is well documented that codon usage can affect translation, and most likely mRNA stability in eukaryotes, remarkably few quantitative studies explore the impact of watermarking on transcription, protein expression and physiology in the popular model and industrial yeast Saccharomyces cerevisiae. The present study, using S. cerevisiae as eukaryotic paradigm, designed, implemented and experimentally validated a systematic strategy to watermark DNA with minimal alteration of yeast physiology. The thirteen genes encoding proteins involved in the major pathway for sugar utilization (i.e glycolysis and alcoholic fermentation) were simultaneously watermarked in a yeast strain using the previously published pathway swapping strategy. Carefully swapping codons of these naturally codon optimized, highly expressed genes, did not affect yeast physiology and did not alter transcript abundance, protein abundance and protein activity besides a mild effect on Gpm1. The markerQuant bioinformatics method could reliably discriminate native from watermarked genes and transcripts. Furthermore, presence of watermarks enabled selective CRISPR/Cas genome editing, specifically targeting the native gene copy while leaving the synthetic, watermarked variant intact. This study offers a validated strategy to simply watermark genes in S. cerevisiae.}, }
@article {pmid32411384, year = {2020}, author = {Lin, J and Feng, M and Zhang, H and She, Q}, title = {Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase.}, journal = {Cell discovery}, volume = {6}, number = {}, pages = {29}, doi = {10.1038/s41421-020-0160-4}, pmid = {32411384}, issn = {2056-5968}, abstract = {Antiviral defense by type III CRISPR-Cas systems relies on two distinct activities of their effectors: the RNA-activated DNA cleavage and synthesis of cyclic oligoadenylate. Both activities are featured as indiscriminate nucleic acid cleavage and subjected to the spatiotemporal regulation. To yield further insights into the involved mechanisms, we reconstituted LdCsm, a lactobacilli III-A system in Escherichia coli. Upon activation by target RNA, this immune system mediates robust DNA degradation but lacks the synthesis of cyclic oligoadenylates. Mutagenesis of the Csm3 and Cas10 conserved residues revealed that Csm3 and multiple structural domains in Cas10 function in the allosteric regulation to yield an active enzyme. Target RNAs carrying various truncations in the 3' anti-tag were designed and tested for their influence on DNA binding and DNA cleavage of LdCsm. Three distinct states of ternary LdCsm complexes were identified. In particular, binding of target RNAs carrying a single nucleotide in the 3' anti-tag to LdCsm yielded an active LdCsm DNase regardless whether the nucleotide shows a mismatch, as in the cognate target RNA (CTR), or a match, as in the noncognate target RNA (NTR), to the 5' tag of crRNA. In addition, further increasing the number of 3' anti-tag in CTR facilitated the substrate binding and enhanced the substrate degradation whereas doing the same as in NTR gradually decreased the substrate binding and eventually shut off the DNA cleavage by the enzyme. Together, these results provide the mechanistic insights into the allosteric activation and repression of LdCsm enzymes.}, }
@article {pmid32409581, year = {2020}, author = {Collins, MP and Stransky, LA and Forgac, M}, title = {AKT Ser/Thr kinase increases V-ATPase-dependent lysosomal acidification in response to amino acid starvation in mammalian cells.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1074/jbc.RA120.013223}, pmid = {32409581}, issn = {1083-351X}, abstract = {The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that is essential for cellular homeostasis. V-ATPase activity is controlled by regulated assembly of the enzyme from its component V1 and V0 domains. We previously reported that amino acid starvation rapidly increases V-ATPase assembly and activity in mammalian lysosomes, but the signaling pathways controlling this effect are unknown. In testing inhibitors of pathways important for controlling cellular metabolism, we found here that the cAMP-dependent protein kinase (PKA) inhibitor H89 increases lysosomal V-ATPase activity and blocks any further change upon starvation. The AMP-activated protein kinase (AMPK) inhibitor dorsomorphin decreased lysosomal V-ATPase activity and also blocked any increase upon starvation. However, CRISPR-mediated gene editing revealed that PKA and AMPK are not required for the starvation-dependent increase in lysosomal V-ATPase activity, indicating that H89 and dorsomorphin modify V-ATPase activity through other cellular targets. We next found that the AKT Ser/Thr kinase (AKT) inhibitor MK2206 blocks the starvation-dependent increase in lysosomal V-ATPase activity without altering basal activity. Expression of AKT1 or AKT3, but not AKT2, was required for increased lysosomal V-ATPase activity in response to amino acid starvation in mouse fibroblasts. Finally, HEK293T cells expressing only AKT1 responded normally to starvation, whereas cells expressing only AKT2 displayed a significantly reduced increase in V-ATPase activity and assembly upon starvation. These results show that AKT is required for controlling the rapid response of lysosomal V-ATPase activity to changes in amino acid availability and that this response depends on specific AKT isoforms.}, }
@article {pmid32409578, year = {2020}, author = {Morimoto, K and Suzuki, N and Tanida, I and Kakuta, S and Furuta, Y and Uchiyama, Y and Hanada, K and Suzuki, Y and Yamaji, T}, title = {Blood group P1 antigen-bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1074/jbc.RA120.013926}, pmid = {32409578}, issn = {1083-351X}, abstract = {Shiga toxin (STx) is a virulence factor produced by enterohemorrhagic Escherichia coli. STx is taken up by mammalian host cells by binding to the glycosphingolipid (GSL) globotriaosylceramide (Gb3, Galα1-4Galβ1-4Glc-ceramide) and causes cell death after its retrograde membrane transport. However, the contribution of the hydrophobic portion of Gb3 (ceramide) to STx transport remains unclear. In pigeons, blood group P1 glycan antigens (Galα1-4Galβ1-4GlcNAc-) are expressed on glycoproteins that are synthesized by α1,4-galactosyltransferase 2 (pA4GalT2). To examine whether these glycoproteins can also function as STx receptors, here we constructed glycan-remodeled HeLa cell variants lacking Gb3 expression but instead expressing pA4GalT2-synthesized P1 glycan antigens on glycoproteins. We compared STx binding and sensitivity of these variants with those of the parental, Gb3-expressing HeLa cells. The glycan-remodeled cells bound STx1 via N-glycans of glycoproteins and were sensitive to STx1 even without Gb3 expression, indicating that P1-containing glycoproteins also function as STx receptors. However, these variants were significantly less sensitive to STx than the parent cells. Fluorescence microscopy and correlative light-EM revealed that the STx1 B subunit accumulates less in the Golgi apparatus after glycoprotein-mediated than after Gb3-mediated uptake, but instead accumulates in vacuole-like structures probably derived from early endosomes. Furthermore, co-expression of Galα1-4Gal on both glycoproteins and GSLs reduced the sensitivity of cells to STx1 compared with those expressing Galα1-4Gal only on GSLs, probably because of competition for STx binding or internalization. We conclude that lipid-based receptors are much more effective in STx retrograde transport and mediate greater STx cytotoxicity than protein-based receptors.}, }
@article {pmid32408568, year = {2020}, author = {Pan, M and Nethery, MA and Hidalgo-Cantabrana, C and Barrangou, R}, title = {Comprehensive Mining and Characterization of CRISPR-Cas Systems in Bifidobacterium.}, journal = {Microorganisms}, volume = {8}, number = {5}, pages = {}, doi = {10.3390/microorganisms8050720}, pmid = {32408568}, issn = {2076-2607}, support = {probiotics research//North Carolina Agricultural Foundation/ ; Nutrition &amp; Health//DuPont/ ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated cas) systems constitute the adaptive immune system in prokaryotes, which provides resistance against bacteriophages and invasive genetic elements. The landscape of applications in bacteria and eukaryotes relies on a few Cas effector proteins that have been characterized in detail. However, there is a lack of comprehensive studies on naturally occurring CRISPR-Cas systems in beneficial bacteria, such as human gut commensal Bifidobacterium species. In this study, we mined 954 publicly available Bifidobacterium genomes and identified CRIPSR-Cas systems in 57% of these strains. A total of five CRISPR-Cas subtypes were identified as follows: Type I-E, I-C, I-G, II-A, and II-C. Among the subtypes, Type I-C was the most abundant (23%). We further characterized the CRISPR RNA (crRNA), tracrRNA, and PAM sequences to provide a molecular basis for the development of new genome editing tools for a variety of applications. Moreover, we investigated the evolutionary history of certain Bifidobacterium strains through visualization of acquired spacer sequences and demonstrated how these hypervariable CRISPR regions can be used as genotyping markers. This extensive characterization will enable the repurposing of endogenous CRISPR-Cas systems in Bifidobacteria for genome engineering, transcriptional regulation, genotyping, and screening of rare variants.}, }
@article {pmid31727171, year = {2019}, author = {Zhang, S and Zhang, F and Chen, Q and Wan, C and Xiong, J and Xu, J}, title = {CRISPR/Cas9-mediated knockout of NSD1 suppresses the hepatocellular carcinoma development via the NSD1/H3/Wnt10b signaling pathway.}, journal = {Journal of experimental &amp; clinical cancer research : CR}, volume = {38}, number = {1}, pages = {467}, pmid = {31727171}, issn = {1756-9966}, mesh = {Animals ; CRISPR-Cas Systems ; Carcinoma, Hepatocellular/genetics/metabolism/pathology ; Cell Line, Tumor ; Cell Movement/physiology ; Cell Proliferation/physiology ; Female ; Hep G2 Cells ; Heterografts ; Histone-Lysine N-Methyltransferase/genetics/*metabolism ; Histones/genetics/*metabolism ; Humans ; Liver Neoplasms/genetics/*metabolism/pathology ; Male ; Methylation ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Middle Aged ; Promoter Regions, Genetic ; Proto-Oncogene Proteins/genetics/*metabolism ; Signal Transduction ; Wnt Proteins/genetics/*metabolism ; }, abstract = {BACKGROUND: The NSD family of histone lysine methyltransferases have emerged as important biomarkers that participate in a variety of malignancies. Recent evidence has indicated that somatic dysregulation of the nuclear receptor binding SET domain-containing protein 1 (NSD1) is associated with the tumorigenesis in HCC, suggesting that NSD1 may serve as a prognostic target for this malignant tumor. However, its mechanism in human hepatocellular carcinoma (HCC), the major primary malignant tumor in the human liver, remains unclear. Hence, we investigated how NSD1 regulated HCC progression via regulation of the Wnt/β-catenin signaling pathway.<br><br>METHODS: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis was performed to identify the expression of NSD1 in HCC cells and clinically obtained tissues. The relationship between NSD1 expression and prognosis was analyzed by Kaplan-Meier survival curve. Further, a NSD1 knockout cell line was constructed by CRISPR/Cas9 genomic editing system, which was investigated in a battery of assays such as HCC cell proliferation, migration and invasion, followed by the investigation into NSD1 regulation on histone H3, Wnt10b and Wnt/β-catenin signaling pathway via ChIP. Finally, a nude mouse xenograft model was conducted in order to assess tumorigenesis affected by NSD1 knockout in vivo.<br><br>RESULTS: NSD1 was overexpressed in HCC tissues and cell lines in association with poor prognosis. Knockout of NSD1 inhibited the proliferation, migration and invasion abilities of HCC cells. CRISPR/Cas9-mediated knockout of NSD1 promoted methylation of H3K27me3 and reduced methylation of H3K36me2, which inhibited Wnt10b expression. The results thereby indicated an inactivation of the Wnt/β-catenin signaling pathway suppressed cell proliferation, migration and invasion in HCC. Moreover, these in vitro findings were reproduced in vivo on tumor xenograft in nude mice.<br><br>CONCLUSION: In conclusion, the study provides evidence that CRISPR/Cas9-mediated NSD1 knockout suppresses HCC cell proliferation and migration via the NSD1/H3/Wnt10b signaling pathway, suggesting that NSD1, H3 and Wnt10b may serve as potential targets for HCC.}, }
@article {pmid31418136, year = {2019}, author = {Ji, X and Wang, D and Gao, C}, title = {CRISPR editing-mediated antiviral immunity: a versatile source of resistance to combat plant virus infections.}, journal = {Science China. Life sciences}, volume = {62}, number = {9}, pages = {1246-1249}, doi = {10.1007/s11427-019-9722-2}, pmid = {31418136}, issn = {1869-1889}, mesh = {Antiviral Agents/*metabolism ; CRISPR-Cas Systems/*genetics/*immunology ; DNA, Plant/genetics ; Gene Editing ; Gene Expression ; Genome, Bacterial/genetics ; Infections/metabolism ; Mutation ; Plant Diseases/*genetics/virology ; Plant Viruses/genetics/*metabolism ; Virus Diseases/*prevention &amp; control ; }, }
@article {pmid31060819, year = {2019}, author = {Xu, RG and Wang, X and Shen, D and Sun, J and Qiao, HH and Wang, F and Liu, LP and Ni, JQ}, title = {Perspectives on gene expression regulation techniques in Drosophila.}, journal = {Journal of genetics and genomics = Yi chuan xue bao}, volume = {46}, number = {4}, pages = {213-220}, doi = {10.1016/j.jgg.2019.03.006}, pmid = {31060819}, issn = {1673-8527}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Drosophila melanogaster/*genetics ; Gene Expression ; Genetic Engineering/*methods ; RNA Interference ; Transcriptional Activation ; }, abstract = {Gene expression regulation, including loss-of-function and gain-of-function assays, is a powerful method to study developmental and disease mechanisms. Drosophila melanogaster is an ideal model system particularly well-equipped with many genetic tools. In this review, we describe and discuss the gene expression regulation techniques recently developed and their applications, including the CRISPR/Cas9-triggered heritable mutation system, CRISPR/dCas9-based transcriptional activation (CRISPRa) system, and CRISPR/dCas9-based transcriptional repression (CRISPRi) system, as well as the next-generation transgenic RNAi system. The main purpose of this review is to provide the fly research community with an updated summary of newly developed gene expression regulation techniques and help the community to select appropriate methods and optimize the research strategy.}, }
@article {pmid30593611, year = {2019}, author = {Liu, Q and Qi, Y and Liang, Q and Song, J and Liu, J and Li, W and Shu, Y and Tao, M and Zhang, C and Qin, Q and Wang, J and Liu, S}, title = {Targeted disruption of tyrosinase causes melanin reduction in Carassius auratus cuvieri and its hybrid progeny.}, journal = {Science China. Life sciences}, volume = {62}, number = {9}, pages = {1194-1202}, doi = {10.1007/s11427-018-9404-7}, pmid = {30593611}, issn = {1869-1889}, mesh = {Animals ; Breeding/methods ; CRISPR-Cas Systems/genetics ; Female ; Gene Expression Regulation ; Gene Knockout Techniques ; Goldfish ; Hybridization, Genetic ; Male ; Melanins/*metabolism ; Monophenol Monooxygenase/*genetics/*metabolism ; Mutation ; Pigmentation/genetics ; RNA, Messenger ; Sequence Analysis, RNA ; }, abstract = {The white crucian carp (Carassius auratus cuvieri, WCC) not only is one of the most economically important fish in Asia, characterized by strong reproductive ability and rapid growth rates, but also represents a good germplasm to produce hybrid progenies with heterosis. Gene knockout technique provides a safe and acceptant way for fish breeding. Achieving gene knockout in WCC and its hybrid progeny will be of great importance for both genetic studies and hybridization breeding. Tyrosinase (TYR) is a key enzyme in melanin synthesis. Depletion of tyr in zebrafish and mice results in mosaic pigmentation or total albinism. Here, we successfully used CRISPR-Cas9 to target tyr in WCC and its hybrid progeny (WR) derived from the cross of WCC (♀) and red crucian carp (Carassius auratus red var., RCC, ♂). The level of TYR protein was significantly reduced in mutant WCC. Both the mutant WCC and the mutant WR showed different degrees of melanin reduction compared with the wild-type sibling control fish, resulting from different mutation efficiency ranging from 60% to 90%. In addition, the transcriptional expression profiles of a series of pivotal pigment synthesis genes, i.e. tyrp1, mitfa, mitfb, dct and sox10, were down-regulated in tyr-CRISPR WCC, which ultimately caused a reduction in melanin synthesis. These results demonstrated that tyr plays a key role in melanin synthesis in WCC and WR, and CRISPR-Cas9 is an effective tool for modifying the genome of economical fish. Furthermore, the tyr-CRISPR models could be valuable in understanding fundamental mechanisms of pigment formation in non-model fish.}, }
@article {pmid31917956, year = {2020}, author = {Zhang, L and Xu, W and Gao, X and Li, W and Qi, S and Guo, D and Ajayi, OE and Ding, SW and Wu, Q}, title = {lncRNA Sensing of a Viral Suppressor of RNAi Activates Non-canonical Innate Immune Signaling in Drosophila.}, journal = {Cell host &amp; microbe}, volume = {27}, number = {1}, pages = {115-128.e8}, doi = {10.1016/j.chom.2019.12.006}, pmid = {31917956}, issn = {1934-6069}, mesh = {Animals ; Antimicrobial Cationic Peptides/genetics/metabolism ; CRISPR-Cas Systems ; Carrier Proteins/*metabolism ; Dicistroviridae/genetics/*immunology/pathogenicity ; Drosophila Proteins/*metabolism ; Drosophila melanogaster/genetics/*immunology ; Gene Knockdown Techniques ; Immunity, Innate ; RNA Interference/immunology ; *RNA, Long Noncoding/genetics/immunology/metabolism ; Signal Transduction ; Viral Proteins/metabolism ; Virulence Factors/metabolism ; }, abstract = {Antiviral immunity in insects is mediated by the RNA interference (RNAi) pathway. Viruses evade antiviral RNAi by expressing virulence factors known as viral suppressors of RNAi (VSR). Here, we report the identification of VINR, a Drosophila VSR-interacting long non-coding (lnc) RNA that activates non-canonical innate immune signaling upon detection of the dsRNA-binding VSR of Drosophila C virus (DCV). VINR is required for the induction of antimicrobial peptide (AMP) genes but dispensable for antiviral RNAi. VINR functions by preventing the ubiquitin proteasome-dependent degradation of Cactin, a coiled-coil and arginine-serine-rich domain-containing protein that regulates a non-cannonical antimicrobial pathway for AMP induction. CRISPR-Cas9 knockout of VINR in Drosophila cells enhances DCV replication independently of antiviral RNAi, and VINR-knockout adult flies exhibit enhanced disease susceptibility to DCV and bacteria. Our findings reveal a counter counter-defense strategy activated by a lncRNA in response to the viral suppression of the primary antiviral RNAi immunity.}, }
@article {pmid31789463, year = {2020}, author = {Pizon, V and Gaudin, N and Poteau, M and Cifuentes-Diaz, C and Demdou, R and Heyer, V and Reina San Martin, B and Azimzadeh, J}, title = {hVFL3/CCDC61 is a component of mother centriole subdistal appendages required for centrosome cohesion and positioning.}, journal = {Biology of the cell}, volume = {112}, number = {1}, pages = {22-37}, doi = {10.1111/boc.201900038}, pmid = {31789463}, issn = {1768-322X}, support = {//CNRS/ ; RS16/75-105//Ligue Contre le Cancer/ ; //ANR/ ; }, mesh = {Animals ; CRISPR-Cas Systems ; Carrier Proteins/genetics/*metabolism ; Cell Line ; *Centrioles/metabolism/ultrastructure ; *Centrosome/metabolism/ultrastructure ; Cilia/ultrastructure ; Cytoskeletal Proteins/metabolism ; Cytoskeleton/ultrastructure ; Humans ; Microscopy, Electron ; Microtubules/metabolism/ultrastructure ; RNA, Small Interfering ; Tubulin/*metabolism ; }, abstract = {BACKGROUND: The centrosome regulates cell spatial organisation by controlling the architecture of the microtubule (MT) cytoskeleton. Conversely, the position of the centrosome within the cell depends on cytoskeletal networks it helps organizing. In mammalian cells, centrosome positioning involves a population of MT stably anchored at centrioles, the core components of the centrosome. An MT-anchoring complex containing the proteins ninein and Cep170 is enriched at subdistal appendages (SAP) that decorate the older centriole (called mother centriole) and at centriole proximal ends. Here, we studied the role played at the centrosome by hVFL3/CCDC61, the human ortholog of proteins required for anchoring distinct sets of cytoskeletal fibres to centrioles in unicellular eukaryotes.<br><br>RESULTS: We show that hVFL3 co-localises at SAP and at centriole proximal ends with components of the MT-anchoring complex, and physically interacts with Cep170. Depletion of hVFL3 increased the distance between mother and daughter centrioles without affecting the assembly of a filamentous linker that tethers the centrioles and contains the proteins rootletin and C-Nap1. When the linker was disrupted by inactivating C-Nap1, hVFL3-depletion exacerbated centriole splitting, a phenotype also observed following depletion of other SAP components. This supported that hVFL3 is required for SAP function, which we further established by showing that centrosome positioning is perturbed in hVFL3-depleted interphase cells. Finally, we found that hVFL3 is an MT-binding protein.<br><br>CONCLUSIONS AND SIGNIFICANCE: Together, our results support that hVFL3 is required for anchoring MT at SAP during interphase and ensuring proper centrosome cohesion and positioning. The role of the VFL3 family of proteins thus appears to have been conserved in evolution despite the great variation in the shape of centriole appendages in different eukaryotic species.}, }
@article {pmid31786675, year = {2020}, author = {Dong, L and Lin, X and Yu, D and Huang, L and Wang, B and Pan, L}, title = {High-level expression of highly active and thermostable trehalase from Myceliophthora thermophila in Aspergillus niger by using the CRISPR/Cas9 tool and its application in ethanol fermentation.}, journal = {Journal of industrial microbiology &amp; biotechnology}, volume = {47}, number = {1}, pages = {133-144}, pmid = {31786675}, issn = {1476-5535}, support = {31871736//National Natural Science Foundation of China/ ; 2018B020205002//the Research and Development Plan in Key Areas of Guangdong Province/ ; 2017B030302002//Guangdong Provincial Key Laboratory of Advanced Biofermentation Technology Enterprise in Flavoring &amp; Food/ ; }, mesh = {Aspergillus niger/genetics/*metabolism ; CRISPR-Cas Systems ; Enzyme Stability ; Ethanol/*metabolism ; Fermentation ; Hot Temperature ; Sordariales/genetics/*metabolism ; Trehalase/genetics/*metabolism ; }, abstract = {Trehalase catalyzes the hydrolysis of the non-reducing disaccharide trehalose. The highly active trehalase MthT from Myceliophthora thermophila was screened from the trehalase genes of six species of filamentous fungi. An ingenious multi-copy knock-in expression strategy mediated by the CRISPR/Cas9 tool and medium optimization were used to improve MthT production in Aspergillus niger, up to 1698.83 U/mL. The protein background was dramatically abated due to insertion. The recombinant MthT showed optimal activity at pH 5.5 and 60 °C, and exhibited prominent thermal stability between 50 and 60 °C under acid conditions (pH 4.5-6.5). The ethanol conversion rate (ethanol yield/total glucose) was significantly improved by addition of MthT (51.88%) compared with MthT absence (34.38%), using 30% starch saccharification liquid. The results of this study provided an effective strategy, established a convenient platform for heterologous expression in A. niger and showed a potential strategy to decrease production costs in industrial ethanol production.}, }
@article {pmid31768773, year = {2020}, author = {Li, ZH and Meng, H and Ma, B and Tao, X and Liu, M and Wang, FQ and Wei, DZ}, title = {Immediate, multiplexed and sequential genome engineering facilitated by CRISPR/Cas9 in Saccharomyces cerevisiae.}, journal = {Journal of industrial microbiology &amp; biotechnology}, volume = {47}, number = {1}, pages = {83-96}, pmid = {31768773}, issn = {1476-5535}, support = {31500043//National Natural Science Foundation of China/ ; 19ZR1473000//Natural Science Foundation of Shanghai (CN)/ ; 22221818014//Fundamental Research Funds for the Central Universities/ ; none//Open Funding Project of the State Key Laboratory of Bioreactor Engineering/ ; }, mesh = {CRISPR-Cas Systems ; Genome, Fungal ; Plasmids/genetics ; RNA, Fungal/genetics ; RNA, Guide/genetics ; Saccharomyces cerevisiae/*genetics ; }, abstract = {A method called Cas-3P allowing for immediate, multiplexed and sequential genome engineering was developed using one plasmid expressing Cas9 and three marked plasmid backbones (P1, P2 and P3) for guide RNA (gRNA) expression. The three marked gRNA plasmid backbones were recurred in a P1-P2-P3 order for sequential gene targeting, without construction of any additional plasmid and elimination of gRNA plasmid by induction in each round. The efficiency of direct gRNA plasmid curing mediated by Cas-3P was more than 40% in sequential gene targeting. Besides, Cas-3P allowed single-, double- and triple-loci gene targeting with an efficiency of 75%, 36.8% and 8.2% within 3-4 days, respectively. Through three sequential rounds of gene targeting within 10 days, S. cerevisiae was optimized for the production of patchoulol by replacing one promoter, overexpressing three genes and disrupting four genes. The work is important for practical application in the cell factory engineering of S. cerevisiae.}, }
@article {pmid31680172, year = {2019}, author = {Cali, CP and Park, DS and Lee, EB}, title = {Targeted DNA methylation of neurodegenerative disease genes via homology directed repair.}, journal = {Nucleic acids research}, volume = {47}, number = {22}, pages = {11609-11622}, pmid = {31680172}, issn = {1362-4962}, support = {R01 NS095793/NS/NINDS NIH HHS/United States ; }, mesh = {Amyloid beta-Protein Precursor/*genetics ; Amyotrophic Lateral Sclerosis/*genetics ; C9orf72 Protein/*genetics ; CRISPR-Cas Systems ; Cell Line ; DNA Breaks, Double-Stranded ; DNA Damage/genetics ; DNA Methylation/*genetics ; DNA Repair/genetics ; DNA-Cytosine Methylases/*metabolism ; Frontotemporal Dementia/*genetics ; Gene Knockout Techniques ; Genome, Human/genetics ; HEK293 Cells ; Histones/metabolism ; Humans ; RNA, Guide ; }, abstract = {DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5' regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.}, }
@article {pmid31672877, year = {2019}, author = {Cohen, J}, title = {Parents weigh promise and risks of germline editing.}, journal = {Science (New York, N.Y.)}, volume = {366}, number = {6465}, pages = {564-565}, doi = {10.1126/science.366.6465.564}, pmid = {31672877}, issn = {1095-9203}, mesh = {*CRISPR-Cas Systems ; Connexins/genetics ; Deafness/genetics/*therapy ; *Gene Editing ; *Genetic Therapy ; Germ Cells ; Humans ; Mutation ; *Parents ; }, }
@article {pmid31331444, year = {2019}, author = {Bilousova, G}, title = {Gene Therapy for Skin Fragility Diseases: The New Generation.}, journal = {The Journal of investigative dermatology}, volume = {139}, number = {8}, pages = {1634-1637}, doi = {10.1016/j.jid.2019.04.001}, pmid = {31331444}, issn = {1523-1747}, mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Epidermolysis Bullosa Dystrophica ; Genetic Therapy ; Humans ; Transcription Activator-Like Effector Nucleases ; }, abstract = {Ex vivo gene therapy is a promising approach to treat devastating skin fragility diseases. March et al. and Takashima et al. report that programmable nucleases-TALENs and CRISPR/Cas9-can safely and efficiently correct genetic defects in cultured adult skin cells, paving the way for broader clinical applications of gene therapies in dermatology.}, }
@article {pmid31323192, year = {2019}, author = {Nimura, T and Itoh, T and Hagio, H and Hayashi, T and Di Donato, V and Takeuchi, M and Itoh, T and Inoguchi, F and Sato, Y and Yamamoto, N and Katsuyama, Y and Del Bene, F and Shimizu, T and Hibi, M}, title = {Role of Reelin in cell positioning in the cerebellum and the cerebellum-like structure in zebrafish.}, journal = {Developmental biology}, volume = {455}, number = {2}, pages = {393-408}, doi = {10.1016/j.ydbio.2019.07.010}, pmid = {31323192}, issn = {1095-564X}, mesh = {Animals ; CRISPR-Cas Systems ; Cell Adhesion Molecules, Neuronal/genetics/*physiology ; Cell Movement ; Cerebellum/cytology/*embryology ; Extracellular Matrix Proteins/genetics/*physiology ; Kinesin/metabolism ; Mutation ; Nerve Tissue Proteins/genetics/*physiology ; Purkinje Cells/cytology ; Serine Endopeptidases/genetics/*physiology ; Signal Transduction ; Zebrafish/anatomy &amp; histology/*embryology ; Zebrafish Proteins/genetics/*physiology ; }, abstract = {The cerebellum and the cerebellum-like structure in the mesencephalic tectum in zebrafish contain multiple cell types, including principal cells (i.e., Purkinje cells and type I neurons) and granule cells, that form neural circuits in which the principal cells receive and integrate inputs from granule cells and other neurons. It is largely unknown how these cells are positioned and how neural circuits form. While Reelin signaling is known to play an important role in cell positioning in the mammalian brain, its role in the formation of other vertebrate brains remains elusive. Here we found that zebrafish with mutations in Reelin or in the Reelin-signaling molecules Vldlr or Dab1a exhibited ectopic Purkinje cells, eurydendroid cells (projection neurons), and Bergmann glial cells in the cerebellum, and ectopic type I neurons in the tectum. The ectopic Purkinje cells and type I neurons received aberrant afferent fibers in these mutants. In wild-type zebrafish, reelin transcripts were detected in the internal granule cell layer, while Reelin protein was localized to the superficial layer of the cerebellum and the tectum. Laser ablation of the granule cell axons perturbed the localization of Reelin, and the mutation of both kif5aa and kif5ba, which encode major kinesin I components in the granule cells, disrupted the elongation of granule cell axons and the Reelin distribution. Our findings suggest that in zebrafish, (1) Reelin is transported from the granule cell soma to the superficial layer by axonal transport; (2) Reelin controls the migration of neurons and glial cells from the ventricular zone; and (3) Purkinje cells and type I neurons attract afferent axons during the formation of the cerebellum and the cerebellum-like structure.}, }
@article {pmid31228551, year = {2019}, author = {Wang, F and Chang, C and Li, R and Zhang, Z and Jiang, H and Zeng, N and Li, D and Chen, L and Xiao, Y and Chen, W and Wang, Q}, title = {Retinol binding protein 4 mediates MEHP-induced glucometabolic abnormalities in HepG2 cells.}, journal = {Toxicology}, volume = {424}, number = {}, pages = {152236}, doi = {10.1016/j.tox.2019.06.007}, pmid = {31228551}, issn = {1879-3185}, mesh = {Apoptosis/drug effects ; CRISPR-Cas Systems ; Diethylhexyl Phthalate/*analogs &amp; derivatives/toxicity ; Gene Deletion ; Gluconeogenesis/drug effects ; Glucose/*metabolism ; Hep G2 Cells ; Homeostasis/drug effects ; Humans ; Insulin Resistance/genetics ; Liver/drug effects/metabolism ; Plasticizers/*toxicity ; Retinol-Binding Proteins, Plasma/genetics/*metabolism ; Signal Transduction/genetics ; }, abstract = {Epidemiological and experimental data have implicated the role of di(2-ethylhexyl) phthalate (DEHP) and its metabolite mono(2-ethylhexyl) phthalate (MEHP) in the pathogenesis of metabolic syndrome, including the impairment of hepatic glucose metabolism. To elucidate the underlying mechanism by which DEHP or MEHP perturbs hepatic glucose homeostasis, we compared the effect of DEHP (0-200 μM) and MEHP (0-200 μM) on glucose metabolism in HepG2 cells. In this study, we found that MEHP can induce more severe impairments in glucose homeostasis than DEHP can; these include increased hepatic gluconeogenesis via receptor substrate-1/protein kinase B/fork-head box protein O1 (IRS-1/AKT/FOXO1)-mediated phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6PC) up-regulation, as well as decreased hepatic glycogen synthesis via glucokinase (GCK) inhibition and IRS-1/AKT/glycogen synthase kinase-3β (GSK-3β)-mediated glycogen synthase (GYS) inactivation. Additionally, our results demonstrated that retinol binding protein 4 (RBP4), an insulin resistance-inducing factor, plays a critical role in the MEHP-induced disorder of glucose homeostasis and the dysfunction of insulin signaling transduction, whereas the deletion of RBP4 by the clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR/Cas9) significantly reversed these toxic effects. Although these should be interpreted with caution in view of limited in vivo evidence, the present study provides the first in vitro evidence for potential involvements of RBP4 in disturbance of glucose homeostasis in the MEHP-treated HepG2 cells.}, }
@article {pmid31218907, year = {2019}, author = {Zha, S and Li, Z and Chen, C and Du, Z and Tay, JC and Wang, S}, title = {Beta-2 microglobulin knockout K562 cell-based artificial antigen presenting cells for ex vivo expansion of T lymphocytes.}, journal = {Immunotherapy}, volume = {11}, number = {11}, pages = {967-982}, doi = {10.2217/imt-2018-0211}, pmid = {31218907}, issn = {1750-7448}, mesh = {Adoptive Transfer ; Antigen-Presenting Cells/cytology/*immunology ; Antigens, CD19/genetics/immunology ; CRISPR-Cas Systems ; *Cell Culture Techniques ; *Cell Proliferation ; *Gene Knockout Techniques ; Humans ; K562 Cells ; T-Lymphocytes, Cytotoxic/cytology/*immunology ; beta 2-Microglobulin/*genetics/immunology ; }, abstract = {Aim: The human K562 leukemia cell line as a scaffold of artificial antigen presenting cells (aAPCs) for ex vivo lymphocyte expansion does not usually express major histocompatibility complex (MHC) molecules. However, when stimulated by supernatants from human T lymphocyte cultures, K562 cells upregulate β-2 microglobulin (B2M) and MHC class I expression, which would induce allo-specific T cells. Methods: We disrupted the B2M locus in K562 cells by CRISPR/Cas9 and achieved MHC class I-negative K562 cells. Results: We further generated K562-based MHC class I-negative aAPC line by zinc-finger nuclease mediated insertion of costimulatory molecules into the AAVS1 locus. This aAPC line could attenuate allogeneic immune responses but support robust antigen-independent and CD19 antigen-specific chimeric antigen receptor-T cell expansion in vitro. Conclusion: B2M-knockout K562 cells provide a new scaffold for aAPC construction and broader application in adoptive immunotherapies.}, }
@article {pmid30979834, year = {2019}, author = {Wang, T and Wang, M and Zhang, Q and Cao, S and Li, X and Qi, Z and Tan, Y and You, Y and Bi, Y and Song, Y and Yang, R and Du, Z}, title = {Reversible Gene Expression Control in Yersinia pestis by Using an Optimized CRISPR Interference System.}, journal = {Applied and environmental microbiology}, volume = {85}, number = {12}, pages = {}, pmid = {30979834}, issn = {1098-5336}, mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Expression ; Gene Knockdown Techniques/*methods ; Streptococcus pyogenes ; Yersinia pestis/*genetics ; }, abstract = {Many genes in the bacterial pathogen Yersinia pestis, the causative agent of three plague pandemics, remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been shown to be an effective tool for gene knockdown in model bacteria. In this system, a catalytically dead Cas9 (dCas9) and a small guide RNA (sgRNA) form a complex, binding to the specific DNA target through base pairing, thereby impeding RNA polymerase binding and causing target gene repression. Here, we introduce an optimized CRISPRi system using Streptococcus pyogenes Cas9-derived dCas9 for gene knockdown in Y. pestis Multiple genes harbored on either the chromosome or plasmids of Y. pestis were efficiently knocked down (up to 380-fold) in a strictly anhydrotetracycline-inducible manner using this CRISPRi approach. Knockdown of hmsH (responsible for biofilm formation) or cspB (encoding a cold shock protein) resulted in greatly decreased biofilm formation or impaired cold tolerance in in vitro phenotypic assays. Furthermore, silencing of the virulence-associated genes yscB or ail using this CRISPRi system resulted in attenuation of virulence in HeLa cells and mice similar to that previously reported for yscB and ail null mutants. Taken together, our results confirm that this optimized CRISPRi system can reversibly and efficiently repress the expression of target genes in Y. pestis, providing an alternative to conventional gene knockdown techniques, as well as a strategy for high-throughput phenotypic screening of Y. pestis genes with unknown functions.IMPORTANCEYersiniapestis is a lethal pathogen responsible for millions of human deaths in history. It has also attracted much attention for potential uses as a bioweapon or bioterrorism agent, against which new vaccines are desperately needed. However, many Y. pestis genes remain uncharacterized, greatly hampering the development of measures for plague prevention and control. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been successfully used in a variety of bacteria in functional genomic studies, but no such genetic tool has been reported in Y. pestis Here, we systematically optimized the CRISPRi approach for use in Y. pestis, which ultimately repressed target gene expression with high efficiency in a reversible manner. Knockdown of functional genes using this method produced phenotypes that were readily detected by in vitro assays, cell infection assays, and mouse infection experiments. This is a report of a CRISPRi approach in Y. pestis and highlights the potential use of this approach in high-throughput functional genomics studies of this pathogen.}, }
@article {pmid30793514, year = {2019}, author = {Bergeron, F and Boulende Sab, A and Bouchard, MF and Taniguchi, H and Souchkova, O and Brousseau, C and Tremblay, JJ and Pilon, N and Viger, RS}, title = {Phosphorylation of GATA4 serine 105 but not serine 261 is required for testosterone production in the male mouse.}, journal = {Andrology}, volume = {7}, number = {3}, pages = {357-372}, doi = {10.1111/andr.12601}, pmid = {30793514}, issn = {2047-2927}, support = {MOP-14796//Canadian Institutes of Health Research (CIHR)/International ; //Fonds de la Recherche du Québec - Santé (FRQS)/International ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; GATA4 Transcription Factor/*metabolism ; Male ; Mice ; Phosphorylation ; Serine/*metabolism ; Testis/*metabolism ; Testosterone/*biosynthesis ; }, abstract = {BACKGROUND: GATA4 is a transcription factor essential for male sex determination, testicular differentiation during fetal development, and male fertility in the adult. GATA4 exerts part of its function by regulating multiple genes in the steroidogenic enzyme pathway. In spite of these crucial roles, how the activity of this factor is regulated remains unclear.<br><br>OBJECTIVES: Studies in gonadal cell lines have shown that GATA4 is phosphorylated on at least two serine residues-serine 105 (S105) and serine 261 (S261)-and that this phosphorylation is important for GATA4 activity. The objective of the present study is to characterize the endogenous role of GATA4 S105 and S261 phosphorylation in the mouse testis.<br><br>MATERIALS AND METHODS: We examined both previously described GATA4 S105A mice and a novel GATA4 S261A knock-in mouse that we generated by CRISPR/Cas9 gene editing. The male phenotype of the mutants was characterized by assessing androgen-dependent organ weights, hormonal profiles, and expression of multiple testicular target genes using standard biochemical and molecular biology techniques.<br><br>RESULTS: The fecundity of crosses between GATA4 S105A mice was reduced but without a change in sex ratio. The weight of androgen-dependent organs was smaller when compared to wild-type controls. Plasma testosterone levels showed a 70% decrease in adult GATA4 S105A males. This decrease was associated with a reduction in Cyp11a1, Cyp17a1, and Hsd17b3 expression. GATA4 S261A mice were viable and testis morphology appeared normal. Testosterone production and steroidogenic enzyme expression were not altered in GATA4 S261A males.<br><br>DISCUSSION AND CONCLUSION: Our analysis showed that blocking GATA4 S105 phosphorylation is associated with decreased androgen production in males. In contrast, S261 phosphorylation by itself is dispensable for GATA4 function. These results confirm that endogenous GATA4 action is essential for normal steroid production in males and that this activity requires phosphorylation on at least one serine residue.}, }
@article {pmid30739529, year = {2019}, author = {West, KL and Byrum, SD and Mackintosh, SG and Edmondson, RD and Taverna, SD and Tackett, AJ}, title = {Proteomic characterization of the arsenic response locus in S. cerevisiae.}, journal = {Epigenetics}, volume = {14}, number = {2}, pages = {130-145}, pmid = {30739529}, issn = {1559-2308}, support = {R01 GM118760/GM/NIGMS NIH HHS/United States ; }, mesh = {Arsenate Reductases/genetics ; Arsenic/*pharmacology ; Basic-Leucine Zipper Transcription Factors/genetics ; CRISPR-Cas Systems ; Gene Expression Profiling ; Gene Expression Regulation, Fungal/drug effects ; Membrane Transport Proteins/genetics ; Promoter Regions, Genetic ; Proteomics/*methods ; Repressor Proteins/genetics ; Saccharomyces cerevisiae/*growth &amp; development/metabolism ; Saccharomyces cerevisiae Proteins/genetics/*metabolism ; }, abstract = {Arsenic exposure is a global health problem. Millions of people encounter arsenic through contaminated drinking water, consumption, and inhalation. The arsenic response locus in budding yeast is responsible for the detoxification of arsenic and its removal from the cell. This locus constitutes a conserved pathway ranging from prokaryotes to higher eukaryotes. The goal of this study was to identify how transcription from the arsenic response locus is regulated in an arsenic dependent manner. An affinity enrichment strategy called CRISPR-Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) was used, which provides for the proteomic characterization of a targeted locus. CRISPR-ChAP-MS was applied to the promoter regions of the activated arsenic response locus and uncovered 40 nuclear-annotated proteins showing enrichment. Functional assays identified the histone acetyltransferase SAGA and the chromatin remodelling complex SWI/SNF to be required for activation of the locus. Furthermore, SAGA and SWI/SNF were both found to specifically organize the chromatin structure at the arsenic response locus for activation of gene transcription. This study provides the first proteomic characterization of an arsenic response locus and key insight into the mechanisms of transcriptional activation that are necessary for detoxification of arsenic from the cell.}, }
@article {pmid32402073, year = {2020}, author = {Yi, H and Huang, L and Yang, B and Gomez, J and Zhang, H and Yin, Y}, title = {AcrFinder: genome mining anti-CRISPR operons in prokaryotes and their viruses.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkaa351}, pmid = {32402073}, issn = {1362-4962}, abstract = {Anti-CRISPR (Acr) proteins encoded by (pro)phages/(pro)viruses have a great potential to enable a more controllable genome editing. However, genome mining new Acr proteins is challenging due to the lack of a conserved functional domain and the low sequence similarity among experimentally characterized Acr proteins. We introduce here AcrFinder, a web server (http://bcb.unl.edu/AcrFinder) that combines three well-accepted ideas used by previous experimental studies to pre-screen genomic data for Acr candidates. These ideas include homology search, guilt-by-association (GBA), and CRISPR-Cas self-targeting spacers. Compared to existing bioinformatics tools, AcrFinder has the following unique functions: (i) it is the first online server specifically mining genomes for Acr-Aca operons; (ii) it provides a most comprehensive Acr and Aca (Acr-associated regulator) database (populated by GBA-based Acr and Aca datasets); (iii) it combines homology-based, GBA-based, and self-targeting approaches in one software package; and (iv) it provides a user-friendly web interface to take both nucleotide and protein sequence files as inputs, and output a result page with graphic representation of the genomic contexts of Acr-Aca operons. The leave-one-out cross-validation on experimentally characterized Acr-Aca operons showed that AcrFinder had a 100% recall. AcrFinder will be a valuable web resource to help experimental microbiologists discover new Anti-CRISPRs.}, }
@article {pmid32401064, year = {2020}, author = {Xiang, X and Qian, K and Zhang, Z and Lin, F and Xie, Y and Liu, Y and Yang, Z}, title = {CRISPR-Cas Systems Based Molecular Diagnostic Tool for Infectious Diseases and Emerging 2019 Novel Coronavirus (COVID-19) Pneumonia.}, journal = {Journal of drug targeting}, volume = {}, number = {}, pages = {1-10}, doi = {10.1080/1061186X.2020.1769637}, pmid = {32401064}, issn = {1029-2330}, abstract = {Emerging infectious diseases, the persistent potential for destabilizing pandemics, remain a global threat leading to excessive morbidity and mortality. The current outbreak of pneumonia caused by 2019 novel coronavirus (COVID-19) illustrated difficulties in lack of effective drugs for treatment. Accurate and rapid diagnostic tools are essential for early recognition and treatment of infectious diseases, allowing timely implementation of infection control, improved clinical care and other public health measures to stop the spread of the disease. CRISPR-Cas technology speed up the development of infectious disease diagnostics with high rapid and accurate. In this review, we summarize current advance regarding diverse CRISPR-Cas systems, including CRISPR-Cas9, CRISPR-Cas12 and CRISPR-Cas13, in the development of fast, accurate and portable diagnostic tests and highlight the potential of CRISPR-Cas13 in COVID-19 Pneumonia and other emerging infectious diseases diagnosis.}, }
@article {pmid32400327, year = {2020}, author = {Rösner, J and Wellmeyer, B and Merzendorfer, H}, title = {Tribolium castaneum: a model for investigating the mode of action of insecticides and mechanisms of resistance.}, journal = {Current pharmaceutical design}, volume = {}, number = {}, pages = {}, doi = {10.2174/1381612826666200513113140}, pmid = {32400327}, issn = {1873-4286}, abstract = {The red flour beetle, Tribolium castaneum, is a worldwide insect pest of stored products, particularly food grains, and a powerful model organism for developmental, physiological and applied entomological research on coleopteran species. Among coleopterans, T. castaneum has the most fully sequenced and annotated genome and consequently provides the most advanced genetic model of a coleopteran pest. The beetle is also easy to culture and has a short generation time. Research on this beetle is further assisted by the availability of expressed sequence tags and transcriptomic data. Most importantly, it exhibits a very robust response to systemic RNA interference (RNAi), and a database of RNAi phenotypes (iBeetle) is available. Finally, classical transposon-based techniques together with CRISPR/Cas-mediated gene knockout and genome editing allow the creation of transgenic lines. As T. castaneum develops resistance rapidly to many classes of insecticides including organophosphates, methyl carbamates, pyrethroids, neonicotinoids and insect growth regulators such as chitin synthesis inhibitors, it is further a suitable test system for studying resistance mechanisms. In this review, we will summarize recent advances in research focusing on the mode of action of insecticides and mechanisms of resistance identified using T. castaneum as a pest model.}, }
@article {pmid32397339, year = {2020}, author = {Wang, L and Lim, CK and Klotz, MG}, title = {High Synteny and Sequence Identity between Genomes of Nitrosococcus oceani Strains Isolated from Different Oceanic Gyres Reveals Genome Economization and Autochthonous Clonal Evolution.}, journal = {Microorganisms}, volume = {8}, number = {5}, pages = {}, doi = {10.3390/microorganisms8050693}, pmid = {32397339}, issn = {2076-2607}, abstract = {The ammonia-oxidizing obligate aerobic chemolithoautotrophic gammaproteobacterium, Nitrosococcus oceani, is omnipresent in the world's oceans and as such important to the global nitrogen cycle. We generated and compared high quality draft genome sequences of N. oceani strains isolated from the Northeast (AFC27) and Southeast (AFC132) Pacific Ocean and the coastal waters near Barbados at the interface between the Caribbean Sea and the North Atlantic Ocean (C-27) with the recently published Draft Genome Sequence of N. oceani Strain NS58 (West Pacific Ocean) and the complete genome sequence of N. oceani C-107, the type strain (ATCC 19707) isolated from the open North Atlantic, with the goal to identify indicators for the evolutionary origin of the species. The genomes of strains C-107, NS58, C-27, and AFC27 were highly conserved in content and synteny, and these four genomes contained one nearly sequence-identical plasmid. The genome of strain AFC132 revealed the presence of genetic inventory unknown from other marine ammonia-oxidizing bacteria such as genes encoding NiFe-hydrogenase and a non-ribosomal peptide synthetase (NRPS)-like siderophore biosynthesis module. Comparative genome analysis in context with the literature suggests that AFC132 represents a metabolically more diverse ancestral lineage to the other strains with C-107 and NS58 potentially being the youngest. The results suggest that the N. oceani species evolved by genome economization characterized by the loss of genes encoding catabolic diversity while acquiring a higher redundancy in inventory dedicated to nitrogen catabolism, both of which could have been facilitated by their rich complements of CRISPR/Cas and Restriction Modification systems.}, }
@article {pmid32397082, year = {2020}, author = {Zhao, Y and Li, G and Chen, Y and Lu, Y}, title = {Challenges and Advances in Genome Editing Technologies in Streptomyces.}, journal = {Biomolecules}, volume = {10}, number = {5}, pages = {}, doi = {10.3390/biom10050734}, pmid = {32397082}, issn = {2218-273X}, support = {31970083 and 31400083//National Natural Science Foundation of China/ ; 2019YFA0905400//the National Key R&amp;D Program/ ; 2018ZX09711001-006-012//the National Mega-project for Innovative Drugs/ ; NY2018015//the Key R&amp;D Plan of Zhenjiang City/ ; NY2017001//the Key R&amp;D Plan of Dantu District/ ; }, abstract = {The genome of Streptomyces encodes a high number of natural product (NP) biosynthetic gene clusters (BGCs). Most of these BGCs are not expressed or are poorly expressed (commonly called silent BGCs) under traditional laboratory experimental conditions. These NP BGCs represent an unexplored rich reservoir of natural compounds, which can be used to discover novel chemical compounds. To activate silent BGCs for NP discovery, two main strategies, including the induction of BGCs expression in native hosts and heterologous expression of BGCs in surrogate Streptomyces hosts, have been adopted, which normally requires genetic manipulation. So far, various genome editing technologies have been developed, which has markedly facilitated the activation of BGCs and NP overproduction in their native hosts, as well as in heterologous Streptomyces hosts. In this review, we summarize the challenges and recent advances in genome editing tools for Streptomyces genetic manipulation with a focus on editing tools based on clustered regularly interspaced short palindrome repeat (CRISPR)/CRISPR-associated protein (Cas) systems. Additionally, we discuss the future research focus, especially the development of endogenous CRISPR/Cas-based genome editing technologies in Streptomyces.}, }
@article {pmid31369818, year = {2019}, author = {Tanic, J and Wang, Y and Lee, W and Coelho, NM and Glogauer, M and McCulloch, CA}, title = {Adseverin modulates morphology and invasive function of MCF7 cells.}, journal = {Biochimica et biophysica acta. Molecular basis of disease}, volume = {1865}, number = {10}, pages = {2716-2725}, doi = {10.1016/j.bbadis.2019.07.015}, pmid = {31369818}, issn = {1879-260X}, support = {MOP-142250//CIHR/Canada ; }, mesh = {Actins/metabolism ; Breast Neoplasms/metabolism ; CRISPR-Cas Systems ; Cell Movement ; Collagen/metabolism ; Fibronectins/metabolism ; Gelsolin/genetics/*metabolism ; Humans ; MCF-7 Cells/*metabolism ; Phagocytosis ; }, abstract = {Adseverin (Ads) is a Ca2+-dependent actin-capping and severing protein that is highly expressed in gastric, prostate and bladder cancer cells. Currently it is unknown whether Ads contributes to the subcortical actin remodeling associated with the formation of cell extensions and matrix invasion in cancer. We compared cell extension formation and matrix degradation in Ads wildtype and Ads-null MCF7 breast cancer cells generated by CRISPR/Cas9. Compared with wildtype, Ads-null cells plated on fibronectin or collagen exhibited a more circular morphology with shorter cell extensions (37% reduction on fibronectin; p < 0.001). Reconstitution of Ads in Ads-null cells restored the formation of cell extensions (p < 0.05). While cell migration on two-dimensional matrices was unchanged by Ads deletion, the formation of cell extensions across Transwell membranes was reduced (~40% reduction, p < 0.05). When plated on fibrillar collagen, compared with wildtype, Ads-null cells showed reduced expression of MT1-MMP, collagen degradation (p < 0.05) and phagocytosis of collagen-coated beads (25% reduction; p = 0.001). We conclude that Ads is involved in the formation of cell extensions and collagen degradation in MCF7 cells, which may in turn affect matrix invasion and metastasis.}, }
@article {pmid31348989, year = {2019}, author = {Qu, Z and Yimer, TA and Xie, S and Wong, F and Yu, S and Liu, X and Han, S and Ma, J and Lu, Z and Hu, X and Qin, Y and Huang, Y and Lv, Y and Li, J and Tang, Z and Liu, F and Liu, M}, title = {Knocking out lca5 in zebrafish causes cone-rod dystrophy due to impaired outer segment protein trafficking.}, journal = {Biochimica et biophysica acta. Molecular basis of disease}, volume = {1865}, number = {10}, pages = {2694-2705}, doi = {10.1016/j.bbadis.2019.07.009}, pmid = {31348989}, issn = {1879-260X}, mesh = {Animals ; CRISPR-Cas Systems ; Cilia/metabolism ; Cone-Rod Dystrophies/*genetics ; Disease Models, Animal ; Eye Proteins/metabolism ; Gene Knockout Techniques ; Genetic Predisposition to Disease/*genetics ; Humans ; Leber Congenital Amaurosis/*genetics/*metabolism/pathology ; Microtubule-Associated Proteins ; Phenotype ; Protein Transport/*physiology ; Retina/metabolism ; Retinal Cone Photoreceptor Cells/pathology ; Retinal Degeneration/genetics/pathology ; Tumor Suppressor Proteins/genetics/metabolism ; Zebrafish/*genetics ; }, abstract = {Leber congenital amaurosis (LCA) is the most serious form of inherited retinal dystrophy that leads to blindness or severe visual impairment within a few months after birth. Approximately 1-2% of the reported cases are caused by mutations in the LCA5 gene. This gene encodes a ciliary protein called LCA5 that is localized to the connecting cilium of photoreceptors. The retinal phenotypes caused by LCA5 mutations and the underlying pathological mechanisms are still not well understood. In this study, we knocked out the lca5 gene in zebrafish using CRISPR/Cas9 technology. An early onset visual defect is detected by the ERG in 7 dpf lca5-/- zebrafish. Histological analysis by HE staining and immunofluorescence reveal progressive degeneration of rod and cone photoreceptors, with a pattern that cones are more severely affected than rods. In addition, ultrastructural analysis by transmission electron microscopy shows disordered and broken membrane discs in rods' and cones' outer segments, respectively. In our lca5-/- zebrafish, the red-cone opsin and cone α-transducin are selectively mislocalized to the inner segment and synaptic terminal. Moreover, we found that Ift88, a key component of the intraflagellar transport complex, is retained in the outer segments. These data suggest that the intraflagellar transport complex-mediated outer segment protein trafficking might be impaired due to lca5 deletion, which finally leads to a type of retinal degeneration mimicking the phenotype of cone-rod dystrophy in human. Our work provides a novel animal model to study the physiological function of LCA5 and develop potential treatments of LCA.}, }
@article {pmid31321991, year = {2019}, author = {Lake, F}, title = {Putting the spotlight on CRISPR.}, journal = {BioTechniques}, volume = {67}, number = {2}, pages = {41}, doi = {10.2144/btn-2019-0084}, pmid = {31321991}, issn = {1940-9818}, mesh = {Animals ; *CRISPR-Cas Systems ; Cell Engineering ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Neoplasms/genetics ; }, }
@article {pmid32396992, year = {2020}, author = {Baerwald, MR and Goodbla, AM and Nagarajan, RP and Gootenberg, JS and Abudayyeh, OO and Zhang, F and Schreier, AD}, title = {Rapid and accurate species identification for ecological studies and monitoring using CRISPR-based SHERLOCK.}, journal = {Molecular ecology resources}, volume = {}, number = {}, pages = {}, doi = {10.1111/1755-0998.13186}, pmid = {32396992}, issn = {1755-0998}, abstract = {One of the most foundational aspects of ecological research and monitoring is accurate species identification, but cryptic speciation and observer error can confound phenotype-based identification. The CRISPR-Cas toolkit has facilitated remarkable advances in many scientific disciplines, but the fields of ecology and conservation biology have yet to fully embrace this powerful technology. The recently developed CRISPR-Cas13a platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) enables highly accurate taxonomic identification and has all the characteristics needed to transition to ecological and environmental disciplines. Here we conducted a series of &quot;proof of principle&quot; experiments to characterize SHERLOCK's ability to accurately, sensitively, and rapidly distinguish three fish species of management interest co-occurring in the San Francisco Estuary that are easily misidentified in the field. We improved SHERLOCK's ease of field deployment by combining the previously demonstrated rapid isothermal amplification and CRISPR genetic identification with a minimally invasive and extraction-free DNA collection protocol, as well as the option of instrument-free lateral flow detection. This approach opens the door for redefining how, where, and by whom genetic identifications occur in the future.}, }
@article {pmid32395634, year = {2020}, author = {Pirona, AC and Oktriani, R and Boettcher, M and Hoheisel, JD}, title = {Process for an efficient lentiviral cell transduction.}, journal = {Biology methods &amp; protocols}, volume = {5}, number = {1}, pages = {bpaa005}, doi = {10.1093/biomethods/bpaa005}, pmid = {32395634}, issn = {2396-8923}, abstract = {The combination of lentiviruses with techniques such as CRISPR-Cas9 has resulted in efficient and precise processes for targeted genome modification. An often-limiting aspect, however, is the efficiency of cell transduction. Low efficiencies with particular cell types and/or the high complexity of lentiviral libraries can cause insufficient representation. Here, we present a protocol that yielded substantial increases in transduction efficiency in various cell lines in comparison to several other procedures.}, }
@article {pmid32395632, year = {2020}, author = {Culp, E and Richman, C and Sharanya, D and Jhaveri, N and van den Berg, W and Gupta, BP}, title = {Genome editing in the nematode Caenorhabditis briggsae using the CRISPR/Cas9 system.}, journal = {Biology methods &amp; protocols}, volume = {5}, number = {1}, pages = {bpaa003}, doi = {10.1093/biomethods/bpaa003}, pmid = {32395632}, issn = {2396-8923}, abstract = {The CRISPR/Cas system has recently emerged as a powerful tool to engineer the genome of an organism. The system is adopted from bacteria where it confers immunity against invading foreign DNA. This work reports the first successful use of the CRISPR/Cas system in Caenorhabditis briggsae (a cousin of the well-known nematode C. elegans), to generate mutations via non-homologous end joining. We recovered deletion alleles of several conserved genes by microinjecting plasmids that express Cas9 endonuclease and an engineered CRISPR RNA corresponding to the DNA sequence to be cleaved. Evidence for somatic mutations and off-target mutations are also reported. Our approach allows for the generation of loss-of-function mutations in C. briggsae genes thereby facilitating a comparative study of gene function.}, }
@article {pmid32393110, year = {2020}, author = {Zhou, Y and Tang, Y and Pan, F and Dongxing, T and Yu, L and Huang, Y and Li, G and Li, M and Wang, Y and Yang, Z and Xu, X and Yin, Z and Zhou, D and Poirel, L and Jiang, X}, title = {The type I-E CRISPR-Cas system influences the acquisition of blaKPC-IncF plasmid in Klebsiella pneumonia.}, journal = {Emerging microbes &amp; infections}, volume = {}, number = {}, pages = {1-36}, doi = {10.1080/22221751.2020.1763209}, pmid = {32393110}, issn = {2222-1751}, abstract = {Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) have disseminated worldwide and emerged as major threats to public health. Of epidemiological significance, the international pandemic of KPC-KP is primarily associated with CG258 isolates and the related blaKPC -IncF plasmids (the predominant blaKPC plasmids). The CRISPR-Cas system is an adaptive immune system that can hinder gene expansion driven by horizontal gene transfer (HGT), including the spread of antibiotic resistance. Because of blaKPC-IncF plasmids are favored by CG258 K. pneumoniae, it was of interest to examine the co-distribution of CRISPR and acquired blaKPC-IncF plasmids in CG258 K. pneumoniae lineages. We collected 459 clinical K. pneumoniae isolates in China and collected 203 global whole-genome sequences in GenBank to determine the prevalence of CRISPR-Cas systems. We observed that the type I-E CRISPR-Cas system was significantly scarce in the CG258 lineage (p < 0.0001) and this feature was also observed while analyzing the blaKPC -positive isolates. Furthermore, the results of conjugation and plasmid stability assay fully demonstrated the CRIPSR-Cas system in K. pneumoniae could effectively hindered blaKPC- IncF plasmids invasion and existence. Notably, most blaKPC- IncF plasmids in this study were also proved to be good targets of CRISPR owing to carry matched and functional protospacers and PAMs. Overall, our work suggests that type I-E CRISPR-Cas systems could impact the spread of blaKPC in K. pneumoniae populations, and the scarcity of type I-E CRISPR-Cas system was one of the potential factors leading to the propagation of blaKPC-IncF plasmids in K. pneumoniae CG258 lineage.}, }
@article {pmid32390420, year = {2020}, author = {Yin, K and Ding, X and Li, Z and Zhao, H and Cooper, K and Liu, C}, title = {Dynamic aqueous multiphase reaction system for one-pot CRISPR-Cas12a based ultrasensitive and quantitative molecular diagnosis.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.0c01459}, pmid = {32390420}, issn = {1520-6882}, abstract = {Recently, CRISPR-Cas technology has opened a new era of nucleic acid-based molecular diagnostics. However, current CRISPR-Cas-based nucleic acid biosensing has a lack of the quantitative detection ability and typically requires separate manual operations. Herein, we reported a dynamic aqueous multiphase reaction (DAMR) system for simple, sensitive and quantitative one-pot CRISPR-Cas12a based molecular diagnosis by taking advantage of density difference of sucrose concentration. In the DAMR system, recombinase polymerase amplification (RPA) and CRISPR-Cas12a derived fluorescent detection occurred in spatially separated but connected aqueous phases. Our DAMR system was utilized to quantitatively detect human papillomavirus (HPV) 16 and 18 DNAs with sensitivities of 10 and 100 copies within less than one hour. Multiplex detection of HPV16/18 in clinical human swab samples were successfully achieved in the DAMR system using 3D-printed microfluidic device. Furthermore, we demonstrated that target DNA in real human plasma samples can be directly amplified and detected in the DAMR system without complicated sample pre-treatment. As demonstrated, the DAMR system has shown great potential for development of next-generation point-of-care molecular diagnostics.}, }
@article {pmid31961902, year = {2020}, author = {Mata López, S and Balog-Alvarez, C and Vitha, S and Bettis, AK and Canessa, EH and Kornegay, JN and Nghiem, PP}, title = {Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMD genetic mutation in canine Duchenne muscular dystrophy.}, journal = {PloS one}, volume = {15}, number = {1}, pages = {e0228072}, pmid = {31961902}, issn = {1932-6203}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Dogs ; Dystrophin/*genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; *Muscular Dystrophy, Duchenne/genetics/therapy ; Mutation ; Myoblasts/cytology/*metabolism ; Transcription Activator-Like Effector Nucleases/genetics ; }, abstract = {Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.}, }
@article {pmid31843888, year = {2020}, author = {Liu, X and Xiao, W and Zhang, Y and Wiley, SE and Zuo, T and Zheng, Y and Chen, N and Chen, L and Wang, X and Zheng, Y and Huang, L and Lin, S and Murphy, AN and Dixon, JE and Xu, P and Guo, X}, title = {Reversible phosphorylation of Rpn1 regulates 26S proteasome assembly and function.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {1}, pages = {328-336}, pmid = {31843888}, issn = {1091-6490}, support = {R01 GM074830/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Enzyme Assays ; Gene Knock-In Techniques ; Humans ; Membrane Proteins/genetics/*metabolism ; Mice ; Mice, Knockout ; Mice, Transgenic ; Mitochondria/metabolism ; Nuclear Proteins/genetics/*metabolism ; Oxidative Stress ; Phosphoprotein Phosphatases/genetics/*metabolism ; Phosphorylation/physiology ; Proteasome Endopeptidase Complex/genetics/*metabolism ; Protein Subunits/genetics/*metabolism ; Protein-Serine-Threonine Kinases/genetics/*metabolism ; RNA, Small Interfering/metabolism ; Serine/metabolism ; Trans-Activators/genetics/metabolism ; }, abstract = {The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.}, }
@article {pmid31818368, year = {2019}, author = {Bastin-Héline, L and de Fouchier, A and Cao, S and Koutroumpa, F and Caballero-Vidal, G and Robakiewicz, S and Monsempes, C and François, MC and Ribeyre, T and Maria, A and Chertemps, T and de Cian, A and Walker, WB and Wang, G and Jacquin-Joly, E and Montagné, N}, title = {A novel lineage of candidate pheromone receptors for sex communication in moths.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {31818368}, issn = {2050-084X}, support = {ANR-16-CE02-0003-01//Agence Nationale de la Recherche/International ; ANR-16-CE21-0002-01//Agence Nationale de la Recherche/International ; 31725023//National Natural Science Foundation of China/International ; 31621064//National Natural Science Foundation of China/International ; }, mesh = {Animals ; Behavior, Animal ; CRISPR-Cas Systems ; Drosophila/genetics/metabolism ; Female ; Gene Expression Regulation ; Gene Knockout Techniques ; Insect Proteins/genetics/*metabolism ; Lepidoptera/genetics/metabolism ; Male ; Moths/genetics/*metabolism ; Receptors, Odorant ; Receptors, Pheromone/classification/genetics/*metabolism ; Sex Attractants/*metabolism ; Spodoptera/genetics/metabolism ; Transcriptome ; Xenopus/genetics/metabolism ; }, abstract = {Sex pheromone receptors (PRs) are key players in chemical communication between mating partners in insects. In the highly diversified insect order Lepidoptera, male PRs tuned to female-emitted type I pheromones (which make up the vast majority of pheromones identified) form a dedicated subfamily of odorant receptors (ORs). Here, using a combination of heterologous expression and in vivo genome editing methods, we bring functional evidence that at least one moth PR does not belong to this subfamily but to a distantly related OR lineage. This PR, identified in the cotton leafworm Spodoptera littoralis, is highly expressed in male antennae and is specifically tuned to the major sex pheromone component emitted by females. Together with a comprehensive phylogenetic analysis of moth ORs, our functional data suggest two independent apparitions of PRs tuned to type I pheromones in Lepidoptera, opening up a new path for studying the evolution of moth pheromone communication.}, }
@article {pmid31794381, year = {2019}, author = {Bendriem, RM and Singh, S and Aleem, AA and Antonetti, DA and Ross, ME}, title = {Tight junction protein occludin regulates progenitor Self-Renewal and survival in developing cortex.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {31794381}, issn = {2050-084X}, support = {Training Fellowship (T32HD060600)/NH/NIH HHS/United States ; R01 NS105477/NS/NINDS NIH HHS/United States ; P01 HD067244/HD/NICHD NIH HHS/United States ; P01HD067244/NH/NIH HHS/United States ; R01NS105477/NH/NIH HHS/United States ; }, mesh = {Aneuploidy ; Animals ; Apoptosis ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Proliferation ; Centrosome/metabolism ; Cerebral Cortex/*growth &amp; development/*metabolism/pathology ; Disease Models, Animal ; Gene Editing ; Humans ; Mice ; Mice, Knockout ; Microcephaly/genetics/pathology ; Microtubules/metabolism ; Mutagenesis ; Mutation ; Neurogenesis/genetics/physiology ; Occludin/genetics/*metabolism ; Spindle Apparatus/metabolism ; Tight Junctions/genetics/*metabolism ; }, abstract = {Occludin (OCLN) mutations cause human microcephaly and cortical malformation. A tight junction component thought absent in neuroepithelium after neural tube closure, OCLN isoform-specific expression extends into corticogenesis. Full-length and truncated isoforms localize to neuroprogenitor centrosomes, but full-length OCLN transiently localizes to plasma membranes while only truncated OCLN continues at centrosomes throughout neurogenesis. Mimicking human mutations, full-length OCLN depletion in mouse and in human CRISPR/Cas9-edited organoids produce early neuronal differentiation, reduced progenitor self-renewal and increased apoptosis. Human neural progenitors were more severely affected, especially outer radial glial cells, which mouse embryonic cortex lacks. Rodent and human mutant progenitors displayed reduced proliferation and prolonged M-phase. OCLN interacted with mitotic spindle regulators, NuMA and RAN, while full-length OCLN loss impaired spindle pole morphology, astral and mitotic microtubule integrity. Thus, early corticogenesis requires full-length OCLN to regulate centrosome organization and dynamics, revealing a novel role for this tight junction protein in early brain development.}, }
@article {pmid31789594, year = {2019}, author = {Oh, HS and Neuhausser, WM and Eggan, P and Angelova, M and Kirchner, R and Eggan, KC and Knipe, DM}, title = {Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {31789594}, issn = {2050-084X}, support = {P01 AI098681/NH/NIH HHS/United States ; R21 AI135423/NH/NIH HHS/United States ; Q-FASTR Award//Harvard Medical School/International ; P01 AI098681/NH/NIH HHS/United States ; R21 AI135423/NH/NIH HHS/United States ; }, mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line ; DNA Repair/genetics ; DNA, Viral/genetics ; *Gene Editing ; Gene Expression Regulation, Viral ; *Genes, Viral ; Herpesviridae/*genetics ; Herpesvirus 1, Human/genetics ; Humans ; Models, Genetic ; Mutagenesis/genetics ; RNA, Guide/genetics ; Virus Replication/genetics ; }, abstract = {Herpes simplex virus (HSV) establishes lifelong latent infection and can cause serious human disease, but current antiviral therapies target lytic but not latent infection. We screened for sgRNAs that cleave HSV-1 DNA sequences efficiently in vitro and used these sgRNAs to observe the first editing of quiescent HSV-1 DNA. The sgRNAs targeted lytic replicating viral DNA genomes more efficiently than quiescent genomes, consistent with the open structure of lytic chromatin. Editing of latent genomes caused short indels while editing of replicating genomes produced indels, linear molecules, and large genomic sequence loss around the gRNA target site. The HSV ICP0 protein and viral DNA replication increased the loss of DNA sequences around the gRNA target site. We conclude that HSV, by promoting open chromatin needed for viral gene expression and by inhibiting the DNA damage response, makes the genome vulnerable to a novel form of editing by CRISPR-Cas9 during lytic replication.}, }
@article {pmid31739790, year = {2019}, author = {Alexander, J and Findlay, GM and Kircher, M and Shendure, J}, title = {Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement.}, journal = {BMC biology}, volume = {17}, number = {1}, pages = {90}, pmid = {31739790}, issn = {1741-7007}, support = {F30 CA213728/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; DP1 HG007811/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Ctenophora/*genetics ; Epigenome/genetics ; Gene Editing/*methods ; Genome/*genetics ; }, abstract = {BACKGROUND: Recent advances in genome editing have facilitated the direct manipulation of not only the genome, but also the epigenome. Genome editing is typically performed by introducing a single CRISPR/Cas9-mediated double-strand break (DSB), followed by non-homologous end joining (NHEJ)- or homology-directed repair-mediated repair. Epigenome editing, and in particular methylation of CpG dinucleotides, can be performed using catalytically inactive Cas9 (dCas9) fused to a methyltransferase domain. However, for investigations of the role of methylation in gene silencing, studies based on dCas9-methyltransferase have limited resolution and are potentially confounded by the effects of binding of the fusion protein. As an alternative strategy for epigenome editing, we tested CRISPR/Cas9 dual cutting of the genome in the presence of in vitro methylated exogenous DNA, with the aim of driving replacement of the DNA sequence intervening the dual cuts via NHEJ.<br><br>RESULTS: In a proof of concept at the HPRT1 promoter, successful replacement events with heavily methylated alleles of a CpG island resulted in functional silencing of the HPRT1 gene. Although still limited in efficiency, our study demonstrates concurrent epigenome and genome editing in a single event.<br><br>CONCLUSIONS: This study opens the door to investigations of the functional consequences of methylation patterns at single CpG dinucleotide resolution. Our results furthermore support the conclusion that promoter methylation is sufficient to functionally silence gene expression.}, }
@article {pmid31566294, year = {2019}, author = {Kim, J and Lana, B and Torelli, S and Ryan, D and Catapano, F and Ala, P and Luft, C and Stevens, E and Konstantinidis, E and Louzada, S and Fu, B and Paredes-Redondo, A and Chan, AE and Yang, F and Stemple, DL and Liu, P and Ketteler, R and Selwood, DL and Muntoni, F and Lin, YY}, title = {A new patient-derived iPSC model for dystroglycanopathies validates a compound that increases glycosylation of α-dystroglycan.}, journal = {EMBO reports}, volume = {20}, number = {11}, pages = {e47967}, pmid = {31566294}, issn = {1469-3178}, support = {RG130417//Royal Society/International ; SG/14-15/14//Newlife - The Charity for Disabled Children/International ; 115582//EC|Seventh Framework Programme (FP7)/International ; 2012-305121//EC|Seventh Framework Programme (FP7)/International ; MC_U12266B//UK Research and Innovation|Medical Research Council (MRC)/International ; 92-963//UK Research and Innovation|Medical Research Council (MRC)/International ; 098051/WT_/Wellcome Trust/United Kingdom ; }, mesh = {Base Sequence ; CRISPR-Cas Systems ; Cells, Cultured ; *Drug Evaluation, Preclinical/methods ; Dystroglycans/genetics/*metabolism ; Gene Editing ; Gene Targeting ; Genetic Loci ; Glycosylation/drug effects ; High-Throughput Nucleotide Sequencing ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Molecular Imaging ; Muscular Dystrophies/drug therapy/etiology/metabolism ; Mutation ; N-Acetylglucosaminyltransferases/genetics/metabolism ; Neural Stem Cells/metabolism ; Neurons/metabolism ; Pentosyltransferases/genetics/metabolism ; }, abstract = {Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.}, }
@article {pmid31559572, year = {2019}, author = {Lu, Y and Shou, J and Jia, Z and Wu, Y and Li, J and Guo, Y and Wu, Q}, title = {Genetic evidence for asymmetric blocking of higher-order chromatin structure by CTCF/cohesin.}, journal = {Protein &amp; cell}, volume = {10}, number = {12}, pages = {914-920}, pmid = {31559572}, issn = {1674-8018}, mesh = {Animals ; CCCTC-Binding Factor/*genetics ; CRISPR-Cas Systems ; Cadherins/*genetics ; Cell Cycle Proteins/*genetics ; Cell Line, Tumor ; Chromatin/*genetics ; Chromosomal Proteins, Non-Histone/*genetics ; Humans ; }, }
@article {pmid31535025, year = {2019}, author = {Oleinik, N and Kim, J and Roth, BM and Selvam, SP and Gooz, M and Johnson, RH and Lemasters, JJ and Ogretmen, B}, title = {Mitochondrial protein import is regulated by p17/PERMIT to mediate lipid metabolism and cellular stress.}, journal = {Science advances}, volume = {5}, number = {9}, pages = {eaax1978}, pmid = {31535025}, issn = {2375-2548}, support = {R01 CA088932/CA/NCI NIH HHS/United States ; R01 CA214461/CA/NCI NIH HHS/United States ; R01 CA173687/CA/NCI NIH HHS/United States ; R01 DE016572/DE/NIDCR NIH HHS/United States ; P01 CA203628/CA/NCI NIH HHS/United States ; C06 RR015455/RR/NCRR NIH HHS/United States ; P30 CA138313/CA/NCI NIH HHS/United States ; P30 GM103339/GM/NIGMS NIH HHS/United States ; S10 OD018113/OD/NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Ceramides/metabolism ; Endoplasmic Reticulum/*metabolism/pathology ; Humans ; Lipid Metabolism ; Membrane Proteins/*physiology ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mitochondria/metabolism/*pathology ; Mitochondrial Membranes/*metabolism ; Mitochondrial Proteins/antagonists &amp; inhibitors/*physiology ; *Mitophagy ; Protein Transport ; Sphingosine N-Acyltransferase/*physiology ; *Stress, Physiological ; }, abstract = {How lipid metabolism is regulated at the outer mitochondrial membrane (OMM) for transducing stress signaling remains largely unknown. We show here that this process is controlled by trafficking of ceramide synthase 1 (CerS1) from the endoplasmic reticulum (ER) to the OMM by a previously uncharacterized p17, which is now renamed protein that mediates ER-mitochondria trafficking (PERMIT). Data revealed that p17/PERMIT associates with newly translated CerS1 on the ER surface to mediate its trafficking to the OMM. Cellular stress induces Drp1 nitrosylation/activation, releasing p17/PERMIT to retrieve CerS1 for its OMM trafficking, resulting in mitochondrial ceramide generation, mitophagy and cell death. In vivo, CRISPR-Cas9-dependent genetic ablation of p17/PERMIT prevents acute stress-mediated CerS1 trafficking to OMM, attenuating mitophagy in p17/PERMIT-/- mice, compared to controls, in various metabolically active tissues, including brain, muscle, and pancreas. Thus, these data have implications in diseases associated with accumulation of damaged mitochondria such as cancer and/or neurodegeneration.}, }
@article {pmid31464370, year = {2019}, author = {Brown, KR and Mair, B and Soste, M and Moffat, J}, title = {CRISPR screens are feasible in TP53 wild-type cells.}, journal = {Molecular systems biology}, volume = {15}, number = {8}, pages = {e8679}, pmid = {31464370}, issn = {1744-4292}, mesh = {CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism ; Cell Line ; Cell Line, Transformed ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21/genetics/metabolism ; Epithelial Cells/cytology/*metabolism ; Gene Editing/methods/*standards ; Gene Expression Regulation ; HCT116 Cells ; HeLa Cells ; Humans ; Neuroglia/metabolism/pathology ; Organ Specificity ; Proto-Oncogene Proteins/genetics/metabolism ; Proto-Oncogene Proteins c-mdm2/genetics/metabolism ; Quality Control ; RNA, Guide/*genetics/metabolism ; Retinal Pigment Epithelium/cytology/metabolism ; Retinoblastoma Binding Proteins/genetics/metabolism ; Tumor Suppressor Protein p53/*genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; }, abstract = {A recent study by Haapaniemi et al (2018) reported that intact p53 signaling hampers CRISPR-based functional genomic screens. Brown et al report good performance of genome-scale screens in TP53 wild-type cells and reiterate best practices for CRISPR screening.}, }
@article {pmid31402173, year = {2019}, author = {El Khattabi, L and Zhao, H and Kalchschmidt, J and Young, N and Jung, S and Van Blerkom, P and Kieffer-Kwon, P and Kieffer-Kwon, KR and Park, S and Wang, X and Krebs, J and Tripathi, S and Sakabe, N and Sobreira, DR and Huang, SC and Rao, SSP and Pruett, N and Chauss, D and Sadler, E and Lopez, A and Nóbrega, MA and Aiden, EL and Asturias, FJ and Casellas, R}, title = {A Pliable Mediator Acts as a Functional Rather Than an Architectural Bridge between Promoters and Enhancers.}, journal = {Cell}, volume = {178}, number = {5}, pages = {1145-1158.e20}, doi = {10.1016/j.cell.2019.07.011}, pmid = {31402173}, issn = {1097-4172}, support = {DP2 OD008540/OD/NIH HHS/United States ; UM1 HG009375/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; CD4-Positive T-Lymphocytes/cytology/metabolism ; CRISPR-Cas Systems/genetics ; Cell Cycle Proteins/metabolism ; Cells, Cultured ; Chromosomal Proteins, Non-Histone/metabolism ; Cryoelectron Microscopy ; Enhancer Elements, Genetic ; Gene Editing ; Humans ; Male ; Mediator Complex/chemistry/genetics/*metabolism ; Mice ; Mice, Inbred C57BL ; Mouse Embryonic Stem Cells/cytology/metabolism ; Promoter Regions, Genetic ; Protein Structure, Quaternary ; RNA Polymerase II/genetics/*metabolism ; Saccharomyces cerevisiae/metabolism ; Saccharomyces cerevisiae Proteins/metabolism ; }, abstract = {While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.}, }
@article {pmid31355948, year = {2019}, author = {Wang, L and Rubio, MC and Xin, X and Zhang, B and Fan, Q and Wang, Q and Ning, G and Becana, M and Duanmu, D}, title = {CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation.}, journal = {The New phytologist}, volume = {224}, number = {2}, pages = {818-832}, doi = {10.1111/nph.16077}, pmid = {31355948}, issn = {1469-8137}, mesh = {*CRISPR-Cas Systems ; Gene Deletion ; Gene Expression Regulation, Enzymologic/genetics/physiology ; Gene Expression Regulation, Plant/genetics/*physiology ; Isoenzymes/genetics/metabolism ; Leghemoglobin/*genetics/metabolism ; Lotus/genetics/*metabolism ; Nitrogen Fixation/genetics/*physiology ; Plant Root Nodulation/genetics/physiology ; Superoxide Dismutase ; }, abstract = {Legume nodules contain high concentrations of leghemoglobins (Lbs) encoded by several genes. The reason for this multiplicity is unknown. CRISPR/Cas9 technology was used to generate stable mutants of the three Lbs of Lotus japonicus. The phenotypes were characterized at the physiological, biochemical and molecular levels. Nodules of the triple mutants were examined by electron microscopy and subjected to RNA-sequencing (RNA-seq) analysis. Complementation studies revealed that Lbs function synergistically to maintain optimal N2 fixation. The nodules of the triple mutants overproduced superoxide radicals and hydrogen peroxide, which was probably linked to activation of NADPH oxidases and changes in superoxide dismutase isoforms expression. The mutant nodules showed major ultrastructural alterations, including vacuolization, accumulation of poly-β-hydroxybutyrate and disruption of mitochondria. RNA-seq of c. 20 000 genes revealed significant changes in expression of carbon and nitrogen metabolism genes, transcription factors, and proteinases. Lb-deficient nodules had c. 30-50-fold less heme but similar transcript levels of heme biosynthetic genes, suggesting a post-translational regulatory mechanism of heme synthesis. We conclude that Lbs act additively in nodules and that the lack of Lbs results in early nodule senescence. Our observations also provide insight into the reprogramming of the gene expression network associated with Lb deficiency, probably as a result of uncontrolled intracellular free O2 concentration.}, }
@article {pmid31243729, year = {2019}, author = {Yan, M and Li, J}, title = {The evolving CRISPR technology.}, journal = {Protein &amp; cell}, volume = {10}, number = {11}, pages = {783-786}, pmid = {31243729}, issn = {1674-8018}, mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; }, }
@article {pmid31076406, year = {2019}, author = {Izaguirre-Carbonell, J and Christiansen, L and Burns, R and Schmitz, J and Li, C and Mokry, RL and Bluemn, T and Zheng, Y and Shen, J and Carlson, KS and Rao, S and Wang, D and Zhu, N}, title = {Critical role of Jumonji domain of JMJD1C in MLL-rearranged leukemia.}, journal = {Blood advances}, volume = {3}, number = {9}, pages = {1499-1511}, pmid = {31076406}, issn = {2473-9537}, support = {R00 CA168996/CA/NCI NIH HHS/United States ; R01 CA204231/CA/NCI NIH HHS/United States ; R01 HL130724/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Gene Editing ; Histone-Lysine N-Methyltransferase/*genetics ; Histones/metabolism ; Humans ; Interleukin-3/metabolism ; Jumonji Domain-Containing Histone Demethylases/chemistry/genetics/*metabolism ; Leukemia, Myeloid, Acute/genetics/*pathology ; Mice ; Mice, Inbred C57BL ; Myeloid-Lymphoid Leukemia Protein/*genetics ; Oxidoreductases, N-Demethylating/chemistry/genetics/*metabolism ; Protein Domains ; RNA, Guide/metabolism ; Signal Transduction ; Transplantation, Heterologous ; Zinc Fingers/genetics ; }, abstract = {JMJD1C, a member of the lysine demethylase 3 family, is aberrantly expressed in mixed lineage leukemia (MLL) gene-rearranged (MLLr) leukemias. We have shown previously that JMJD1C is required for self-renewal of acute myeloid leukemia (AML) leukemia stem cells (LSCs) but not normal hematopoietic stem cells. However, the domains within JMJD1C that promote LSC self-renewal are unknown. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) negative-selection screening and identified a requirement for the catalytic Jumonji (JmjC) domain and zinc finger domain for leukemia cell survival in vitro and in vivo. In addition, we found that histone H3 lysine 36 methylation (H3K36me) is a marker for JMJD1C activity at gene loci. Moreover, we performed single cell transcriptome analysis of mouse leukemia cells harboring a single guide RNA (sgRNA) against the JmjC domain and identified increased activation of RAS/MAPK and the JAK-STAT pathway in cells harboring the JmjC sgRNA. We discovered that upregulation of interleukin 3 (IL-3) receptor genes mediates increased activation of IL-3 signaling upon JMJD1C loss or mutation. Along these lines, we observed resistance to JMJD1C loss in MLLr AML bearing activating RAS mutations, suggesting that RAS pathway activation confers resistance to JMJD1C loss. Overall, we discovered the functional importance of the JMJD1C JmjC domain in AML leukemogenesis and a novel interplay between JMJD1C and the IL-3 signaling pathway as a potential resistance mechanism to targeting JMJD1C catalytic activity.}, }
@article {pmid31018108, year = {2019}, author = {Himeda, CL and Jones, PL}, title = {The Genetics and Epigenetics of Facioscapulohumeral Muscular Dystrophy.}, journal = {Annual review of genomics and human genetics}, volume = {20}, number = {}, pages = {265-291}, doi = {10.1146/annurev-genom-083118-014933}, pmid = {31018108}, issn = {1545-293X}, mesh = {CRISPR-Cas Systems ; Chromatin/chemistry ; Chromosomal Proteins, Non-Histone/*genetics/metabolism ; Chromosomes, Human, Pair 4 ; DNA (Cytosine-5-)-Methyltransferases/*genetics/metabolism ; DNA Methylation ; *Epigenesis, Genetic ; Gene Editing ; Genetic Loci ; Genome, Human ; Homeodomain Proteins/*genetics/metabolism ; Humans ; Muscle, Skeletal/metabolism/pathology ; Muscular Dystrophy, Facioscapulohumeral/classification/*genetics/metabolism/pathology ; Mutation ; Severity of Illness Index ; }, abstract = {Facioscapulohumeral muscular dystrophy (FSHD), a progressive myopathy that afflicts individuals of all ages, provides a powerful model of the complex interplay between genetic and epigenetic mechanisms of chromatin regulation. FSHD is caused by dysregulation of a macrosatellite repeat, either by contraction of the repeat or by mutations in silencing proteins. Both cases lead to chromatin relaxation and, in the context of a permissive allele, aberrant expression of the DUX4 gene in skeletal muscle. DUX4 is a pioneer transcription factor that activates a program of gene expression during early human development, after which its expression is silenced in most somatic cells. When misexpressed in FSHD skeletal muscle, the DUX4 program leads to accumulated muscle pathology. Epigenetic regulators of the disease locus represent particularly attractive therapeutic targets for FSHD, as many are not global modifiers of the genome, and altering their expression or activity should allow correction of the underlying defect.}, }
@article {pmid30767107, year = {2019}, author = {Jia, K and Lu, Z and Zhou, F and Xiong, Z and Zhang, R and Liu, Z and Ma, Y and He, L and Li, C and Zhu, Z and Pan, D and Lian, Z}, title = {Multiple sgRNAs facilitate base editing-mediated i-stop to induce complete and precise gene disruption.}, journal = {Protein &amp; cell}, volume = {10}, number = {11}, pages = {832-839}, pmid = {30767107}, issn = {1674-8018}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Knockout Techniques/*methods ; Mice ; Mice, Knockout ; RNA, Guide/*genetics ; }, }
@article {pmid30702927, year = {2019}, author = {Du, L and Yang, F and Fang, H and Sun, H and Chen, Y and Xu, Y and Li, H and Zheng, L and Zhou, BS}, title = {AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {33}, number = {3}, pages = {4525-4537}, doi = {10.1096/fj.201801559RR}, pmid = {30702927}, issn = {1530-6860}, mesh = {AMP-Activated Protein Kinases/deficiency/genetics/physiology ; Aminoimidazole Carboxamide/*analogs &amp; derivatives/antagonists &amp; inhibitors/pharmacology/toxicity ; Apoptosis/drug effects ; CRISPR-Cas Systems ; Cell Cycle Checkpoints/drug effects ; Cell Line, Tumor ; DNA Breaks, Double-Stranded/drug effects ; DNA Replication/drug effects ; Deoxyribonucleotides/metabolism ; Drug Screening Assays, Antitumor ; Gene Knockout Techniques ; Genes, p53 ; Genes, rRNA ; Humans ; Nucleotides/*metabolism ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*drug therapy/metabolism/pathology ; RNA, Ribosomal/biosynthesis ; Ribonucleotides/antagonists &amp; inhibitors/metabolism/*pharmacology/toxicity ; Transcription, Genetic/drug effects ; Tumor Suppressor Protein p53/deficiency/physiology ; Uridine/pharmacology ; }, abstract = {It has been shown that 5-amino-4-imidazolecarboxamide riboside (AICAr) can inhibit cell proliferation and induce apoptosis in childhood acute lymphoblastic leukemia (ALL) cells. Although AICAr could regulate cellular energy metabolism by activating AMPK, the cytotoxic mechanisms of AICAr are still unclear. Here, we knocked out TP53 or PRKAA1 gene (encoding AMPKα1) in NALM-6 and Reh cells by using the clustered regularly interspaced short palindromic repeats/Cas9 system and found that AICAr-induced proliferation inhibition was independent of AMPK activation but dependent on p53. Liquid chromatography-mass spectrometry analysis of nucleotide metabolites indicated that AICAr caused an increase in adenosine triphosphate, deoxyadenosine triphosphate, and deoxyguanosine triphosphate levels by up-regulating purine biosynthesis, while AICAr led to a decrease in cytidine triphosphate, uridine triphosphate, deoxycytidine triphosphate, and deoxythymidine triphosphate levels because of reduced phosphoribosyl pyrophosphate production, which consequently impaired the pyrimidine biosynthesis. Ribonucleoside triphosphate (NTP) pool imbalances suppressed the rRNA transcription efficiency. Furthermore, deoxy-ribonucleoside triphosphate (dNTP) pool imbalances induced DNA replication stress and DNA double-strand breaks, followed by cell cycle arrest and apoptosis in ALL cells. Exogenous uridine could rebalance the NTP and dNTP pools by supplementing pyrimidine and then attenuate AICAr-induced cytotoxicity. Our data indicate that RNA transcription inhibition and DNA replication stress induced by NTP and dNTP pool imbalances might play a key role in AICAr-mediated cytotoxic effects on ALL cells, suggesting a potential clinical application of AICAr in future ALL therapy.-Du, L., Yang, F., Fang, H., Sun, H., Chen, Y., Xu, Y., Li, H., Zheng, L., Zhou, B.-B. S. AICAr suppresses cell proliferation by inducing NTP and dNTP pool imbalances in acute lymphoblastic leukemia cells.}, }
@article {pmid30683899, year = {2019}, author = {van de Vrugt, HJ and Harmsen, T and Riepsaame, J and Alexantya, G and van Mil, SE and de Vries, Y and Bin Ali, R and Huijbers, IJ and Dorsman, JC and Wolthuis, RMF and Te Riele, H}, title = {Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair.}, journal = {Scientific reports}, volume = {9}, number = {1}, pages = {768}, pmid = {30683899}, issn = {2045-2322}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cells, Cultured ; DNA Repair ; Ear ; Fanconi Anemia/*genetics/*therapy ; Fanconi Anemia Complementation Group F Protein/*genetics ; Fibroblasts ; Gene Editing/*methods ; Genetic Therapy/*methods ; Mice ; Mouse Embryonic Stem Cells ; }, abstract = {Fanconi anemia (FA) is a cancer predisposition syndrome characterized by congenital abnormalities, bone marrow failure, and hypersensitivity to aldehydes and crosslinking agents. For FA patients, gene editing holds promise for therapeutic applications aimed at functionally restoring mutated genes in hematopoietic stem cells. However, intrinsic FA DNA repair defects may obstruct gene editing feasibility. Here, we report on the CRISPR/Cas9-mediated correction of a disruptive mutation in Fancf. Our experiments revealed that gene editing could effectively restore Fancf function via error-prone end joining resulting in a 27% increased survival in the presence of mitomycin C. In addition, templated gene correction could be achieved after double strand or single strand break formation. Although templated gene editing efficiencies were low (≤6%), FA corrected embryonic stem cells acquired a strong proliferative advantage over non-corrected cells, even without imposing genotoxic stress. Notably, Cas9 nickase activity resulted in mono-allelic gene editing and avoidance of undesired mutagenesis. In conclusion: DNA repair defects associated with FANCF deficiency do not prohibit CRISPR/Cas9 gene correction. Our data provide a solid basis for the application of pre-clinical models to further explore the potential of gene editing against FA, with the eventual aim to obtain therapeutic strategies against bone marrow failure.}, }
@article {pmid30655567, year = {2019}, author = {Supharattanasitthi, W and Carlsson, E and Sharif, U and Paraoan, L}, title = {CRISPR/Cas9-mediated one step bi-allelic change of genomic DNA in iPSCs and human RPE cells in vitro with dual antibiotic selection.}, journal = {Scientific reports}, volume = {9}, number = {1}, pages = {174}, pmid = {30655567}, issn = {2045-2322}, mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Cystatin C/*genetics ; DNA/*genetics ; Epithelial Cells/cytology ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/cytology ; }, abstract = {CRISPR/Cas9 causes double-stranded DNA breaks that can undergo DNA repair either via non-homologous end joining (NHEJ) or, in the presence of a template, homology-directed repair (HDR). HDR is typically used to insert a specific genetic modification into the genome but has low efficiency compared to NHEJ, which is lowered even further when trying to create a homozygous change. In this study we devised a novel approach for homozygous single base editing based on utilising simultaneously two donor DNA templates cloned in plasmids with different antibiotic resistant genes. The donor templates were co-transfected alongside the CRISPR/Cas9 machinery into cells and a double antibiotic selection was optimised and allowed the isolation of viable desired clones. We applied the method for obtaining isogenic cells homozygous for variant B cystatin C, a recessive risk factor for age-related macular degeneration and Alzheimer's disease, in both induced Pluripotent Stem Cells (iPSCs) and a human RPE cell line. Bi-allelic gene edited clones were validated by sequencing, demonstrating that the double antibiotic templates approach worked efficiently for both iPSCs and human differentiated cells. We propose that this one step gene editing approach can be used to improve the specificity and frequency of introducing homozygous modifications in mammalian cells.}, }
@article {pmid30640520, year = {2019}, author = {Fan, HH and Yu, IS and Lin, YH and Wang, SY and Liaw, YH and Chen, PL and Yang, TL and Lin, SW and Chen, YT}, title = {P53 ICE CRIM mouse: a tool to generate mutant allelic series in somatic cells and germ lines for cancer studies.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {33}, number = {4}, pages = {5571-5584}, doi = {10.1096/fj.201802027R}, pmid = {30640520}, issn = {1530-6860}, mesh = {Alleles ; Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Gene Targeting/methods ; Germ Cells ; Mice ; Mice, Transgenic/genetics ; Mutagenesis/genetics ; Mutation/*genetics ; Neoplasms/*genetics ; Oncogenes/genetics ; RNA, Guide/genetics ; Tumor Suppressor Protein p53/*genetics ; }, abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology facilitates somatic genome editing to reveal cooperative genetic interactions at the cellular level without extensive breeding between different mutant animals. Here we propose a transgenic inducible Cas9 effector-CRISPR mutagen (ICE CRIM) mouse model in which CRISPR/Cas9-mediated somatic mutagenesis events can occur in response to Cre expression. The well-known tumor suppressor gene, Trp53, and 2 important DNA mismatch repair genes, Mlh1 and Msh2, were selected to be our somatic mutagenesis targets. Amplicon-based sequencing was performed to validate the editing efficiency and to identify the mutant allelic series. Crossed with various Cre lines, the Trp53 ICE CRIM alleles were activated to generate targeted cancer gene somatic or germ line mutant variants. We provide experimental evidence to show that an activated ICE CRIM can mutate both targeted alleles within a cell. Simultaneous disruption of multiple genes was also achieved when there were multiple single-guide RNA expression cassettes embedded within an activated ICE CRIM. Our mouse model can be used to generate mutant pools in vivo, which enables a functional screen to be performed in situ. Our results also provide evidence to support a monoclonal origin of hematopoietic neoplasms and to indicate that DNA mismatch repair deficiency accelerates tumorigenesis in Trp53 mutant genetic background.-Fan, H.-H., Yu, I.-S., Lin, Y.-H., Wang, S.-Y., Liaw, Y.-H., Chen, P.-L., Yang, T.-L., Lin, S.-W., Chen, Y.-T. P53 ICE CRIM mouse: a tool to generate mutant allelic series in somatic cells and germ lines for cancer studies.}, }
@article {pmid30540494, year = {2019}, author = {Violante, S and Achetib, N and van Roermund, CWT and Hagen, J and Dodatko, T and Vaz, FM and Waterham, HR and Chen, H and Baes, M and Yu, C and Argmann, CA and Houten, SM}, title = {Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {33}, number = {3}, pages = {4355-4364}, pmid = {30540494}, issn = {1530-6860}, support = {R01 DK113172/DK/NIDDK NIH HHS/United States ; }, mesh = {ATP-Binding Cassette Transporters/deficiency/genetics/*physiology ; Animals ; CRISPR-Cas Systems ; Carnitine/analogs &amp; derivatives/metabolism ; Carnitine O-Palmitoyltransferase/antagonists &amp; inhibitors/deficiency/physiology ; Fatty Acids/*metabolism ; HEK293 Cells ; Humans ; Lauric Acids/metabolism ; Membrane Proteins/metabolism ; Mice ; Mice, Knockout ; Mitochondria/enzymology ; Oxidation-Reduction ; Palmitic Acid/metabolism ; Peroxisomal Bifunctional Enzyme/deficiency ; Peroxisomal Multifunctional Protein-2/deficiency/genetics/*physiology ; Peroxisomes/*enzymology ; Recombinant Proteins/metabolism ; }, abstract = {Peroxisomes are essential organelles for the specialized oxidation of a wide variety of fatty acids, but they are also able to degrade fatty acids that are typically handled by mitochondria. Using a combination of pharmacological inhibition and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 genome editing technology to simultaneously manipulate peroxisomal and mitochondrial fatty acid β-oxidation (FAO) in HEK-293 cells, we identified essential players in the metabolic crosstalk between these organelles. Depletion of carnitine palmitoyltransferase (CPT)2 activity through pharmacological inhibition or knockout (KO) uncovered a significant residual peroxisomal oxidation of lauric and palmitic acid, leading to the production of peroxisomal acylcarnitine intermediates. Generation and analysis of additional single- and double-KO cell lines revealed that the D-bifunctional protein (HSD17B4) and the peroxisomal ABC transporter ABCD3 are essential in peroxisomal oxidation of lauric and palmitic acid. Our results indicate that peroxisomes not only accept acyl-CoAs but can also oxidize acylcarnitines in a similar biochemical pathway. By using an Hsd17b4 KO mouse model, we demonstrated that peroxisomes contribute to the plasma acylcarnitine profile after acute inhibition of CPT2, proving in vivo relevance of this pathway. We summarize that peroxisomal FAO is important when mitochondrial FAO is defective or overloaded.-Violante, S., Achetib, N., van Roermund, C. W. T., Hagen, J., Dodatko, T., Vaz, F. M., Waterham, H. R., Chen, H., Baes, M., Yu, C., Argmann, C. A., Houten, S. M. Peroxisomes can oxidize medium- and long-chain fatty acids through a pathway involving ABCD3 and HSD17B4.}, }
@article {pmid32387678, year = {2020}, author = {Ding, N and Lee, S and Lieber-Kotz, M and Yang, J and Gao, X}, title = {Advances in genome editing for genetic hearing loss.}, journal = {Advanced drug delivery reviews}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.addr.2020.05.001}, pmid = {32387678}, issn = {1872-8294}, abstract = {According to the World Health Organization, hearing loss affects over 466 million people worldwide and is the most common human sensory impairment. It is estimated that genetic factors contribute to the causation of approximately 50% of congenital hearing loss. Yet, curative approaches to reversing or preventing genetic hearing impairment are still limited. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) systems enable programmable and targeted gene editing in highly versatile manners and offer new gene therapy strategies for genetic hearing loss. Here, we summarize the most common deafness-associated genes, illustrate recent strategies undertaken by using CRISPR-Cas 9 systems for targeted gene editing and further compare the CRISPR strategies to non-CRISPR gene therapies. We also examine the merits of different vehicles and delivery forms of genome editing agents. Lastly, we describe the development of animal models that could facilitate the eventual clinical applications of the CRISPR technology to the treatment of genetic hearing diseases.}, }
@article {pmid32387190, year = {2020}, author = {Mukama, O and de Dieu Habimana, J and Meng, X and Ting, Y and Songwe, F and Al Farga, A and Mugisha, S and Rwibasira, P and Li, Z and Zeng, L}, title = {Synergetic performance of isothermal amplification techniques and lateral flow approach for Nucleic acid diagnostics.}, journal = {Analytical biochemistry}, volume = {}, number = {}, pages = {113762}, doi = {10.1016/j.ab.2020.113762}, pmid = {32387190}, issn = {1096-0309}, abstract = {The advancement in developing sensitive, rapid, and specific sensing tools is crucial in diagnostics and biotechnological applications. Although various isothermal amplification approaches exist for the detection and identification of nucleic acids, post-amplicon analysis is still based on traditional methods such as gel electrophoresis, colorimetry, turbidity, which could be non-specific and inconvenient. Thus, this review will first elaborate various isothermal amplification techniques (principle, merits, and demerits) and their potentials when combined with lateral flow approach for point-of-care nucleic acid diagnostics. Different methods for monitoring carryover contamination resulting from amplification product contamination will be discussed. Then, we will present recent advances in diagnostics with both target pre-amplification and CRISPR-Cas systems, which exhibit collateral cleavage of target nucleic acid and a reporter single strand nucleic acid within the vicinity. When the reporter is fluorophore-labelled, it provides a detectable signal by fluorescence or lateral flow biosensors. Lastly, we will discuss how CRISPR-Cas system based diagnostics could be more effective, affordable and portable for on-site detection.}, }
@article {pmid32385085, year = {2020}, author = {Sun, D and Mao, X and Fei, M and Chen, Z and Zhu, T and Qiu, J}, title = {Histone-like nucleoid-structuring protein (H-NS) paralogue StpA activates the type I-E CRISPR-Cas system against natural transformation in Escherichia coli.}, journal = {Applied and environmental microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1128/AEM.00731-20}, pmid = {32385085}, issn = {1098-5336}, abstract = {Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (P cas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli Although H-NS paralogue StpA also binds P cas , its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli We first documented that although the activated type I-E CRISPR-Cas system due to hns deletion interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of P cas Third, by comparing transcriptional activities of the intact P cas and the P cas with disrupted H-NS binding site in the hns and hns stpA null-deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in P cas Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of P cas Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coliImportance StpA is normally considered as a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double stranded-DNA (dsDNA) entry during natural transformation of E. coli In this study, we show that StpA plays a role opposite to its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids, but also sheds new lights on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share the same DNA binding site but play opposite roles in transcriptional regulation implicates higher order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.}, }
@article {pmid32383796, year = {2020}, author = {Common, J and Walker-Sünderhauf, D and van Houte, S and Westra, ER}, title = {Diversity in CRISPR-based immunity protects susceptible genotypes by restricting phage spread and evolution.}, journal = {Journal of evolutionary biology}, volume = {}, number = {}, pages = {}, doi = {10.1111/jeb.13638}, pmid = {32383796}, issn = {1420-9101}, abstract = {Diversity in host resistance often associates with reduced pathogen spread. This may result from ecological and evolutionary processes, likely with feedback between them. Theory and experiments on bacteria-phage interactions have shown that genetic diversity of the bacterial adaptive immune system can limit phage evolution to overcome resistance. Using the CRISPR-Cas bacterial immune system and lytic phage, we engineered a host-pathogen system where each bacterial host genotype could be infected by only one phage genotype. With this model system, we explored how CRISPR diversity impacts the spread of phage when they can overcome a resistance allele, how immune diversity affects the evolution of the phage to increase its host range, and if there was feedback between these processes. We show that increasing CRISPR diversity benefits susceptible bacteria via a dilution effect, which limits the spread of the phage. We suggest that this ecological effect impacts the evolution of novel phage genotypes, which then feeds back into phage population dynamics.}, }
@article {pmid32382982, year = {2020}, author = {Zhang, Q and Yin, K and Liu, G and Li, S and Li, M and Qiu, JL}, title = {Fusing T5 exonuclease with Cas9 and Cas12a increases the frequency and size of deletion at target sites.}, journal = {Science China. Life sciences}, volume = {}, number = {}, pages = {}, doi = {10.1007/s11427-020-1671-6}, pmid = {32382982}, issn = {1869-1889}, abstract = {CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deletions usually requires the use of pairs of guide RNAs. Here we show that it is possible to create such deletions with a single guide RNA by fusing Cas9 or Cas12a with T5 exonuclease (T5exo). These fusion constructs were found to increase both the frequency and size of deletions at target loci in rice protoplasts and seedlings. Moreover, the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease. These T5exo-Cas fusions expand the CRISPR toolbox, and facilitate knockout of regulatory and non-coding DNA sequences. From a wider standpoint, our results suggest a general strategy for producing larger deletions using other Cas nucleases.}, }
@article {pmid32036448, year = {2020}, author = {Leonard, F and Curtis, LT and Hamed, AR and Zhang, C and Chau, E and Sieving, D and Godin, B and Frieboes, HB}, title = {Nonlinear response to cancer nanotherapy due to macrophage interactions revealed by mathematical modeling and evaluated in a murine model via CRISPR-modulated macrophage polarization.}, journal = {Cancer immunology, immunotherapy : CII}, volume = {69}, number = {5}, pages = {731-744}, pmid = {32036448}, issn = {1432-0851}, support = {METAvivor Foundation Early Career Investigator Award//Houston Methodist Research Institute/ ; R15CA203605/CA/NCI NIH HHS/United States ; R15 CA203605/CA/NCI NIH HHS/United States ; Cardiovascular Nanomedicine Grant//Dr. Cyrus and Myrtle Katzen Cancer Research Center, George Washington University (US)/ ; Department of Nanomedicine Innovative Grant Award//Houston Methodist Research Institute/ ; }, mesh = {Albumin-Bound Paclitaxel/*administration &amp; dosage ; Animals ; Breast Neoplasms/immunology/pathology/*therapy ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics/immunology ; Cell Engineering ; Cell Line, Tumor/transplantation ; Coculture Techniques ; Disease Models, Animal ; Female ; Humans ; Liposomes ; Liver Neoplasms/immunology/secondary/*therapy ; Macrophage Activation/*genetics ; Macrophages/*immunology ; Mice ; *Models, Biological ; Nanoparticles ; Spheroids, Cellular ; Treatment Outcome ; Tumor Microenvironment/drug effects/genetics/immunology ; }, abstract = {Tumor-associated macrophages (TAMs) have been shown to both aid and hinder tumor growth, with patient outcomes potentially hinging on the proportion of M1, pro-inflammatory/growth-inhibiting, to M2, growth-supporting, phenotypes. Strategies to stimulate tumor regression by promoting polarization to M1 are a novel approach that harnesses the immune system to enhance therapeutic outcomes, including chemotherapy. We recently found that nanotherapy with mesoporous particles loaded with albumin-bound paclitaxel (MSV-nab-PTX) promotes macrophage polarization towards M1 in breast cancer liver metastases (BCLM). However, it remains unclear to what extent tumor regression can be maximized based on modulation of the macrophage phenotype, especially for poorly perfused tumors such as BCLM. Here, for the first time, a CRISPR system is employed to permanently modulate macrophage polarization in a controlled in vitro setting. This enables the design of 3D co-culture experiments mimicking the BCLM hypovascularized environment with various ratios of polarized macrophages. We implement a mathematical framework to evaluate nanoparticle-mediated chemotherapy in conjunction with TAM polarization. The response is predicted to be not linearly dependent on the M1:M2 ratio. To investigate this phenomenon, the response is simulated via the model for a variety of M1:M2 ratios. The modeling indicates that polarization to an all-M1 population may be less effective than a combination of both M1 and M2. Experimental results with the CRISPR system confirm this model-driven hypothesis. Altogether, this study indicates that response to nanoparticle-mediated chemotherapy targeting poorly perfused tumors may benefit from a fine-tuned M1:M2 ratio that maintains both phenotypes in the tumor microenvironment during treatment.}, }
@article {pmid31150757, year = {2019}, author = {Lu, Y and Liang, M and Zhang, Q and Liu, Z and Song, Y and Lai, L and Li, Z}, title = {Mutations of GADD45G in rabbits cause cleft lip by the disorder of proliferation, apoptosis and epithelial-mesenchymal transition (EMT).}, journal = {Biochimica et biophysica acta. Molecular basis of disease}, volume = {1865}, number = {9}, pages = {2356-2367}, doi = {10.1016/j.bbadis.2019.05.015}, pmid = {31150757}, issn = {1879-260X}, mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems/genetics ; Cell Proliferation ; Cleft Lip/genetics/*pathology/veterinary ; Embryo, Mammalian/cytology/metabolism/pathology ; Embryonic Development ; Epithelial-Mesenchymal Transition ; Gene Knockout Techniques/methods ; Homozygote ; Intracellular Signaling Peptides and Proteins/chemistry/*genetics/metabolism ; Lip/pathology ; Mutation ; Protein Structure, Tertiary ; Rabbits ; }, abstract = {The cleft lip with or without cleft palate (CL/P) is one of the most common congenital defects in humans. Genome-wide association studies (GWAS) have been widely used for identifying candidate genes, and different genes or chromosomal regions have shown strong evidence for the presence of causal genes in CL/P. To date, two independent GWAS have identified GADD45G as influencing risk for CL/P. However, there is no animal model evidence about GADD45G related to CL/P. Here, we reported the generation of a novel GADD45G mutated rabbit model by CRISPR/Cas9 and CRISPR-based BE4-Gam systems. The homozygous (GADD45G-/-) while not heterozygous (GADD45G+/-) pups died after birth due to severe craniofacial defects of unilateral or bilateral cleft lip (CL). Moreover, the disorder of proliferation, apoptosis and epithelial-mesenchymal transition (EMT) were also determined in the medial and lateral nasal processes (MNP and LNP) of the embryonic day 13 (E13) GADD45G-/- rabbits, which compared with the normal wild type (WT) rabbits. Thus, our study confirmed for the first time that loss of GADD45G lead to CL at the animal level and provided new insights into the crucial role of GADD45G for upper lip formation and fusion.}, }
@article {pmid32376630, year = {2020}, author = {Specht, DA and Xu, Y and Lambert, G}, title = {Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {}, number = {}, pages = {}, doi = {10.1073/pnas.1918685117}, pmid = {32376630}, issn = {1091-6490}, abstract = {The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has led to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable nonspecific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of nuclease-dead Cas12a (dCas12a) from Francisella novicida as it inspects and binds to its DNA target. Our results reveal concealed thermodynamic factors affecting dCas12a DNA binding, which should guide the design and optimization of crRNA that limits off-target effects, including the crucial role of an extended protospacer adjacent motif (PAM) sequence and the impact of the specific base composition of crRNA-DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.}, }
@article {pmid32060484, year = {2020}, author = {Mäkelä, J and Sherratt, DJ}, title = {Catching an invader.}, journal = {Nature reviews. Microbiology}, volume = {18}, number = {4}, pages = {194}, doi = {10.1038/s41579-020-0337-8}, pmid = {32060484}, issn = {1740-1534}, support = {200782/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; }, mesh = {*CRISPR-Cas Systems ; DNA, Viral ; Escherichia coli/metabolism/virology ; Streptococcus pyogenes/metabolism/virology ; }, }
@article {pmid31942067, year = {2020}, author = {Athukoralage, JS and McMahon, SA and Zhang, C and Grüschow, S and Graham, S and Krupovic, M and Whitaker, RJ and Gloster, TM and White, MF}, title = {An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity.}, journal = {Nature}, volume = {577}, number = {7791}, pages = {572-575}, doi = {10.1038/s41586-019-1909-5}, pmid = {31942067}, issn = {1476-4687}, support = {BB/G011400/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R008035/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {Adenine Nucleotides/chemistry/metabolism ; CRISPR-Associated Proteins/chemistry/metabolism ; CRISPR-Cas Systems/*immunology ; DNA, Viral/metabolism ; Endonucleases/chemistry/*metabolism ; Host Microbial Interactions/*immunology ; Models, Molecular ; Nucleotides, Cyclic/chemistry/metabolism ; Oligoribonucleotides/chemistry/metabolism ; Phylogeny ; Signal Transduction ; Sulfolobus/genetics/immunology/metabolism/*virology ; Viral Proteins/chemistry/classification/*metabolism ; Viruses/*enzymology/immunology ; }, abstract = {The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3-5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7-10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host-pathogen interactions11-13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize the type III CRISPR defence system. The AcrIII-1 family has a broad host range, as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.}, }
@article {pmid31874627, year = {2019}, author = {Bradford, J and Perrin, D}, title = {Improving CRISPR guide design with consensus approaches.}, journal = {BMC genomics}, volume = {20}, number = {Suppl 9}, pages = {931}, doi = {10.1186/s12864-019-6291-z}, pmid = {31874627}, issn = {1471-2164}, mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Software ; }, abstract = {BACKGROUND: CRISPR-based systems are playing an important role in modern genome engineering. A large number of computational methods have been developed to assist in the identification of suitable guides. However, there is only limited overlap between the guides that each tool identifies. This can motivate further development, but also raises the question of whether it is possible to combine existing tools to improve guide design.<br><br>RESULTS: We considered nine leading guide design tools, and their output when tested using two sets of guides for which experimental validation data is available. We found that consensus approaches were able to outperform individual tools. The best performance (with a precision of up to 0.912) was obtained when combining four of the tools and accepting all guides selected by at least three of them.<br><br>CONCLUSIONS: These results can be used to improve CRISPR-based studies, but also to guide further tool development. However, they only provide a short-term solution as the time and computational resources required to run four tools may be impractical in certain applications.}, }
@article {pmid31595153, year = {2019}, author = {Li, P and Meng, Y and Wang, Y and Li, J and Lam, M and Wang, L and Di, LJ}, title = {Nuclear localization of Desmoplakin and its involvement in telomere maintenance.}, journal = {International journal of biological sciences}, volume = {15}, number = {11}, pages = {2350-2362}, pmid = {31595153}, issn = {1449-2288}, mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Nucleus/*metabolism ; DNA Damage ; Desmoplakins/chemistry/metabolism/*physiology ; HEK293 Cells ; Humans ; Telomerase/metabolism ; Telomere/metabolism ; *Telomere Homeostasis ; Telomere Shortening ; Telomere-Binding Proteins/chemistry/metabolism/*physiology ; }, abstract = {The interaction between genomic DNA and protein fundamentally determines the activity and the function of DNA elements. Capturing the protein complex and identifying the proteins associated with a specific DNA locus is difficult. Herein, we employed CRISPR, the well-known gene-targeting tool in combination with the proximity-dependent labeling tool BioID to capture a specific genome locus associated proteins and to uncover the novel functions of these proteins. By applying this research tool on telomeres, we identified DSP, out of many others, as a convincing telomere binding protein validated by both biochemical and cell-biological approaches. We also provide evidence to demonstrate that the C-terminal domain of DSP is required for its binding to telomere after translocating to the nucleus mediated by NLS sequence of DSP. In addition, we found that the telomere binding of DSP is telomere length dependent as hTERT inhibition or knockdown caused a decrease of telomere length and diminished DSP binding to the telomere. Knockdown of TRF2 also negatively influenced DSP binding to the telomere. Functionally, loss of DSP resulted in the shortened telomere DNA and induced the DNA damage response and cell apoptosis. In conclusion, our studies identified DSP as a novel potential telomere binding protein and highlighted its role in protecting against telomere DNA damage and resultant cell apoptosis.}, }
@article {pmid31504824, year = {2019}, author = {Mao, S and Ying, Y and Wu, X and Krueger, CJ and Chen, AK}, title = {CRISPR/dual-FRET molecular beacon for sensitive live-cell imaging of non-repetitive genomic loci.}, journal = {Nucleic acids research}, volume = {47}, number = {20}, pages = {e131}, pmid = {31504824}, issn = {1362-4962}, mesh = {*CRISPR-Cas Systems ; Fluorescence Resonance Energy Transfer/*methods ; Fluorescent Dyes/chemistry ; *Genetic Loci ; HeLa Cells ; Humans ; RNA, Guide/chemistry/metabolism ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based genomic imaging systems predominantly rely on fluorescent protein reporters, which lack the optical properties essential for sensitive dynamic imaging. Here, we modified the CRISPR single-guide RNA (sgRNA) to carry two distinct molecular beacons (MBs) that can undergo fluorescence resonance energy transfer (FRET) and demonstrated that the resulting system, CRISPR/dual-FRET MB, enables dynamic imaging of non-repetitive genomic loci with only three unique sgRNAs.}, }
@article {pmid31479876, year = {2019}, author = {Malerba, N and Benzoni, P and Squeo, GM and Milanesi, R and Giannetti, F and Sadleir, LG and Poke, G and Augello, B and Croce, AI and Barbuti, A and Merla, G}, title = {Generation of the induced human pluripotent stem cell lines CSSi009-A from a patient with a GNB5 pathogenic variant, and CSSi010-A from a CRISPR/Cas9 engineered GNB5 knock-out human cell line.}, journal = {Stem cell research}, volume = {40}, number = {}, pages = {101547}, doi = {10.1016/j.scr.2019.101547}, pmid = {31479876}, issn = {1876-7753}, mesh = {CRISPR-Cas Systems ; Cell Differentiation ; Cell Line/cytology/*metabolism ; Cells, Cultured ; Child ; Female ; Fibroblasts/cytology/metabolism ; Frameshift Mutation ; GTP-Binding Protein beta Subunits/*genetics/metabolism ; Gene Editing ; Gene Knockout Techniques ; Genetic Diseases, Inborn/*genetics/metabolism/physiopathology ; Genetic Engineering ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Male ; Middle Aged ; }, abstract = {GNB5 loss-of-function pathogenic variants cause IDDCA, a rare autosomal recessive human genetic disease characterized by infantile onset of intellectual disability, sinus bradycardia, hypotonia, visual abnormalities, and epilepsy. We generated human induced pluripotent stem cells (hiPSCs) from skin fibroblasts of a patient with the homozygous c.136delG frameshift variant, and a GNB5 knock-out (KO) line by CRISPR/Cas9 editing. hiPSCs express common pluripotency markers and differentiate into the three germ layers. These lines represent a powerful cellular model to study the molecular basis of GNB5-related disorders as well as offer an in vitro model for drug screening.}, }
@article {pmid32372485, year = {2020}, author = {Weng, X and Yang, S and Wu, F and Peng, S and Wang, F and Chen, Y and Yuan, Y and Zhou, X}, title = {A m6A sensing method by its impact on the stability of RNA double helix.}, journal = {Chemistry &amp; biodiversity}, volume = {}, number = {}, pages = {}, doi = {10.1002/cbdv.202000050}, pmid = {32372485}, issn = {1612-1880}, abstract = {N6-methyladenosine (m6A) is one of the most important RNA modifications in epigenetics. The development of detection method for m6A is limited by its abundance and structure. Although it has been previously reported that its presence has an impact on the complementary pairing of RNA, few assays have been developed using this finding. We used this discovery and designed a detection method based on cas13a system, which has different fluorescence signals for target RNAs containing m6A modification and target RNAs without m6A modification. We verified the fact that the presence of m6A could cause the instability of dsRNA using the Cas13a system and provided a new direction and strategy for the development of m6A detection methods in the future.}, }
@article {pmid32371471, year = {2020}, author = {Bezuidt, OKI and Lebre, PH and Pierneef, R and León-Sobrino, C and Adriaenssens, EM and Cowan, DA and Van de Peer, Y and Makhalanyane, TP}, title = {Phages Actively Challenge Niche Communities in Antarctic Soils.}, journal = {mSystems}, volume = {5}, number = {3}, pages = {}, doi = {10.1128/mSystems.00234-20}, pmid = {32371471}, issn = {2379-5077}, abstract = {By modulating the structure, diversity, and trophic outputs of microbial communities, phages play crucial roles in many biomes. In oligotrophic polar deserts, the effects of katabatic winds, constrained nutrients, and low water availability are known to limit microbial activity. Although phages may substantially govern trophic interactions in cold deserts, relatively little is known regarding the precise ecological mechanisms. Here, we provide the first evidence of widespread antiphage innate immunity in Antarctic environments using metagenomic sequence data from hypolith communities as model systems. In particular, immunity systems such as DISARM and BREX are shown to be dominant systems in these communities. Additionally, we show a direct correlation between the CRISPR-Cas adaptive immunity and the metavirome of hypolith communities, suggesting the existence of dynamic host-phage interactions. In addition to providing the first exploration of immune systems in cold deserts, our results suggest that phages actively challenge niche communities in Antarctic polar deserts. We provide evidence suggesting that the regulatory role played by phages in this system is an important determinant of bacterial host interactions in this environment.IMPORTANCE In Antarctic environments, the combination of both abiotic and biotic stressors results in simple trophic levels dominated by microbiomes. Although the past two decades have revealed substantial insights regarding the diversity and structure of microbiomes, we lack mechanistic insights regarding community interactions and how phages may affect these. By providing the first evidence of widespread antiphage innate immunity, we shed light on phage-host dynamics in Antarctic niche communities. Our analyses reveal several antiphage defense systems, including DISARM and BREX, which appear to dominate in cold desert niche communities. In contrast, our analyses revealed that genes which encode antiphage adaptive immunity were underrepresented in these communities, suggesting lower infection frequencies in cold edaphic environments. We propose that by actively challenging niche communities, phages play crucial roles in the diversification of Antarctic communities.}, }
@article {pmid32371470, year = {2020}, author = {Li, T and Zhang, Y and Dong, K and Kuo, CJ and Li, C and Zhu, YQ and Qin, J and Li, QT and Chang, YF and Guo, X and Zhu, Y}, title = {Isolation and Characterization of the Novel Phage JD032 and Global Transcriptomic Response during JD032 Infection of Clostridioides difficile Ribotype 078.}, journal = {mSystems}, volume = {5}, number = {3}, pages = {}, doi = {10.1128/mSystems.00017-20}, pmid = {32371470}, issn = {2379-5077}, abstract = {Insights into the interaction between phages and their bacterial hosts are crucial for the development of phage therapy. However, only one study has investigated global gene expression of Clostridioides (formerly Clostridium) difficile carrying prophage, and transcriptional reprogramming during lytic infection has not been studied. Here, we presented the isolation, propagation, and characterization of a newly discovered 35,109-bp phage, JD032, and investigated the global transcriptomes of both JD032 and C. difficile ribotype 078 (RT078) strain TW11 during JD032 infection. Transcriptome sequencing (RNA-seq) revealed the progressive replacement of bacterial host mRNA with phage transcripts. The expressed genes of JD032 were clustered into early, middle, and late temporal categories that were functionally similar. Specifically, a gene (JD032_orf016) involved in the lysis-lysogeny decision was identified as an early expression gene. Only 17.7% (668/3,781) of the host genes were differentially expressed, and more genes were downregulated than upregulated. The expression of genes involved in host macromolecular synthesis (DNA/RNA/proteins) was altered by JD032 at the level of transcription. In particular, the expression of the ropA operon was downregulated. Most noteworthy is that the gene expression of some antiphage systems, including CRISPR-Cas, restriction-modification, and toxin-antitoxin systems, was suppressed by JD032 during infection. In addition, bacterial sporulation, adhesion, and virulence factor genes were significantly downregulated. This study provides the first description of the interaction between anaerobic spore-forming bacteria and phages during lytic infection and highlights new aspects of C. difficile phage-host interactions.IMPORTANCEC. difficile is one of the most clinically significant intestinal pathogens. Although phages have been shown to effectively control C. difficile infection, the host responses to phage predation have not been fully studied. In this study, we reported the isolation and characterization of a new phage, JD032, and analyzed the global transcriptomic changes in the hypervirulent RT078 C. difficile strain, TW11, during phage JD032 infection. We found that bacterial host mRNA was progressively replaced with phage transcripts, three temporal categories of JD032 gene expression, the extensive interplay between phage-bacterium, antiphage-like responses of the host and phage evasion, and decreased expression of sporulation- and virulence-related genes of the host after phage infection. These findings confirmed the complexity of interactions between C. difficile and phages and suggest that phages undergoing a lytic cycle may also cause different phenotypes in hosts, similar to prophages, which may inspire phage therapy for the control of C. difficile.}, }
@article {pmid32101536, year = {2020}, author = {Liu, C and Zhao, J and Lu, W and Dai, Y and Hockings, J and Zhou, Y and Nussinov, R and Eng, C and Cheng, F}, title = {Individualized genetic network analysis reveals new therapeutic vulnerabilities in 6,700 cancer genomes.}, journal = {PLoS computational biology}, volume = {16}, number = {2}, pages = {e1007701}, pmid = {32101536}, issn = {1553-7358}, support = {HHSN261200800001E/CA/NCI NIH HHS/United States ; }, mesh = {Antineoplastic Agents/therapeutic use ; BRCA2 Protein/genetics ; Biomarkers, Tumor ; CRISPR-Cas Systems ; Carcinogenesis ; Cell Line, Tumor ; Computational Biology/*methods ; Exome ; *Gene Regulatory Networks ; Genetic Testing ; Genomics ; Humans ; Inhibitory Concentration 50 ; Models, Theoretical ; Mutation ; Neoplasms/drug therapy/*genetics ; Pharmacogenetics ; Precision Medicine ; Protein Interaction Mapping ; Survival Rate ; Tumor Suppressor Protein p53/genetics ; }, abstract = {Tumor-specific genomic alterations allow systematic identification of genetic interactions that promote tumorigenesis and tumor vulnerabilities, offering novel strategies for development of targeted therapies for individual patients. We develop an Individualized Network-based Co-Mutation (INCM) methodology by inspecting over 2.5 million nonsynonymous somatic mutations derived from 6,789 tumor exomes across 14 cancer types from The Cancer Genome Atlas. Our INCM analysis reveals a higher genetic interaction burden on the significantly mutated genes, experimentally validated cancer genes, chromosome regulatory factors, and DNA damage repair genes, as compared to human pan-cancer essential genes identified by CRISPR-Cas9 screenings on 324 cancer cell lines. We find that genes involved in the cancer type-specific genetic subnetworks identified by INCM are significantly enriched in established cancer pathways, and the INCM-inferred putative genetic interactions are correlated with patient survival. By analyzing drug pharmacogenomics profiles from the Genomics of Drug Sensitivity in Cancer database, we show that the network-predicted putative genetic interactions (e.g., BRCA2-TP53) are significantly correlated with sensitivity/resistance of multiple therapeutic agents. We experimentally validated that afatinib has the strongest cytotoxic activity on BT474 (IC50 = 55.5 nM, BRCA2 and TP53 co-mutant) compared to MCF7 (IC50 = 7.7 μM, both BRCA2 and TP53 wild type) and MDA-MB-231 (IC50 = 7.9 μM, BRCA2 wild type but TP53 mutant). Finally, drug-target network analysis reveals several potential druggable genetic interactions by targeting tumor vulnerabilities. This study offers a powerful network-based methodology for identification of candidate therapeutic pathways that target tumor vulnerabilities and prioritization of potential pharmacogenomics biomarkers for development of personalized cancer medicine.}, }
@article {pmid31763806, year = {2019}, author = {Tang, Q and Liu, J and Jiang, Y and Zhang, M and Mao, L and Wang, M}, title = {Cell-Selective Messenger RNA Delivery and CRISPR/Cas9 Genome Editing by Modulating the Interface of Phenylboronic Acid-Derived Lipid Nanoparticles and Cellular Surface Sialic Acid.}, journal = {ACS applied materials &amp; interfaces}, volume = {11}, number = {50}, pages = {46585-46590}, doi = {10.1021/acsami.9b17749}, pmid = {31763806}, issn = {1944-8252}, mesh = {Boronic Acids/chemistry ; CRISPR-Cas Systems/drug effects/genetics ; Gene Editing/methods ; *Gene Transfer Techniques ; Genetic Therapy ; HeLa Cells ; Humans ; Lipids/chemistry/*pharmacology ; N-Acetylneuraminic Acid/chemistry/metabolism ; Nanoparticles/*chemistry ; RNA, Messenger/chemistry/*pharmacology ; Transfection/methods ; }, abstract = {Messenger RNA (mRNA) represents an emerging class of nucleic acid therapeutics for genome editing and genetic disease treatment. Delivering exogenous mRNA selectively to cells, however, remains a main challenge to broaden the biomedical application of mRNA and develop targeted gene therapy. Herein, we report cell-selective mRNA delivery and CRISPR/Cas9 genome editing by modulating the interface of phenylboronic acid (PBA) derived lipid nanoparticles (NPs) and cellular surface sialic acid (SA). We design a cationic lipid featuring a PBA group, PBA-BADP, to self-assemble with mRNA into nanoparticles via electrostatic interactions. Importantly, these nanoparticles present free PBA groups on their surface, showing an enhanced cellular uptake by SA-overexpressing cancer cells via the interfacial PBA/SA interaction. It is shown that PBA-BADP/mRNA NPs transfection results in 300 times higher luciferase reporter gene expression in cancer cells than that in noncancer cells. Moreover, we demonstrate that the delivery of tumor suppressor p53 mRNA using PBA-BADP selectively prohibits cancer cell growth, while PBA-BADP/Cas9 mRNA NPs delivery knocks out gene expression of HeLa cancer cells in a much higher efficiency than noncancer cells. We believe these findings could further extend the modulation of PBA and cellular SA interface to advance mRNA delivery and genome editing for new gene therapy.}, }
@article {pmid31526944, year = {2019}, author = {Han, HJ and Han, HW and Seo, HH and Kim, JH}, title = {Generation of a KSCBi005-A-5(TLR8KO-A10) homozygous knockout human induced pluripotent stem cell line using CRISPR/Cas9.}, journal = {Stem cell research}, volume = {40}, number = {}, pages = {101561}, doi = {10.1016/j.scr.2019.101561}, pmid = {31526944}, issn = {1876-7753}, mesh = {CRISPR-Cas Systems ; Cell Line/cytology/*metabolism ; Cellular Reprogramming ; Gene Editing ; Gene Knockout Techniques ; Homozygote ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Male ; Middle Aged ; Toll-Like Receptor 8/*genetics/metabolism ; }, abstract = {The Toll like Receptor (TLR) family plays an essential role in pathogen recognition and innate immunity activation. TLR8, an endosomal receptor, can recognize single-stranded RNA viruses, such as influenza virus, Sendai virus, Coxsackie B virus, HIV, and HCV. TLR8 binding to the viral RNA recruits MyD88 and leads to activation of the transcription factor NF-kB and antiviral response. We generated biallelic mutants of the TLR8 gene using a CRISPR-Cas9 genome editing method in human induced pluripotent stem cells (hiPSCs). The TLR8 homozygous-knockout hiPSCs retained normal morphology, gene expression, and in vivo differentiation potential.}, }
@article {pmid31499408, year = {2019}, author = {Lee, Y and Choi, HY and Kwon, A and Park, H and Park, M and Kim, YO and Kwak, S and Koo, SK}, title = {Generation of a NESTIN-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-1, using CRISPR/Cas9 nuclease.}, journal = {Stem cell research}, volume = {40}, number = {}, pages = {101554}, doi = {10.1016/j.scr.2019.101554}, pmid = {31499408}, issn = {1876-7753}, mesh = {CRISPR-Cas Systems ; Cell Differentiation ; Cell Line/cytology/*metabolism ; Genes, Reporter ; Green Fluorescent Proteins/*genetics/metabolism ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Male ; Nestin/*genetics/metabolism ; }, abstract = {NESTIN, an intermediate filament, is a neuroectodermal marker involved in induced pluripotent stem cell (iPSC) differentiation toward neural lineages. Here, we introduced an EGFP reporter into the C-terminus of NESTIN in KSCBi005-A hiPSCs through homologous recombination using CRISPR/Cas9 nuclease. The successfully edited line was confirmed by sequencing and had a normal karyotype. It expressed EGFP upon induction of neural differentiation and exhibited potential for differentiation into three germ layers. KSCBi005-A-1 cells could be used to monitor the expression of NESTIN in differentiated cell types. This cell line is available at the National Stem Cell Bank, Korea National Institute of Health.}, }
@article {pmid32368102, year = {2020}, author = {Gholizadeh, P and Köse, Ş and Dao, S and Ganbarov, K and Tanomand, A and Dal, T and Aghazadeh, M and Ghotaslou, R and Ahangarzadeh Rezaee, M and Yousefi, B and Samadi Kafil, H}, title = {How CRISPR-Cas System Could Be Used to Combat Antimicrobial Resistance.}, journal = {Infection and drug resistance}, volume = {13}, number = {}, pages = {1111-1121}, doi = {10.2147/IDR.S247271}, pmid = {32368102}, issn = {1178-6973}, abstract = {Rapid emergence of antibiotic-resistant bacteria has made it harder for us to combat infectious diseases and to develop new antibiotics. The clustered regularly interspaced short palindromic repeats - CRISPR-associated (CRISPR-Cas) system, as a bacterial adaptive immune system, is recognized as one of the new strategies for controlling antibiotic-resistant strains. The programmable Cas nuclease of this system used against bacterial genomic sequences could be lethal or could help reduce resistance of bacteria to antibiotics. Therefore, this study aims to review using the CRISPR-Cas system to promote sensitizing bacteria to antibiotics. We envision that CRISPR-Cas approaches may open novel ways for the development of smart antibiotics, which could eliminate multidrug-resistant (MDR) pathogens and differentiate between beneficial and pathogenic microorganisms. These systems can be exploited to quantitatively and selectively eliminate individual bacterial strains based on a sequence-specific manner, creating opportunities in the treatment of MDR infections, the study of microbial consortia, and the control of industrial fermentation.}, }
@article {pmid32347065, year = {2020}, author = {Wang, K and An, W and Liu, Y and Liu, D and Feng, L and Wang, J and Huang, J and Liu, X and Liang, W}, title = {[Disruption of OsRhoGDI2 by CRISPR/Cas9 technology leads to semi-dwarf in rice].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {36}, number = {4}, pages = {707-715}, doi = {10.13345/j.cjb.190333}, pmid = {32347065}, issn = {1872-2075}, mesh = {CRISPR-Cas Systems ; *Genes, Plant/genetics ; *Oryza/genetics/growth &amp; development ; Plants, Genetically Modified ; rho Guanine Nucleotide Dissociation Inhibitor beta/genetics ; }, abstract = {OsRhoGDI2 was isolated as a putative partner of Rho protein family member OsRacD from rice panicles by yeast two-hybrid, but its function remains unknown. In order to identify the function of OsRhoGDI2, OsRhoGDI2 knockout mutants were created by CRISPR/Cas9 technology. The results showed that two different homozygous mutants were obtained in T0 generation, and eight kinds homozygous mutants were identified in T1 generation. Sequence analysis revealed that the base substitution or base deletion occurred near the editing targets of the gene in knockout rice, and it could be expected that the truncated OsRhoGDI2 proteins lacking the RhoGDI conserved domain would be generated. Phenotype analysis showed that the OsRhoGDI2 knockout rice plants were significantly lower than the control plants. Statistical analysis confirmed that the significant decrease of plant height was due to the shortening of the second and third internodes, suggesting that OsRhoGDI2 gene may be related with rice height control.}, }
@article {pmid32169520, year = {2020}, author = {Satyam, R and Bhardwaj, T and Jha, NK and Jha, SK and Nand, P}, title = {Toward a chimeric vaccine against multiple isolates of Mycobacteroides - An integrative approach.}, journal = {Life sciences}, volume = {250}, number = {}, pages = {117541}, doi = {10.1016/j.lfs.2020.117541}, pmid = {32169520}, issn = {1879-0631}, mesh = {Alleles ; B-Lymphocytes/immunology ; Bacterial Vaccines/*chemistry ; Bacteriophages ; CRISPR-Cas Systems ; Computational Biology ; Drug Resistance, Bacterial ; Epitopes ; Epitopes, T-Lymphocyte/genetics ; Gastrointestinal Microbiome ; Genome, Bacterial ; Genomics ; Gram-Positive Bacterial Infections/*prevention &amp; control ; Histocompatibility Antigens Class I/metabolism ; Histocompatibility Antigens Class II/metabolism ; Humans ; Immunotherapy ; Molecular Docking Simulation ; Molecular Dynamics Simulation ; Mycobacteriaceae/*genetics/pathogenicity ; Proteome ; *Vaccinology ; Virulence ; }, abstract = {AIM: Nontuberculous mycobacterial (NTM) infection such as endophthalmitis, dacryocystitis, and canaliculitis are pervasive across the globe and are currently managed by antibiotics. However, the recent cases of Mycobacteroides developing drug resistance reported along with the improper practice of medicine intrigued us to explore its genomic and proteomic canvas at a global scale and develop a chimeric vaccine against Mycobacteroides.<br><br>MAIN METHODS: We carried out a vivid genomic study on five recently sequenced strains of Mycobacteroides and explored their Pan-core genome/proteome in three different phases. The promiscuous antigenic proteins were identified via a subtractive proteomics approach that qualified for virulence causation, resistance and essentiality factors for this notorious bacterium. An integrated pipeline was developed for the identification of B-Cell, MHC (Major histocompatibility complex) class I and II epitopes.<br><br>KEY FINDINGS: Phase I identified the shreds of evidence of reductive evolution and propensity of the Pan-genome of Mycobacteroides getting closed soon. Phase II and Phase III produced 8 vaccine constructs. Our final vaccine construct, V6 qualified for all tests such as absence for allergenicity, presence of antigenicity, etc. V6 contains β-defensin as an adjuvant, linkers, Lysosomal-associated membrane protein 1 (LAMP1) signal peptide, and PADRE (Pan HLA-DR epitopes) amino acid sequence. Besides, V6 also interacts with a maximum number of MHC molecules and the TLR4/MD2 (Toll-like receptor 4/Myeloid differentiation factor 2) complex confirmed by docking and molecular dynamics simulation studies.<br><br>SIGNIFICANCE: The knowledge harnessed from the current study can help improve the current treatment regimens or in an event of an outbreak and propel further related studies.}, }
@article {pmid32109227, year = {2020}, author = {Champer, J and Lee, E and Yang, E and Liu, C and Clark, AG and Messer, PW}, title = {A toxin-antidote CRISPR gene drive system for regional population modification.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {1082}, pmid = {32109227}, issn = {2041-1723}, support = {F32 AI138476/AI/NIAID NIH HHS/United States ; R01 GM127418/GM/NIGMS NIH HHS/United States ; R21 AI130635/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; Animals, Genetically Modified ; *Antidotes ; CRISPR-Cas Systems/genetics ; Drosophila/genetics ; Drosophila Proteins/*genetics ; Female ; Gene Drive Technology/*methods ; Genetics, Population ; Heterozygote ; *Models, Genetic ; Mutation ; Protein Engineering/*methods ; }, abstract = {Engineered gene drives based on a homing mechanism could rapidly spread genetic alterations through a population. However, such drives face a major obstacle in the form of resistance against the drive. In addition, they are expected to be highly invasive. Here, we introduce the Toxin-Antidote Recessive Embryo (TARE) drive. It functions by disrupting a target gene, forming recessive lethal alleles, while rescuing drive-carrying individuals with a recoded version of the target. Modeling shows that such drives will have threshold-dependent invasion dynamics, spreading only when introduced above a fitness-dependent frequency. We demonstrate a TARE drive in Drosophila with 88-95% transmission by female heterozygotes. This drive was able to spread through a large cage population in just six generations following introduction at 24% frequency without any apparent evolution of resistance. Our results suggest that TARE drives constitute promising candidates for the development of effective, flexible, and regionally confinable drives for population modification.}, }
@article {pmid32032363, year = {2020}, author = {Drews, K and Calgi, MP and Harrison, WC and Drews, CM and Costa-Pinheiro, P and Shaw, JJP and Jobe, KA and Han, JD and Fox, TE and White, JM and Kester, M}, title = {Glucosylceramide synthase maintains influenza virus entry and infection.}, journal = {PloS one}, volume = {15}, number = {2}, pages = {e0228735}, pmid = {32032363}, issn = {1932-6203}, support = {R01 AI114776/AI/NIAID NIH HHS/United States ; T32 CA009109/CA/NCI NIH HHS/United States ; T32 GM008715/GM/NIGMS NIH HHS/United States ; }, mesh = {A549 Cells ; CRISPR-Cas Systems/genetics ; Gene Editing ; Glucosyltransferases/deficiency/*genetics ; HEK293 Cells ; Humans ; Influenza A virus/*physiology ; Macrolides/pharmacology ; Virus Internalization/drug effects ; }, abstract = {Influenza virus is an enveloped virus wrapped in a lipid bilayer derived from the host cell plasma membrane. Infection by influenza virus is dependent on these host cell lipids, which include sphingolipids. Here we examined the role of the sphingolipid, glucosylceramide, in influenza virus infection by knocking out the enzyme responsible for its synthesis, glucosylceramide synthase (UGCG). We observed diminished influenza virus infection in HEK 293 and A549 UGCG knockout cells and demonstrated that this is attributed to impaired viral entry. We also observed that entry mediated by the glycoproteins of other enveloped viruses that enter cells by endocytosis is also impaired in UGCG knockout cells, suggesting a broader role for UGCG in viral entry by endocytosis.}, }
@article {pmid32029687, year = {2020}, author = {Stadtmauer, EA and Fraietta, JA and Davis, MM and Cohen, AD and Weber, KL and Lancaster, E and Mangan, PA and Kulikovskaya, I and Gupta, M and Chen, F and Tian, L and Gonzalez, VE and Xu, J and Jung, IY and Melenhorst, JJ and Plesa, G and Shea, J and Matlawski, T and Cervini, A and Gaymon, AL and Desjardins, S and Lamontagne, A and Salas-Mckee, J and Fesnak, A and Siegel, DL and Levine, BL and Jadlowsky, JK and Young, RM and Chew, A and Hwang, WT and Hexner, EO and Carreno, BM and Nobles, CL and Bushman, FD and Parker, KR and Qi, Y and Satpathy, AT and Chang, HY and Zhao, Y and Lacey, SF and June, CH}, title = {CRISPR-engineered T cells in patients with refractory cancer.}, journal = {Science (New York, N.Y.)}, volume = {367}, number = {6481}, pages = {}, doi = {10.1126/science.aba7365}, pmid = {32029687}, issn = {1095-9203}, support = {R01 CA120409/CA/NCI NIH HHS/United States ; P01 CA214278/CA/NCI NIH HHS/United States ; U01 AG066100/AG/NIA NIH HHS/United States ; }, mesh = {*Adoptive Transfer ; Aged ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Cell Engineering ; Female ; *Gene Editing ; Humans ; Male ; Middle Aged ; Programmed Cell Death 1 Receptor/genetics ; Receptors, Antigen, T-Cell, alpha-beta/*genetics ; T-Lymphocytes/*immunology/*transplantation ; Transgenes ; }, abstract = {CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRβ (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.}, }
@article {pmid31937945, year = {2020}, author = {}, title = {Towards better base editing.}, journal = {Nature biomedical engineering}, volume = {4}, number = {1}, pages = {1}, doi = {10.1038/s41551-020-0515-z}, pmid = {31937945}, issn = {2157-846X}, mesh = {Base Pairing/*genetics ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; }, }
@article {pmid31937939, year = {2020}, author = {Kim, HK and Lee, S and Kim, Y and Park, J and Min, S and Choi, JW and Huang, TP and Yoon, S and Liu, DR and Kim, HH}, title = {High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells.}, journal = {Nature biomedical engineering}, volume = {4}, number = {1}, pages = {111-124}, doi = {10.1038/s41551-019-0505-1}, pmid = {31937939}, issn = {2157-846X}, support = {RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Deep Learning ; Gene Editing/*methods ; Genetic Vectors/genetics ; HEK293 Cells ; Humans ; Lentivirus/physiology ; Streptococcus pyogenes/genetics ; }, abstract = {The applications of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing can be limited by a lack of compatible protospacer adjacent motifs (PAMs), insufficient on-target activity and off-target effects. Here, we report an extensive comparison of the PAM-sequence compatibilities and the on-target and off-target activities of Cas9 from Streptococcus pyogenes (SpCas9) and the SpCas9 variants xCas9 and SpCas9-NG (which are known to have broader PAM compatibility than SpCas9) at 26,478 lentivirally integrated target sequences and 78 endogenous target sites in human cells. We found that xCas9 has the lowest tolerance for mismatched target sequences and that SpCas9-NG has the broadest PAM compatibility. We also show, on the basis of newly identified non-NGG PAM sequences, that SpCas9-NG and SpCas9 can edit six previously unedited endogenous sites associated with genetic diseases. Moreover, we provide deep-learning models that predict the activities of xCas9 and SpCas9-NG at the target sequences. The resulting deeper understanding of the activities of xCas9, SpCas9-NG and SpCas9 in human cells should facilitate their use.}, }
@article {pmid31933285, year = {2019}, author = {Hirsch, F and Lemaitre, C and Chneiweiss, H and Montoliu, L}, title = {Genome Editing: Promoting Responsible Research.}, journal = {Pharmaceutical medicine}, volume = {33}, number = {3}, pages = {187-191}, pmid = {31933285}, issn = {1178-2595}, mesh = {Animals ; CRISPR-Cas Systems ; Ethics, Research ; Gene Editing/*ethics/*legislation &amp; jurisprudence ; Genome, Human ; Humans ; }, abstract = {For more than 40 years, scientists have been developing tools and technologies for genome modification; however, initial progress was slow and few outside of the molecular biology community took an interest in the field. Everything has dramatically changed with the recent appearance of the so-called precision approaches, and especially with the 'CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) revolution'. With great powers come great responsibilities. CRISPR-derived technologies have been proven efficient, cheap, rather easy and fast, and provided universal access to genome modification techniques beyond the leading research centers and reference laboratories. The popularization of techniques to manipulate the human genome and that of all other living beings consequently raises many essential questions, on the ethical and legal sides, both for the scientific community and the lay public. In order to mitigate excessive hype and concern among citizens, a call for the mobilization of the various stakeholders is now urgent through a global governance of genome editing.}, }
@article {pmid31888216, year = {2019}, author = {Yoshimura, T and Nakamura, K and Li, C and Fujisawa, M and Shiina, T and Imamura, M and Li, T and Mukaida, N and Matsukawa, A}, title = {Cancer Cell-Derived Granulocyte-Macrophage Colony-Stimulating Factor Is Dispensable for the Progression of 4T1 Murine Breast Cancer.}, journal = {International journal of molecular sciences}, volume = {20}, number = {24}, pages = {}, pmid = {31888216}, issn = {1422-0067}, support = {None//Extramural Collaborative Research Grant of Cancer Research Institute, Kanazawa University/ ; JP18K08571//Japan Society for the Promotion of Science/ ; }, mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation ; Clone Cells ; *Disease Progression ; Female ; Gene Expression Regulation, Neoplastic ; Granulocyte-Macrophage Colony-Stimulating Factor/genetics/*metabolism ; Granulocytes/metabolism ; Macrophages/metabolism ; Mammary Neoplasms, Animal/genetics/*metabolism/*pathology ; Mice, Inbred BALB C ; Neoplasm Proteins/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Splenomegaly/pathology ; }, abstract = {We previously reported that 4T1 murine breast cancer cells produce GM-CSF that up-regulates macrophage expression of several cancer promoting genes, including Mcp-1/Ccl2, Ccl17 and Rankl, suggesting a critical role of cancer cell-derived GM-CSF in cancer progression. Here, we attempted to define whether 4T1 cell-derived GM-CSF contributes to the expression of these genes by 4T1tumors, and their subsequent progression. Intraperitoneal injection of anti-GM-CSF neutralizing antibody did not decrease the expression of Mcp-1, Ccl17 or Rankl mRNA by 4T1 tumors. To further examine the role of cancer cell-derived GM-CSF, we generated GM-CSF-deficient 4T1 cells by using the Crisper-Cas9 system. As previously demonstrated, 4T1 cells are a mixture of cells and cloning of cells by itself significantly reduced tumor growth and lung metastasis. By contrast, GM-CSF-deficiency did not affect tumor growth, lung metastasis or the expression of these chemokine and cytokine genes in tumor tissues. By in-situ hybridization, the expression of Mcp-1 mRNA was detected in both F4/80-expressing and non-expressing cells in tumors of GM-CSF-deficient cells. These results indicate that cancer cell-derived GM-CSF is dispensable for the tuning of the 4T1 tumor microenvironment and the production of MCP-1, CCL17 or RANKL in the 4T1 tumor microenvironment is likely regulated by redundant mechanisms.}, }
@article {pmid31871154, year = {2020}, author = {Ng, SR and Rideout, WM and Akama-Garren, EH and Bhutkar, A and Mercer, KL and Schenkel, JM and Bronson, RT and Jacks, T}, title = {CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {1}, pages = {513-521}, pmid = {31871154}, issn = {1091-6490}, support = {/HHMI/Howard Hughes Medical Institute/United States ; P30 CA014051/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Proliferation/genetics ; Disease Models, Animal ; Disease Progression ; Feasibility Studies ; Gene Editing/*methods ; *Gene Expression Regulation, Neoplastic ; *Genes, Tumor Suppressor ; Humans ; Loss of Function Mutation ; Lung/pathology ; Lung Neoplasms/*genetics/pathology ; Mice ; Mice, Transgenic ; Neoplasm Staging ; Retinoblastoma-Like Protein p107/genetics ; Retinoblastoma-Like Protein p130/genetics ; Small Cell Lung Carcinoma/*genetics/pathology ; Tumor Burden/genetics ; Tumor Suppressor Protein p53/genetics ; }, abstract = {Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer that remains among the most lethal of solid tumor malignancies. Recent genomic sequencing studies have identified many recurrently mutated genes in human SCLC tumors. However, the functional roles of most of these genes remain to be validated. Here, we have adapted the CRISPR-Cas9 system to a well-established murine model of SCLC to rapidly model loss-of-function mutations in candidate genes identified from SCLC sequencing studies. We show that loss of the gene p107 significantly accelerates tumor progression. Notably, compared with loss of the closely related gene p130, loss of p107 results in fewer but larger tumors as well as earlier metastatic spread. In addition, we observe differences in proliferation and apoptosis as well as altered distribution of initiated tumors in the lung, resulting from loss of p107 or p130 Collectively, these data demonstrate the feasibility of using the CRISPR-Cas9 system to model loss of candidate tumor suppressor genes in SCLC, and we anticipate that this approach will facilitate efforts to investigate mechanisms driving tumor progression in this deadly disease.}, }
@article {pmid31775372, year = {2019}, author = {Nakamura, S and Watanabe, S and Ando, N and Ishihara, M and Sato, M}, title = {Transplacental Gene Delivery (TPGD) as a Noninvasive Tool for Fetal Gene Manipulation in Mice.}, journal = {International journal of molecular sciences}, volume = {20}, number = {23}, pages = {}, pmid = {31775372}, issn = {1422-0067}, mesh = {Animals ; *CRISPR-Cas Systems ; DNA/*administration &amp; dosage ; Female ; Fetus/*metabolism ; *Gene Editing ; *Gene Targeting ; Genetic Engineering ; *Genetic Therapy ; Mice ; Placenta/*metabolism ; Pregnancy ; }, abstract = {Transplacental gene delivery (TPGD) is a technique for delivering nucleic acids to fetal tissues via tail-vein injections in pregnant mice. After transplacental transport, administered nucleic acids enter fetal circulation and are distributed among fetal tissues. TPGD was established in 1995 by Tsukamoto et al., and its mechanisms, and potential applications have been further characterized since. Recently, discoveries of sequence specific nucleases, such as zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9), have revolutionized genome editing. In 2019, we demonstrated that intravenous injection of plasmid DNA containing CRISPR/Cas9 produced indels in fetal myocardial cells, which are comparatively amenable to transfection with exogenous DNA. In the future, this unique technique will allow manipulation of fetal cell functions in basic studies of fetal gene therapy. In this review, we describe developments of TPGD and discuss their applications to the manipulation of fetal cells.}, }
@article {pmid31763447, year = {2019}, author = {Zhang, Q and Wen, F and Zhang, S and Jin, J and Bi, L and Lu, Y and Li, M and Xi, XG and Huang, X and Shen, B and Sun, B}, title = {The post-PAM interaction of RNA-guided spCas9 with DNA dictates its target binding and dissociation.}, journal = {Science advances}, volume = {5}, number = {11}, pages = {eaaw9807}, pmid = {31763447}, issn = {2375-2548}, mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; *CRISPR-Cas Systems ; DNA/chemistry/genetics/*metabolism ; Gene Editing/methods ; Models, Genetic ; Nucleic Acid Conformation ; Nucleotide Motifs/genetics ; Protein Binding ; RNA, Guide/chemistry/genetics/*metabolism ; Sequence Analysis, DNA/methods ; }, abstract = {Cas9 is an RNA-guided endonuclease that targets complementary DNA for cleavage and has been repurposed for many biological usages. Cas9 activities are governed by its direct interactions with DNA. However, information about this interplay and the mechanism involved in its direction of Cas9 activity remain obscure. Using a single-molecule approach, we probed Cas9/sgRNA/DNA interactions along the DNA sequence and found two stable interactions flanking the protospacer adjacent motif (PAM). Unexpectedly, one of them is located approximately 14 base pairs downstream of the PAM (post-PAM interaction), which is beyond the apparent footprint of Cas9 on DNA. Loss or occupation of this interaction site on DNA impairs Cas9 binding and cleavage. Consistently, a downstream helicase could readily displace DNA-bound Cas9 by disrupting this relatively weak post-PAM interaction. Our work identifies a critical interaction of Cas9 with DNA that dictates its binding and dissociation, which may suggest distinct strategies to modulate Cas9 activity.}, }
@article {pmid31757214, year = {2019}, author = {Hong, W and Guo, F and Yang, M and Xu, D and Zhuang, Z and Niu, B and Bai, Q and Li, X}, title = {Hydroxysteroid sulfotransferase 2B1 affects gastric epithelial function and carcinogenesis induced by a carcinogenic agent.}, journal = {Lipids in health and disease}, volume = {18}, number = {1}, pages = {203}, pmid = {31757214}, issn = {1476-511X}, mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Carcinogenesis/drug effects/*genetics/metabolism/pathology ; Cell Line, Tumor ; Cell Proliferation ; Cholesterol/analogs &amp; derivatives/metabolism ; Gastric Mucosa/drug effects/enzymology/pathology ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; Humans ; Hydroxycholesterols/metabolism ; Methylcholanthrene/administration &amp; dosage ; Mice ; Mice, Knockout ; Phosphatidylinositol 3-Kinases/*genetics/metabolism ; Proto-Oncogene Proteins c-akt/*genetics/metabolism ; RNA, Small Interfering/genetics/metabolism ; Signal Transduction ; Stomach Neoplasms/chemically induced/enzymology/*genetics/mortality ; Sulfotransferases/antagonists &amp; inhibitors/deficiency/*genetics ; Survival Analysis ; }, abstract = {BACKGROUND: A healthy gastric mucosal epithelium exhibits tumor-suppressive properties. Gastric epithelial cell dysfunction contributes to gastric cancer development. Oxysterols provided from food or cholesterol oxidation in the gastric epithelium may be further sulfated by hydroxysteroid sulfotransferase 2B1 (SULT2B1), which is highly abundant in the gastric epithelium. However, the effects of SULT2B1 on gastric epithelial function and gastric carcinogenesis are unclear.<br><br>METHODS: A mouse gastric tumor model was established using carcinogenic agent 3-methylcholanthrene (3-MCA). A SULT2B1 deletion (SULT2B1-/-) human gastric epithelial line GES-1 was constructed by CRISPR/CAS9 genome editing system.<br><br>RESULTS: The gastric tumor incidence was higher in the SULT2B1-/- mice than in the wild-type (WT) mice. In gastric epithelial cells, adenovirus-mediated SULT2B1b overexpression reduced the levels of oxysterols, such as 24(R/S),25-epoxycholesterol (24(R/S),25-EC) and 27-hydroxycholesterol (27HC). This condition also increased PI3K/AKT signaling to promote gastric epithelial cell proliferation, epithelization, and epithelial development. However, SULT2B1 deletion or SULT2B1 knockdown suppressed PI3K/AKT signaling, epithelial cell epithelization, and wound healing and induced gastric epithelial cell malignant transition upon 3-MCA induction.<br><br>CONCLUSIONS: The abundant SULT2B1 expression in normal gastric epithelium might maintain epithelial function via the PI3K/AKT signaling pathway and suppress gastric carcinogenesis induced by a carcinogenic agent.}, }
@article {pmid31723604, year = {2019}, author = {Kim, HK and Kim, Y and Lee, S and Min, S and Bae, JY and Choi, JW and Park, J and Jung, D and Yoon, S and Kim, HH}, title = {SpCas9 activity prediction by DeepSpCas9, a deep learning-based model with high generalization performance.}, journal = {Science advances}, volume = {5}, number = {11}, pages = {eaax9249}, pmid = {31723604}, issn = {2375-2548}, mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; *Deep Learning ; Gene Editing/methods ; Humans ; Internet ; Mutation ; RNA, Guide/genetics/*metabolism ; Reproducibility of Results ; }, abstract = {We evaluated SpCas9 activities at 12,832 target sequences using a high-throughput approach based on a human cell library containing single-guide RNA-encoding and target sequence pairs. Deep learning-based training on this large dataset of SpCas9-induced indel frequencies led to the development of a SpCas9 activity-predicting model named DeepSpCas9. When tested against independently generated datasets (our own and those published by other groups), DeepSpCas9 showed high generalization performance. DeepSpCas9 is available at http://deepcrispr.info/DeepSpCas9.}, }
@article {pmid31636200, year = {2019}, author = {Zhang, M and Hill, MC and Kadow, ZA and Suh, JH and Tucker, NR and Hall, AW and Tran, TT and Swinton, PS and Leach, JP and Margulies, KB and Ellinor, PT and Li, N and Martin, JF}, title = {Long-range Pitx2c enhancer-promoter interactions prevent predisposition to atrial fibrillation.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {45}, pages = {22692-22698}, pmid = {31636200}, issn = {1091-6490}, support = {R01 HL118761/HL/NHLBI NIH HHS/United States ; U54 HD083092/HD/NICHD NIH HHS/United States ; U54 HG006348/HG/NHGRI NIH HHS/United States ; F30 HL145908/HL/NHLBI NIH HHS/United States ; F31 HL136065/HL/NHLBI NIH HHS/United States ; K01 HL140187/HL/NHLBI NIH HHS/United States ; R01 HL127717/HL/NHLBI NIH HHS/United States ; R01 HL130804/HL/NHLBI NIH HHS/United States ; R01 DE023177/DE/NIDCR NIH HHS/United States ; }, mesh = {Animals ; Atrial Fibrillation/*genetics ; CRISPR-Cas Systems ; Chromosome Mapping ; *Enhancer Elements, Genetic ; Epigenesis, Genetic ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; Homeodomain Proteins/*genetics ; Mice ; Mice, Knockout ; *Promoter Regions, Genetic ; Transcription Factors/*genetics ; }, abstract = {Genome-wide association studies found that increased risk for atrial fibrillation (AF), the most common human heart arrhythmia, is associated with noncoding sequence variants located in proximity to PITX2 Cardiomyocyte-specific epigenomic and comparative genomics uncovered 2 AF-associated enhancers neighboring PITX2 with varying conservation in mice. Chromosome conformation capture experiments in mice revealed that the Pitx2c promoter directly contacted the AF-associated enhancer regions. CRISPR/Cas9-mediated deletion of a 20-kb topologically engaged enhancer led to reduced Pitx2c transcription and AF predisposition. Allele-specific chromatin immunoprecipitation sequencing on hybrid heterozygous enhancer knockout mice revealed that long-range interaction of an AF-associated region with the Pitx2c promoter was required for maintenance of the Pitx2c promoter chromatin state. Long-range looping was mediated by CCCTC-binding factor (CTCF), since genetic disruption of the intronic CTCF-binding site caused reduced Pitx2c expression, AF predisposition, and diminished active chromatin marks on Pitx2 AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory functions directed at PITX2.}, }
@article {pmid31633062, year = {2019}, author = {Ikeda, A and Fujii, W and Sugiura, K and Naito, K}, title = {High-fidelity endonuclease variant HypaCas9 facilitates accurate allele-specific gene modification in mouse zygotes.}, journal = {Communications biology}, volume = {2}, number = {}, pages = {371}, pmid = {31633062}, issn = {2399-3642}, mesh = {Animals ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/physiology ; Female ; Gene Editing/*methods ; Male ; Mice, Inbred C57BL ; Mice, Transgenic ; Mutation ; Polymorphism, Single Nucleotide ; *Zygote/metabolism ; }, abstract = {CRISPR/Cas9 has been widely used for the efficient generation of genetically modified animals; however, this system could have unexpected off-target effects. In the present study, we confirmed the validity of a high-fidelity Cas9 variant, HypaCas9, for accurate genome editing in mouse zygotes. HypaCas9 efficiently modified the target locus while minimizing off-target effects even in a single-nucleotide mismatched sequence. Furthermore, by applying HypaCas9 to the discrimination of SNP in hybrid strain-derived zygotes, we accomplished allele-specific gene modifications and successfully generated mice with a monoallelic mutation in an essential gene. These results suggest that the improved accuracy of HypaCas9 facilitates the generation of genetically modified animals.}, }
@article {pmid31493565, year = {2019}, author = {Xu, Z and Xu, X and Gong, Q and Li, Z and Li, Y and Wang, S and Yang, Y and Ma, W and Liu, L and Zhu, B and Zou, L and Chen, G}, title = {Engineering Broad-Spectrum Bacterial Blight Resistance by Simultaneously Disrupting Variable TALE-Binding Elements of Multiple Susceptibility Genes in Rice.}, journal = {Molecular plant}, volume = {12}, number = {11}, pages = {1434-1446}, doi = {10.1016/j.molp.2019.08.006}, pmid = {31493565}, issn = {1752-9867}, mesh = {Base Sequence ; CRISPR-Cas Systems/genetics ; Disease Resistance/*genetics ; Gene Editing/*methods ; Genetic Predisposition to Disease ; Mutation ; Oryza/*genetics/immunology/*microbiology ; Plant Diseases/immunology/*microbiology ; Transcription Activator-Like Effectors/*metabolism ; }, abstract = {Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight of rice, employs the transcription activator-like effectors (TALEs) to induce the expression of the OsSWEET family of putative sugar transporter genes, which function in conferring disease susceptibility (S) in rice plants. To engineer broad-spectrum bacterial blight resistance, we used CRISPR/Cas9-mediated gene editing to disrupt the TALE-binding elements (EBEs) of two S genes, OsSWEET11 and OsSWEET14, in rice cv. Kitaake, which harbors the recessive resistance allele of Xa25/OsSWEET13. The engineered rice line MS14K exhibited broad-spectrum resistance to most Xoo strains with a few exceptions, suggesting that the compatible strains may contain new TALEs. We identified two PthXo2-like TALEs, Tal5LN18 and Tal7PXO61, as major virulence factors in the compatible Xoo strains LN18 and PXO61, respectively, and found that Xoo encodes at least five types of PthXo2-like effectors. Given that PthXo2/PthXo2.1 target OsSWEET13 for transcriptional activation, the genomes of 3000 rice varieties were analyzed for EBE variationsin the OsSWEET13 promoter, and 10 Xa25-like haplotypes were identified. We found that Tal5LN18 and Tal7PXO61 bind slightly different EBE sequences in the OsSWEET13 promoter to activate its expression. CRISPR/Cas9 technology was then used to generate InDels in the EBE of the OsSWEET13 promoter in MS14K to creat a new germplasm with three edited OsSWEET EBEs and broad-spectrum resistance against all Xoo strains tested. Collectively, our findings illustrate how to disarm TALE-S co-evolved loci to generate broad-spectrum resistance through the loss of effector-triggered susceptibility in plants.}, }
@article {pmid31181342, year = {2019}, author = {Deyell, M and Ameta, S and Nghe, P}, title = {Large scale control and programming of gene expression using CRISPR.}, journal = {Seminars in cell &amp; developmental biology}, volume = {96}, number = {}, pages = {124-132}, doi = {10.1016/j.semcdb.2019.05.013}, pmid = {31181342}, issn = {1096-3634}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Expression Regulation/*genetics ; Gene Regulatory Networks/genetics ; Humans ; }, abstract = {The control of gene expression in cells and organisms allows to unveil gene to function relationships and to reprogram biological responses. Several systems, such as Zinc fingers, TALE (Transcription activator-like effectors), and siRNAs (small-interfering RNAs), have been exploited to achieve this. However, recent advances in Clustered Regularly Interspaced Short Palindromic Repeats and Cas9 (CRISPR-Cas9) have overshadowed them due to high specificity, compatibility with many different organisms, and design flexibility. In this review we summarize state-of-the art for CRISPR-Cas9 technology for large scale gene perturbation studies, including single gene and multiple genes knock-out, knock-down, knock-up libraries, and their associated screening assays. We feature in particular the combination of these methods with single-cell transcriptomics approaches. Finally, we highlight the application of CRISPR-Cas9 systems in building synthetic circuits that can be interfaced with gene networks to control cellular states.}, }
@article {pmid31150758, year = {2019}, author = {Mirza, Z and Karim, S}, title = {Advancements in CRISPR/Cas9 technology-Focusing on cancer therapeutics and beyond.}, journal = {Seminars in cell &amp; developmental biology}, volume = {96}, number = {}, pages = {13-21}, doi = {10.1016/j.semcdb.2019.05.026}, pmid = {31150758}, issn = {1096-3634}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Humans ; Neoplasms/genetics/*therapy ; }, abstract = {&quot;CRISPR&quot; is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats, which are a characteristic of the bacterial defense system and Cas9 (or &quot;CRISPR-associated&quot;) is a RNA-guided DNA endonuclease or molecular scissor, capable of cutting DNA strands. Both together forms the basis for CRISPR-Cas9 targeted genome editing technology and enables highly specific genomic modifications to an organism's DNA. Recent advent of high-throughput genomics has revolutionizing personalized medicine and enhanced our molecular understanding of human cancers. The development of the CRISPR/Cas9 tool has unveiled advancement of new, simplistic and efficient in vivo model systems in oncology. The usage of CRISPR/Cas9 gene editing systems for curing various cancers promises to be the next great biotechnological breakthrough in medicine. However, urgent attention is needed to assess the functional relevance of novel cancer-associated mutations and translate our molecular knowledge to therapeutics. Herein, we will review the development and applications of the exciting uses of the CRISPR/Cas9 technique for cancer research and therapy with focus on origin, progress, clinical trials, implications, and challenges ahead. Major ethical and safety concerns are perhaps unknown long term consequences of DNA manipulation and irreversibility of this procedure.}, }
@article {pmid31131955, year = {2019}, author = {Tang, J and Lee, T and Sun, T}, title = {Single-nucleotide editing: From principle, optimization to application.}, journal = {Human mutation}, volume = {40}, number = {12}, pages = {2171-2183}, pmid = {31131955}, issn = {1098-1004}, support = {R01 MH083680/MH/NIMH NIH HHS/United States ; R01-MH083680//NIH/NIMH/International ; 81471152 and 31771141//National Natural Science Foundation of China/International ; }, mesh = {Aminohydrolases/*metabolism ; Animals ; CRISPR-Cas Systems ; Cytosine Deaminase/*metabolism ; Gene Editing/*methods ; Gene Silencing ; Humans ; Mice ; Polymorphism, Single Nucleotide ; }, abstract = {Cytosine base editors (CBEs) and adenine base editors (ABEs), which are generally composed of an engineered deaminase and a catalytically impaired CRISPR-Cas9 variant, are new favorite tools for single base substitution in cells and organisms. In this review, we summarize the principle of base-editing systems and elaborate on the evolution of different platforms of CBEs and ABEs, including their deaminase, Cas9 variants, and editing outcomes. Moreover, we highlight their applications in mouse and human cells and discuss the challenges and prospects of base editors. The ABE- and CBE systems have been used in gene silencing, pathogenic gene correction, and functional genetic screening. Single base editing is becoming a new promising genetic tool in biomedical research and gene therapy.}, }
@article {pmid31113472, year = {2019}, author = {Bharathy, N and Berlow, NE and Wang, E and Abraham, J and Settelmeyer, TP and Hooper, JE and Svalina, MN and Bajwa, Z and Goros, MW and Hernandez, BS and Wolff, JE and Pal, R and Davies, AM and Ashok, A and Bushby, D and Mancini, M and Noakes, C and Goodwin, NC and Ordentlich, P and Keck, J and Hawkins, DS and Rudzinski, ER and Mansoor, A and Perkins, TJ and Vakoc, CR and Michalek, JE and Keller, C}, title = {Preclinical rationale for entinostat in embryonal rhabdomyosarcoma.}, journal = {Skeletal muscle}, volume = {9}, number = {1}, pages = {12}, pmid = {31113472}, issn = {2044-5040}, support = {R01 CA143082/CA/NCI NIH HHS/United States ; R01 CA189299/CA/NCI NIH HHS/United States ; }, mesh = {Adolescent ; Animals ; Antineoplastic Agents, Phytogenic/administration &amp; dosage ; Antineoplastic Combined Chemotherapy Protocols/administration &amp; dosage ; Benzamides/administration &amp; dosage/*therapeutic use ; CRISPR-Cas Systems ; Cell Differentiation/drug effects ; Cell Line, Tumor ; Cellular Reprogramming/drug effects/genetics ; Child ; Child, Preschool ; Drug Screening Assays, Antitumor ; Female ; Histone Deacetylase 1/antagonists &amp; inhibitors/genetics ; Histone Deacetylase Inhibitors/administration &amp; dosage/*therapeutic use ; Humans ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Pyridines/administration &amp; dosage/*therapeutic use ; RNA-Seq ; Rhabdomyosarcoma, Alveolar/drug therapy/enzymology/pathology ; Rhabdomyosarcoma, Embryonal/*drug therapy/enzymology/pathology ; Tumor Burden/drug effects ; Tumor Microenvironment/drug effects/genetics ; Vincristine/administration &amp; dosage ; Xenograft Model Antitumor Assays ; }, abstract = {BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in the pediatric cancer population. Survival among metastatic RMS patients has remained dismal yet unimproved for years. We previously identified the class I-specific histone deacetylase inhibitor, entinostat (ENT), as a pharmacological agent that transcriptionally suppresses the PAX3:FOXO1 tumor-initiating fusion gene found in alveolar rhabdomyosarcoma (aRMS), and we further investigated the mechanism by which ENT suppresses PAX3:FOXO1 oncogene and demonstrated the preclinical efficacy of ENT in RMS orthotopic allograft and patient-derived xenograft (PDX) models. In this study, we investigated whether ENT also has antitumor activity in fusion-negative eRMS orthotopic allografts and PDX models either as a single agent or in combination with vincristine (VCR).<br><br>METHODS: We tested the efficacy of ENT and VCR as single agents and in combination in orthotopic allograft and PDX mouse models of eRMS. We then performed CRISPR screening to identify which HDAC among the class I HDACs is responsible for tumor growth inhibition in eRMS. To analyze whether ENT treatment as a single agent or in combination with VCR induces myogenic differentiation, we performed hematoxylin and eosin (H&amp;E) staining in tumors.<br><br>RESULTS: ENT in combination with the chemotherapy VCR has synergistic antitumor activity in a subset of fusion-negative eRMS in orthotopic &quot;allografts,&quot; although PDX mouse models were too hypersensitive to the VCR dose used to detect synergy. Mechanistic studies involving CRISPR suggest that HDAC3 inhibition is the primary mechanism of cell-autonomous cytoreduction in eRMS. Following cytoreduction in vivo, residual tumor cells in the allograft models treated with chemotherapy undergo a dramatic, entinostat-induced (70-100%) conversion to non-proliferative rhabdomyoblasts.<br><br>CONCLUSION: Our results suggest that the targeting class I HDACs may provide a therapeutic benefit for selected patients with eRMS. ENT's preclinical in vivo efficacy makes ENT a rational drug candidate in a phase II clinical trial for eRMS.}, }
@article {pmid31112800, year = {2019}, author = {Singh, DD and Hawkins, RD and Lahesmaa, R and Tripathi, SK}, title = {CRISPR/Cas9 guided genome and epigenome engineering and its therapeutic applications in immune mediated diseases.}, journal = {Seminars in cell &amp; developmental biology}, volume = {96}, number = {}, pages = {32-43}, doi = {10.1016/j.semcdb.2019.05.007}, pmid = {31112800}, issn = {1096-3634}, support = {R01 AR065952/AR/NIAMS NIH HHS/United States ; R01 DK103667/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Epigenesis, Genetic/*genetics ; *Genetic Engineering ; Humans ; Immune System Diseases/*drug therapy/*genetics ; }, abstract = {Recent developments in the nucleic acid editing technologies have provided a powerful tool to precisely engineer the genome and epigenome for studying many aspects of immune cell differentiation and development as well as several immune mediated diseases (IMDs) including autoimmunity and cancer. Here, we discuss the recent technological achievements of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based RNA-guided genome and epigenome editing toolkit and provide an insight into how CRISPR/Cas9 (CRISPR Associated Protein 9) toolbox could be used to examine genetic and epigenetic mechanisms underlying IMDs. In addition, we will review the progress in CRISPR/Cas9-based genome-wide genome and epigenome screens in various cell types including immune cells. Finally, we will discuss the potential of CRISPR/Cas9 in defining the molecular function of disease associated SNPs overlapping gene regulatory elements.}, }
@article {pmid31101673, year = {2019}, author = {Moffett, HF and Harms, CK and Fitzpatrick, KS and Tooley, MR and Boonyaratanakornkit, J and Taylor, JJ}, title = {B cells engineered to express pathogen-specific antibodies protect against infection.}, journal = {Science immunology}, volume = {4}, number = {35}, pages = {}, pmid = {31101673}, issn = {2470-9468}, support = {T32 AI118690/AI/NIAID NIH HHS/United States ; T32 GM095421/GM/NIGMS NIH HHS/United States ; }, mesh = {3T3 Cells ; Adoptive Transfer/methods ; Animals ; Antibodies, Monoclonal/*immunology ; Antibodies, Neutralizing/*immunology ; Antibodies, Viral/*immunology ; B-Lymphocytes/*immunology ; CRISPR-Cas Systems ; Female ; HEK293 Cells ; Homeodomain Proteins/genetics ; Humans ; Male ; Metabolic Engineering/*methods ; Mice ; Mice, Inbred BALB C ; Mice, Knockout ; Respiratory Syncytial Virus Infections/*therapy/virology ; Respiratory Syncytial Virus, Human/*immunology ; }, abstract = {Effective vaccines inducing lifelong protection against many important infections such as respiratory syncytial virus (RSV), HIV, influenza virus, and Epstein-Barr virus (EBV) are not yet available despite decades of research. As an alternative to a protective vaccine, we developed a genetic engineering strategy in which CRISPR-Cas9 was used to replace endogenously encoded antibodies with antibodies targeting RSV, HIV, influenza virus, or EBV in primary human B cells. The engineered antibodies were expressed efficiently in primary B cells under the control of endogenous regulatory elements, which maintained normal antibody expression and secretion. Using engineered mouse B cells, we demonstrated that a single transfer of B cells engineered to express an antibody against RSV resulted in potent and durable protection against RSV infection in RAG1-deficient mice. This approach offers the opportunity to achieve sterilizing immunity against pathogens for which traditional vaccination has failed to induce or maintain protective antibody responses.}, }
@article {pmid31054324, year = {2019}, author = {Kaushik, I and Ramachandran, S and Srivastava, SK}, title = {CRISPR-Cas9: A multifaceted therapeutic strategy for cancer treatment.}, journal = {Seminars in cell &amp; developmental biology}, volume = {96}, number = {}, pages = {4-12}, pmid = {31054324}, issn = {1096-3634}, support = {R01 CA129038/CA/NCI NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/*genetics ; Humans ; *Immunotherapy ; Neoplasms/*genetics/immunology/*therapy ; }, abstract = {CRISPR-Cas9 is an RNA guided endonuclease that has revolutionized the ability to edit genome and introduce desired manipulations in the target genomic sequence. It is a flexible methodology and is capable of targeting multiple loci simultaneously. Owing to the fact that cancer is an amalgamation of several genetic mutations, application of CRISPR-Cas9 technology is considered as a novel strategy to combat cancer. Genetic and epigenetic modulations in cancer leads to development of resistance to conventional therapy options. Given the abundance of transcriptomic and genomic alterations in cancer, developing a strategy to decipher these alterations is critical. CRISPR-Cas9 system has proven to be a promising tool in generating cellular and animal models to mimic the mutations and understand their role in tumorigenesis. CRISPR-Cas9 is an upheaval in the field of cancer immunotherapy. Furthermore, CRISPR-Cas9 plays an important role in the development of whole genome libraries for cancer patients. This approach will help understand the diversity in genome variation among the patients and also, will provide multiple variables to scientists to investigate and improvise cancer therapy. This review will focus on the discovery of CRISPR-Cas9 system, mechanisms behind CRISPR technique and its current status as a potential tool for investigating the genomic mutations associated with all cancer types.}, }
@article {pmid30996081, year = {2019}, author = {Alapati, D and Zacharias, WJ and Hartman, HA and Rossidis, AC and Stratigis, JD and Ahn, NJ and Coons, B and Zhou, S and Li, H and Singh, K and Katzen, J and Tomer, Y and Chadwick, AC and Musunuru, K and Beers, MF and Morrisey, EE and Peranteau, WH}, title = {In utero gene editing for monogenic lung disease.}, journal = {Science translational medicine}, volume = {11}, number = {488}, pages = {}, pmid = {30996081}, issn = {1946-6242}, support = {R01 HL119436/HL/NHLBI NIH HHS/United States ; UL1 TR001878/TR/NCATS NIH HHS/United States ; T32 HL007586/HL/NHLBI NIH HHS/United States ; U01 HL134745/HL/NHLBI NIH HHS/United States ; K08 HL140178/HL/NHLBI NIH HHS/United States ; I01 BX001176/BX/BLRD VA/United States ; R01 HL132999/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Epithelial Cells/metabolism ; Gene Editing/methods ; Humans ; Lung Diseases/*genetics ; Mice ; Mutation/genetics ; Pulmonary Surfactant-Associated Protein C/genetics ; }, abstract = {Monogenic lung diseases that are caused by mutations in surfactant genes of the pulmonary epithelium are marked by perinatal lethal respiratory failure or chronic diffuse parenchymal lung disease with few therapeutic options. Using a CRISPR fluorescent reporter system, we demonstrate that precisely timed in utero intra-amniotic delivery of CRISPR-Cas9 gene editing reagents during fetal development results in targeted and specific gene editing in fetal lungs. Pulmonary epithelial cells are predominantly targeted in this approach, with alveolar type 1, alveolar type 2, and airway secretory cells exhibiting high and persistent gene editing. We then used this in utero technique to evaluate a therapeutic approach to reduce the severity of the lethal interstitial lung disease observed in a mouse model of the human SFTPCI73T mutation. Embryonic expression of SftpcI73T alleles is characterized by severe diffuse parenchymal lung damage and rapid demise of mutant mice at birth. After in utero CRISPR-Cas9-mediated inactivation of the mutant SftpcI73T gene, fetuses and postnatal mice showed improved lung morphology and increased survival. These proof-of-concept studies demonstrate that in utero gene editing is a promising approach for treatment and rescue of monogenic lung diseases that are lethal at birth.}, }
@article {pmid30986568, year = {2019}, author = {Verma, R and Sahu, R and Singh, DD and Egbo, TE}, title = {A CRISPR/Cas9 based polymeric nanoparticles to treat/inhibit microbial infections.}, journal = {Seminars in cell &amp; developmental biology}, volume = {96}, number = {}, pages = {44-52}, doi = {10.1016/j.semcdb.2019.04.007}, pmid = {30986568}, issn = {1096-3634}, mesh = {Bacterial Infections/*genetics/*therapy ; CRISPR-Cas Systems/*genetics ; *Drug Delivery Systems ; Gene Editing/*methods ; Humans ; Nanomedicine ; Nanoparticles/*administration &amp; dosage ; Polymers/*administration &amp; dosage ; }, abstract = {The latest breakthrough towards the adequate and decisive methods of gene editing tools provided by CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR Associated System), has been repurposed into a tool for genetically engineering eukaryotic cells and now considered as the major innovation in gene-related disorders. Nanotechnology has provided an alternate way to overcome the conventional problems where methods to deliver therapeutic agents have failed. The use of nanotechnology has the potential to safe-side the CRISPR/Cas9 components delivery by using customized polymeric nanoparticles for safety and efficacy. The pairing of two (CRISPR/Cas9 and nanotechnology) has the potential for opening new avenues in therapeutic use. In this review, we will discuss the most recent advances in developing nanoparticle-based CRISPR/Cas9 gene editing cargo delivery with a focus on several polymeric nanoparticles including fabrication proposals to combat microbial infections.}, }
@article {pmid30951893, year = {2019}, author = {Jyoti, A and Kaushik, S and Srivastava, VK and Datta, M and Kumar, S and Yugandhar, P and Kothari, SL and Rai, V and Jain, A}, title = {The potential application of genome editing by using CRISPR/Cas9, and its engineered and ortholog variants for studying the transcription factors involved in the maintenance of phosphate homeostasis in model plants.}, journal = {Seminars in cell &amp; developmental biology}, volume = {96}, number = {}, pages = {77-90}, doi = {10.1016/j.semcdb.2019.03.010}, pmid = {30951893}, issn = {1096-3634}, mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; Genetic Variation/genetics ; Homeostasis/*genetics ; *Models, Biological ; Phosphates/*metabolism ; Plants/*genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; Transcription Factors/*genetics/*metabolism ; }, abstract = {Phosphorus (P), an essential macronutrient, is pivotal for growth and development of plants. Availability of phosphate (Pi), the only assimilable P, is often suboptimal in rhizospheres. Pi deficiency triggers an array of spatiotemporal adaptive responses including the differential regulation of several transcription factors (TFs). Studies on MYB TF PHR1 in Arabidopsis thaliana (Arabidopsis) and its orthologs OsPHRs in Oryza sativa (rice) have provided empirical evidence of their significant roles in the maintenance of Pi homeostasis. Since the functional characterization of PHR1 in 2001, several other TFs have now been identified in these model plants. This raised a pertinent question whether there are any likely interactions across these TFs. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has provided an attractive paradigm for editing genome in plants. Here, we review the applications and challenges of this technique for genome editing of the TFs for deciphering the function and plausible interactions across them. This technology could thus provide a much-needed fillip towards engineering TFs for generating Pi use efficient plants for sustainable agriculture. Furthermore, we contemplate whether this technology could be a viable alternative to the controversial genetically modified (GM) rice or it may also eventually embroil into a limbo.}, }
@article {pmid30949579, year = {2019}, author = {Pan, Y and Yang, J and Luan, X and Liu, X and Li, X and Yang, J and Huang, T and Sun, L and Wang, Y and Lin, Y and Song, Y}, title = {Near-infrared upconversion-activated CRISPR-Cas9 system: A remote-controlled gene editing platform.}, journal = {Science advances}, volume = {5}, number = {4}, pages = {eaav7199}, pmid = {30949579}, issn = {2375-2548}, mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems/*genetics/radiation effects ; Carcinoma, Squamous Cell/genetics/pathology/*therapy ; Cell Proliferation ; Drug Carriers/*chemistry ; Female ; Gene Editing/*methods ; Gene Transfer Techniques ; Humans ; *Infrared Rays ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Mouth Neoplasms/genetics/pathology/*therapy ; Nanoparticles/*administration &amp; dosage/chemistry ; RNA, Guide/genetics/radiation effects ; Tumor Cells, Cultured ; Xenograft Model Antitumor Assays ; }, abstract = {As an RNA-guided nuclease, CRISPR-Cas9 offers facile and promising solutions to mediate genome modification with respect to versatility and high precision. However, spatiotemporal manipulation of CRISPR-Cas9 delivery remains a daunting challenge for robust effectuation of gene editing both in vitro and in vivo. Here, we designed a near-infrared (NIR) light-responsive nanocarrier of CRISPR-Cas9 for cancer therapeutics based on upconversion nanoparticles (UCNPs). The UCNPs served as &quot;nanotransducers&quot; that can convert NIR light (980 nm) into local ultraviolet light for the cleavage of photosensitive molecules, thereby resulting in on-demand release of CRISPR-Cas9. In addition, by preparing a single guide RNA targeting a tumor gene (polo-like kinase-1), our strategies have successfully inhibited the proliferation of tumor cell via NIR light-activated gene editing both in vitro and in vivo. Overall, this exogenously controlled method presents enormous potential for targeted gene editing in deep tissues and treatment of a myriad of diseases.}, }
@article {pmid30786106, year = {2019}, author = {Zarei, A and Razban, V and Hosseini, SE and Tabei, SMB}, title = {Creating cell and animal models of human disease by genome editing using CRISPR/Cas9.}, journal = {The journal of gene medicine}, volume = {21}, number = {4}, pages = {e3082}, doi = {10.1002/jgm.3082}, pmid = {30786106}, issn = {1521-2254}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Genetic Therapy/*methods ; Humans ; Models, Animal ; }, abstract = {A set of unique sequences in bacterial genomes, responsible for protecting bacteria against bacteriophages, has recently been used for the genetic manipulation of specific points in the genome. These systems consist of one RNA component and one enzyme component, known as CRISPR (&quot;clustered regularly interspaced short palindromic repeats&quot;) and Cas9, respectively. The present review focuses on the applications of CRISPR/Cas9 technology in the development of cellular and animal models of human disease. Making a desired genetic alteration depends on the design of RNA molecules that guide endonucleases to a favorable genomic location. With the discovery of CRISPR/Cas9 technology, researchers are able to achieve higher levels of accuracy because of its advantages over alternative methods for editing genome, including a simple design, a high targeting efficiency and the ability to create simultaneous alterations in multiple sequences. These factors allow the researchers to apply this technology to creating cellular and animal models of human diseases by knock-in, knock-out and Indel mutation strategies, such as for Huntington's disease, cardiovascular disorders and cancers. Optimized CRISPR/Cas9 technology will facilitate access to valuable novel cellular and animal genetic models with respect to the development of innovative drug discovery and gene therapy.}, }
@article {pmid30771271, year = {2019}, author = {Wang, H and Wang, X and Li, X and Wang, Q and Qing, S and Zhang, Y and Gao, MQ}, title = {A novel long non-coding RNA regulates the immune response in MAC-T cells and contributes to bovine mastitis.}, journal = {The FEBS journal}, volume = {286}, number = {9}, pages = {1780-1795}, doi = {10.1111/febs.14783}, pmid = {30771271}, issn = {1742-4658}, mesh = {Animals ; CRISPR-Cas Systems ; Caseins/metabolism ; Cattle/*genetics/immunology ; Cell Line, Transformed ; Epithelial Cells/*immunology/metabolism ; Escherichia coli/physiology ; Female ; Gene Expression Regulation ; Gene Knockout Techniques ; High-Throughput Nucleotide Sequencing ; Inflammation ; Mammary Glands, Animal/cytology ; Mastitis, Bovine/*genetics ; Organ Specificity ; Quantitative Trait Loci ; RNA, Guide/genetics ; RNA, Long Noncoding/genetics/isolation &amp; purification/*physiology ; Staphylococcus aureus/physiology ; Transforming Growth Factor beta1/metabolism ; Tubulin/genetics ; }, abstract = {The long non-coding RNAs (lncRNAs) are known to transcriptionally regulate a wide spectrum of diseases. Here, we screened for potentially functional lncRNAs in a mammary epithelial cell model of bovine mastitis by RNA-Seq technology and identified a class of previously undetected mastitis-related lncRNAs. A novel lncRNA was widely expressed in a variety of bovine tissues with diverse relative abundance and had a relatively low expression in mammary tissue. Given its predicted target gene is TUBA1C, we name it lncRNA-TUB. We found a higher expression of lncRNA-TUB in mammary epithelial cells that received a proinflammatory stimulus compared to normal cells. Knockout of lncRNA-TUB by the CRISPR/Cas9 system revealed that it plays crucial roles in the morphological shape, proliferation, migration and β-casein secretion of mammary epithelial cells. In addition, lncRNA-TUB mediates Escherichia coli-induced inflammatory factor secretion and Staphylococcus aureus adhesion to epithelial cells. Our results suggest that the lncRNAs identified here function in bovine mastitis, and that lncRNA-TUB affects the basic biological characteristics and functions of bovine mammary epithelial cells in inflammatory conditions, providing valuable insights into the mechanisms of bovine mastitis.}, }
@article {pmid30702425, year = {2019}, author = {McVeigh, P and Maule, AG}, title = {Can CRISPR help in the fight against parasitic worms?.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {30702425}, issn = {2050-084X}, support = {NC/N001486/1//National Centre for the Replacement, Refinement and Reduction of Animals in Research/United Kingdom ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing ; Helminths/*genetics ; }, abstract = {The first reports of CRISPR/Cas9 genome editing in flatworms could usher in a new era of research on these dangerous human parasites.}, }
@article {pmid30682322, year = {2019}, author = {Starkuviene, V and Kallenberger, SM and Beil, N and Lisauskas, T and Schumacher, BS and Bulkescher, R and Wajda, P and Gunkel, M and Beneke, J and Erfle, H}, title = {High-Density Cell Arrays for Genome-Scale Phenotypic Screening.}, journal = {SLAS discovery : advancing life sciences R &amp; D}, volume = {24}, number = {3}, pages = {274-283}, doi = {10.1177/2472555218818757}, pmid = {30682322}, issn = {2472-5560}, mesh = {CRISPR-Cas Systems ; Cell Line ; Cytological Techniques/*methods ; Endocytosis ; Epidermal Growth Factor/metabolism ; *Genome, Human ; Humans ; Miniaturization ; Phenotype ; RNA Interference ; Robotics ; }, abstract = {Due to high associated costs and considerable time investments of cell-based screening, there is a strong demand for new technologies that enable preclinical development and tests of diverse biologicals in a cost-saving and time-efficient manner. For those reasons we developed the high-density cell array (HD-CA) platform, which miniaturizes cell-based screening in the form of preprinted and ready-to-run screening arrays. With the HD-CA technology, up to 24,576 samples can be tested in a single experiment, thereby saving costs and time for microscopy-based screening by 75%. Experiments on the scale of the entire human genome can be addressed in a real parallel manner, with screening campaigns becoming more comfortable and devoid of robotics infrastructure on the user side. The high degree of miniaturization enables working with expensive reagents and rare and difficult-to-obtain cell lines. We have also optimized an automated imaging procedure for HD-CA and demonstrate the applicability of HD-CA to CRISPR-Cas9- and RNAi-mediated phenotypic assessment of the gene function.}, }
@article {pmid30672298, year = {2019}, author = {Qiang, J and Ma, Z and Xie, X and Shi, L and Geng, Y and Hu, J and Liu, R and Liu, N and Zhang, Y}, title = {Multi-omic Analyses Reveal Minimal Impact of the CRISPR-Cas9 Nuclease on Cultured Human Cells.}, journal = {Journal of proteome research}, volume = {18}, number = {3}, pages = {1054-1063}, doi = {10.1021/acs.jproteome.8b00751}, pmid = {30672298}, issn = {1535-3907}, mesh = {CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Epigenesis, Genetic/genetics ; Gene Editing/methods ; Gene Expression Regulation, Enzymologic/genetics ; Histone Code/genetics ; Humans ; Protein Interaction Maps/genetics ; Proteome/*genetics ; Transcriptome/*genetics ; }, abstract = {The CRISPR-Cas9 system is a genomic editing tool widely used in basic research and under investigation for potential applications in gene therapies for human diseases. To accomplish genomic editing, the system requires the expression of a prokaryotic DNA endonuclease enzyme, Cas9, in host cells. Previous studies have mainly focused on the specificity of Cas9 on the host genome, and thus it is unclear whether this bacterium-derived enzyme affects the protein homeostasis of host cells. Here we applied multi-omic analyses, including transcriptome, proteome, phosphoproteome, Cas9-associated protein interactome, protein synthesis, and histone epigenetic modification, to investigate the cellular response of human cells upon the expression of Cas9. We demonstrate that Cas9 has minimal impact on host cells. Our assessment of intracellular effects of Cas9 paves a path for its broad applications in biological studies and potential clinical translations.}, }
@article {pmid30655324, year = {2019}, author = {Pegg, HJ and Harrison, H and Rogerson, C and Shore, P}, title = {The RUNX Transcriptional Coregulator, CBFβ, Suppresses Migration of ER+ Breast Cancer Cells by Repressing ERα-Mediated Expression of the Migratory Factor TFF1.}, journal = {Molecular cancer research : MCR}, volume = {17}, number = {5}, pages = {1015-1023}, doi = {10.1158/1541-7786.MCR-18-1039}, pmid = {30655324}, issn = {1557-3125}, support = {MR/L011840/1/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Breast Neoplasms/*genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Core Binding Factor Alpha 2 Subunit/genetics/*metabolism ; Core Binding Factor beta Subunit/*genetics ; Estrogen Receptor alpha/genetics/*metabolism ; Female ; HeLa Cells ; Humans ; MCF-7 Cells ; *Mutation, Missense ; Trefoil Factor-1/genetics/*metabolism ; }, abstract = {Core binding factor β (CBFβ), the essential coregulator of RUNX transcription factors, is one of the most frequently mutated genes in estrogen receptor-positive (ER+) breast cancer. Many of these mutations are nonsense mutations and are predicted to result in loss of function, suggesting a tumor suppressor role for CBFβ. However, the impact of missense mutations and the loss of CBFβ in ER+ breast cancer cells have not been determined. Here we demonstrate that missense mutations in CBFβ accumulate near the Runt domain-binding region. These mutations inhibit the ability of CBFβ to form CBFβ-Runx-DNA complexes. We further show that deletion of CBFβ, using CRISPR-Cas9, in ER+ MCF7 cells results in an increase in cell migration. This increase in migration is dependent on the presence of ERα. Analysis of the potential mechanism revealed that the increase in migration is driven by the coregulation of Trefoil factor 1 (TFF1) by CBFβ and ERα. RUNX1-CBFβ acts to repress ERα-activated expression of TFF1. TFF1 is a motogen that stimulates migration and we show that knockdown of TFF1 in CBFβ-/- cells inhibits the migratory phenotype. Our findings reveal a new mechanism by which RUNX1-CBFβ and ERα combine to regulate gene expression and a new role for RUNX1-CBFβ in the prevention of cell migration by suppressing the expression of the motogen TFF1. IMPLICATIONS: Mutations in CBFβ contribute to the development of breast cancer by inducing a metastatic phenotype that is dependent on ER.}, }
@article {pmid30442540, year = {2019}, author = {Claes, C and Van Den Daele, J and Boon, R and Schouteden, S and Colombo, A and Monasor, LS and Fiers, M and Ordovás, L and Nami, F and Bohrmann, B and Tahirovic, S and De Strooper, B and Verfaillie, CM}, title = {Human stem cell-derived monocytes and microglia-like cells reveal impaired amyloid plaque clearance upon heterozygous or homozygous loss of TREM2.}, journal = {Alzheimer's &amp; dementia : the journal of the Alzheimer's Association}, volume = {15}, number = {3}, pages = {453-464}, doi = {10.1016/j.jalz.2018.09.006}, pmid = {30442540}, issn = {1552-5279}, mesh = {Amyloid beta-Protein Precursor/genetics/metabolism ; Animals ; Brain ; CRISPR-Cas Systems ; Cells, Cultured ; Escherichia coli ; Membrane Glycoproteins/*deficiency/genetics ; Mice, Transgenic ; Microglia/*metabolism ; Monocytes/*metabolism ; Phagocytosis ; Plaque, Amyloid/*metabolism ; Pluripotent Stem Cells ; Presenilin-1/genetics/metabolism ; Receptors, Immunologic/*deficiency/genetics ; }, abstract = {INTRODUCTION: Murine microglia expressing the Alzheimer's disease-linked TREM2R47H mutation display variable decrease in phagocytosis, while impaired phagocytosis is reported following loss of TREM2. However, no data exist on TREM2+/R47H human microglia. Therefore, we created human pluripotent stem cell (hPSC) monocytes and transdifferentiated microglia-like cells (tMGs) to examine the effect of the TREM2+/R47H mutation and loss of TREM2 on phagocytosis.<br><br>METHODS: We generated isogenic TREM2+/R47H, TREM2+/-, and TREM2-/- hPSCs using CRISPR/Cas9. Following differentiation to monocytes and tMGs, we studied the uptake of Escherichia coli fragments and analyzed amyloid plaque clearance from cryosections of APP/PS1+/- mouse brains.<br><br>RESULTS: We demonstrated that tMGs resemble cultured human microglia. TREM2+/- and TREM2-/- hPSC monocytes and tMGs phagocytosed significantly less E. coli fragments and cleared less amyloid plaques than wild-type hPSC progeny, with no difference for TREM2+/R47H progeny.<br><br>DISCUSSION: In vitro phagocytosis of hPSC monocytes and tMGs was not affected by the TREM2+/R47H mutation but was significantly impaired in TREM2+/- and TREM2-/- progeny.}, }
@article {pmid32364943, year = {2020}, author = {Mukama, O and Wu, J and Li, Z and Liang, Q and Yi, Z and Lu, X and Liu, Y and Liu, Y and Hussain, M and Makafe, GG and Liu, J and Xu, N and Zeng, L}, title = {An ultrasensitive and specific point-of-care CRISPR/Cas12 based lateral flow biosensor for the rapid detection of nucleic acids.}, journal = {Biosensors &amp; bioelectronics}, volume = {159}, number = {}, pages = {112143}, doi = {10.1016/j.bios.2020.112143}, pmid = {32364943}, issn = {1873-4235}, abstract = {CRISPR/Cas systems have displayed remarkable potential in developing novel biosensing applications for nucleic acid detection owing to the collateral cleavage activity of Cas effector proteins (Cas12, Cas13, etc.). Despite tremendous progress in recent years, the existing CRISPR/Cas based biosensing platforms have several limitations, including reliance on proper amplification methods, expensive fluorescence detection equipment, or lateral flow biosensor (LFB). Herein, we report a simple, inexpensive, and ultrasensitive DNA probe based LFB with CRISPR/Cas and loop-mediated Isothermal Amplification (namely CIA). The concept behind this approach is a non-detectable test line on the LFB when the Cas effector protein collaterally cleaves the cognate target and an ssDNA reporter sequence. The CIA based LFB can detect as low as a single copy cloned Pseudomonas aeruginosa acyltransferase gene, 1 cfu/ml plasmid containing E. coli DH5α pure cultures, as well as clinical samples without DNA extraction/purification or advanced apparatuses. No cross-reactivity with other non-target bacteria was observed. The naked eye result readout was obtained in 15 min of LAMP amplification, 30 min of Cas12 reaction, and 5 min of LFB readout. This platform is robust and of low cost for on-site testing.}, }
@article {pmid32363186, year = {2020}, author = {Whelan, AI and Gutti, P and Lema, MA}, title = {Gene Editing Regulation and Innovation Economics.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {8}, number = {}, pages = {303}, doi = {10.3389/fbioe.2020.00303}, pmid = {32363186}, issn = {2296-4185}, abstract = {Argentina was the first country that enacted regulatory criteria to assess if organisms resulting from new breeding techniques (NBTs) are to be regarded as genetically modified organisms (GMOs) or not. The country has now accumulated 4 year of experience applying such criteria, reaching a considerable number of cases, composed mostly of gene-edited plants, animals, and microorganisms of agricultural use. This article explores the effects on economic innovation of such regulatory experience. This is done by comparing the cases of products derived from gene editing and other NBTs that have been presented to the regulatory system, against the cases of GMOs that have been deregulated in the country. Albeit preliminary, this analysis suggests that products from gene editing will have different profiles and market release rates compared with the first wave of products from the so called &quot;modern biotechnology.&quot; Gene editing products seems to follow a much faster development rate from bench to market. Such development is driven by a more diverse group of developers, and led mostly by small and medium enterprises (SMEs) and public research institutions. In addition, product profiles are also more diversified in terms of traits and organisms. The inferences of these findings for the agricultural and biotechnology sectors, particularly in developing countries, are discussed.}, }
@article {pmid32360576, year = {2020}, author = {Song, X and Liu, C and Wang, N and Huang, H and He, S and Gong, C and Wei, Y}, title = {Delivery of CRISPR/Cas systems for cancer gene therapy and immunotherapy.}, journal = {Advanced drug delivery reviews}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.addr.2020.04.010}, pmid = {32360576}, issn = {1872-8294}, abstract = {The clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems are efficient and versatile gene editing tools, which offer enormous potential to treat cancer by editing genome, transcriptome or epigenome of tumor cells and/or immune cells. A large body of works have been done with CRISPR/Cas systems for genetic modification, and 16 clinical trials were conducted to treat cancer by ex vivo or in vivo gene editing approaches. Now, promising preclinical works have begun using CRISPR/Cas systems in vivo. However, efficient and safe delivery of CRISPR/Cas systems in vivo is still a critical challenge for their clinical applications. This article summarizes delivery of CRISPR/Cas systems by physical methods, viral vectors and non-viral vectors for cancer gene therapy and immunotherapy. The prospects for the development of physical methods, viral vectors and non-viral vectors for delivery of CRISPR/Cas systems are reviewed, and promising advances in cancer treatment using CRISPR/Cas systems are discussed.}, }
@article {pmid32359477, year = {2020}, author = {Soleimany, AP and Bhatia, SN}, title = {Activity-Based Diagnostics: An Emerging Paradigm for Disease Detection and Monitoring.}, journal = {Trends in molecular medicine}, volume = {26}, number = {5}, pages = {450-468}, doi = {10.1016/j.molmed.2020.01.013}, pmid = {32359477}, issn = {1471-499X}, abstract = {Diagnostics to accurately detect disease and monitor therapeutic response are essential for effective clinical management. Bioengineering, chemical biology, molecular biology, and computer science tools are converging to guide the design of diagnostics that leverage enzymatic activity to measure or produce biomarkers of disease. We review recent advances in the development of these 'activity-based diagnostics' (ABDx) and their application in infectious and noncommunicable diseases. We highlight efforts towards both molecular probes that respond to disease-specific catalytic activity to produce a diagnostic readout, as well as diagnostics that use enzymes as an engineered component of their sense-and-respond cascade. These technologies exemplify how integrating techniques from multiple disciplines with preclinical validation has enabled ABDx that may realize the goals of precision medicine.}, }
@article {pmid32358986, year = {2020}, author = {Pröbsting, M and Schenke, D and Hossain, R and Häder, C and Thurau, T and Wighardt, L and Schuster, A and Zhou, Z and Ye, W and Rietz, S and Leckband, G and Cai, D}, title = {Loss-of-function of CRT1a (Calreticulin) reduces plant susceptibility to Verticillium longisporum in both Arabidopsis thaliana and oilseed rape (Brassica napus).}, journal = {Plant biotechnology journal}, volume = {}, number = {}, pages = {}, doi = {10.1111/pbi.13394}, pmid = {32358986}, issn = {1467-7652}, abstract = {Brassica napus is highly susceptible towards Verticillium longisporum (Vl43) with no effective genetic resistance. It is believed that the fungus reprograms plant physiological processes by up-regulation of so-called susceptibility factors to establish a compatible interaction. By transcriptome analysis we identified genes, which were activated/upregulated in rapeseed after Vl43 infection. To test if one of these genes is functionally involved in the infection process and loss-of-function would lead to decreased susceptibility, we firstly challenged KO lines of corresponding Arabidopsis orthologs with Vl43 and compared them with wild type plants. Here, we report that the KO of AtCRT1a results in drastically reduced susceptibility of plants to Vl43. To prove crt1a mutation also decreases susceptibility in B. napus, we identified 10 mutations in a TILLING population. Three T3-mutants displayed increased resistance as compared to the wild type. To validate the results, we generated CRISPR/Cas-induced BnCRT1a mutants, challenged T2-plants with Vl43 and observed an overall reduced susceptibility in 3 out of 4 independent lines. Genotyping by allele-specific sequencing suggests a major effect of mutations in the CRT1a A-genome copy, while the C-genome copy appears to have no significant impact on plant susceptibility when challenged with Vl43. As revealed by transcript analysis, the loss-of-function of CRT1a results in activation of the ethylene signaling pathway, which may contribute to reduced susceptibility. Furthermore, this study demonstrates a novel strategy with great potential to improve plant disease resistance.}, }
@article {pmid32358068, year = {2020}, author = {Rowe, JB and Taghon, GJ and Kapolka, NJ and Morgan, WM and Isom, DG}, title = {CRISPR-addressable yeast strains with applications in human G protein-coupled receptor profiling and synthetic biology.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1074/jbc.RA120.013066}, pmid = {32358068}, issn = {1083-351X}, abstract = {Genome stability is essential for engineering cell-based devices and reporter systems. With the advent of CRISPR technology, it is now possible to build such systems by installing the necessary genetic parts directly into an organism's genome. Here, we used this approach to build a set of 10 versatile yeast-based reporter strains for studying human G protein-coupled receptors (GPCRs), the largest class of membrane receptors in humans. These reporter strains contain the necessary genetically encoded parts for studying human GPCR signaling in yeast, as well as four CRISPR-addressable expression cassettes, i.e. landing pads, installed at known safe-harbor sites in the yeast genome. We showcase the utility of these strains in two applications. First, we demonstrate that increasing GPCR expression by incrementally increasing GPCR gene copy number potentiates Gα coupling of the pharmacologically dark receptor GPR68. Second, we used two CRISPR-addressable landing pads for autocrine activation of a GPCR (the somatostatin receptor SSTR5) with its peptide agonist SRIF-14. The utility of these reporter strains can be extended far beyond these select examples to include applications such as nanobody development, mutational analysis, drug discovery, and studies of GPCR chaperoning. Additionally, we present a BY4741 yeast strain created for broad applications in the yeast and synthetic biology communities that contains only the four CRISPR-addressable landing pads. The general utility of these yeast strains provides an inexpensive, scalable, and easy means of installing and expressing genes directly from the yeast genome to build genome-barcoded sensors, reporter systems, and cell-based factories.}, }
@article {pmid32358065, year = {2020}, author = {Traughber, CA and Opoku, E and Brubaker, G and Major, J and Lu, H and Lorkowski, SW and Neumann, C and Hardaway, A and Chung, YM and Gulshan, K and Sharifi, N and Brown, JM and Smith, JD}, title = {Uptake of high-density lipoprotein by scavenger receptor class B type 1 is associated with prostate cancer proliferation and tumor progression in mice.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1074/jbc.RA120.013694}, pmid = {32358065}, issn = {1083-351X}, abstract = {High-density lipoprotein (HDL) metabolism is facilitated in part by scavenger receptor class B, type 1 (SR-B1) that mediates HDL uptake into cells. Higher levels of HDL have been associated with protection in other diseases, however its role in prostate cancer is not definitive. SR-B1 is up-regulated in prostate cancer tissue, suggesting a possible role of this receptor in tumor progression. Here, we report that knockout (KO) of SR-B1 in both human and mouse prostate cancer cell lines through CRISPR/Cas9-mediated genome editing reduces HDL uptake into the prostate cancer cells and reduces their proliferation in response to HDL. In vivo studies using syngeneic SR-B1 WT (SR-B1+/+) and SR-B1 KO (SR-B1-/-) prostate cancer cells in WT and apolipoprotein-AI KO (apoA1-KO) C57BL/6J mice revealed that WT hosts, containing higher levels of total and HDL-cholesterol, grew larger tumors than apoA1-KO hosts with lower levels of total and HDL-cholesterol. Furthermore, SR-B1-/- prostate cancer cells formed smaller tumors in WT hosts than SR-B1+/+ cells in the same host model. Increased tumor volume was overall associated with reduced survival. We conclude that knocking out SR-B1 in prostate cancer tumors reduces HDL-associated increases in prostate cancer cell proliferation and disease progression.}, }
@article {pmid32357907, year = {2020}, author = {Perez Kerkvliet, C and Dwyer, AR and Diep, CH and Oakley, RH and Liddle, C and Cidlowski, JA and Lange, CA}, title = {Glucocorticoid receptors are required effectors of TGFβ1-induced p38 MAPK signaling to advanced cancer phenotypes in triple-negative breast cancer.}, journal = {Breast cancer research : BCR}, volume = {22}, number = {1}, pages = {39}, doi = {10.1186/s13058-020-01277-8}, pmid = {32357907}, issn = {1465-542X}, support = {R01 CA192178/CA/NCI NIH HHS/United States ; T32 CA009138/CA/NCI NIH HHS/United States ; T32 GM008244/GM/NIGMS NIH HHS/United States ; F30 CA228261/CA/NCI NIH HHS/United States ; UL1TR002494//National Institute of Health/ ; UL1TR002494//National Institute of Health/ ; ES090057//National Institute of Health/ ; }, abstract = {BACKGROUND: Altered signaling pathways typify breast cancer and serve as direct inputs to steroid hormone receptor sensors. We previously reported that phospho-Ser134-GR (pS134-GR) species are elevated in triple-negative breast cancer (TNBC) and cooperate with hypoxia-inducible factors, providing a novel avenue for activation of GR in response to local or cellular stress.<br><br>METHODS: We probed GR regulation by factors (cytokines, growth factors) that are rich within the tumor microenvironment (TME). TNBC cells harboring endogenous wild-type (wt) or S134A-GR species were created by CRISPR/Cas knock-in and subjected to transwell migration, invasion, soft-agar colony formation, and tumorsphere assays. RNA-seq was employed to identify pS134-GR target genes that are regulated both basally (intrinsic) or by TGFβ1 in the absence of exogenously added GR ligands. Regulation of selected basal and TGFβ1-induced pS134-GR target genes was validated by qRT-PCR and chromatin immunoprecipitation assays. Bioinformatics tools were used to probe public data sets for expression of pS134-GR 24-gene signatures.<br><br>RESULTS: In the absence of GR ligands, GR is transcriptionally activated via p38-dependent phosphorylation of Ser134 as a mechanism of homeostatic stress-sensing and regulated upon exposure of TNBC cells to TME-derived agents. The ligand-independent pS134-GR transcriptome encompasses TGFβ1 and MAPK signaling gene sets associated with TNBC cell survival and migration/invasion. Accordingly, pS134-GR was essential for TNBC cell anchorage-independent growth in soft-agar, migration, invasion, and tumorsphere formation, an in vitro readout of cancer stemness properties. Both pS134-GR and expression of the MAPK-scaffolding molecule 14-3-3ζ were essential for a functionally intact p38 MAPK signaling pathway downstream of MAP3K5/ASK1, indicative of a feedforward signaling loop wherein self-perpetuated GR phosphorylation enables cancer cell autonomy. A 24-gene pS134-GR-dependent signature induced by TGFβ1 predicts shortened overall survival in breast cancer patients.<br><br>CONCLUSIONS: Phospho-S134-GR is a critical downstream effector of p38 MAPK signaling and TNBC migration/invasion, survival, and stemness properties. Our studies define a ligand-independent role for GR as a homeostatic &quot;sensor&quot; of intrinsic stimuli as well as extrinsic factors rich within the TME (TGFβ1) that enable potent activation of the p38 MAPK stress-sensing pathway and nominate pS134-GR as a therapeutic target in aggressive TNBC.}, }
@article {pmid31392536, year = {2019}, author = {Permyakova, NV and Sidorchuk, YV and Marenkova, TV and Khozeeva, SA and Kuznetsov, VV and Zagorskaya, AA and Rozov, SM and Deineko, EV}, title = {CRISPR/Cas9-mediated gfp gene inactivation in Arabidopsis suspension cells.}, journal = {Molecular biology reports}, volume = {46}, number = {6}, pages = {5735-5743}, pmid = {31392536}, issn = {1573-4978}, support = {17-14-01099//Russian Science Foundation/ ; }, mesh = {*Arabidopsis/cytology/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/methods ; Cells, Cultured ; DNA, Plant/genetics ; *Gene Silencing ; Genes, Reporter/genetics ; Green Fluorescent Proteins/*genetics/metabolism ; Mutagenesis, Site-Directed/methods ; Mutation/genetics ; Sequence Analysis, DNA ; }, abstract = {Targeted genome editing using CRISPR/Cas9 is a promising technology successfully verified in various plant species; however, it has hardly been used in plant cell suspension cultures. Here, we describe a successful knockout of a green fluorescent protein (gfp) reporter gene in Arabidopsis cell culture. We transformed seven transgenic suspension cell lines carrying one to three gfp gene copies with a binary vector containing genes coding for Cas9 and guide RNAs targeting the gfp gene. We detected the site-specific mutations by restriction analysis of a gfp amplicon. DNA sequencing of the PCR products confirmed high diversity of insertion-deletion mutations in the cell lines after the editing. We also analyzed gfp mRNA expression by real-time PCR and observed a decrease in gfp transcription after the target site modification. We can conclude that the CRISPR/Cas9 system can be successfully used for introducing site-specific mutations into the genome of cultured suspension cells of Arabidopsis.}, }
@article {pmid31286070, year = {2019}, author = {Horikoshi, N and Sharma, D and Leonard, F and Pandita, RK and Charaka, VK and Hambarde, S and Horikoshi, NT and Gaur Khaitan, P and Chakraborty, S and Cote, J and Godin, B and Hunt, CR and Pandita, TK}, title = {Pre-existing H4K16ac levels in euchromatin drive DNA repair by homologous recombination in S-phase.}, journal = {Communications biology}, volume = {2}, number = {}, pages = {253}, pmid = {31286070}, issn = {2399-3642}, support = {R01 CA129537/CA/NCI NIH HHS/United States ; R01 GM109768/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Chromosome Structures/chemistry ; *DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *DNA Repair ; Euchromatin/*chemistry ; HEK293 Cells ; HeLa Cells ; Heterochromatin ; Histones/*chemistry ; *Homologous Recombination ; Humans ; Kinetics ; Protein Processing, Post-Translational ; RNA, Guide/genetics ; RNA, Small Interfering/genetics ; }, abstract = {The homologous recombination (HR) repair pathway maintains genetic integrity after DNA double-strand break (DSB) damage and is particularly crucial for maintaining fidelity of expressed genes. Histone H4 acetylation on lysine 16 (H4K16ac) is associated with transcription, but how pre-existing H4K16ac directly affects DSB repair is not known. To answer this question, we used CRISPR/Cas9 technology to introduce I-SceI sites, or repair pathway reporter cassettes, at defined locations within gene-rich (high H4K16ac/euchromatin) and gene-poor (low H4K16ac/heterochromatin) regions. The frequency of DSB repair by HR is higher in gene-rich regions. Interestingly, artificially targeting H4K16ac at specific locations using gRNA/dCas9-MOF increases HR frequency in euchromatin. Finally, inhibition/depletion of RNA polymerase II or Cockayne syndrome B protein leads to decreased recruitment of HR factors at DSBs. These results indicate that the pre-existing H4K16ac status at specific locations directly influences the repair of local DNA breaks, favoring HR in part through the transcription machinery.}, }
@article {pmid31286062, year = {2019}, author = {Görlich, S and Pawolski, D and Zlotnikov, I and Kröger, N}, title = {Control of biosilica morphology and mechanical performance by the conserved diatom gene Silicanin-1.}, journal = {Communications biology}, volume = {2}, number = {}, pages = {245}, pmid = {31286062}, issn = {2399-3642}, mesh = {CRISPR-Cas Systems ; Cell Wall/*physiology ; Diatoms/genetics/*physiology ; Membrane Proteins/genetics/*physiology ; Microscopy, Atomic Force ; Morphogenesis ; Mutagenesis ; *Mutation ; Phenotype ; Plasmids/genetics ; Promoter Regions, Genetic ; Silicon Dioxide/*chemistry ; }, abstract = {The species-specifically patterned biosilica cell walls of diatoms are paradigms for biological mineral morphogenesis and the evolution of lightweight materials with exceptional mechanical performance. Biosilica formation is a membrane-mediated process that occurs in intracellular compartments, termed silica deposition vesicles (SDVs). Silicanin-1 (Sin1) is a highly conserved protein of the SDV membrane, but its role in biosilica formation has remained elusive. Here we generate Sin1 knockout mutants of the diatom Thalassiosira pseudonana. Although the mutants grow normally, they exhibit reduced biosilica content and morphological aberrations, which drastically compromise the strength and stiffness of their cell walls. These results identify Sin1 as essential for the biogenesis of mechanically robust diatom cell walls, thus providing an explanation for the conservation of this gene throughout the diatom realm. This insight paves the way for genetic engineering of silica architectures with desired structures and mechanical performance.}, }
@article {pmid31206027, year = {2019}, author = {Liu, C and Wan, T and Wang, H and Zhang, S and Ping, Y and Cheng, Y}, title = {A boronic acid-rich dendrimer with robust and unprecedented efficiency for cytosolic protein delivery and CRISPR-Cas9 gene editing.}, journal = {Science advances}, volume = {5}, number = {6}, pages = {eaaw8922}, pmid = {31206027}, issn = {2375-2548}, mesh = {Animals ; Boronic Acids/*chemistry ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Dendrimers/*chemistry/metabolism ; Drug Delivery Systems/*methods ; Fluorescein-5-isothiocyanate/chemistry ; Fluorescent Dyes/chemistry ; Gene Editing/*methods ; *Gene Transfer Techniques ; Green Fluorescent Proteins/chemistry/genetics ; HeLa Cells ; Hemoglobins/chemistry/genetics ; Humans ; Mice ; Models, Molecular ; NIH 3T3 Cells ; Protein Structure, Secondary ; RNA, Guide/genetics/metabolism ; Staining and Labeling/methods ; beta Catenin/chemistry/genetics ; }, abstract = {Cytosolic protein delivery is of central importance for the development of protein-based biotechnologies and therapeutics; however, efficient intracellular delivery of native proteins remains a challenge. Here, we reported a boronic acid-rich dendrimer with unprecedented efficiency for cytosolic delivery of native proteins. The dendrimer could bind with both negatively and positively charged proteins and efficiently delivered 13 cargo proteins into the cytosol of living cells. All the delivered proteins kept their bioactivities after cytosolic delivery. The dendrimer ensures efficient intracellular delivery of Cas9 protein into various cell lines and showed high efficiency in CRISPR-Cas9 genome editing. The rationally designed boronic acid-rich dendrimer permits the development of an efficient platform with high generality for the delivery of native proteins.}, }
@article {pmid31149642, year = {2019}, author = {Aksoy, YA and Nguyen, DT and Chow, S and Chung, RS and Guillemin, GJ and Cole, NJ and Hesselson, D}, title = {Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos.}, journal = {Communications biology}, volume = {2}, number = {}, pages = {198}, pmid = {31149642}, issn = {2399-3642}, mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; *Gene Editing ; Genotype ; Green Fluorescent Proteins/metabolism ; RNA/metabolism ; Recombinational DNA Repair ; Zebrafish/*embryology/*genetics ; }, abstract = {Precise genome editing is limited by the inefficiency of homology-directed repair (HDR) compared to the non-homologous end-joining (NHEJ) of double strand breaks (DSBs). The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system generates precise, locus-specific DSBs that can serve as substrates for HDR. We developed an in vivo visual reporter assay to quantify HDR-mediated events at single-cell resolution in zebrafish and used this system to identify small-molecule modulators that shift the DNA repair equilibrium in favor of HDR. By further optimizing the reaction environment and repair template, we achieved dramatic enhancement of HDR-mediated repair efficiency in zebrafish. Accordingly, under optimized conditions, inhibition of NHEJ with NU7441 enhanced HDR-mediated repair up to 13.4-fold. Importantly, we demonstrate that the increase in somatic HDR events correlates directly with germline transmission, permitting the efficient recovery of large seamlessly integrated DNA fragments in zebrafish.}, }
@article {pmid30988165, year = {2019}, author = {Watanabe, S and Tsuchiya, K and Nishimura, R and Shirasaki, T and Katsukura, N and Hibiya, S and Okamoto, R and Nakamura, T and Watanabe, M}, title = {TP53 Mutation by CRISPR System Enhances the Malignant Potential of Colon Cancer.}, journal = {Molecular cancer research : MCR}, volume = {17}, number = {7}, pages = {1459-1467}, doi = {10.1158/1541-7786.MCR-18-1195}, pmid = {30988165}, issn = {1557-3125}, mesh = {CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics ; Cell Line, Tumor ; Cell Proliferation/genetics ; Colonic Neoplasms/drug therapy/*genetics/pathology ; Drug Resistance, Neoplasm/genetics ; Exons/genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Lithostathine/*genetics ; Mutation/genetics ; Neoplasm Invasiveness/genetics/pathology ; Neoplastic Stem Cells/metabolism/pathology ; Tumor Suppressor Protein p53/*genetics ; }, abstract = {Tumor protein p53 (TP53) mutation is a well-known occurrence at the late phase of carcinogenesis during the adenoma-carcinoma sequence of a sporadic colon cancer. Although numerous reports about clinical information of the patients with colon cancer have suggested that TP53 mutation might be related to various types of malignant potential, the direct effects of this mutation on the malignant potential of colon cancer remain unknown. Notably, no previous report has described a relationship between TP53 mutation and cancer stemness. We therefore aimed to assess the function of a TP53 mutant induced by the CRISPR-Cas9 system in colon cancer cells. In this study, two TP53 mutations, corresponding to exon 3 (TP53E3) and 10 (TP53E10), were generated in LS174T cells derived from a wild-type TP53 human colon cancer via a lentiviral CRISPR-Cas9 system. The loss of function of TP53 resulting from both mutations manifested as resistance to Nutlin3a-induced apoptosis and the downregulation of target genes of TP53. TP53 mutants exhibited an enhanced malignant potential, characterized by accelerated cell growth, invasiveness, chemoresistance, and cancer stemness. Interestingly, TP53E10 but not TP53E3 cells exhibited aberrant transcriptional activity of regenerating family member 1-α (REG1A) and expression of REG1A, resulting in the acquisition of enhanced malignant potential. In conclusion, we demonstrated for the first time that TP53 genomic mutation into human colon cancer cells affects the malignant potential. IMPLICATIONS: These findings suggest that both a loss of function and an aberrant gain of function of TP53 might promote high malignant potentials at the late phase of carcinogenesis in colon cancer.}, }
@article {pmid32353627, year = {2020}, author = {Li, J and Li, H and Chen, J and Yan, L and Xia, L}, title = {Toward precision genome editing in crop plants.}, journal = {Molecular plant}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.molp.2020.04.008}, pmid = {32353627}, issn = {1752-9867}, }
@article {pmid32353388, year = {2020}, author = {Mitić, D and Radovčić, M and Markulin, D and Ivančić-Baće, I}, title = {StpA represses CRISPR-Cas immunity in H-NS deficient Escherichia coli.}, journal = {Biochimie}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.biochi.2020.04.020}, pmid = {32353388}, issn = {1638-6183}, abstract = {Functional CRISPR-Cas systems provide many bacteria and most archaea with adaptive immunity against invading DNA elements. CRISPR arrays store DNA fragments of previous infections while products of cas genes provide immunity by integrating new DNA fragments and using this information to recognize and destroy invading DNA. Escherichia coli contains the CRISPR-Cas type I-E system in which foreign DNA targets are recognized by Cascade, a crRNA-guided complex comprising five proteins (CasA, CasB, CasC, CasD, CasE), and degraded by Cas3. In E. coli the CRISPR-Cas type I-E system is repressed by the histone-like nucleoid-structuring protein H-NS. H-NS repression can be relieved either by inactivation of the hns gene or by elevated levels of the H-NS antagonist LeuO, which induces higher transcript levels of cas genes than was observed for Δhns cells. This suggests that derepression in Δhns cells is incomplete and that an additional repressor could be involved in the silencing. One such candidate is the H-NS paralog protein StpA, which has DNA binding preferences similar to those of H-NS. Here we show that overexpression of StpA in Δhns cells containing anti-lambda spacers abolishes resistance to λvir infection and reduces transcription of the casA gene. In cells lacking hns and stpA genes, the transcript levels of the casA gene are higher than Δhns and similar to wt cells overexpressing LeuO. Taken together, these results suggest that Cascade genes in E. coli are repressed by the StpA protein when H-NS is absent.}, }
@article {pmid32350510, year = {2020}, author = {Landmesser, U and Poller, W and Tsimikas, S and Most, P and Paneni, F and Lüscher, TF}, title = {From traditional pharmacological towards nucleic acid-based therapies for cardiovascular diseases.}, journal = {European heart journal}, volume = {}, number = {}, pages = {}, doi = {10.1093/eurheartj/ehaa229}, pmid = {32350510}, issn = {1522-9645}, abstract = {Nucleic acid-based therapeutics are currently developed at large scale for prevention and management of cardiovascular diseases (CVDs), since: (i) genetic studies have highlighted novel therapeutic targets suggested to be causal for CVD; (ii) there is a substantial recent progress in delivery, efficacy, and safety of nucleic acid-based therapies; (iii) they enable effective modulation of therapeutic targets that cannot be sufficiently or optimally addressed using traditional small molecule drugs or antibodies. Nucleic acid-based therapeutics include (i) RNA-targeted therapeutics for gene silencing; (ii) microRNA-modulating and epigenetic therapies; (iii) gene therapies; and (iv) genome-editing approaches (e.g. CRISPR-Cas-based): (i) RNA-targeted therapeutics: several large-scale clinical development programmes, using antisense oligonucleotides (ASO) or short interfering RNA (siRNA) therapeutics for prevention and management of CVD have been initiated. These include ASO and/or siRNA molecules to lower apolipoprotein (a) [apo(a)], proprotein convertase subtilisin/kexin type 9 (PCSK9), apoCIII, ANGPTL3, or transthyretin (TTR) for prevention and treatment of patients with atherosclerotic CVD or TTR amyloidosis. (ii) MicroRNA-modulating and epigenetic therapies: novel potential therapeutic targets are continually arising from human non-coding genome and epigenetic research. First microRNA-based therapeutics or therapies targeting epigenetic regulatory pathways are in clinical studies. (iii) Gene therapies: EMA/FDA have approved gene therapies for non-cardiac monogenic diseases and LDL receptor gene therapy is currently being examined in patients with homozygous hypercholesterolaemia. In experimental studies, gene therapy has significantly improved cardiac function in heart failure animal models. (iv) Genome editing approaches: these technologies, such as using CRISPR-Cas, have proven powerful in stem cells, however, important challenges are remaining, e.g. low rates of homology-directed repair in somatic cells such as cardiomyocytes. In summary, RNA-targeted therapies (e.g. apo(a)-ASO and PCSK9-siRNA) are now in large-scale clinical outcome trials and will most likely become a novel effective and safe therapeutic option for CVD in the near future. MicroRNA-modulating, epigenetic, and gene therapies are tested in early clinical studies for CVD. CRISPR-Cas-mediated genome editing is highly effective in stem cells, but major challenges are remaining in somatic cells, however, this field is rapidly advancing.}, }
@article {pmid32350113, year = {2020}, author = {McDonald, IM and Graves, LM}, title = {Enigmatic MELK: The controversy surrounding its complex role in cancer.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1074/jbc.REV120.013433}, pmid = {32350113}, issn = {1083-351X}, abstract = {The Ser/Thr protein kinase MELK (maternal embryonic leucine zipper kinase) has been considered an attractive therapeutic target for managing cancer since 2005. Studies using expression analysis have indicated that MELK expression is higher in numerous cancer cells and tissues than in their normal, non-neoplastic counterparts. Further, RNAi-mediated MELK depletion impairs proliferation of multiple cancers, including triple-negative breast cancer (TNBC), and these growth defects can be rescued with exogenous wild-type MELK, but not kinase-dead MELK complementation. Pharmacological MELK inhibition with OTS167 (alternatively called OTSSP167) and NVS-MELK8a, among other small molecules, also impairs cancer cell growth. These collective results led to MELK being classified as essential for cancer proliferation. More recently, in 2017, the proliferation of TNBC and other cancer cell lines was reported to be unaffected by genetic CRISPR/Cas9-mediated MELK deletion, calling into question the essentiality of this kinase in cancer. To date, the requirement of MELK in cancer remains controversial, and mechanisms underlying the disparate growth effects observed with RNAi, pharmacological inhibition, and CRISPR remain unclear. Our objective with this review is to highlight the evidence on both sides of this controversy, to provide commentary on the purported requirement of MELK in cancer, and to emphasize the need for continued elucidation of the functions of MELK.}, }
@article {pmid32349666, year = {2020}, author = {Brandt, K and Nethery, MA and O'Flaherty, S and Barrangou, R}, title = {Genomic characterization of Lactobacillus fermentum DSM 20052.}, journal = {BMC genomics}, volume = {21}, number = {1}, pages = {328}, doi = {10.1186/s12864-020-6740-8}, pmid = {32349666}, issn = {1471-2164}, support = {Probiotics research//DuPont/ ; Probiotics research//North Carolina Ag Foundation/ ; Internal funds//College of Agriculture and Life Sciences, North Carolina State University/ ; }, abstract = {BACKGROUND: Lactobacillus fermentum, a member of the lactic acid bacteria complex, has recently garnered increased attention due to documented antagonistic properties and interest in assessing the probiotic potential of select strains that may provide human health benefits. Here, we genomically characterize L. fermentum using the type strain DSM 20052 as a canonical representative of this species.<br><br>RESULTS: We determined the polished whole genome sequence of this type strain and compared it to 37 available genome sequences within this species. Results reveal genetic diversity across nine clades, with variable content encompassing mobile genetic elements, CRISPR-Cas immune systems and genomic islands, as well as numerous genome rearrangements. Interestingly, we determined a high frequency of occurrence of diverse Type I, II, and III CRISPR-Cas systems in 72% of the genomes, with a high level of strain hypervariability.<br><br>CONCLUSIONS: These findings provide a basis for the genetic characterization of L. fermentum strains of scientific and commercial interest. Furthermore, our study enables genomic-informed selection of strains with specific traits for commercial product formulation, and establishes a framework for the functional characterization of features of interest.}, }
@article {pmid31874693, year = {2020}, author = {Lin, WR and Ng, IS}, title = {Development of CRISPR/Cas9 system in Chlorella vulgaris FSP-E to enhance lipid accumulation.}, journal = {Enzyme and microbial technology}, volume = {133}, number = {}, pages = {109458}, doi = {10.1016/j.enzmictec.2019.109458}, pmid = {31874693}, issn = {1879-0909}, mesh = {Agrobacterium tumefaciens/genetics ; Biofuels ; Biomass ; *CRISPR-Cas Systems ; Chlorella vulgaris/*genetics/*metabolism ; Fatty Acid Desaturases/genetics ; Green Fluorescent Proteins/genetics ; *Lipid Metabolism ; Lipids/*analysis ; Microalgae/genetics/metabolism ; Plasmids/genetics ; }, abstract = {Microalgae biorefinery is an alternative, sustainable and promising trend to solve the problem of fossil oil depletion and carbon dioxide emission. However, considering the innate limitation of cell growth and oil content in microalgae, to accelerate metabolic balance by CRISPR/Cas9 system is attractive. At first, plasmid based from Agrobacterium tumefaciens and a fragment of mGFP was transformed into Chlorella sorokiniana and Chlorella vulgaris FSP-E by electroporation, respectively. Selected colonies were tested by spectrophotometer and inverted fluorescence microscopy (IFM), and an increase of fluorescent was observed by 67% compared with that in wild type, which proved the Agrobacterium-mediated plasmid is suitable for gene insertion in Chlorella species. Consequently, plasmid with similar structure as mentioned previously containing fragment of Cas9 with sgRNA designed on omega-3 fatty acid desaturase (fad3) gene was constructed and showed a higher accumulation of lipid content by 46% (w/w) in C. vulgaris FSP-E. This is first-time to use CRISPR/Cas9 based technology for gene manipulation in Chlorella.}, }
@article {pmid31874686, year = {2020}, author = {Salazar-Cerezo, S and Kun, RS and de Vries, RP and Garrigues, S}, title = {CRISPR/Cas9 technology enables the development of the filamentous ascomycete fungus Penicillium subrubescens as a new industrial enzyme producer.}, journal = {Enzyme and microbial technology}, volume = {133}, number = {}, pages = {109463}, doi = {10.1016/j.enzmictec.2019.109463}, pmid = {31874686}, issn = {1879-0909}, mesh = {*CRISPR-Cas Systems ; Enzymes/*biosynthesis ; *Gene Editing ; Genome, Fungal ; Industrial Microbiology/methods ; Penicillium/*enzymology/*genetics ; Phenotype ; }, abstract = {Penicillium subrubescens is an ascomycete fungus with an enriched content of specific carbohydrate-active enzyme families involved in plant biomass degradation, which makes this strain a promising industrial cell factory for enzyme production. The development of tools that allow genetic manipulation is crucial for further strain improvement and the functional characterization of its genes. In this context, the CRISPR/Cas9 system represents an excellent option for genome editing due to its high efficiency and versatility. To establish CRISPR/Cas9 genome editing in P. subrubescens, first a method for protoplast generation and transformation was developed, using hygromycin as selection marker. Then the CRISPR/Cas9 system was established in P. subrubescens by successfully deleting the ku70 gene, which is involved in the non-homologous end joining DNA repair mechanism. Phenotypic characterization of the mutants showed that ku70 mutation did not affect P. subrubescens growth at optimal temperature and Δku70 strains showed similar protein production pattern to the wild type.}, }
@article {pmid31506018, year = {2019}, author = {Hustedt, N and Álvarez-Quilón, A and McEwan, A and Yuan, JY and Cho, T and Koob, L and Hart, T and Durocher, D}, title = {A consensus set of genetic vulnerabilities to ATR inhibition.}, journal = {Open biology}, volume = {9}, number = {9}, pages = {190156}, pmid = {31506018}, issn = {2046-2441}, support = {P30 CA016672/CA/NCI NIH HHS/United States ; FDN143343//CIHR/Canada ; }, mesh = {Ataxia Telangiectasia Mutated Proteins/antagonists &amp; inhibitors/*genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; Gene Expression ; Gene Knockdown Techniques ; Gene Silencing ; Gene Targeting ; Genes, Reporter ; Genetic Association Studies ; *Genetic Variation ; Humans ; RNA Interference ; RNA, Guide ; }, abstract = {The response to DNA replication stress in eukaryotes is under the control of the ataxia-telangiectasia and Rad3-related (ATR) kinase. ATR responds to single-stranded (ss) DNA to stabilize distressed DNA replication forks, modulate DNA replication firing and prevent cells with damaged DNA or incomplete DNA replication from entering into mitosis. Furthermore, inhibitors of ATR are currently in clinical development either as monotherapies or in combination with agents that perturb DNA replication. To gain a genetic view of the cellular pathways requiring ATR kinase function, we mapped genes whose mutation causes hypersensitivity to ATR inhibitors with genome-scale CRISPR/Cas9 screens. We delineate a consensus set of 117 genes enriched in DNA replication, DNA repair and cell cycle regulators that promote survival when ATR kinase activity is suppressed. We validate 14 genes from this set and report genes not previously described to modulate response to ATR inhibitors. In particular we found that the loss of the POLE3/POLE4 proteins, which are DNA polymerase ε accessory subunits, results in marked hypersensitivity to ATR inhibition. We anticipate that this 117-gene set will be useful for the identification of genes involved in the regulation of genome integrity and the characterization of new biological processes involving ATR, and may reveal biomarkers of ATR inhibitor response in the clinic.}, }
@article {pmid31493817, year = {2019}, author = {Li, Y and Xu, W and Jerman, S and Sun, Z}, title = {In vivo analysis of renal epithelial cells in zebrafish.}, journal = {Methods in cell biology}, volume = {154}, number = {}, pages = {163-181}, doi = {10.1016/bs.mcb.2019.04.016}, pmid = {31493817}, issn = {0091-679X}, support = {R01 DK113135/DK/NIDDK NIH HHS/United States ; R01 HL125885/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Polarity ; Cilia/metabolism/ultrastructure ; Embryo, Nonmammalian/anatomy &amp; histology/*cytology/metabolism ; Epithelial Cells/*cytology/metabolism ; Female ; Gene Editing/*methods ; Immunohistochemistry/*methods ; Kidney/*cytology/embryology/metabolism ; Male ; Organogenesis/genetics ; RNA, Guide/genetics/metabolism ; Zebrafish ; Zebrafish Proteins/deficiency/*genetics ; }, abstract = {The zebrafish kidney has been used effectively for studying kidney development, repair and disease. New gene editing capability makes it a more versatile in vivo vertebrate model system to investigate renal epithelial cells in their native environment. In this chapter we focus on dissecting gene function in basic cellular biology of renal epithelial cells, including lumen formation and cell polarity, in intact zebrafish embryos.}, }
@article {pmid31493814, year = {2019}, author = {Drake, KA and Fessler, AR and Carroll, TJ}, title = {Methods for renal lineage tracing: In vivo and beyond.}, journal = {Methods in cell biology}, volume = {154}, number = {}, pages = {121-143}, doi = {10.1016/bs.mcb.2019.06.002}, pmid = {31493814}, issn = {0091-679X}, mesh = {Animals ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Lineage/*genetics ; Cell Tracking/*methods ; Doxycycline/pharmacology ; Epithelial Cells/cytology/metabolism ; Fluorescent Antibody Technique/*methods ; Gene Expression/drug effects ; *Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; Homeostasis/genetics ; Integrases/*genetics/metabolism ; Kidney/*cytology/growth &amp; development/metabolism ; Luminescent Proteins/genetics/metabolism ; Mice ; Mice, Transgenic ; Organogenesis/genetics ; Pluripotent Stem Cells/cytology/metabolism ; beta-Galactosidase/genetics/metabolism ; }, abstract = {Lineage tracing has resulted in fundamental discoveries in kidney development and disease and remains a powerful technique to study mechanisms of organogenesis, homeostasis, and repair/regeneration. Following decades of research on the cellular and molecular regulation of renal organogenesis, the kidney has become one of the most well-characterized organs, resulting in exciting advancements in pluripotent stem cell differentiation, tissue bioengineering, and the potential for developing novel regenerative therapies for kidney disease. Lineage tracing, or the labeling of progeny cells arising from a single cell or group of cells, allows for spatial and temporal analyses of dynamic in vivo and in vitro processes. As lineage tracing techniques expand across disciplines of developmental biology, stem cell biology, and regenerative medicine, careful experimental design and interpretation, along with an understanding of the basic principles and technical limitations, are essential for utilizing genetically complex lineage tracing models to further understand kidney development and disease.}, }
@article {pmid31455158, year = {2019}, author = {Littler, S and Sloss, O and Geary, B and Pierce, A and Whetton, AD and Taylor, SS}, title = {Oncogenic MYC amplifies mitotic perturbations.}, journal = {Open biology}, volume = {9}, number = {8}, pages = {190136}, pmid = {31455158}, issn = {2046-2441}, support = {MR/M008959/1/MRC_/Medical Research Council/United Kingdom ; C1422/A19842/CRUK_/Cancer Research UK/United Kingdom ; C5759/A25254/CRUK_/Cancer Research UK/United Kingdom ; }, mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Transformation, Neoplastic/*genetics ; Chromosomal Instability ; Chromosome Segregation ; *Gene Amplification ; Gene Expression Regulation ; Genomic Instability ; Humans ; Mitosis/*genetics ; Mutagenesis ; Proto-Oncogene Proteins c-myc/*genetics ; }, abstract = {The oncogenic transcription factor MYC modulates vast arrays of genes, thereby influencing numerous biological pathways including biogenesis, metabolism, proliferation, apoptosis and pluripotency. When deregulated, MYC drives genomic instability via several mechanisms including aberrant proliferation, replication stress and ROS production. Deregulated MYC also promotes chromosome instability, but less is known about how MYC influences mitosis. Here, we show that deregulating MYC modulates multiple aspects of mitotic chromosome segregation. Cells overexpressing MYC have altered spindle morphology, take longer to align their chromosomes at metaphase and enter anaphase sooner. When challenged with a variety of anti-mitotic drugs, cells overexpressing MYC display more anomalies, the net effect of which is increased micronuclei, a hallmark of chromosome instability. Proteomic analysis showed that MYC modulates multiple networks predicted to influence mitosis, with the mitotic kinase PLK1 identified as a central hub. In turn, we show that MYC modulates several PLK1-dependent processes, namely mitotic entry, spindle assembly and SAC satisfaction. These observations thus underpin the pervasive nature of oncogenic MYC and provide a mechanistic rationale for MYC's ability to drive chromosome instability.}, }
@article {pmid31450191, year = {2019}, author = {Zeng, R and Sidik, H and Robinson, KS and Zhong, FL and Reversade, B and Pouladi, MA}, title = {Generation of four H1 hESC sublines carrying a hemizygous knock-out/mutant MECP2.}, journal = {Stem cell research}, volume = {40}, number = {}, pages = {101533}, doi = {10.1016/j.scr.2019.101533}, pmid = {31450191}, issn = {1876-7753}, mesh = {CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Embryonic Stem Cells/*cytology/metabolism ; Exons ; *Gene Editing ; Humans ; Karyotype ; Methyl-CpG-Binding Protein 2/*genetics ; Rett Syndrome/genetics/pathology ; }, abstract = {Rett syndrome (RTT) is a childhood neurodevelopmental disorder caused by mutations in MECP2. To study the molecular mechanisms underlying RTT, four sublines of H1 hESCs were generated, carrying a hemizygous knockout or mutant allele of MECP2. Exons 3 and 4 of MECP2 were targeted using the CRISPR/Cas9 nuclease system.}, }
@article {pmid31445394, year = {2019}, author = {Han, HJ and Seo, HH and Han, HW and Kim, JH}, title = {Generation of a TLR7 homozygous knockout human induced pluripotent stem cell line using CRISPR/Cas9.}, journal = {Stem cell research}, volume = {40}, number = {}, pages = {101520}, doi = {10.1016/j.scr.2019.101520}, pmid = {31445394}, issn = {1876-7753}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Embryoid Bodies/cytology/metabolism ; Gene Editing ; Homozygote ; Humans ; Induced Pluripotent Stem Cells/*cytology/metabolism/transplantation ; Karyotype ; Male ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Teratoma/pathology ; Toll-Like Receptor 7/*genetics ; }, abstract = {Toll Like Receptor (TLR) family plays an important role in the activation of innate immunity against pathogens. TLR7 mediates the recognition of single-stranded RNA viruses, such as human immunodeficiency virus, hepatitis C virus, and influenza virus in endosomes. Here, we generated a TLR7 homozygous knockout human induced pluripotent cell (hiPSC) line, hiPSC-TLR7KO-A59, using the CRISPR/Cas9 genome editing method. The hiPSC-TLR7KO-A59 line maintains normal morphology, pluripotency, and differentiation capacity into three germ layers.}, }
@article {pmid31107171, year = {2019}, author = {Newby, GA}, title = {Base Editing: Efficient Installation of Point Mutations with Minimal Byproducts.}, journal = {Stem cells and development}, volume = {28}, number = {11}, pages = {712-713}, doi = {10.1089/scd.2019.0080}, pmid = {31107171}, issn = {1557-8534}, mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Hematologic Diseases/therapy ; Humans ; Polymorphism, Single Nucleotide/genetics ; }, }
@article {pmid31090512, year = {2019}, author = {Bak, RO}, title = {The Potential of CRISPR/Cas9 in Hematotherapy.}, journal = {Stem cells and development}, volume = {28}, number = {11}, pages = {710-711}, doi = {10.1089/scd.2019.0079}, pmid = {31090512}, issn = {1557-8534}, mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Humans ; Immunotherapy, Adoptive/*methods ; }, }
@article {pmid31090491, year = {2019}, author = {Cong, L}, title = {Take Risks and Constantly Challenge the Status Quo.}, journal = {Stem cells and development}, volume = {28}, number = {11}, pages = {709}, doi = {10.1089/scd.2019.0082}, pmid = {31090491}, issn = {1557-8534}, mesh = {CRISPR-Cas Systems/*genetics ; Cell- and Tissue-Based Therapy/*methods ; Gene Editing/methods ; Genetic Therapy/*methods ; Humans ; }, }
@article {pmid31081744, year = {2019}, author = {Mussolino, C}, title = {Hematotherapy.}, journal = {Stem cells and development}, volume = {28}, number = {11}, pages = {714}, doi = {10.1089/scd.2019.0078}, pmid = {31081744}, issn = {1557-8534}, mesh = {CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Hematopoietic Stem Cell Transplantation ; Humans ; Immunotherapy/*methods ; }, }
@article {pmid30858339, year = {2019}, author = {Howden, SE and Vanslambrouck, JM and Wilson, SB and Tan, KS and Little, MH}, title = {Reporter-based fate mapping in human kidney organoids confirms nephron lineage relationships and reveals synchronous nephron formation.}, journal = {EMBO reports}, volume = {20}, number = {4}, pages = {}, pmid = {30858339}, issn = {1469-3178}, support = {UH2 DK107344/DK/NIDDK NIH HHS/United States ; UH3 DK107344/DK/NIDDK NIH HHS/United States ; }, mesh = {Biomarkers ; CRISPR-Cas Systems ; Cell Culture Techniques ; Cell Differentiation ; *Chromosome Mapping ; *Gene Expression Profiling ; *Genes, Reporter ; Homeodomain Proteins/genetics ; Humans ; Nephrons/*embryology/*metabolism ; Nerve Tissue Proteins/genetics ; Organogenesis/*genetics ; Organoids ; Pluripotent Stem Cells/cytology/metabolism ; Single-Cell Analysis ; }, abstract = {Nephron formation continues throughout kidney morphogenesis in both mice and humans. Lineage tracing studies in mice identified a self-renewing Six2-expressing nephron progenitor population able to give rise to the full complement of nephrons throughout kidney morphogenesis. To investigate the origin of nephrons within human pluripotent stem cell-derived kidney organoids, we performed a similar fate-mapping analysis of the SIX2-expressing lineage in induced pluripotent stem cell (iPSC)-derived kidney organoids to explore the feasibility of investigating lineage relationships in differentiating iPSCs in vitro Using CRISPR/Cas9 gene-edited lineage reporter lines, we show that SIX2-expressing cells give rise to nephron epithelial cell types but not to presumptive ureteric epithelium. The use of an inducible (CreERT2) line revealed a declining capacity for SIX2+ cells to contribute to nephron formation over time, but retention of nephron-forming capacity if provided an exogenous WNT signal. Hence, while human iPSC-derived kidney tissue appears to maintain lineage relationships previously identified in developing mouse kidney, unlike the developing kidney in vivo, kidney organoids lack a nephron progenitor niche capable of both self-renewal and ongoing nephrogenesis.}, }
@article {pmid30854433, year = {2019}, author = {Min, YL and Li, H and Rodriguez-Caycedo, C and Mireault, AA and Huang, J and Shelton, JM and McAnally, JR and Amoasii, L and Mammen, PPA and Bassel-Duby, R and Olson, EN}, title = {CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion mutations in mice and human cells.}, journal = {Science advances}, volume = {5}, number = {3}, pages = {eaav4324}, pmid = {30854433}, issn = {2375-2548}, support = {R01 AR067294/AR/NIAMS NIH HHS/United States ; R01 HL130253/HL/NHLBI NIH HHS/United States ; R01 HL138426/HL/NHLBI NIH HHS/United States ; U54 HD087351/HD/NICHD NIH HHS/United States ; }, mesh = {Animals ; *CRISPR-Cas Systems ; Dependovirus/genetics ; Disease Models, Animal ; Dystrophin/*genetics ; *Exons ; Gene Editing ; Gene Expression ; Gene Targeting ; Gene Transfer Techniques ; Genetic Vectors/genetics ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Mice ; Muscular Dystrophy, Duchenne/drug therapy/*genetics ; Myocytes, Cardiac/metabolism ; RNA, Guide ; *Sequence Deletion ; Transduction, Genetic ; }, abstract = {Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), which is characterized by lethal degeneration of cardiac and skeletal muscles. Mutations that delete exon 44 of the dystrophin gene represent one of the most common causes of DMD and can be corrected in ~12% of patients by editing surrounding exons, which restores the dystrophin open reading frame. Here, we present a simple and efficient strategy for correction of exon 44 deletion mutations by CRISPR-Cas9 gene editing in cardiomyocytes obtained from patient-derived induced pluripotent stem cells and in a new mouse model harboring the same deletion mutation. Using AAV9 encoding Cas9 and single guide RNAs, we also demonstrate the importance of the dosages of these gene editing components for optimal gene correction in vivo. Our findings represent a significant step toward possible clinical application of gene editing for correction of DMD.}, }
@article {pmid32345641, year = {2020}, author = {DeWerff, SJ and Bautista, MA and Pauly, M and Zhang, C and Whitaker, RJ}, title = {Killer Archaea: Virus-Mediated Antagonism to CRISPR-Immune Populations Results in Emergent Virus-Host Mutualism.}, journal = {mBio}, volume = {11}, number = {2}, pages = {}, doi = {10.1128/mBio.00404-20}, pmid = {32345641}, issn = {2150-7511}, abstract = {Theory, simulation, and experimental evolution demonstrate that diversified CRISPR-Cas immunity to lytic viruses can lead to stochastic virus extinction due to a limited number of susceptible hosts available to each potential new protospacer escape mutation. Under such conditions, theory predicts that to evade extinction, viruses evolve toward decreased virulence and promote vertical transmission and persistence in infected hosts. To better understand the evolution of host-virus interactions in microbial populations with active CRISPR-Cas immunity, we studied the interaction between CRISPR-immune Sulfolobus islandicus cells and immune-deficient strains that are infected by the chronic virus SSV9. We demonstrate that Sulfolobus islandicus cells infected with SSV9, and with other related SSVs, kill uninfected, immune strains through an antagonistic mechanism that is a protein and is independent of infectious virus. Cells that are infected with SSV9 are protected from killing and persist in the population. We hypothesize that this infection acts as a form of mutualism between the host and the virus by removing competitors in the population and ensuring continued vertical transmission of the virus within populations with diversified CRISPR-Cas immunity.IMPORTANCE Multiple studies, especially those focusing on the role of lytic viruses in key model systems, have shown the importance of viruses in shaping microbial populations. However, it has become increasingly clear that viruses with a long host-virus interaction, such as those with a chronic lifestyle, can be important drivers of evolution and have large impacts on host ecology. In this work, we describe one such interaction with the acidic crenarchaeon Sulfolobus islandicus and its chronic virus Sulfolobus spindle-shaped virus 9. Our work expands the view in which this symbiosis between host and virus evolved, describing a killing phenotype which we hypothesize has evolved in part due to the high prevalence and diversity of CRISPR-Cas immunity seen in natural populations. We explore the implications of this phenotype in population dynamics and host ecology, as well as the implications of mutualism between this virus-host pair.}, }
@article {pmid32344896, year = {2020}, author = {Xu, X and Hulshoff, MS and Tan, X and Zeisberg, M and Zeisberg, EM}, title = {CRISPR/Cas Derivatives as Novel Gene Modulating Tools: Possibilities and In Vivo Applications.}, journal = {International journal of molecular sciences}, volume = {21}, number = {9}, pages = {}, doi = {10.3390/ijms21093038}, pmid = {32344896}, issn = {1422-0067}, abstract = {The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy.}, }
@article {pmid32340238, year = {2020}, author = {Seys, FM and Rowe, P and Bolt, EL and Humphreys, CM and Minton, NP}, title = {A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences.}, journal = {Genes}, volume = {11}, number = {4}, pages = {}, doi = {10.3390/genes11040458}, pmid = {32340238}, issn = {2073-4425}, support = {BB/L013940/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/K00283X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/L502030/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, abstract = {Phenotypic complementation of gene knockouts is an essential step in establishing function. Here, we describe a simple strategy for 'gold standard' complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) 'bookmark' sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele. The bookmark comprises a 23 nt Cas9 target sequence plus an additional nt to ensure the deletion is in-frame. Here, bookmarks are tailored to Streptococcus pyogenes CRISPR/Cas9 but could be designed for any CRISPR/Cas system. For proof of concept, nine bookmarks were tested in Clostridium autoethanogenum. Complementation efficiencies reached 91%. As complemented strains are indistinguishable from their progenitors, concerns over contamination may be satisfied by the incorporation of 'watermark' sequences into the complementing genes.}, }
@article {pmid32298586, year = {2020}, author = {Choudhury, A and Fankhauser, RG and Freed, EF and Oh, EJ and Morgenthaler, AB and Bassalo, MC and Copley, SD and Kaar, JL and Gill, RT}, title = {Determinants for Efficient Editing with Cas9-Mediated Recombineering in Escherichia coli.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.9b00440}, pmid = {32298586}, issn = {2161-5063}, abstract = {In E. coli, editing efficiency with Cas9-mediated recombineering varies across targets due to differences in the level of Cas9:gRNA-mediated DNA double-strand break (DSB)-induced cell death. We found that editing efficiency with the same gRNA and repair template can also change with target position, cas9 promoter strength, and growth conditions. Incomplete editing, off-target activity, nontargeted mutations, and failure to cleave target DNA even if Cas9 is bound also compromise editing efficiency. These effects on editing efficiency were gRNA-specific. We propose that differences in the efficiency of Cas9:gRNA-mediated DNA DSBs, as well as possible differences in binding of Cas9:gRNA complexes to their target sites, account for the observed variations in editing efficiency between gRNAs. We show that editing behavior using the same gRNA can be modified by mutating the gRNA spacer, which changes the DNA DSB activity. Finally, we discuss how variable editing with different gRNAs could limit high-throughput applications and provide strategies to overcome these limitations.}, }
@article {pmid32029609, year = {2020}, author = {Couzin-Frankel, J}, title = {CRISPR takes on cancer.}, journal = {Science (New York, N.Y.)}, volume = {367}, number = {6478}, pages = {616}, doi = {10.1126/science.367.6478.616}, pmid = {32029609}, issn = {1095-9203}, mesh = {Antigens, Neoplasm/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Knockdown Techniques ; Humans ; Immunotherapy, Adoptive/*methods ; Membrane Proteins/genetics ; T-Lymphocytes/*immunology/*transplantation ; }, }
@article {pmid31337724, year = {2019}, author = {Lum, KK and Howard, TR and Pan, C and Cristea, IM}, title = {Charge-Mediated Pyrin Oligomerization Nucleates Antiviral IFI16 Sensing of Herpesvirus DNA.}, journal = {mBio}, volume = {10}, number = {4}, pages = {}, pmid = {31337724}, issn = {2150-7511}, support = {F31 GM120936/GM/NIGMS NIH HHS/United States ; R01 GM114141/GM/NIGMS NIH HHS/United States ; T32 GM007388/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; Cells, Cultured ; Cytokines/immunology ; DNA, Viral/genetics/*metabolism ; Fibroblasts/*immunology/virology ; HEK293 Cells ; Herpesvirus 1, Human/*genetics/physiology ; Host-Pathogen Interactions ; Humans ; Immunity, Innate ; Mutation ; Nuclear Proteins/genetics/*metabolism ; Phosphoproteins/genetics/*metabolism ; Protein Domains ; Protein Multimerization ; Proteomics ; Pyrin/genetics/*metabolism ; Virus Replication ; }, abstract = {The formation of multimerized protein assemblies has emerged as a core component of immune signal amplification, yet the biochemical basis of this phenomenon remains unclear for many mammalian proteins within host defense pathways. The interferon-inducible protein 16 (IFI16) is a viral DNA sensor that oligomerizes upon binding to nuclear viral DNA and induces downstream antiviral responses. Here, we identify the pyrin domain (PYD) residues that mediate IFI16 oligomerization in a charge-dependent manner. Based on structure modeling, these residues are predicted to be surface exposed within distinct α-helices. By generating oligomerization-deficient mutants, we demonstrate that IFI16 homotypic clustering is necessary for its assembly onto parental viral genomes at the nuclear periphery upon herpes simplex virus 1 (HSV-1) infection. Preventing oligomerization severely hampered the capacity of IFI16 to induce antiviral cytokine expression, suppress viral protein levels, and restrict viral progeny production. Restoring oligomerization via residue-specific charge mimics partially rescued IFI16 antiviral roles. We show that pyrin domains from PYHIN proteins are functionally interchangeable, facilitating cooperative assembly with the IFI16 HINs, highlighting an inherent role for pyrin domains in antiviral response. Using immunoaffinity purification and targeted mass spectrometry, we establish that oligomerization promotes IFI16 interactions with proteins involved in transcriptional regulation, including PAF1C, UBTF, and ND10 bodies. We further discover PAF1C as an HSV-1 restriction factor. Altogether, our study uncovers intrinsic properties that govern IFI16 oligomerization, which serves as a signal amplification platform to activate innate immune responses and to recruit transcriptional regulatory proteins that suppress HSV-1 replication.IMPORTANCE The ability of mammalian cells to detect the genomes of nuclear-replicating viruses via cellular DNA sensors is fundamental to innate immunity. Recently, mounting evidence is supporting the universal role of polymerization in these host defense factors as a signal amplification strategy. Yet, what has remained unclear are the intrinsic properties that govern their immune signal transmission. Here, we uncover the biochemical basis for oligomerization of the nuclear DNA sensor, IFI16. Upon infection with herpes simplex virus 1 (HSV-1) in human fibroblasts, we characterize the contribution of IFI16 oligomerization to downstream protein interactions and antiviral functions, including cytokine induction and suppression of HSV-1 replication. Until now, the global characterization of oligomerization-dependent protein interactions for an immune receptor has never been explored. Our integrative quantitative proteomics, molecular CRISPR/Cas9-based assays, mutational analyses, and confocal microscopy shed light on the dynamics of immune signaling cascades activated against pathogens.}, }
@article {pmid30908848, year = {2019}, author = {Carnes, RM and Mobley, JA and Crossman, DK and Liu, H and Korf, BR and Kesterson, RA and Wallis, D}, title = {Multi-Omics Profiling for NF1 Target Discovery in Neurofibromin (NF1) Deficient Cells.}, journal = {Proteomics}, volume = {19}, number = {11}, pages = {e1800334}, doi = {10.1002/pmic.201800334}, pmid = {30908848}, issn = {1615-9861}, mesh = {*CRISPR-Cas Systems ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Neurofibromin 1/*genetics ; Neurogenesis ; Proteomics/*methods ; Signal Transduction ; *Transcriptome ; }, abstract = {Loss of NF1 is an oncogenic driver. In efforts to define pathways responsible for the development of neurofibromas and other cancers, transcriptomic and proteomic changes are evaluated in a non-malignant NF1 null cell line. NF1 null HEK293 cells were created using CRISPR/Cas9 technology and they are compared to parental cells that express neurofibromin. A total of 1222 genes and 132 proteins are found to be differentially expressed. The analysis is integrated to identify eight transcripts/proteins that are differentially regulated in both analyses. Metacore Pathway analysis identifies Neurogenesis NGF/TrkA MAPK-mediated signaling alterations. Next, the data set is compared with other published studies that involve analysis of cells or tumors deficient for NF1 and it is found that 141 genes recur in the sample and others; only thirteen of these genes recur in two or more studies. Genes/proteins of interest are validated via q-RT-PCR or Western blot. It is shown that KRT8 and 14-3-3σ protein levels respond to exogenously introduced mNf1 cDNA. Hence, transcripts/proteins that respond to neurofibromin levels are identified and they can potentially be used as biomarkers.}, }
@article {pmid30741685, year = {2019}, author = {Mohamed, NV and Larroquette, F and Beitel, LK and Fon, EA and Durcan, TM}, title = {One Step Into the Future: New iPSC Tools to Advance Research in Parkinson's Disease and Neurological Disorders.}, journal = {Journal of Parkinson's disease}, volume = {9}, number = {2}, pages = {265-281}, pmid = {30741685}, issn = {1877-718X}, mesh = {Biomedical Research ; CRISPR-Cas Systems ; Coculture Techniques ; Drug Discovery ; Gene Editing ; Humans ; In Vitro Techniques ; *Induced Pluripotent Stem Cells ; Lab-On-A-Chip Devices ; Organoids ; *Parkinson Disease ; }, abstract = {Studying Parkinson's disease (PD) in the laboratory presents many challenges, the main one being the limited availability of human cells and tissue from affected individuals. As PD is characterized by a loss of dopaminergic (DA) neurons in the brain, it is nearly impossible for researchers to access and extract these cells from living patients. Thus, in the past PD research has focused on the use of patients' post-mortem tissues, animal models, or immortalized cell lines to dissect cellular pathways of interest. While these strategies deepened our knowledge of pathological mechanisms in PD, they failed to faithfully capture key mechanisms at play in the human brain. The emergence of induced pluripotent stem cell (iPSC) technology is revolutionizing PD research, as it allows for the differentiation and growth of human DA neurons in vitro, holding immense potential not only for modelling PD, but also for identifying novel therapies. However, to reproduce the complexity of the brain's environment, researchers are recognizing the need to further develop and refine iPSC-based tools. In this review, we provide an overview of different systems now available for the study of PD, with a particular emphasis on the potential and limitations of iPSC as research tools to generate more relevant models of PD pathophysiology and advance the drug discovery process.}, }
@article {pmid32054864, year = {2020}, author = {Wang, Y and Maeda, Y and Liu, YS and Takada, Y and Ninomiya, A and Hirata, T and Fujita, M and Murakami, Y and Kinoshita, T}, title = {Cross-talks of glycosylphosphatidylinositol biosynthesis with glycosphingolipid biosynthesis and ER-associated degradation.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {860}, pmid = {32054864}, issn = {2041-1723}, mesh = {Acyltransferases/deficiency/genetics/metabolism ; CRISPR-Cas Systems ; *Endoplasmic Reticulum-Associated Degradation/genetics ; Galactosyltransferases/deficiency/genetics/metabolism ; Gene Knockout Techniques ; Glycosphingolipids/*biosynthesis/genetics ; Glycosylphosphatidylinositols/*biosynthesis/genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Models, Molecular ; Recombinant Fusion Proteins/chemistry/genetics/metabolism ; }, abstract = {Glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipids interact with each other in the mammalian plasma membranes, forming dynamic microdomains. How their interaction starts in the cells has been unclear. Here, based on a genome-wide CRISPR-Cas9 genetic screen for genes required for GPI side-chain modification by galactose in the Golgi apparatus, we report that β1,3-galactosyltransferase 4 (B3GALT4), the previously characterized GM1 ganglioside synthase, additionally functions in transferring galactose to the N-acetylgalactosamine side-chain of GPI. Furthermore, B3GALT4 requires lactosylceramide for the efficient GPI side-chain galactosylation. Thus, our work demonstrates previously unexpected functional relationships between GPI-anchored proteins and glycosphingolipids in the Golgi. Through the same screening, we also show that GPI biosynthesis in the endoplasmic reticulum (ER) is severely suppressed by ER-associated degradation to prevent GPI accumulation when the transfer of synthesized GPI to proteins is defective. Our data demonstrates cross-talks of GPI biosynthesis with glycosphingolipid biosynthesis and the ER quality control system.}, }
@article {pmid32029722, year = {2020}, author = {DeWeirdt, PC and Sangree, AK and Hanna, RE and Sanson, KR and Hegde, M and Strand, C and Persky, NS and Doench, JG}, title = {Genetic screens in isogenic mammalian cell lines without single cell cloning.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {752}, pmid = {32029722}, issn = {2041-1723}, mesh = {Apoptosis/genetics ; CRISPR-Cas Systems ; Cell Line ; Clone Cells ; Gene Knockout Techniques ; Gene Library ; Gene Regulatory Networks ; Genetic Testing/*methods ; Humans ; Multigene Family ; Myeloid Cell Leukemia Sequence 1 Protein/antagonists &amp; inhibitors/deficiency/genetics ; Poly (ADP-Ribose) Polymerase-1/antagonists &amp; inhibitors/deficiency/genetics ; Single-Cell Analysis ; bcl-X Protein/antagonists &amp; inhibitors/deficiency/genetics ; }, abstract = {Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs of mammalian cells, however, is labor-intensive, time-consuming, and, in some cell types, essentially impossible. Here, we present an approach to create isogenic pairs of cells that avoids single cell cloning, and screen these pairs with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We query the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, identifying both expected and uncharacterized buffering and synthetic lethal interactions. Additionally, we compare acute CRISPR-based knockout, single cell clones, and small-molecule inhibition. We observe that, while the approaches provide largely overlapping information, differences emerge, highlighting an important consideration when employing genetic screens to identify and characterize potential drug targets. We anticipate that this methodology will be broadly useful to comprehensively study gene function across many contexts.}, }
@article {pmid32004468, year = {2020}, author = {Verma, R and Mohl, D and Deshaies, RJ}, title = {Harnessing the Power of Proteolysis for Targeted Protein Inactivation.}, journal = {Molecular cell}, volume = {77}, number = {3}, pages = {446-460}, doi = {10.1016/j.molcel.2020.01.010}, pmid = {32004468}, issn = {1097-4164}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genetic Engineering/*methods/*trends ; Humans ; Morpholinos/genetics ; Protein Engineering/*methods/*trends ; Protein Transport ; Proteolysis ; }, abstract = {Two decades into the twenty-first century, a confluence of breakthrough technologies wielded at the molecular level is presenting biologists with unique opportunities to unravel the complexities of the cellular world. CRISPR/Cas9 allows gene knock-outs, knock-ins, and single-base editing at chromosomal loci. RNA-based tools such as siRNA, antisense oligos, and morpholinos can be used to silence expression of specific genes. Meanwhile, protein knockdown tools that draw inspiration from natural regulatory mechanisms and facilitate elimination of native or degron-tagged proteins from cells are rapidly emerging. The acute and reversible reduction in protein levels enabled by these methods allows for precise determination of loss-of-function phenotypes free from secondary effects or compensatory adaptation that can confound nucleic-acid-based methods that involve slow depletion or permanent loss of a protein. In this Review, we summarize the ingenious ways biologists have exploited natural mechanisms for protein degradation to direct the elimination of specific proteins at will. This has led to advancements not only in basic research but also in the therapeutic space with the introduction of PROTACs into clinical trials for cancer patients.}, }
@article {pmid32001375, year = {2020}, author = {Espino-Saldaña, AE and Durán-Ríos, K and Olivares-Hernandez, E and Rodríguez-Ortiz, R and Arellano-Carbajal, F and Martínez-Torres, A}, title = {Temporal and spatial expression of zebrafish mctp genes and evaluation of frameshift alleles of mctp2b.}, journal = {Gene}, volume = {738}, number = {}, pages = {144371}, doi = {10.1016/j.gene.2020.144371}, pmid = {32001375}, issn = {1879-0038}, mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Calcium/metabolism ; Embryonic Development/genetics ; Endoplasmic Reticulum/metabolism ; Frameshift Mutation/genetics ; Gene Expression Regulation, Developmental/genetics ; HEK293 Cells ; Humans ; Membrane Proteins/genetics/*metabolism ; Receptors, Calcium-Sensing/genetics ; Zebrafish/genetics ; Zebrafish Proteins/genetics ; }, abstract = {MCTPs (multiple C2 domain proteins with two transmembrane regions) have been proposed as novel endoplasmic reticulum calcium sensors; however, their function remains largely unknown. Here we report the structure of the four mctp genes from zebrafish (mctp1a, mctp1b, mctp2a and mctp2b), their diversity, expression pattern during embryonic development and in adult tissue and the effect of knocking down the expression of Mctp2b by CRISPR/Cas9. The four mctp genes are expressed from early development and exhibit differential expression patterns but are found mainly in the nervous and muscular systems. Mctp2b tagged with fluorescent proteins and expressed in HEK-293 cells and neurons of the fish spinal cord localized mostly in the endoplasmic reticulum but also in lysosomes and late and recycling endosomes. Knocking down mctp2b expression impaired embryonic development, suggesting that the functional participation of this gene is relevant, at least during the early stages of development.}, }
@article {pmid31945142, year = {2020}, author = {Morio, F and Lombardi, L and Butler, G}, title = {The CRISPR toolbox in medical mycology: State of the art and perspectives.}, journal = {PLoS pathogens}, volume = {16}, number = {1}, pages = {e1008201}, pmid = {31945142}, issn = {1553-7374}, mesh = {Aspergillus fumigatus/genetics ; *CRISPR-Cas Systems ; Cryptococcus neoformans/genetics ; Forecasting ; Gene Editing ; Humans ; Mucorales/genetics ; Mycology/*methods ; }, abstract = {Fungal pathogens represent a major human threat affecting more than a billion people worldwide. Invasive infections are on the rise, which is of considerable concern because they are accompanied by an escalation of antifungal resistance. Deciphering the mechanisms underlying virulence traits and drug resistance strongly relies on genetic manipulation techniques such as generating mutant strains carrying specific mutations, or gene deletions. However, these processes have often been time-consuming and cumbersome in fungi due to a number of complications, depending on the species (e.g., diploid genomes, lack of a sexual cycle, low efficiency of transformation and/or homologous recombination, lack of cloning vectors, nonconventional codon usage, and paucity of dominant selectable markers). These issues are increasingly being addressed by applying clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 mediated genetic manipulation to medically relevant fungi. Here, we summarize the state of the art of CRISPR-Cas9 applications in four major human fungal pathogen lineages: Candida spp., Cryptococcus neoformans, Aspergillus fumigatus, and Mucorales. We highlight the different ways in which CRISPR has been customized to address the critical issues in different species, including different strategies to deliver the CRISPR-Cas9 elements, their transient or permanent expression, use of codon-optimized CAS9, and methods of marker recycling and scarless editing. Some approaches facilitate a more efficient use of homology-directed repair in fungi in which nonhomologous end joining is more commonly used to repair double-strand breaks (DSBs). Moreover, we highlight the most promising future perspectives, including gene drives, programmable base editors, and nonediting applications, some of which are currently available only in model fungi but may be adapted for future applications in pathogenic species. Finally, this review discusses how the further evolution of CRISPR technology will allow mycologists to tackle the multifaceted issue of fungal pathogenesis.}, }
@article {pmid31911736, year = {2019}, author = {Eissenberg, JC}, title = {The Brave New World of Gene Editing and Molecular Medicine.}, journal = {Missouri medicine}, volume = {116}, number = {6}, pages = {497-502}, pmid = {31911736}, issn = {0026-6620}, mesh = {*CRISPR-Cas Systems ; Gene Editing/*ethics ; Genetic Therapy/*ethics ; Humans ; Molecular Medicine/trends ; }, abstract = {Gene therapy has long been a promise of molecular biology. So far, that promise has largely been unrealized. The advent of gene editing using technology adapted from bacteria may finally usher in the era of gene therapy.}, }
@article {pmid31881433, year = {2020}, author = {Mushtaq, M and Mukhtar, S and Sakina, A and Dar, AA and Bhat, R and Deshmukh, R and Molla, K and Kundoo, AA and Dar, MS}, title = {Tweaking genome-editing approaches for virus interference in crop plants.}, journal = {Plant physiology and biochemistry : PPB}, volume = {147}, number = {}, pages = {242-250}, doi = {10.1016/j.plaphy.2019.12.022}, pmid = {31881433}, issn = {1873-2690}, mesh = {*CRISPR-Cas Systems ; *Crops, Agricultural/virology ; *Gene Editing/methods ; *Genome, Plant ; *Plant Viruses/physiology ; Reproducibility of Results ; }, abstract = {Plant viruses infect various economically important crops and cause a serious threat to agriculture. As of now, conventional strategies employed are inadequate to circumvent the proliferation of rapidly evolving plant viruses. In this regard, recent advancement in genome-editing approach looks promising to produce plants resistant to DNA/RNA virus infections. Clustered regularly interspaced palindromic repeats (CRISPR) system has been emerged as a promising genome-editing tool that has received special interest because of its ease, competence and reproducibility. Recent studies have demonstrated that CRISPR/Cas9 system has great potential to confer plant immunity by either directly targeting or cleaving the viral genome in both RNA and DNA viruses. Similarly, the approach can be used for targeting the host susceptibility genes more particularly in case of RNA viruses. In the present review, different approaches and strategies being used to improve plant resistance against devastating viruses are discussed in view of recent advances in CRISPR systems. This review also describes the major pitfalls of CRISPR/Cas9 system that utilizes highly efficient and novel platforms to engineer interference to single and multiple plant RNA viruses.}, }
@article {pmid31853092, year = {2019}, author = {}, title = {The scientific events that shaped the decade.}, journal = {Nature}, volume = {576}, number = {7787}, pages = {337-338}, doi = {10.1038/d41586-019-03857-x}, pmid = {31853092}, issn = {1476-4687}, mesh = {*Artificial Intelligence ; Bias ; Big Data ; *CRISPR-Cas Systems ; *Climate Change/statistics &amp; numerical data ; Conservation of Energy Resources ; *Environmental Policy ; Female ; *Gene Editing ; Genomics ; Gravitation ; Humans ; Infant, Newborn ; *Microbiota ; *Physics ; Social Change ; }, }
@article {pmid31853090, year = {2019}, author = {Castelvecchi, D and Cyranoski, D and Gibney, E and Ledford, H and Maxmen, A and Morello, L and Stoye, E and Subbaraman, N and Tollefson, J and Witze, A}, title = {The science news events that shaped 2019.}, journal = {Nature}, volume = {576}, number = {7787}, pages = {350-353}, doi = {10.1038/d41586-019-03838-0}, pmid = {31853090}, issn = {1476-4687}, mesh = {*Agriculture/trends ; Animals ; Artificial Intelligence ; *Astronomy ; Bullying ; CRISPR-Cas Systems ; *Embryo Research ; *Environmental Policy/legislation &amp; jurisprudence/trends ; European Union/organization &amp; administration ; Extinction, Biological ; *Gene Editing/ethics ; *Global Warming/legislation &amp; jurisprudence/prevention &amp; control ; HIV Infections/therapy/virology ; Hemorrhagic Fever, Ebola/drug therapy/prevention &amp; control ; Humans ; Induced Pluripotent Stem Cells/transplantation ; Leadership ; Mars ; Measles/epidemiology ; Meteoroids ; Minor Planets ; Moon ; Politics ; Sexual Harassment ; *Space Flight ; Stem Cell Research ; Strikes, Employee ; United States ; }, }
@article {pmid31852823, year = {2020}, author = {Wu, Q and Xu, F and Liu, L and Char, SN and Ding, Y and Je, BI and Schmelz, E and Yang, B and Jackson, D}, title = {The maize heterotrimeric G protein β subunit controls shoot meristem development and immune responses.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {117}, number = {3}, pages = {1799-1805}, pmid = {31852823}, issn = {1091-6490}, mesh = {Arabidopsis/genetics/metabolism ; Autoimmunity/physiology ; CRISPR-Cas Systems ; GTP-Binding Protein beta Subunits/chemistry/genetics/*metabolism ; Gene Knockout Techniques ; Meristem/cytology/*growth &amp; development/immunology ; Phenotype ; Plant Immunity/*physiology ; Plant Shoots/cytology/*growth &amp; development/immunology ; Plants, Genetically Modified/genetics/metabolism ; Signal Transduction ; Transcriptome ; Zea mays/*metabolism ; }, abstract = {Heterotrimeric G proteins are important transducers of receptor signaling, functioning in plants with CLAVATA receptors in controlling shoot meristem size and with pathogen-associated molecular pattern receptors in basal immunity. However, whether specific members of the heterotrimeric complex potentiate cross-talk between development and defense, and the extent to which these functions are conserved across species, have not yet been addressed. Here we used CRISPR/Cas9 to knock out the maize G protein β subunit gene (Gβ) and found that the mutants are lethal, differing from those in Arabidopsis, in which homologous mutants have normal growth and fertility. We show that lethality is caused not by a specific developmental arrest, but by autoimmunity. We used a genetic diversity screen to suppress the lethal Gβ phenotype and also identified a maize Gβ allele with weak autoimmune responses but strong development phenotypes. Using these tools, we show that Gβ controls meristem size in maize, acting epistatically with G protein α subunit gene (Gα), suggesting that Gβ and Gα function in a common signaling complex. Furthermore, we used an association study to show that natural variation in Gβ influences maize kernel row number, an important agronomic trait. Our results demonstrate the dual role of Gβ in immunity and development in a cereal crop and suggest that it functions in cross-talk between these competing signaling networks. Therefore, modification of Gβ has the potential to optimize the trade-off between growth and defense signaling to improve agronomic production.}, }
@article {pmid31827283, year = {2019}, author = {Wei, J and Long, L and Zheng, W and Dhungana, Y and Lim, SA and Guy, C and Wang, Y and Wang, YD and Qian, C and Xu, B and Kc, A and Saravia, J and Huang, H and Yu, J and Doench, JG and Geiger, TL and Chi, H}, title = {Targeting REGNASE-1 programs long-lived effector T cells for cancer therapy.}, journal = {Nature}, volume = {576}, number = {7787}, pages = {471-476}, doi = {10.1038/s41586-019-1821-z}, pmid = {31827283}, issn = {1476-4687}, support = {AI140761/NH/NIH HHS/United States ; R01 AI131703/AI/NIAID NIH HHS/United States ; CA221290/NH/NIH HHS/United States ; AI150241/NH/NIH HHS/United States ; R01 AI150514/AI/NIAID NIH HHS/United States ; AI131703/NH/NIH HHS/United States ; R37 AI105887/AI/NIAID NIH HHS/United States ; R01 AI140761/AI/NIAID NIH HHS/United States ; AI105887/NH/NIH HHS/United States ; AI150514/NH/NIH HHS/United States ; R01 CA221290/CA/NCI NIH HHS/United States ; R01 AI150241/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; Basic-Leucine Zipper Transcription Factors/deficiency/metabolism ; CD8-Positive T-Lymphocytes/cytology/*immunology ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Female ; Gene Deletion ; Humans ; Immunotherapy, Adoptive/*methods ; Leukemia/genetics/*immunology/metabolism/*therapy ; Lymphocytes, Tumor-Infiltrating/immunology/metabolism ; Melanoma/genetics/*immunology/metabolism/*therapy ; Mice ; Mitochondria/metabolism ; *Molecular Targeted Therapy ; Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics/metabolism ; Reproducibility of Results ; Ribonucleases/deficiency/genetics/immunology/*metabolism ; Suppressor of Cytokine Signaling 1 Protein/genetics/metabolism ; Tumor Microenvironment/immunology ; }, abstract = {Adoptive cell therapy represents a new paradigm in cancer immunotherapy, but it can be limited by the poor persistence and function of transferred T cells1. Here we use an in vivo pooled CRISPR-Cas9 mutagenesis screening approach to demonstrate that, by targeting REGNASE-1, CD8+ T cells are reprogrammed to long-lived effector cells with extensive accumulation, better persistence and robust effector function in tumours. REGNASE-1-deficient CD8+ T cells show markedly improved therapeutic efficacy against mouse models of melanoma and leukaemia. By using a secondary genome-scale CRISPR-Cas9 screening, we identify BATF as the key target of REGNASE-1 and as a rheostat that shapes antitumour responses. Loss of BATF suppresses the increased accumulation and mitochondrial fitness of REGNASE-1-deficient CD8+ T cells. By contrast, the targeting of additional signalling factors-including PTPN2 and SOCS1-improves the therapeutic efficacy of REGNASE-1-deficient CD8+ T cells. Our findings suggest that T cell persistence and effector function can be coordinated in tumour immunity and point to avenues for improving the efficacy of adoptive cell therapy for cancer.}, }
@article {pmid31727847, year = {2019}, author = {Fei, T and Li, W and Peng, J and Xiao, T and Chen, CH and Wu, A and Huang, J and Zang, C and Liu, XS and Brown, M}, title = {Deciphering essential cistromes using genome-wide CRISPR screens.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {50}, pages = {25186-25195}, pmid = {31727847}, issn = {1091-6490}, support = {R01 HG008728/HG/NHGRI NIH HHS/United States ; }, mesh = {Binding Sites ; Breast Neoplasms/genetics/metabolism ; CCCTC-Binding Factor/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Genome, Human ; Hepatocyte Nuclear Factor 3-alpha/genetics/*metabolism ; Humans ; Male ; Promoter Regions, Genetic ; Prostatic Neoplasms/genetics/metabolism ; *Regulatory Elements, Transcriptional ; }, abstract = {Although millions of transcription factor binding sites, or cistromes, have been identified across the human genome, defining which of these sites is functional in a given condition remains challenging. Using CRISPR/Cas9 knockout screens and gene essentiality or fitness as the readout, we systematically investigated the essentiality of over 10,000 FOXA1 and CTCF binding sites in breast and prostate cancer cells. We found that essential FOXA1 binding sites act as enhancers to orchestrate the expression of nearby essential genes through the binding of lineage-specific transcription factors. In contrast, CRISPR screens of the CTCF cistrome revealed 2 classes of essential binding sites. The first class of essential CTCF binding sites act like FOXA1 sites as enhancers to regulate the expression of nearby essential genes, while a second class of essential CTCF binding sites was identified at topologically associated domain (TAD) boundaries and display distinct characteristics. Using regression methods trained on our screening data and public epigenetic profiles, we developed a model to predict essential cis-elements with high accuracy. The model for FOXA1 essentiality correctly predicts noncoding variants associated with cancer risk and progression. Taken together, CRISPR screens of cis-regulatory elements can define the essential cistrome of a given factor and can inform the development of predictive models of cistrome function.}, }
@article {pmid31712408, year = {2019}, author = {Lewis, JJ and Geltman, RC and Pollak, PC and Rondem, KE and Van Belleghem, SM and Hubisz, MJ and Munn, PR and Zhang, L and Benson, C and Mazo-Vargas, A and Danko, CG and Counterman, BA and Papa, R and Reed, RD}, title = {Parallel evolution of ancient, pleiotropic enhancers underlies butterfly wing pattern mimicry.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {48}, pages = {24174-24183}, pmid = {31712408}, issn = {1091-6490}, mesh = {Adaptation, Physiological/genetics ; Animals ; Butterflies/*physiology ; CRISPR-Cas Systems ; Chimera ; *Enhancer Elements, Genetic ; Evolution, Molecular ; *Genetic Pleiotropy ; Genome, Insect ; Genome-Wide Association Study ; Insect Proteins/genetics ; Phylogeny ; Pigmentation/genetics/*physiology ; Promoter Regions, Genetic ; Regulatory Sequences, Nucleic Acid ; Wings, Animal/*physiology ; }, abstract = {Color pattern mimicry in Heliconius butterflies is a classic case study of complex trait adaptation via selection on a few large effect genes. Association studies have linked color pattern variation to a handful of noncoding regions, yet the presumptive cis-regulatory elements (CREs) that control color patterning remain unknown. Here we combine chromatin assays, DNA sequence associations, and genome editing to functionally characterize 5 cis-regulatory elements of the color pattern gene optix We were surprised to find that the cis-regulatory architecture of optix is characterized by pleiotropy and regulatory fragility, where deletion of individual cis-regulatory elements has broad effects on both color pattern and wing vein development. Remarkably, we found orthologous cis-regulatory elements associate with wing pattern convergence of distantly related comimics, suggesting that parallel coevolution of ancestral elements facilitated pattern mimicry. Our results support a model of color pattern evolution in Heliconius where changes to ancient, multifunctional cis-regulatory elements underlie adaptive radiation.}, }
@article {pmid31455654, year = {2019}, author = {Louradour, I and Ghosh, K and Inbar, E and Sacks, DL}, title = {CRISPR/Cas9 Mutagenesis in Phlebotomus papatasi: the Immune Deficiency Pathway Impacts Vector Competence for Leishmania major.}, journal = {mBio}, volume = {10}, number = {4}, pages = {}, pmid = {31455654}, issn = {2150-7511}, mesh = {Alleles ; Amino Acid Sequence ; Animals ; *CRISPR-Cas Systems ; Disease Vectors ; Female ; Gene Editing ; Humans ; Insect Proteins/genetics/*metabolism ; Leishmania major/*physiology ; Male ; Mutagenesis ; Mutation ; Phlebotomus/*genetics/immunology/parasitology/physiology ; Sequence Alignment ; }, abstract = {Sand flies are the natural vectors for the Leishmania species that produce a spectrum of diseases in their mammalian hosts, including humans. Studies of sand fly/Leishmania interactions have been limited by the absence of genome editing techniques applicable to these insects. In this report, we adapted CRISPR (clustered regularly interspaced palindromic repeat)/Cas9 (CRISPR-associated protein 9) technology to the Phlebotomus papatasi sand fly, a natural vector for Leishmania major, targeting the sand fly immune deficiency (IMD) pathway in order to decipher its contribution to vector competence. We established a protocol for transformation in P. papatasi and were able to generate transmissible null mutant alleles for Relish (Rel), the only transcription factor of the IMD pathway. While the maintenance of a homozygous mutant stock was severely compromised, we were able to establish in an early generation their greater susceptibility to infection with L. major Flies carrying different heterozygous mutant alleles variably displayed a more permissive phenotype, presenting higher loads of parasites or greater numbers of infective-stage promastigotes. Together, our data show (i) the successful adaptation of the CRISPR/Cas9 technology to sand flies and (ii) the impact of the sand fly immune response on vector competence for Leishmania parasites.IMPORTANCE Sand flies are the natural vectors of Leishmania parasites. Studies of sand fly/Leishmania interactions have been limited by the lack of successful genomic manipulation of these insects. This paper shows the first example of successful targeted mutagenesis in sand flies via adaptation of the CRISPR/Cas9 editing technique. We generated transmissible null mutant alleles of relish, a gene known to be essential for the control of immune response in other insects. In addition to the expected higher level of susceptibility to bacteria, the mutant flies presented higher loads of parasites when infected with L. major, showing that the sand fly immune response impacts its vector competence for this pathogen.}, }
@article {pmid31404748, year = {2019}, author = {Menges, J and Cremanns, M and Steenpass, L}, title = {Generation of two H1 hESC sublines carrying deletions of RB1 exon 1/promoter in heterozygous or compound heterozygous state.}, journal = {Stem cell research}, volume = {39}, number = {}, pages = {101517}, doi = {10.1016/j.scr.2019.101517}, pmid = {31404748}, issn = {1876-7753}, mesh = {Alleles ; CRISPR-Cas Systems/genetics ; Exons/*genetics ; Fluorescent Antibody Technique ; Heterozygote ; Humans ; Karyotyping ; Male ; Microsatellite Repeats/genetics ; Promoter Regions, Genetic/*genetics ; Retinoblastoma/*genetics ; Retinoblastoma Binding Proteins/genetics ; Ubiquitin-Protein Ligases/genetics ; }, abstract = {Biallelic inactivation of the retinoblastoma tumor suppressor gene (RB1) causes formation of retinoblastoma, a retinal eye tumor occurring in early childhood. Using the CRISPR/Cas9 nickase system, exon 1 of RB1 was deleted, including the RB1 promoter. As a result, sublines were generated carrying deletions of RB1 exon 1/promoter on one or both alleles.}, }
@article {pmid31332481, year = {2020}, author = {Yuan, L and Liu, Q and Wang, Z and Hou, J and Xu, P}, title = {EI24 tethers endoplasmic reticulum and mitochondria to regulate autophagy flux.}, journal = {Cellular and molecular life sciences : CMLS}, volume = {77}, number = {8}, pages = {1591-1606}, pmid = {31332481}, issn = {1420-9071}, mesh = {Animals ; Apoptosis Regulatory Proteins/genetics/*metabolism ; *Autophagy ; CRISPR-Cas Systems ; Cells, Cultured ; Endoplasmic Reticulum/*metabolism ; Female ; HEK293 Cells ; Humans ; Insulin-Secreting Cells/cytology/*metabolism ; Male ; Mice ; Mitochondria/*metabolism ; Nuclear Proteins/genetics/*metabolism ; Voltage-Dependent Anion Channel 1/metabolism ; }, abstract = {Etoposide-induced protein 2.4 (EI24), located on the endoplasmic reticulum (ER) membrane, has been proposed to be an essential autophagy protein. Specific ablation of EI24 in neuronal and liver tissues causes deficiency of autophagy flux. However, the molecular mechanism of the EI24-mediated autophagy process is still poorly understood. Like neurons and hepatic cells, pancreatic β cells are also secretory cells. Pancreatic β cells contain large amounts of ER and continuously synthesize and secrete insulin to maintain blood glucose homeostasis. Yet, the effect of EI24 on autophagy of pancreatic β cells has not been reported. Here, we show that the autophagy process is inhibited in EI24-deficient primary pancreatic β cells. Further mechanistic studies demonstrate that EI24 is enriched at the ER-mitochondria interface and that the C-terminal domain of EI24 is important for the integrity of the mitochondria-associated membrane (MAM) and autophagy flux. Overexpression of EI24, but not the EI24-ΔC mutant, can rescue MAM integrity and decrease the aggregation of p62 and LC3II in the EI24-deficient group. By mass spectrometry-based proteomics following immunoprecipitation, EI24 was found to interact with voltage-dependent anion channel 1 (VDAC1), inositol 1,4,5-trisphosphate receptor (IP3R), and the outer mitochondrial membrane chaperone GRP75. Knockout of EI24 impairs the interaction of IP3R with VDAC1, indicating that these proteins may form a quaternary complex to regulate MAM integrity and the autophagy process.}, }
@article {pmid31327526, year = {2019}, author = {Xu, Y and Zhou, P and Cheng, S and Lu, Q and Nowak, K and Hopp, AK and Li, L and Shi, X and Zhou, Z and Gao, W and Li, D and He, H and Liu, X and Ding, J and Hottiger, MO and Shao, F}, title = {A Bacterial Effector Reveals the V-ATPase-ATG16L1 Axis that Initiates Xenophagy.}, journal = {Cell}, volume = {178}, number = {3}, pages = {552-566.e20}, doi = {10.1016/j.cell.2019.06.007}, pmid = {31327526}, issn = {1097-4172}, mesh = {ADP-Ribosylation ; Autophagy-Related Proteins/deficiency/genetics/*metabolism ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing ; HeLa Cells ; Humans ; *Macroautophagy ; Microtubule-Associated Proteins/metabolism ; Protein Binding ; Salmonella/*metabolism/pathogenicity ; Type III Secretion Systems/metabolism ; Vacuolar Proton-Translocating ATPases/genetics/*metabolism ; Virulence Factors/*genetics/metabolism ; }, abstract = {Antibacterial autophagy (xenophagy) is an important host defense, but how it is initiated is unclear. Here, we performed a bacterial transposon screen and identified a T3SS effector SopF that potently blocked Salmonella autophagy. SopF was a general xenophagy inhibitor without affecting canonical autophagy. S. Typhimurium ΔsopF resembled S. flexneri ΔvirAΔicsB with the majority of intracellular bacteria targeted by autophagy, permitting a CRISPR screen that identified host V-ATPase as an essential factor. Upon bacteria-caused vacuolar damage, the V-ATPase recruited ATG16L1 onto bacteria-containing vacuole, which was blocked by SopF. Mammalian ATG16L1 bears a WD40 domain required for interacting with the V-ATPase. Inhibiting autophagy by SopF promoted S. Typhimurium proliferation in vivo. SopF targeted Gln124 of ATP6V0C in the V-ATPase for ADP-ribosylation. Mutation of Gln124 also blocked xenophagy, but not canonical autophagy. Thus, the discovery of SopF reveals the V-ATPase-ATG16L1 axis that critically mediates autophagic recognition of intracellular pathogen.}, }
@article {pmid31280136, year = {2019}, author = {Giri, S and Purushottam, M and Viswanath, B and Muddashetty, RS}, title = {Generation of a FMR1 homozygous knockout human embryonic stem cell line (WAe009-A-16) by CRISPR/Cas9 editing.}, journal = {Stem cell research}, volume = {39}, number = {}, pages = {101494}, doi = {10.1016/j.scr.2019.101494}, pmid = {31280136}, issn = {1876-7753}, mesh = {Blotting, Western ; CRISPR-Cas Systems/*genetics ; Cell Differentiation/genetics/physiology ; Cell Line ; Embryoid Bodies/cytology/metabolism ; Exons/genetics ; Fragile X Mental Retardation Protein/*genetics ; Genotype ; Human Embryonic Stem Cells/*cytology/*metabolism ; Humans ; Immunohistochemistry ; Karyotype ; RNA-Binding Proteins/genetics ; }, abstract = {Mutations in FMR1 gene is the cause of Fragile X Syndrome (FXS) leading inherited cause of intellectual disability and autism spectrum disorders. FMR1 gene encodes Fragile X Mental Retardation Protein (FMRP) which is a RNA binding protein and play important role in synaptic plasticity and translational regulation in neurons. We have generated a homozygous FMR1 knockout (FMR1-KO) hESC line using CRISPR/Cas9 based genome editing. It created a homozygous 280 nucleotide deletion at exon1, removing the start codon. This FMR1-KO cell line maintains stem cell like morphology, pluripotency, normal karyotype and ability to in-vitro differentiation.}, }
@article {pmid31229900, year = {2019}, author = {Li, C and Wang, Q and Peng, Z and Lin, Y and Liu, H and Yang, X and Li, S and Liu, X and Chen, J}, title = {Generation of FOS gene knockout lines from a human embryonic stem cell line using CRISPR/Cas9.}, journal = {Stem cell research}, volume = {39}, number = {}, pages = {101479}, doi = {10.1016/j.scr.2019.101479}, pmid = {31229900}, issn = {1876-7753}, mesh = {CRISPR-Cas Systems/genetics/*physiology ; Cell Differentiation/genetics/physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Embryonic Stem Cells/metabolism ; Endothelial Cells ; Gene Knockout Techniques ; Hematopoietic Stem Cells/metabolism ; Human Embryonic Stem Cells/metabolism ; Humans ; Karyotype ; }, abstract = {FOS is component of the AP-1 complex and has been reported to be involved in many cellular functions, including cell proliferation, differentiation, survival, angiogenesis, hematopoiesis and cancer progress. To further understand the exact role of FOS in these processes, here we created two FOS knockout human embryonic stem cell lines by CRISPR/Cas9 mediated gene targeting. These cell lines retained normal morphology and karyotype, normal expression of pluripotent markers, and differentiation potential both in vivo and in vitro. These cell lines can be used to verify whether the FOS mutated produces any affect on endothelial cells and hematopoietic progenitor cells during the hematopoietic differentiation.}, }
@article {pmid31125290, year = {2019}, author = {Demireva, EY and Xie, H and Flood, ED and Thompson, JM and Seitz, BM and Watts, SW}, title = {Creation of the 5-hydroxytryptamine receptor 7 knockout rat as a tool for cardiovascular research.}, journal = {Physiological genomics}, volume = {51}, number = {7}, pages = {290-301}, pmid = {31125290}, issn = {1531-2267}, mesh = {Animals ; *Animals, Genetically Modified ; Blood Pressure/drug effects ; Body Weight/genetics ; CRISPR-Cas Systems ; Cardiovascular Diseases/*genetics ; Cardiovascular System/metabolism ; Exons ; Female ; Frameshift Mutation ; Gene Deletion ; *Gene Knockout Techniques ; Genotype ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Serotonin/*genetics ; Serotonin/pharmacology ; }, abstract = {Using CRISPR-Cas9 technology, we created a 5-HT7 receptor global knockout (KO) rat, on a Sprague-Dawley background, for use in cardiovascular physiology studies focused on blood pressure regulation. A stable line carrying indels in exons 1 and 2 of the rat Htr7 locus was established and validated. Surprisingly, 5-HT7 receptor mRNA was still present in the KO rat. However, extensive cDNA and genomic sequencing of KO tissues confirmed an 11 bp deletion in exon 1 and 4 bp deletion in exon 2. The exon 1 deletion resulted in a frameshifted mRNA sequence coding for a nonfunctional protein. While the Htr1B locus was a potential off-target for the guide RNAs designed for exon 2 of Htr7, there were no off-target sequence changes at this locus in the originating founder. When the F2 generation of KO was compared with wild-type (WT) counterparts, neither the male nor female KO rats were different in body size, fat weights, or mass of organs (kidney, heart, and brain) important to blood pressure. Females were smaller in mass than their counterpart males. Clinical measures of plasma from nonfasted rats revealed largely similar values, comparing WT and KO, of glucose, blood urea nitrogen, creatinine, phosphate, calcium, and albumin to name a few. Loss of a functional 5-HT7 receptor was validated by the complete loss of relaxation to the 5-HT1/7 receptor agonist 5-carboxamidotryptamine in the isolated abdominal vena cava. This newly created 5-HT7 receptor KO rat will be of use to investigate the importance of the 5-HT7 receptor in blood pressure regulation.}, }
@article {pmid31001583, year = {2019}, author = {Cai, Y and Cheng, T and Yao, Y and Li, X and Ma, Y and Li, L and Zhao, H and Bao, J and Zhang, M and Qiu, Z and Xue, T}, title = {In vivo genome editing rescues photoreceptor degeneration via a Cas9/RecA-mediated homology-directed repair pathway.}, journal = {Science advances}, volume = {5}, number = {4}, pages = {eaav3335}, pmid = {31001583}, issn = {2375-2548}, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cyclic Nucleotide Phosphodiesterases, Type 6/genetics ; *DNA Repair ; Electroporation ; Gene Editing/*methods ; Humans ; Mice ; Mice, Inbred C57BL ; RNA, Guide/metabolism ; Rec A Recombinases/*metabolism ; Retinal Degeneration/therapy ; Retinal Rod Photoreceptor Cells/metabolism ; }, abstract = {Although Cas9-mediated genome editing has been widely used to engineer alleles in animal models of human inherited diseases, very few homology-directed repair (HDR)-based genetic editing systems have been established in postnatal mouse models for effective and lasting phenotypic rescue. Here, we developed an HDR-based Cas9/RecA system to precisely correct Pde6b mutation with increased HDR efficiency in postnatal rodless (rd1) mice, a retinitis pigmentosa (RP) mutant model characterized by photoreceptor degeneration and loss of vision. The Cas9/RecA system incorporated Cas9 endonuclease enzyme to generate double-strand breaks (DSBs) and bacterial recombinase A (RecA) to increase homologous recombination. Our data revealed that Cas9/RecA treatment significantly promoted the survival of both rod and cone photoreceptors, restored the expression of PDE6B in rod photoreceptors, and enhanced the visual functions of rd1 mice. Thus, this study provides a precise therapeutic strategy for RP and other genetic diseases.}, }
@article {pmid30956114, year = {2019}, author = {Roberts, B and Hendershott, MC and Arakaki, J and Gerbin, KA and Malik, H and Nelson, A and Gehring, J and Hookway, C and Ludmann, SA and Yang, R and Haupt, A and Grancharova, T and Valencia, V and Fuqua, MA and Tucker, A and Rafelski, SM and Gunawardane, RN}, title = {Fluorescent Gene Tagging of Transcriptionally Silent Genes in hiPSCs.}, journal = {Stem cell reports}, volume = {12}, number = {5}, pages = {1145-1158}, pmid = {30956114}, issn = {2213-6711}, mesh = {Actinin/genetics/metabolism ; Amino Acid Sequence ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Line ; DNA End-Joining Repair/genetics ; Gene Editing/*methods ; Gene Expression Regulation ; Green Fluorescent Proteins/genetics/metabolism ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Myocytes, Cardiac/cytology/*metabolism ; Sarcomeres/*genetics/metabolism ; Sequence Homology, Amino Acid ; Troponin I/genetics/metabolism ; }, abstract = {We describe a multistep method for endogenous tagging of transcriptionally silent genes in human induced pluripotent stem cells (hiPSCs). A monomeric EGFP (mEGFP) fusion tag and a constitutively expressed mCherry fluorescence selection cassette were delivered in tandem via homology-directed repair to five genes not expressed in hiPSCs but important for cardiomyocyte sarcomere function: TTN, MYL7, MYL2, TNNI1, and ACTN2. CRISPR/Cas9 was used to deliver the selection cassette and subsequently mediate its excision via microhomology-mediated end-joining and non-homologous end-joining. Most excised clones were effectively tagged, and all properly tagged clones expressed the mEGFP fusion protein upon differentiation into cardiomyocytes, allowing live visualization of these cardiac proteins at the sarcomere. This methodology provides a broadly applicable strategy for endogenously tagging transcriptionally silent genes in hiPSCs, potentially enabling their systematic and dynamic study during differentiation and morphogenesis.}, }
@article {pmid30860365, year = {2019}, author = {Qin, P and Park, M and Alfson, KJ and Tamhankar, M and Carrion, R and Patterson, JL and Griffiths, A and He, Q and Yildiz, A and Mathies, R and Du, K}, title = {Rapid and Fully Microfluidic Ebola Virus Detection with CRISPR-Cas13a.}, journal = {ACS sensors}, volume = {4}, number = {4}, pages = {1048-1054}, doi = {10.1021/acssensors.9b00239}, pmid = {30860365}, issn = {2379-3694}, mesh = {Base Sequence ; *CRISPR-Cas Systems ; Ebolavirus/genetics/*isolation &amp; purification ; Endoribonucleases/*chemistry ; Fluorometry ; Lab-On-A-Chip Devices ; Leptotrichia/enzymology ; Limit of Detection ; Microfluidic Analytical Techniques/instrumentation/*methods ; Nucleic Acid Hybridization ; Point-of-Care Testing ; RNA, Viral/chemistry/genetics ; }, abstract = {Highly infectious illness caused by pathogens is endemic especially in developing nations where there is limited laboratory infrastructure and trained personnel. Rapid point-of-care (POC) serological assays with minimal sample manipulation and low cost are desired in clinical practice. In this study, we report an automated POC system for Ebola RNA detection with RNA-guided RNA endonuclease Cas13a, utilizing its collateral RNA degradation after its activation. After automated microfluidic mixing and hybridization, nonspecific cleavage products of Cas13a are immediately measured by a custom integrated fluorometer which is small in size and convenient for in-field diagnosis. Within 5 min, a detection limit of 20 pfu/mL (5.45 × 107 copies/mL) of purified Ebola RNA is achieved. This isothermal and fully solution-based diagnostic method is rapid, amplification-free, simple, and sensitive, thus establishing a key technology toward a useful POC diagnostic platform.}, }
@article {pmid30805989, year = {2019}, author = {Shi, X and Xiao, Z and Zonta, F and Wang, W and Wan, Y and Li, Y and Wang, N and Kuang, Y and Du, M and Dong, J and Wang, J and Yang, G}, title = {Somatic MIWI2 Hinders Direct Lineage Reprogramming From Fibroblast to Hepatocyte.}, journal = {Stem cells (Dayton, Ohio)}, volume = {37}, number = {6}, pages = {803-812}, pmid = {30805989}, issn = {1549-4918}, mesh = {Albumins/genetics/metabolism ; Animals ; Argonaute Proteins/deficiency/*genetics ; CRISPR-Cas Systems ; Cell Lineage/genetics ; Cell Transdifferentiation/genetics ; Cellular Reprogramming/*genetics ; *Epigenesis, Genetic ; Fibroblasts/cytology/*metabolism ; Gene Regulatory Networks ; Genetic Vectors/chemistry/metabolism ; Hepatocyte Nuclear Factor 1-alpha/genetics/metabolism ; Hepatocyte Nuclear Factor 3-gamma/genetics/metabolism ; Hepatocytes/cytology/*metabolism ; Lentivirus/genetics/metabolism ; Mice ; Mice, Knockout ; RNA, Small Interfering/*genetics/metabolism ; Receptors, Notch/genetics/metabolism ; Signal Transduction ; Transduction, Genetic ; }, abstract = {Remodeling of the gene regulatory network in cells is believed to be a prerequisite for their lineage reprogramming. However, its key regulatory factors are not yet elucidated. In this article, we investigate the role of PIWI proteins and provide evidence that one of them, MIWI2, is elicited during transdifferentiation of fibroblasts into hepatocyte-like cells. In coincidence with the peak expression of MIWI2, we identified the appearance of a unique intermediate epigenetic state characterized by a specific Piwi-interacting RNA (piRNA) profile consisting of 219 novel sequences. Knockout of MIWI2 greatly improved the formation of the induced hepatocytes, whereas overexpression of exogenous MIWI2 completely abolished the stimulated effect. A bioinformatics analysis of piRNA interaction network, followed by experimental validation, revealed the Notch signaling pathway as one of the immediate effectors of MIWI2. Altogether, our results show for the first time that temporal expression of MIWI2 contributes negatively to cell plasticity not only in germline, but also in developed cells, such as mouse fibroblasts. Stem Cells 2019;37:803-812.}, }
@article {pmid30801858, year = {2019}, author = {Yu, J and Lu, W and Ge, T and Huang, R and Chen, B and Ye, M and Bai, Y and Shi, G and Songyang, Z and Ma, W and Huang, J}, title = {Interaction Between Sympk and Oct4 Promotes Mouse Embryonic Stem Cell Proliferation.}, journal = {Stem cells (Dayton, Ohio)}, volume = {37}, number = {6}, pages = {743-753}, doi = {10.1002/stem.2992}, pmid = {30801858}, issn = {1549-4918}, mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Gene Deletion ; Gene Expression Profiling ; *Gene Expression Regulation, Developmental ; Genes, Reporter ; *Genome ; Genomic Instability ; Green Fluorescent Proteins/genetics/metabolism ; Mice ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Nuclear Proteins/deficiency/*genetics ; Octamer Transcription Factor-3/*genetics/metabolism ; Pluripotent Stem Cells/cytology/*metabolism ; Signal Transduction ; Teratoma/genetics/metabolism/pathology ; Tight Junctions/metabolism ; }, abstract = {The scaffold protein Symplekin (Sympk) is involved in cytoplasmic RNA polyadenylation, transcriptional modulation, and the regulation of epithelial differentiation and proliferation via tight junctions. It is highly expressed in embryonic stem cells (ESCs), in which its role remains unknown. In this study, we found Sympk overexpression in mouse ESCs significantly increased colony formation, and Sympk deletion via CRISPR/Cas9 decreased colony formation. Sympk promoted ESC growth and its overexpression sustained ESC pluripotency, as assessed by teratoma and chimeric mouse formation. Genomic stability was preserved in these cells after long-term passage. The domain of unknown function 3453 (DUF3453) in Sympk was required for its interaction with the key pluripotent factor Oct4, and its depletion led to impaired colony formation. Sympk activated proliferation-related genes and suppressed differentiation-related genes. Our results indicate that Sympk interacts with Oct4 to promote self-renewal and pluripotency in ESCs and preserves genome integrity; accordingly, it has potential value for stem cell therapies. Stem Cells 2019;37:743-753.}, }
@article {pmid32334351, year = {2020}, author = {Wang, YQ and Li, W and Zhuang, JL and Liu, YD and Shapleigh, JP}, title = {Bacteriophage-mediated extracellular DNA release is important for the structural stability of aerobic granular sludge.}, journal = {The Science of the total environment}, volume = {726}, number = {}, pages = {138392}, doi = {10.1016/j.scitotenv.2020.138392}, pmid = {32334351}, issn = {1879-1026}, abstract = {The aim of this study was to investigate the microbial characteristics and the structural role of exDNA in different size AGSs. Metagenomic results showed that exDNA has a significantly lower GC content, ~46.0%, than the ~65.0% GC of intracellular DNA (inDNA). Taxonomic predictions showed most of the reads from the exDNA that could be taxonomically assigned were from members of the phyla Bacteroidetes (55.0-64.2% of the total exDNA reads). Assigned inDNA reads were mainly from Proteobacteria (50.9-57.8%) or Actinobacteria (18.0-28.0%). Reads mapping showed that exDNA read depths were similar across all predicted open reading frames from assembled genomes that were assigned as Bacteroidetes which is consistent with cell lysis as a source of exDNA. Enrichment of CRISPR-CAS proteins in exDNA reads and CRISPR spacers in Bacteroidetes associated draft genomes suggested that bacteriophage infection may be an important cause of lysis of these cells. A critical role for this exDNA was found using DNase I digestion experiments which showed that the exDNA was vital for the structural stability of relatively small sized AGS but not for the larger sized AGS. The characteristics of exDNA in AGSs revealed in this work provide a new perspective on AGS components and structural stability.}, }
@article {pmid32334080, year = {2020}, author = {Mizuno-Iijima, S and Ayabe, S and Kato, K and Matoba, S and Ikeda, Y and Thi Huong Tra, D and Thu Le, H and Suzuki, H and Nakashima, K and Hasegawa, Y and Hamada, Y and Tanimoto, Y and Daitoku, Y and Iki, N and Ishida, M and Elsayed Ibrahim Elzeftawy, A and Nakashiba, T and Hamada, M and Murata, K and Miwa, Y and Okada-Iwabu, M and Iwabu, M and Yagami, KI and Ogura, A and Obata, Y and Takahashi, S and Mizuno, S and Yoshiki, A and Sugiyama, F}, title = {Efficient production of large deletion and gene fragment knock-in mice mediated by genome editing with Cas9-mouse Cdt1 in mouse zygotes.}, journal = {Methods (San Diego, Calif.)}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.ymeth.2020.04.007}, pmid = {32334080}, issn = {1095-9130}, abstract = {Genetically modified mouse models are essential for in vivo investigation of gene function and human disease research. Targeted mutations can be introduced into mouse embryos using genome editing technology such as CRISPR-Cas. Although mice with small indel mutations can be produced, the production of mice carrying large deletions or gene fragment knock-in alleles remains inefficient. We introduced the nuclear localisation property of Cdt1 protein into the CRISPR-Cas system for efficient production of genetically engineered mice. Mouse Cdt1-connected Cas9 (Cas9-mC) was present in the nucleus of HEK293T cells and mouse embryos. Cas9-mC induced a bi-allelic full deletion of Dmd, GC-rich fragment knock-in, and floxed allele knock-in with high efficiency compared to standard Cas9. These results indicate that Cas9-mC is a useful tool for producing mouse models carrying targeted mutations.}, }
@article {pmid32330873, year = {2020}, author = {Hewes, AM and Sansbury, BM and Barth, S and Tarcic, G and Kmiec, EB}, title = {gRNA Sequence Heterology Tolerance Catalyzed by CRISPR/Cas in an In Vitro Homology-Directed Repair Reaction.}, journal = {Molecular therapy. Nucleic acids}, volume = {20}, number = {}, pages = {568-579}, doi = {10.1016/j.omtn.2020.03.012}, pmid = {32330873}, issn = {2162-2531}, abstract = {CRISPR and associated Cas nucleases are genetic engineering tools revolutionizing innovative approaches to cancer and inherited diseases. CRISPR-directed gene editing relies heavily on proper DNA sequence alignment between the guide RNA (gRNA)/CRISPR complex and its genomic target. Accurate hybridization of complementary DNA initiates gene editing in human cells, but inherent gRNA sequence variation that could influence the gene editing reaction has been clearly established among diverse genetic populations. As this technology advances toward clinical implementation, it will be essential to assess what degree of gRNA variation generates unwanted and erroneous CRISPR activity. With the use of a system in which a cell-free extract catalyzes nonhomologous end joining (NHEJ) and homology-directed repair (HDR), it is possible to observe a more representative population of all forms of gene editing outcomes. In this manuscript, we demonstrate CRISPR/Cas complexation at heterologous binding sites that facilitate precise and error-prone HDR. The tolerance of mispairing between the gRNA and target site of the DNA to enable HDR is surprisingly high and greatly influenced by polarity of the donor DNA strand in the reaction. These results suggest that some collateral genomic activity could occur at unintended sites in CRISPR-directed gene editing in human cells.}, }
@article {pmid32329776, year = {2020}, author = {Jacobsen, T and Ttofali, F and Liao, C and Manchalu, S and Gray, BN and Beisel, CL}, title = {Characterization of Cas12a nucleases reveals diverse PAM profiles between closely-related orthologs.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkaa272}, pmid = {32329776}, issn = {1362-4962}, abstract = {CRISPR-Cas systems comprise diverse adaptive immune systems in prokaryotes whose RNA-directed nucleases have been co-opted for various technologies. Recent efforts have focused on expanding the number of known CRISPR-Cas subtypes to identify nucleases with novel properties. However, the functional diversity of nucleases within each subtype remains poorly explored. Here, we used cell-free transcription-translation systems and human cells to characterize six Cas12a single-effector nucleases from the V-A subtype, including nucleases sharing high sequence identity. While these nucleases readily utilized each other's guide RNAs, they exhibited distinct PAM profiles and apparent targeting activities that did not track based on phylogeny. In particular, two Cas12a nucleases encoded by Prevotella ihumii (PiCas12a) and Prevotella disiens (PdCas12a) shared over 95% amino-acid identity yet recognized distinct PAM profiles, with PiCas12a but not PdCas12a accommodating multiple G's in PAM positions -2 through -4 and T in position -1. Mutational analyses transitioning PiCas12a to PdCas12a resulted in PAM profiles distinct from either nuclease, allowing more flexible editing in human cells. Cas12a nucleases therefore can exhibit widely varying properties between otherwise related orthologs, suggesting selective pressure to diversify PAM recognition and supporting expansion of the CRISPR toolbox through ortholog mining and PAM engineering.}, }
@article {pmid32327470, year = {2020}, author = {Opsomer, R and Contino, S and Perrin, F and Gualdani, R and Tasiaux, B and Doyen, P and Vergouts, M and Vrancx, C and Doshina, A and Pierrot, N and Octave, JN and Gailly, P and Stanga, S and Kienlen-Campard, P}, title = {Amyloid Precursor Protein (APP) controls the expression of the transcriptional activator Neuronal PAS Domain Protein 4 (NPAS4) and synaptic GABA release.}, journal = {eNeuro}, volume = {}, number = {}, pages = {}, doi = {10.1523/ENEURO.0322-19.2020}, pmid = {32327470}, issn = {2373-2822}, abstract = {The Amyloid Precursor Protein (APP) has been extensively studied as the precursor of the β-amyloid peptide (Aβ) peptide, the major component of the senile plaques found in the brain of Alzheimer's disease (AD) patients. However, the function of APP per se in neuronal physiology remains to be fully elucidated. APP is expressed at high levels in the brain. It resembles a cell adhesion molecule or a membrane receptor, suggesting that its function relies on cell-cell interaction and/or activation of intracellular signaling pathways. In this respect, the APP intracellular domain (AICD) was reported to act as a transcriptional regulator. Here, we used a transcriptome-based approach to identify the genes transcriptionally regulated by APP in the rodent embryonic cortex and upon maturation of primary cortical neurons. Surprisingly, the overall transcriptional changes were subtle, but a more detailed analysis pointed to genes clustered in neuronal-activity dependent pathways. In particular, we observed a decreased transcription of Neuronal PAS domain protein 4 (NPAS4) in APP-/- neurons. NPAS4 is an inducible transcription factor (ITF) regulated by neuronal depolarization. The down-regulation of NPAS4 co-occurs with an increased production of the inhibitory neurotransmitter GABA and a reduced expression of the GABAA receptors alpha1. CRISPR-Cas-mediated silencing of NPAS4 in neurons led to similar observations. Patch-clamp investigation did not reveal any functional decrease of GABAA receptors activity, but LTP measurement supported an increased GABA component in synaptic transmission of APP-/- mice. Together, NPAS4 appears to be a downstream target involved in APP-dependent regulation of inhibitory synaptic transmission.Significance Statement The Amyloid Precursor Protein (APP) is a key player in Alzheimer's disease (AD) pathogenesis. We report the down-regulation of the activity-dependent transcription factor Neuronal PAS domain protein 4 (NPAS4) in APP-deficient neurons, along with an increase in GABAergic neuron markers and GABA release, but not in excitatory glutamatergic markers. We identified NPAS4 as an APP target gene by a transcriptome analysis of APP+/+ versus APP-/- primary cortical neurons at different stages of differentiation. The downregulation of NPAS4 observed in APP-/- neurons was confirmed by APP silencing with a CRISPR/Cas9 approach. CRISPR/Cas9-silencing of NPAS4 mimicked APP deficiency and increased GABAergic markers. The activity-dependent transcription factor NPAS4 is therefore a key downstream target in the synaptic functions regulated by APP.}, }
@article {pmid32326901, year = {2020}, author = {Hahn, F and Korolev, A and Sanjurjo Loures, L and Nekrasov, V}, title = {A modular cloning toolkit for genome editing in plants.}, journal = {BMC plant biology}, volume = {20}, number = {1}, pages = {179}, doi = {10.1186/s12870-020-02388-2}, pmid = {32326901}, issn = {1471-2229}, support = {BB/P016855/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R019827/1//Newton Fund/ ; }, abstract = {BACKGROUND: CRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs.<br><br>RESULTS: Here we present an expanded cloning toolkit that contains 103 modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts.<br><br>CONCLUSIONS: We believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.}, }
@article {pmid32315229, year = {2020}, author = {Davies, K and Barrangou, R}, title = {COVID-19 and the CRISPR Community Response.}, journal = {The CRISPR journal}, volume = {3}, number = {2}, pages = {66-67}, doi = {10.1089/crispr.2020.29092.rba}, pmid = {32315229}, issn = {2573-1602}, mesh = {Betacoronavirus ; Biomedical Research/trends ; *CRISPR-Cas Systems ; Clinical Laboratory Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Coronavirus Infections/*diagnosis/drug therapy/*therapy ; Humans ; Pandemics ; Pneumonia, Viral/*diagnosis/*therapy ; }, }
@article {pmid31162951, year = {2019}, author = {Wang, Y and Kuai, Q and Gao, F and Wang, Y and He, M and Zhou, H and Han, G and Jiang, X and Ren, S and Yu, Q}, title = {Overexpression of TIM-3 in Macrophages Aggravates Pathogenesis of Pulmonary Fibrosis in Mice.}, journal = {American journal of respiratory cell and molecular biology}, volume = {61}, number = {6}, pages = {727-736}, doi = {10.1165/rcmb.2019-0070OC}, pmid = {31162951}, issn = {1535-4989}, mesh = {Adoptive Transfer ; Animals ; Bleomycin/toxicity ; CRISPR-Cas Systems ; Disease Models, Animal ; Hepatitis A Virus Cellular Receptor 2/*blood/deficiency/genetics/*physiology ; Humans ; Idiopathic Pulmonary Fibrosis/chemically induced/genetics/*pathology ; Interleukin-10/biosynthesis ; Lung/pathology ; Macrophages, Alveolar/drug effects/*metabolism/transplantation ; Macrophages, Peritoneal/metabolism/pathology ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; RAW 264.7 Cells ; RNA, Messenger/biosynthesis ; Transforming Growth Factor beta1/biosynthesis ; }, abstract = {Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive lung disorder and lacks effective treatments because of unclear mechanisms. Aberrant function of alveolar macrophages is directly linked to pulmonary fibrosis. Here, we show TIM-3 (T-cell immunoglobulin domain and mucin domain-3), a key regulator of macrophage function, aggravates pulmonary fibrosis. TIM-3 mRNA of patients with IPF was analyzed based on the Gene Expression Omnibus and Array Express databases. Lung pathology and profibrotic molecules were assessed in a bleomycin (BLM)-induced pulmonary fibrosis model using wild-type (WT) and TIM-3 transgenic (TIM-3-TG) mice. Macrophage cells, RAW264.7, were then applied to investigate the effect of macrophage TIM-3 under BLM exposure in vitro. Macrophage depletion and adoptive-transfer experiments were finally performed to examine lung morphology and profibrotic molecules. TIM-3 expression was increased both in patients with IPF and in our BLM-induced mouse model. TIM-3-TG mice developed more serious pathological changes in lung tissue and higher expressions of TGF-β1 (transforming growth factor-β1) and IL-10 than WT mice. After BLM treatment, TGF-β1 and IL-10 expression was significantly decreased in RAW264.7 cells after TIM-3 knock-out, whereas it was increased in TIM-3-TG peritoneal macrophages. The scores of pulmonary fibrosis in WT and TIM-3-TG mice were significantly reduced, and there was no difference between them after macrophage depletion. Furthermore, WT mice receiving adoptive macrophages from TIM-3-TG mice also had more serious lung fibrosis and increased expression of TGF-β1 and IL-10 than those receiving macrophages from WT mice. Our findings revealed that overexpressed TIM-3 in alveolar macrophages aggravated pulmonary fibrosis.}, }
@article {pmid30863962, year = {2019}, author = {Wang, X and Lu, J and Lao, K and Wang, S and Mo, X and Xu, X and Chen, X and Mo, B}, title = {Increasing the efficiency of CRISPR/Cas9-based gene editing by suppressing RNAi in plants.}, journal = {Science China. Life sciences}, volume = {62}, number = {7}, pages = {982-984}, doi = {10.1007/s11427-019-9514-9}, pmid = {30863962}, issn = {1869-1889}, mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing ; Mutation ; Phenotype ; Plant Viruses/genetics ; Plants/*genetics ; Plants, Genetically Modified/*genetics ; Promoter Regions, Genetic ; RNA Interference ; Signal Transduction ; }, }
@article {pmid32325051, year = {2020}, author = {Osuna, BA and Karambelkar, S and Mahendra, C and Sarbach, A and Johnson, MC and Kilcher, S and Bondy-Denomy, J}, title = {Critical Anti-CRISPR Locus Repression by a Bi-functional Cas9 Inhibitor.}, journal = {Cell host &amp; microbe}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.chom.2020.04.002}, pmid = {32325051}, issn = {1934-6069}, abstract = {Bacteriophages must rapidly deploy anti-CRISPR proteins (Acrs) to inactivate the RNA-guided nucleases that enforce CRISPR-Cas adaptive immunity in their bacterial hosts. Listeria monocytogenes temperate phages encode up to three anti-Cas9 proteins, with acrIIA1 always present. AcrIIA1 binds and inhibits Cas9 with its C-terminal domain; however, the function of its highly conserved N-terminal domain (NTD) is unknown. Here, we report that the AcrIIA1NTD is a critical transcriptional repressor of the strong anti-CRISPR promoter. A rapid burst of anti-CRISPR transcription occurs during phage infection and the subsequent negative feedback by AcrIIA1NTD is required for optimal phage replication, even in the absence of CRISPR-Cas immunity. In the presence of CRISPR-Cas immunity, full-length AcrIIA1 uses its two-domain architecture to act as a &quot;Cas9 sensor,&quot; tuning acr expression according to Cas9 levels. Finally, we identify AcrIIA1NTD homologs in other Firmicutes and demonstrate that they have been co-opted by hosts as &quot;anti-anti-CRISPRs,&quot; repressing phage anti-CRISPR deployment.}, }
@article {pmid32325050, year = {2020}, author = {Osuna, BA and Karambelkar, S and Mahendra, C and Christie, KA and Garcia, B and Davidson, AR and Kleinstiver, BP and Kilcher, S and Bondy-Denomy, J}, title = {Listeria Phages Induce Cas9 Degradation to Protect Lysogenic Genomes.}, journal = {Cell host &amp; microbe}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.chom.2020.04.001}, pmid = {32325050}, issn = {1934-6069}, abstract = {Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy phage (viral) DNA. Phages, in turn, have evolved diverse &quot;anti-CRISPR&quot; proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode two to three distinct anti-Cas9 proteins, with acrIIA1 always present. However, the significance of AcrIIA1's pervasiveness and its mechanism are unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain. During lysogeny in Listeria, AcrIIA1 triggers Cas9 degradation. During lytic infection, however, AcrIIA1 fails to block Cas9 due to its multi-step inactivation mechanism. Thus, phages encode an additional Acr that rapidly binds and inactivates Cas9. AcrIIA1 also uniquely inhibits a highly diverged Cas9 found in Listeria (similar to SauCas9) and Type II-C Cas9s, likely due to Cas9 HNH domain conservation. In summary, Listeria phages inactivate Cas9 in lytic growth using variable, narrow-spectrum inhibitors, while the broad-spectrum AcrIIA1 stimulates Cas9 degradation for protection of the lysogenic genome.}, }
@article {pmid32322250, year = {2020}, author = {Yang, L and Li, W and Ujiroghene, OJ and Yang, Y and Lu, J and Zhang, S and Pang, X and Lv, J}, title = {Occurrence and Diversity of CRISPR Loci in Lactobacillus casei Group.}, journal = {Frontiers in microbiology}, volume = {11}, number = {}, pages = {624}, doi = {10.3389/fmicb.2020.00624}, pmid = {32322250}, issn = {1664-302X}, abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is an adaptive immune system that resists foreign genes through nuclease targeting in bacteria and archaea. In this study, we analyzed 68 strains of Lactobacillus casei group from the NCBI GenBank database, and bioinformatic tools were used to investigate the occurrence and diversity of CRISPR system. The results showed that a total of 30 CRISPR loci were identified from 27 strains. Apart from three strains which contained double loci with distinguishable distributed sites, most strains contained only one CRISPR locus. The analysis of direct repeat (DR) sequences showed that all DR could form stable RNA secondary structures. The CRISPR spacers showed diversity, and their origin and evolution were revealed through the investigation of their spacer sequences. In addition, a large number of CRISPR spacers showed perfect homologies to phage and plasmid sequences. Collectively, our results would contribute to researches of resistance in L. casei group, and also provide a new vision on the diversity and evolution of CRISPR/Cas system.}, }
@article {pmid32322010, year = {2020}, author = {Baquero, DP and Contursi, P and Piochi, M and Bartolucci, S and Liu, Y and Cvirkaite-Krupovic, V and Prangishvili, D and Krupovic, M}, title = {New virus isolates from Italian hydrothermal environments underscore the biogeographic pattern in archaeal virus communities.}, journal = {The ISME journal}, volume = {}, number = {}, pages = {}, doi = {10.1038/s41396-020-0653-z}, pmid = {32322010}, issn = {1751-7370}, support = {ANR-17-CE15-0005-01//Agence Nationale de la Recherche (French National Research Agency)/ ; }, abstract = {Viruses of hyperthermophilic archaea represent one of the least understood parts of the virosphere, showing little genomic and morphological similarity to viruses of bacteria or eukaryotes. Here, we investigated virus diversity in the active sulfurous fields of the Campi Flegrei volcano in Pozzuoli, Italy. Virus-like particles displaying eight different morphotypes, including lemon-shaped, droplet-shaped and bottle-shaped virions, were observed and five new archaeal viruses proposed to belong to families Rudiviridae, Globuloviridae and Tristromaviridae were isolated and characterized. Two of these viruses infect neutrophilic hyperthermophiles of the genus Pyrobaculum, whereas the remaining three have rod-shaped virions typical of the family Rudiviridae and infect acidophilic hyperthermophiles belonging to three different genera of the order Sulfolobales, namely, Saccharolobus, Acidianus, and Metallosphaera. Notably, Metallosphaera rod-shaped virus 1 is the first rudivirus isolated on Metallosphaera species. Phylogenomic analysis of the newly isolated and previously sequenced rudiviruses revealed a clear biogeographic pattern, with all Italian rudiviruses forming a monophyletic clade, suggesting geographical structuring of virus communities in extreme geothermal environments. Analysis of the CRISPR spacers suggests that isolated rudiviruses have experienced recent host switching across the genus boundary, potentially to escape the targeting by CRISPR-Cas immunity systems. Finally, we propose a revised classification of the Rudiviridae family, with the establishment of six new genera. Collectively, our results further show that high-temperature continental hydrothermal systems harbor a highly diverse virome and shed light on the evolution of archaeal viruses.}, }
@article {pmid32276445, year = {2020}, author = {Ahmar, S and Gill, RA and Jung, KH and Faheem, A and Qasim, MU and Mubeen, M and Zhou, W}, title = {Conventional and Molecular Techniques from Simple Breeding to Speed Breeding in Crop Plants: Recent Advances and Future Outlook.}, journal = {International journal of molecular sciences}, volume = {21}, number = {7}, pages = {}, doi = {10.3390/ijms21072590}, pmid = {32276445}, issn = {1422-0067}, support = {2018YFD0100601//National Key Research and Development Program/ ; }, mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; *Genome, Plant ; Plant Breeding/*methods ; Plants/genetics ; Plants, Genetically Modified ; }, abstract = {In most crop breeding programs, the rate of yield increment is insufficient to cope with the increased food demand caused by a rapidly expanding global population. In plant breeding, the development of improved crop varieties is limited by the very long crop duration. Given the many phases of crossing, selection, and testing involved in the production of new plant varieties, it can take one or two decades to create a new cultivar. One possible way of alleviating food scarcity problems and increasing food security is to develop improved plant varieties rapidly. Traditional farming methods practiced since quite some time have decreased the genetic variability of crops. To improve agronomic traits associated with yield, quality, and resistance to biotic and abiotic stresses in crop plants, several conventional and molecular approaches have been used, including genetic selection, mutagenic breeding, somaclonal variations, whole-genome sequence-based approaches, physical maps, and functional genomic tools. However, recent advances in genome editing technology using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated (Cas) proteins have opened the door to a new plant breeding era. Therefore, to increase the efficiency of crop breeding, plant breeders and researchers around the world are using novel strategies such as speed breeding, genome editing tools, and high-throughput phenotyping. In this review, we summarize recent findings on several aspects of crop breeding to describe the evolution of plant breeding practices, from traditional to modern speed breeding combined with genome editing tools, which aim to produce crop generations with desired traits annually.}, }
@article {pmid32276422, year = {2020}, author = {Xu, K and Han, CX and Zhou, H and Ding, JM and Xu, Z and Yang, LY and He, C and Akinyemi, F and Zheng, YM and Qin, C and Luo, HX and Meng, H}, title = {Effective MSTN Gene Knockout by AdV-Delivered CRISPR/Cas9 in Postnatal Chick Leg Muscle.}, journal = {International journal of molecular sciences}, volume = {21}, number = {7}, pages = {}, doi = {10.3390/ijms21072584}, pmid = {32276422}, issn = {1422-0067}, support = {zhongzi 2017.2-3//The seed industry project of developing agriculture through Science and Technology of Shanghai/ ; }, mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Chickens ; Gene Editing ; *Gene Expression Profiling ; *Gene Knockout Techniques ; Myostatin/*genetics ; }, abstract = {Muscle growth and development are important aspects of chicken meat production, but the underlying regulatory mechanisms remain unclear and need further exploration. CRISPR has been used for gene editing to study gene function in mice, but less has been done in chick muscles. To verify whether postnatal gene editing could be achieved in chick muscles and determine the transcriptomic changes, we knocked out Myostatin (MSTN), a potential inhibitor of muscle growth and development, in chicks and performed transcriptome analysis on knock-out (KO) muscles and wild-type (WT) muscles at two post-natal days: 3d (3-day-old) and 14d (14-day-old). Large fragment deletions of MSTN (>5 kb) were achieved in all KO muscles, and the MSTN gene expression was significantly downregulated at 14d. The transcriptomic results indicated the presence of 1339 differentially expressed genes (DEGs) between the 3d KO and 3d WT muscles, as well as 597 DEGs between 14d KO and 14d WT muscles. Many DEGs were found to be related to cell differentiation and proliferation, muscle growth and energy metabolism. This method provides a potential means of postnatal gene editing in chicks, and the results presented here could provide a basis for further investigation of the mechanisms involved in muscle growth and development.}, }
@article {pmid32235908, year = {2020}, author = {Burgess, DJ}, title = {CRISPR screens beyond Cas9.}, journal = {Nature reviews. Genetics}, volume = {21}, number = {5}, pages = {273}, doi = {10.1038/s41576-020-0232-1}, pmid = {32235908}, issn = {1471-0064}, mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Exons ; Gene Editing ; Genomics ; }, }
@article {pmid32024985, year = {2020}, author = {Bird, L}, title = {MR1-restricted pan-cancer T cells.}, journal = {Nature reviews. Immunology}, volume = {20}, number = {3}, pages = {141}, doi = {10.1038/s41577-020-0284-7}, pmid = {32024985}, issn = {1474-1741}, mesh = {*CRISPR-Cas Systems ; *Early Detection of Cancer ; Histocompatibility Antigens Class I/genetics ; Minor Histocompatibility Antigens ; }, }
@article {pmid31937772, year = {2020}, author = {Shen, J and Zhou, X and Shan, Y and Yue, H and Huang, R and Hu, J and Xing, D}, title = {Sensitive detection of a bacterial pathogen using allosteric probe-initiated catalysis and CRISPR-Cas13a amplification reaction.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {267}, pmid = {31937772}, issn = {2041-1723}, mesh = {Allosteric Regulation ; Animals ; Aptamers, Nucleotide ; Bacteria/*isolation &amp; purification ; CRISPR-Cas Systems ; Catalysis ; DNA, Bacterial/genetics ; Fluorescence ; Food Microbiology ; Mice ; Microbiological Techniques/*methods ; Milk/microbiology ; Real-Time Polymerase Chain Reaction ; Salmonella Infections/blood/diagnosis ; Salmonella enteritidis/isolation &amp; purification ; }, abstract = {The ability to detect low numbers of microbial cells in food and clinical samples is highly valuable but remains a challenge. Here we present a detection system (called 'APC-Cas') that can detect very low numbers of a bacterial pathogen without isolation, using a three-stage amplification to generate powerful fluorescence signals. APC-Cas involves a combination of nucleic acid-based allosteric probes and CRISPR-Cas13a components. It can selectively and sensitively quantify Salmonella Enteritidis cells (from 1 to 105 CFU) in various types of samples such as milk, showing similar or higher sensitivity and accuracy compared with conventional real-time PCR. Furthermore, APC-Cas can identify low numbers of S. Enteritidis cells in mouse serum, distinguishing mice with early- and late-stage infection from uninfected mice. Our method may have potential clinical applications for early diagnosis of pathogens.}, }
@article {pmid31828084, year = {2019}, author = {Wu, J and Tang, Y and Zhang, CL}, title = {Targeting N-Terminal Huntingtin with a Dual-sgRNA Strategy by CRISPR/Cas9.}, journal = {BioMed research international}, volume = {2019}, number = {}, pages = {1039623}, pmid = {31828084}, issn = {2314-6141}, mesh = {Amino Acid Sequence ; CRISPR-Cas Systems/*genetics ; Cell Line ; Gene Expression/genetics ; HEK293 Cells ; Humans ; Huntingtin Protein/*genetics ; Huntington Disease/genetics ; Nerve Degeneration/genetics ; Protein Biosynthesis/genetics ; RNA/*genetics ; Sequence Alignment ; }, abstract = {Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder, caused by a CAG/polyglutamine (polyQ) repeat expansion in the Huntingtin (HTT) gene. The polyQ tract is located in and transcribed from N-terminal HTT of exon 1. HTT is a large multifaceted protein, which participates in a range of cellular functions. Previous studies have shown that truncated HTT, which lacks N-terminus, retains specific functions that can produce neuroprotective benefits. It gives an insight that it is possible to repair HD by removing deleterious N-terminal HTT with CRISPR/Cas9, without compromising functions of remaining HTT peptides. To successfully generate functional truncated HTT proteins, an alternative downstream ATG start codon that is capable of initiating truncated HTT expression is required. In this study, we searched all possible in-frame ATGs before exon 7 and demonstrated that one of them can indeed initiate the downstream GFP expression in plasmids. We then tried to remove endogenous N-terminal HTT with an optimized dual-sgRNA strategy by CRISPR/Cas9; however, we cannot detect obvious traits of truncated HTT expression. Our results suggest that noncanonical ATGs of N-terminal HTT may not be effective in the genomic context, as in the construct context. Nevertheless, our study examined the therapeutic efficacy of downstream noncanonical ATGs for protein translation and also provided an optimized dual-sgRNA strategy for further genome manipulation of the HTT gene.}, }
@article {pmid31659303, year = {2020}, author = {Burgess, DJ}, title = {CRISPR screens come into sight.}, journal = {Nature reviews. Genetics}, volume = {21}, number = {1}, pages = {1}, doi = {10.1038/s41576-019-0192-5}, pmid = {31659303}, issn = {1471-0064}, mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; RNA, Guide ; }, }
@article {pmid31571107, year = {2019}, author = {Batır, MB and Şahin, E and Çam, FS}, title = {Evaluation of the CRISPR/Cas9 directed mutant TP53 gene repairing effect in human prostate cancer cell line PC-3.}, journal = {Molecular biology reports}, volume = {46}, number = {6}, pages = {6471-6484}, pmid = {31571107}, issn = {1573-4978}, support = {FEF 2017-144//Celal Bayar Üniversitesi/ ; }, mesh = {Apoptosis ; *CRISPR-Cas Systems ; Cell Proliferation ; *DNA Repair ; DNA, Single-Stranded/metabolism ; High-Throughput Nucleotide Sequencing ; Humans ; Male ; *Mutation ; Oligodeoxyribonucleotides/metabolism ; PC-3 Cells ; Prostatic Neoplasms/*genetics ; RNA, Guide/metabolism ; Sequence Analysis, DNA ; Tumor Suppressor Protein p53/*genetics ; }, abstract = {Prostate cancer is a common health problem among men worldwide and most of these prostate cancer cases are related to a dysfunctional mutant Tumor Protein p53 (TP53) gene. However, the CRISPR/Cas9 system can be used for repairing of a dysfunctional mutant TP53 gene in combination with donor single-stranded oligodeoxynucleotide (ssODN) via cells' own homology-directed repair (HDR) mechanism. In this study, we aimed to evaluate the CRISPR/Cas9 repairing efficiency on TP53 414delC (p.K139fs*31) null mutation, located in the TP53 gene, of human prostate cancer cell line PC-3 in combination with ssODNs. According to the next-generation sequencing results, TP53 414delC mutation was repaired with an efficiency of 19.95% and 26.0% at the TP53 414delC position with ssODN1 and ssODN2 accompanied by sgRNA2 guided CRISPR/Cas9, respectively. Besides, qPCR and immunofluorescence analysis showed that PC-3 cells, the TP53 414delC mutation of which were repaired, expressed wild type p53 again. Also, significantly increased number of apoptotic cells, driven by the repaired TP53 gene were detected compared to the control cells by flow cytometry analysis. As a result, sgRNA2 guided CRISPR/Cas9 system accompanied by ssODN was shown to effectively repair the TP53 414delC gene region and inhibit the cell proliferation of PC-3 cells. Therefore, the effects of the TP53 414delC mutation repairment in PC-3 cells will be investigated in the in vivo models for tumor clearance analysis in the near future.}, }
@article {pmid29970486, year = {2019}, author = {Xia, AL and He, QF and Wang, JC and Zhu, J and Sha, YQ and Sun, B and Lu, XJ}, title = {Applications and advances of CRISPR-Cas9 in cancer immunotherapy.}, journal = {Journal of medical genetics}, volume = {56}, number = {1}, pages = {4-9}, doi = {10.1136/jmedgenet-2018-105422}, pmid = {29970486}, issn = {1468-6244}, mesh = {Biomarkers, Tumor ; *CRISPR-Cas Systems ; *Gene Editing ; Genetic Therapy ; Humans ; Immunotherapy ; Neoplasms/*genetics/*immunology/therapy ; }, abstract = {Immunotherapy has emerged as one of the most promising therapeutic strategies in cancer. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR-Cas9) system, as an RNA-guided genome editing technology, is triggering a revolutionary change in cancer immunotherapy. With its versatility and ease of use, CRISPR-Cas9 can be implemented to fuel the production of therapeutic immune cells, such as construction of chimeric antigen receptor T (CAR-T) cells and programmed cell death protein 1 knockout. Therefore, CRISPR-Cas9 technology holds great promise in cancer immunotherapy. In this review, we will introduce the origin, development and mechanism of CRISPR-Cas9. Also, we will focus on its various applications in cancer immunotherapy, especially CAR-T cell-based immunotherapy, and discuss the potential challenges it faces.}, }
@article {pmid32157203, year = {2020}, author = {Fenner, A}, title = {CRISPR-Cas9 ERβ deletion reveals roles in prostate.}, journal = {Nature reviews. Urology}, volume = {17}, number = {4}, pages = {192-193}, doi = {10.1038/s41585-020-0302-3}, pmid = {32157203}, issn = {1759-4820}, mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Estrogen Receptor beta ; Male ; Mice ; Phenotype ; Prostate ; }, }
@article {pmid31707298, year = {2020}, author = {Zoppo, M and Di Luca, M and Franco, M and Rizzato, C and Lupetti, A and Stringaro, A and De Bernardis, F and Schaudinn, C and Barrasa, MI and Bottai, D and Vyas, VK and Tavanti, A}, title = {CpALS4770 and CpALS4780 contribution to the virulence of Candida parapsilosis.}, journal = {Microbiological research}, volume = {231}, number = {}, pages = {126351}, doi = {10.1016/j.micres.2019.126351}, pmid = {31707298}, issn = {1618-0623}, mesh = {Animals ; Biofilms/growth &amp; development ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; *Candida parapsilosis/genetics/pathogenicity ; Candidiasis ; Cell Adhesion/*genetics ; Cell Culture Techniques ; Female ; Fungal Proteins/genetics ; Gene Silencing ; Genes, Fungal ; Humans ; Mice ; Mucous Membrane/microbiology ; Virulence/*genetics ; }, abstract = {The ability of yeast to adhere to biotic and abiotic surfaces represents an essential trait during the early stages of infection. Agglutinin-like sequence (Als) cell-wall proteins play a key role in adhesion of Candida species. Candida parapsilosis genome encompasses 5 ALS members, of which only the role of CPAR2_404800 has been elucidated. The present project was aimed at investigating the contribution of C. parapsilosis Als proteins by generating edited strains lacking functional Als proteins. CPAR2_404770 and CPAR2_404780, further indicated as CpALS4770 and CpALS4780, were selected for the generation of single and double edited strains using an episomal CRISPR/Cas9 technology. Phenotypic characterization of mutant strains revealed that editing of both genes had no impact on the in vitro growth of C. parapsilosis or on morphogenesis. Notably, CpALS4770-edited strain showed a reduction of biofilm formation and adhesive properties to human buccal cells (HBECs). Conversely, single CpALS4780-edited strain did not show any difference compared to the wild-type strain in all the assays performed, while the double CpALS4770-CpALS4780 mutant revealed an increased ability to produce biofilm, a hyper-adhesive phenotype to HBECs, and a marked tendency to form cellular aggregates. Murine vaginal infection experiments indicated a significant reduction in CFUs recovered from BALC/c mice infected with single and double edited strains, compared to those infected with the wild-type strain. These finding clearly indicate that CpAls4770 plays a role in adhesion to biotic and abiotic surfaces, while both CpALS4770 and CpALS4780 genes are required for C. parapsilosis ability to colonize and persist in the vaginal mucosa.}, }
@article {pmid31526854, year = {2019}, author = {Ley, D and Pereira, S and Pedersen, LE and Arnsdorf, J and Hefzi, H and Davy, AM and Ha, TK and Wulff, T and Kildegaard, HF and Andersen, MR}, title = {Reprogramming AA catabolism in CHO cells with CRISPR/Cas9 genome editing improves cell growth and reduces byproduct secretion.}, journal = {Metabolic engineering}, volume = {56}, number = {}, pages = {120-129}, doi = {10.1016/j.ymben.2019.09.005}, pmid = {31526854}, issn = {1096-7184}, mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems ; *Cellular Reprogramming Techniques ; Cricetulus ; *Gene Editing ; Metabolic Networks and Pathways/*genetics ; }, abstract = {Chinese hamster ovary (CHO) cells are the preferred host for producing biopharmaceuticals. Amino acids are biologically important precursors for CHO metabolism; they serve as building blocks for proteogenesis, including synthesis of biomass and recombinant proteins, and are utilized for growth and cellular maintenance. In this work, we studied the physiological impact of disrupting a range of amino acid catabolic pathways in CHO cells. We aimed to reduce secretion of growth inhibiting metabolic by-products derived from amino acid catabolism including lactate and ammonium. To achieve this, we engineered nine genes in seven different amino acid catabolic pathways using the CRISPR-Cas9 genome editing system. For identification of target genes, we used a metabolic network reconstruction of amino acid catabolism to follow transcriptional changes in response to antibody production, which revealed candidate genes for disruption. We found that disruption of single amino acid catabolic genes reduced specific lactate and ammonium secretion while specific growth rate and integral of viable cell density were increased in many cases. Of particular interest were Hpd and Gad2 disruptions, which show unchanged AA uptake rates, while having growth rates increased up to 19%, and integral of viable cell density as much as 50% higher, and up to 26% decrease in specific ammonium production and to a lesser extent (up to 22%) decrease in lactate production. This study demonstrates the broad potential of engineering amino acid catabolism in CHO cells to achieve improved phenotypes for bioprocessing.}, }
@article {pmid30793048, year = {2019}, author = {Simeonov, DR and Brandt, AJ and Chan, AY and Cortez, JT and Li, Z and Woo, JM and Lee, Y and Carvalho, CMB and Indart, AC and Roth, TL and Zou, J and May, AP and Lupski, JR and Anderson, MS and Buaas, FW and Rokhsar, DS and Marson, A}, title = {A large CRISPR-induced bystander mutation causes immune dysregulation.}, journal = {Communications biology}, volume = {2}, number = {}, pages = {70}, pmid = {30793048}, issn = {2399-3642}, support = {T32 AI007334/AI/NIAID NIH HHS/United States ; T32 DK007418/DK/NIDDK NIH HHS/United States ; DP3 DK111914/DK/NIDDK NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; S10 RR029668/RR/NCRR NIH HHS/United States ; S10 RR027303/RR/NCRR NIH HHS/United States ; P30 DK063720/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Cells, Cultured ; DNA Damage ; DNA Repair ; Gene Duplication ; Gene Editing/*methods ; Gene Expression Regulation/immunology ; Interleukin-2 Receptor alpha Subunit/*genetics/immunology ; Mice, Inbred NOD ; *Mutation ; T-Lymphocytes/*immunology/metabolism ; T-Lymphocytes, Regulatory/*immunology/metabolism ; }, abstract = {A persistent concern with CRISPR-Cas9 gene editing has been the potential to generate mutations at off-target genomic sites. While CRISPR-engineering mice to delete a ~360 bp intronic enhancer, here we discovered a founder line that had marked immune dysregulation caused by a 24 kb tandem duplication of the sequence adjacent to the on-target deletion. Our results suggest unintended repair of on-target genomic cuts can cause pathogenic &quot;bystander&quot; mutations that escape detection by routine targeted genotyping assays.}, }
@article {pmid32315755, year = {2020}, author = {Mijanur Rahman, M and Tollefsbol, TO}, title = {Targeting cancer epigenetics with CRISPR-dCAS9: Principles and prospects.}, journal = {Methods (San Diego, Calif.)}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.ymeth.2020.04.006}, pmid = {32315755}, issn = {1095-9130}, abstract = {Cancer therapeutics is an ever-evolving field due to incessant demands for effective and precise treatment options. Over the last few decades, cancer treatment strategies have shifted somewhat from surgery to targeted precision medicine. CRISPR-dCas9 is an emerging version of precision cancer therapy that has been adapted from the prokaryotic CRISPR-Cas system. Once ligated to epigenetic effectors (EE), CRISPR-dCas9 can function as an epigenetic editing tool and CRISPR-dCas9-EE complexes could be exploited to alter cancerous epigenetic features associated with different cancer hallmarks. In this article, we discuss the rationale of epigenetic editing as a therapeutic strategy against cancer. We also outline how sgRNA-dCas9 was derived from the CRISPR-Cas system. In addition, the current status of sgRNA-dCas9 use (in vivo and in vitro) in cancer is updated with a molecular illustration of CRISPR-dCas9-mediated epigenetic and transcriptional modulation. As sgRNA-dCas9 is still at the developmental phase, challenges are inherent to its use. We evaluate major challenges in targeting cancer with sgRNA-dCas9 such as off-target effects, lack of sgRNA designing rubrics, target site selection dilemmas and deficient sgRNA-dCas9 delivery systems. Finally, we appraise the sgRNA-dCas9 as a prospective cancer therapeutic by summarizing ongoing improvements of sgRNA-dCas9 methodology.}, }
@article {pmid32315232, year = {2020}, author = {Shola, DTN and Yang, C and Kewaldar, VS and Kar, P and Bustos, V}, title = {New Additions to the CRISPR Toolbox: CRISPR-CLONInG and CRISPR-CLIP for Donor Construction in Genome Editing.}, journal = {The CRISPR journal}, volume = {3}, number = {2}, pages = {109-122}, doi = {10.1089/crispr.2019.0062}, pmid = {32315232}, issn = {2573-1602}, abstract = {CRISPR-Cas has proven to be the most versatile genetic tinkering system of our time, predominantly as a precision genome editing tool. Here, we demonstrate two additions to the repertoire of CRISPR's application for constructing donor DNA templates: CRISPR-CLONInG and CRISPR-CLIP. CRISPR-CLONInG (CRISPR-Cutting and Ligation Of Nucleic acid In vitro via Gibson) was devised to enable efficient cut-and-paste of multiple complex DNA fragments by using CRISPR-Cas9 as a digestion alternative with precision and exclusivity features, followed by joining the digested products via Gibson Assembly, to construct double-stranded DNA and adeno-associated virus (AAV) donor vectors rapidly without cloning scars. CRISPR-CLIP (CRISPR-Clipped Long ssDNA via Incising Plasmid) was devised as a DNA clipping tool to retrieve long single-stranded DNA (lssDNA) efficiently from plasmid, up to 3.5 kbase, which can be supplied as the donor template for creating genetically engineered mice via Easi-CRISPR. We utilized two different Cas types (Cpf1 and Cas9n) to induce two distinct incisions at the respective ends of the lssDNA cassette junctions on the plasmid, yielding three independent single-stranded DNA units of unique sizes eligible for strand separation, followed by target strand clip-out through gel extraction. The retrieval of the lssDNA donor circumvents involvements of restriction enzymes and DNA polymerase-based steps. Hence, it not only retains sequence fidelity but also carries virtually no restriction on sequence composition, further mitigating limitations on the current Easi-CRISPR method. With the add-on feature of universal DNA-tag sequences of Cpf1-Cas9 duo protospacer adjacent motif, CRISPR-CLIP can be facile and applicable to generate lssDNA templates for any genomic target of choice. Additionally, we demonstrate robust gene editing efficiencies in the neuroblastoma cell line, as well as in mice attained with the AAV and lssDNA donors constructed herein.}, }
@article {pmid32315227, year = {2020}, author = {Liu, Z and Schiel, JA and Maksimova, E and Strezoska, Ž and Zhao, G and Anderson, EM and Wu, Y and Warren, J and Bartels, A and van Brabant Smith, A and Lowe, CE and Forbes, KP}, title = {ErCas12a CRISPR-MAD7 for Model Generation in Human Cells, Mice, and Rats.}, journal = {The CRISPR journal}, volume = {3}, number = {2}, pages = {97-108}, doi = {10.1089/crispr.2019.0068}, pmid = {32315227}, issn = {2573-1602}, abstract = {MAD7 is an engineered class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) system isolated from Eubacterium rectale. Analogous to Cas9, it is an RNA-guided nuclease with demonstrated gene editing activity in Escherichia coli and yeast cells. Here, we report that MAD7 is capable of generating indels and fluorescent gene tagging of endogenous genes in human HCT116 and U2OS cancer cell lines, respectively. In addition, MAD7 is highly proficient in generating indels, small DNA insertions (23 bases), and larger integrations ranging from 1 to 14 kb in size in mouse and rat embryos, resulting in live-born transgenic animals. Due to the different protospacer adjacent motif requirement, small-guide RNA, and highly efficient targeted gene disruption and insertions, MAD7 can expand the CRISPR toolbox for genome enginnering across different systems and model organisms.}, }
@article {pmid32313254, year = {2020}, author = {Wienert, B and Wyman, SK and Yeh, CD and Conklin, BR and Corn, JE}, title = {CRISPR off-target detection with DISCOVER-seq.}, journal = {Nature protocols}, volume = {}, number = {}, pages = {}, doi = {10.1038/s41596-020-0309-5}, pmid = {32313254}, issn = {1750-2799}, support = {310030_188858//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; R01-EY028249//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01-HL130533//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01-HL13535801//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; }, abstract = {DISCOVER-seq (discovery of in situ Cas off-targets and verification by sequencing) is a broadly applicable approach for unbiased CRISPR-Cas off-target identification in cells and tissues. It leverages the recruitment of DNA repair factors to double-strand breaks (DSBs) after genome editing with CRISPR nucleases. Here, we describe a detailed experimental protocol and analysis pipeline with which to perform DISCOVER-seq. The principle of this method is to track the precise recruitment of MRE11 to DSBs by chromatin immunoprecipitation followed by next-generation sequencing. A customized open-source bioinformatics pipeline, BLENDER (blunt end finder), then identifies off-target sequences genome wide. DISCOVER-seq is capable of finding and measuring off-targets in primary cells and in situ. The two main advantages of DISCOVER-seq are (i) low false-positive rates because DNA repair enzyme binding is required for genome edits to occur and (ii) its applicability to a wide variety of systems, including patient-derived cells and animal models. The whole protocol, including the analysis, can be completed within 2 weeks.}, }
@article {pmid32005981, year = {2020}, author = {Kouprina, N and Noskov, VN and Larionov, V}, title = {Selective isolation of large segments from individual microbial genomes and environmental DNA samples using transformation-associated recombination cloning in yeast.}, journal = {Nature protocols}, volume = {15}, number = {3}, pages = {734-749}, doi = {10.1038/s41596-019-0280-1}, pmid = {32005981}, issn = {1750-2799}, mesh = {CRISPR-Cas Systems ; Cloning, Molecular/*methods ; DNA Replication ; DNA, Fungal/*genetics ; Genetic Vectors ; *Genome, Fungal ; Plasmids ; Replication Origin ; Saccharomyces cerevisiae/*genetics ; Transformation, Genetic ; }, abstract = {Here, we describe an extension of our original transformation-associated recombination (TAR) cloning protocol, enabling selective isolation of DNA segments from microbial genomes. The technique is based on the previously described TAR cloning procedure developed for isolation of a desirable region from mammalian genomes that are enriched in autonomously replicating sequence (ARS)-like sequences, elements that function as the origin of replication in yeast. Such sequences are not common in microbial genomes. In this Protocol Extension, an ARS is inserted into the TAR vector along with a counter-selectable marker, allowing for selection of cloning events against vector circularization. Pre-treatment of microbial DNA with CRISPR-Cas9 to generate double-stranded breaks near the targeted sequences greatly increases the yield of region-positive colonies. In comparison to other available methods, this Protocol Extension allows selective isolation of any region from microbial genomes as well as from environmental DNA samples. The entire procedure can be completed in 10 d.}, }
@article {pmid31226583, year = {2019}, author = {Wang, Z and Wang, L and Liu, W and Hu, D and Gao, Y and Ge, Q and Liu, X and Li, L and Wang, Y and Wang, S and Li, C}, title = {Pathogenic mechanism and gene correction for LQTS-causing double mutations in KCNQ1 using a pluripotent stem cell model.}, journal = {Stem cell research}, volume = {38}, number = {}, pages = {101483}, doi = {10.1016/j.scr.2019.101483}, pmid = {31226583}, issn = {1876-7753}, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *KCNQ1 Potassium Channel ; *Long QT Syndrome/genetics/metabolism/pathology/therapy ; *Models, Cardiovascular ; *Mutation ; Myocytes, Cardiac/metabolism/pathology ; }, abstract = {AIMS: To establish a KCNQ1 mutant-specific induced pluripotent stem cell (iPSC) model of a Chinese inherited long QT syndrome (LQTS) patient and to explore the pathogenesis of KCNQ1 mutations.<br><br>METHODS AND RESULTS: (1) Two patient-specific iPSC lines from the proband were obtained. (2) The experiments produced spontaneously beating cardiomyocytes (CMs) from patient iPSCs. Splicing mutation c. 605-2A > G in iPSC-derived cardiomyocytes (iPSC-CMs) resulted in the skipping of exon 4, exons 3-4, or exons 3-6 in KCNQ1 transcription what was observed in the patient's peripheral leukocytes. (3) Action potential duration (APD) at 50% and 90% repolarization (APD50 and APD90) of the patient's iPSC-derived ventricular-like-CMs was significantly longer than that of the control. Moreover, early after depolarization (EAD) and coupled beats were observed only in L1-iPSC-CMs. (4) A c.815G > A corrected iPSC line was obtained by using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) system.<br><br>CONCLUSION: (1) Cardiomyocytes with spontaneous pulsation were successfully differentiated from LQTS patient-specific iPSC lines. (2) For KCNQ1 splicing mutations, there is a chance that splicing patterns in peripheral leukocytes are similar to that in patient iPSC-CMs. (3) The truncated KCNQ1 proteins induced by such splicing mutation might cause Iks decrease, which in turn produced APD prolongation and triggered activities. (4) Our data showed that CRISPR-Cas9 system could be used to rescue the LQTS-related mutations.}, }
@article {pmid31082677, year = {2019}, author = {Howden, S and Hosseini Far, H and Motazedian, A and Elefanty, AG and Stanley, EG and Lamandé, SR and Bateman, JF}, title = {The use of simultaneous reprogramming and gene correction to generate an osteogenesis imperfecta patient COL1A1 c. 3936 G&gt;T iPSC line and an isogenic control iPSC line.}, journal = {Stem cell research}, volume = {38}, number = {}, pages = {101453}, doi = {10.1016/j.scr.2019.101453}, pmid = {31082677}, issn = {1876-7753}, mesh = {Amino Acid Substitution ; *CRISPR-Cas Systems ; Cell Line ; *Cellular Reprogramming Techniques ; *Collagen Type I/genetics/metabolism ; Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *Mutation, Missense ; *Osteogenesis Imperfecta/genetics/metabolism/pathology ; }, abstract = {To develop a disease model for the human 'brittle bone' disease, osteogenesis imperfecta, we used a simultaneous reprogramming and CRISPR-Cas9 genome editing method to produce an iPSC line with the heterozygous patient mutation (COL1A1 c. 3936 G>T) along with an isogenic gene-corrected control iPSC line. Both IPSC lines had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. This osteogenesis imperfecta mutant and isogenic iPSC control line will be of use in exploring disease mechanisms and therapeutic approaches in vitro.}, }
@article {pmid30317623, year = {2019}, author = {Cai, M and Li, S and Shuai, Y and Li, J and Tan, J and Zeng, Q}, title = {Genome-wide CRISPR-Cas9 viability screen reveals genes involved in TNF-α-induced apoptosis of human umbilical vein endothelial cells.}, journal = {Journal of cellular physiology}, volume = {234}, number = {6}, pages = {9184-9193}, doi = {10.1002/jcp.27595}, pmid = {30317623}, issn = {1097-4652}, mesh = {Apoptosis/*drug effects ; Apoptosis Regulatory Proteins/*genetics/metabolism ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Cells, Cultured ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Human Umbilical Vein Endothelial Cells/*drug effects/metabolism/pathology ; Humans ; Mitochondria/drug effects/metabolism/pathology ; Mitochondrial Proteins/genetics/metabolism ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Receptors, Tumor Necrosis Factor, Type I/genetics/metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha/*pharmacology ; bcl-Associated Death Protein/genetics/metabolism ; }, abstract = {Tumor necrosis factor α (TNF-α), a pivotal cytokine in sepsis, protects the host against pathogens by promoting an inflammatory response while simultaneously inducing apoptosis of the vascular endothelium. Unfortunately, inhibitors targeting certain components of the TNF-α signaling pathway to reduce cellular apoptosis have failed to translate into clinical applications, partly due to the adverse effects of excessive immunosuppression. In an attempt to discover potential targets in the TNF-α signaling pathway to modulate moderate inflammation and apoptosis during the development of sepsis, we performed a pooled genome-wide CRISPR/Cas9 knockout screen in human umbilical vein endothelial cells (HUVECs). Tumor necrosis factor receptor superfamily member 1A (TNFRSF1A), B-cell lymphoma 2 (BCL2), Bcl2-associated death promoter (BAD), and NLR family member X1 (NLRX1) deficiencies were identified as the effective genetic suppressors of TNF-α cytotoxicity on a list of candidate regulators. CRISPR-mediated NLRX1 knockout conferred cellular resistance to challenge with TNF-α, and NLRX1 could be induced to colocalize with mitochondria following TNF-α stimulation. Thus, our work demonstrates the advantage of genome-scale screening with Cas9 and validates NLRX1 as a potential modulator of TNF-α-induced vascular endothelial apoptosis during sepsis.}, }
@article {pmid32308928, year = {2020}, author = {Menon, AV and Sohn, JI and Nam, JW}, title = {CGD: Comprehensive guide designer for CRISPR-Cas systems.}, journal = {Computational and structural biotechnology journal}, volume = {18}, number = {}, pages = {814-820}, doi = {10.1016/j.csbj.2020.03.020}, pmid = {32308928}, issn = {2001-0370}, abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas systems, including dead Cas9 (dCas9), Cas9, and Cas12a, have revolutionized genome engineering in mammalian somatic cells. Although computational tools that assess the target sites of CRISPR-Cas systems are inevitably important for designing efficient guide RNAs (gRNAs), they exhibit generalization issues in selecting features and do not provide optimal results in a comprehensive manner. Here, we introduce a Comprehensive Guide Designer (CGD) for four different CRISPR systems, which utilizes the machine learning algorithm, Elastic Net Logistic Regression (ENLOR), to autonomously generalize the models. CGD contains specific models trained with public datasets generated by CRISPRi, CRISPRa, CRISPR-Cas9, and CRISPR-Cas12a (designated as CGDi, CGDa, CGD9, and CGD12a, respectively) in an unbiased manner. The trained CGD models were benchmarked to other regression-based machine learning models, such as ElasticNet Linear Regression (ENLR), Random Forest and Boruta (RFB), and Extreme Gradient Boosting (Xgboost) with inbuilt feature selection. Evaluation with independent test datasets showed that CGD models outperformed the pre-existing methods in predicting the efficacy of gRNAs. All CGD source codes and datasets are available at GitHub (https://github.com/vipinmenon1989/CGD), and the CGD webserver can be accessed at http://big.hanyang.ac.kr:2195/CGD.}, }
@article {pmid32203418, year = {2020}, author = {Zhao, J and Wang, M and Chang, L and Yu, J and Song, A and Liu, C and Huang, W and Zhang, T and Wu, X and Shen, X and Zhu, B and Li, G}, title = {RYBP/YAF2-PRC1 complexes and histone H1-dependent chromatin compaction mediate propagation of H2AK119ub1 during cell division.}, journal = {Nature cell biology}, volume = {22}, number = {4}, pages = {439-452}, doi = {10.1038/s41556-020-0484-1}, pmid = {32203418}, issn = {1476-4679}, mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle Proteins/*genetics/metabolism ; Cell Division ; Epigenesis, Genetic ; Feedback, Physiological ; Gene Deletion ; Gene Editing ; HEK293 Cells ; Histones/*genetics/metabolism ; Humans ; Mice ; Mouse Embryonic Stem Cells/cytology/*metabolism ; Muscle Proteins/*genetics/metabolism ; Nucleosomes/chemistry/metabolism ; Protein Binding ; Protein Isoforms/genetics/metabolism ; *Protein Processing, Post-Translational ; Repressor Proteins/*genetics/metabolism ; Ubiquitination ; }, abstract = {Stable propagation of epigenetic information is important for maintaining cell identity in multicellular organisms. However, it remains largely unknown how mono-ubiquitinated histone H2A on lysine 119 (H2AK119ub1) is established and stably propagated during cell division. In this study, we found that the proteins RYBP and YAF2 each specifically bind H2AK119ub1 to recruit the RYBP-PRC1 or YAF2-PRC1 complex to catalyse the ubiquitination of H2A on neighbouring nucleosomes through a positive-feedback model. Additionally, we demonstrated that histone H1-compacted chromatin enhances the distal propagation of H2AK119ub1, thereby reinforcing the inheritance of H2AK119ub1 during cell division. Moreover, we showed that either disruption of RYBP/YAF2-PRC1 activity or impairment of histone H1-dependent chromatin compaction resulted in a significant defect of the maintenance of H2AK119ub1. Therefore, our results suggest that histone H1-dependent chromatin compaction plays a critical role in the stable propagation of H2AK119ub1 by RYBP/YAF2-PRC1 during cell division.}, }
@article {pmid32056283, year = {2020}, author = {Yang, N and Tian, J and Wang, X and Mei, S and Zou, D and Peng, X and Zhu, Y and Yang, Y and Gong, Y and Ke, J and Zhong, R and Chang, J and Miao, X}, title = {A functional variant in TNXB promoter associates with the risk of esophageal squamous-cell carcinoma.}, journal = {Molecular carcinogenesis}, volume = {59}, number = {4}, pages = {439-446}, doi = {10.1002/mc.23166}, pmid = {32056283}, issn = {1098-2744}, mesh = {CRISPR-Cas Systems ; Carcinoma, Squamous Cell/*genetics/pathology ; Cell Line, Tumor ; Cell Proliferation/genetics ; Esophageal Neoplasms/*genetics/pathology ; Gene Expression Profiling/methods ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks ; Genetic Predisposition to Disease/*genetics ; Humans ; *Polymorphism, Single Nucleotide ; Promoter Regions, Genetic/*genetics ; RNA Interference ; Risk Factors ; Tenascin/*genetics ; }, abstract = {Our previous study identified a tag single-nucleotide polymorphism (SNP) rs204900 in TNXB associated with risk of esophageal squamous-cell carcinoma (ESCC) in the Chinese population. However, the functional role of TNXB and causal variants had not been interrogated in that study. In the present study, we explored the effects of TNXB expression in the development of ESCC and searched for functional variants in this gene. We found TNXB was downregulated in ESCC tumors. Using small interfering RNAs and CRISPR-Cas9 methods, we identified that both knockdown and knockout of TNXB significantly promoted ESCC cell growth in vitro, suggesting a tumor suppressor role of this gene in ESCC. Through further fine-mapping analysis, we identified that a noncoding variant in the promoter of TNXB, rs411337, predisposed to ESCC risk (odds ratio = 1.36, 95% confidence interval: 1.22-1.51, P = 9.10 × 10-9). These findings revealed the functional mechanism of TNXB in the development of ESCC and may contribute to the prevention and treatment of this disease in the future.}, }
@article {pmid32051598, year = {2020}, author = {Doudna, JA}, title = {The promise and challenge of therapeutic genome editing.}, journal = {Nature}, volume = {578}, number = {7794}, pages = {229-236}, doi = {10.1038/s41586-020-1978-5}, pmid = {32051598}, issn = {1476-4687}, mesh = {Anemia, Sickle Cell/*genetics/*therapy ; CRISPR-Cas Systems/genetics ; Gene Editing/ethics/*methods/standards/*trends ; Genome, Human/*genetics ; Germ-Line Mutation/genetics ; Humans ; Muscular Dystrophy, Duchenne/*genetics/*therapy ; Organ Specificity/genetics ; Patient Safety ; beta-Globins/genetics ; }, abstract = {Genome editing, which involves the precise manipulation of cellular DNA sequences to alter cell fates and organism traits, has the potential to both improve our understanding of human genetics and cure genetic disease. Here I discuss the scientific, technical and ethical aspects of using CRISPR (clustered regularly interspaced short palindromic repeats) technology for therapeutic applications in humans, focusing on specific examples that highlight both opportunities and challenges. Genome editing is-or will soon be-in the clinic for several diseases, with more applications under development. The rapid pace of the field demands active efforts to ensure that this breakthrough technology is used responsibly to treat, cure and prevent genetic disease.}, }
@article {pmid32042152, year = {2020}, author = {Mita, P and Sun, X and Fenyö, D and Kahler, DJ and Li, D and Agmon, N and Wudzinska, A and Keegan, S and Bader, JS and Yun, C and Boeke, JD}, title = {BRCA1 and S phase DNA repair pathways restrict LINE-1 retrotransposition in human cells.}, journal = {Nature structural &amp; molecular biology}, volume = {27}, number = {2}, pages = {179-191}, pmid = {32042152}, issn = {1545-9985}, support = {P01 AG051449/AG/NIA NIH HHS/United States ; P50 GM107632/GM/NIGMS NIH HHS/United States ; }, mesh = {BRCA1 Protein/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; DNA Breaks, Double-Stranded ; *DNA Repair ; Fanconi Anemia/genetics/metabolism ; Gene Deletion ; HEK293 Cells ; HeLa Cells ; Homologous Recombination ; Humans ; *Long Interspersed Nucleotide Elements ; Microscopy ; *S Phase ; }, abstract = {Long interspersed element-1 (LINE-1, or L1) is the only autonomous retrotransposon that is active in human cells. Different host factors have been shown to influence L1 mobility; however, systematic analyses of these factors are limited. Here, we developed a high-throughput microscopy-based retrotransposition assay that identified the double-stranded break (DSB) repair and Fanconi anemia (FA) factors active in the S/G2 phase as potent inhibitors and regulators of L1 activity. In particular, BRCA1, an E3 ubiquitin ligase with a key role in several DNA repair pathways, directly affects L1 retrotransposition frequency and structure and plays a distinct role in controlling L1 ORF2 protein translation through L1 mRNA binding. These results suggest the existence of a 'battleground' at the DNA replication fork between homologous recombination (HR) factors and L1 retrotransposons and reveal a potential role for L1 in the genotypic evolution of tumors characterized by BRCA1 and HR repair deficiencies.}, }
@article {pmid31915386, year = {2020}, author = {Si, X and Zhang, H and Wang, Y and Chen, K and Gao, C}, title = {Manipulating gene translation in plants by CRISPR-Cas9-mediated genome editing of upstream open reading frames.}, journal = {Nature protocols}, volume = {15}, number = {2}, pages = {338-363}, doi = {10.1038/s41596-019-0238-3}, pmid = {31915386}, issn = {1750-2799}, mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Open Reading Frames/*genetics ; Plants/*genetics ; Protein Biosynthesis/*genetics ; *Transcription, Genetic ; }, abstract = {Gene expression is regulated by multiple processes, and the translation of mRNAs into proteins is an especially critical step. Upstream open reading frames (uORFs) are widespread cis-elements in eukaryotic genes that usually suppress the translation of downstream primary ORFs (pORFs). Here, we describe a protocol for fine-tuning gene translation in plants by editing endogenous uORFs with the CRISPR-Cas9 system. The method we present readily yields transgene-free uorf mutant offspring. We provide detailed protocols for predicting uORFs and testing their effects on downstream pORFs using a dual-luciferase reporter system, designing and constructing single guide RNA (sgRNA)-Cas9 vectors, identifying transgene-free uorf mutants, and finally comparing the mRNA, protein and phenotypic levels of target genes in uorf mutants and controls. Predicting uORFs and confirming their effects in protoplasts takes only 2-3 weeks, and transgene-free mutants with edited target uORFs controlling different levels of pORF translation can be obtained within 4 months. Unlike previous methods, our strategy achieves fine-tuning of gene translation in transgene-free derivatives, which accelerates the analysis of gene function and the improvement of crop traits.}, }
@article {pmid31740807, year = {2020}, author = {Zhang, ZJ and Pedicord, VA and Peng, T and Hang, HC}, title = {Site-specific acylation of a bacterial virulence regulator attenuates infection.}, journal = {Nature chemical biology}, volume = {16}, number = {1}, pages = {95-103}, doi = {10.1038/s41589-019-0392-5}, pmid = {31740807}, issn = {1552-4469}, support = {R01 GM087544/GM/NIGMS NIH HHS/United States ; R01AT007671//U.S. Department of Health &amp; Human Services | NIH | National Center for Complementary and Integrative Health (NCCIH)/International ; R01GM087544//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/International ; }, mesh = {Animals ; Arginine/chemistry ; Bacterial Proteins/metabolism ; Binding Sites ; Butyrates/chemistry ; CRISPR-Cas Systems ; Fatty Acids/*metabolism ; *Gene Expression Regulation, Bacterial ; Genome, Bacterial ; *Genomic Islands ; Genomics ; Lysine/chemistry ; Mice ; Mice, Inbred C57BL ; Mutagenesis ; Mutation ; Proteomics/methods ; Salmonella typhimurium/*metabolism/pathogenicity ; Trans-Activators/metabolism ; *Virulence ; Virulence Factors/metabolism ; }, abstract = {Microbiota generates millimolar concentrations of short-chain fatty acids (SCFAs) that can modulate host metabolism, immunity and susceptibility to infection. Butyrate in particular can function as a carbon source and anti-inflammatory metabolite, but the mechanism by which it inhibits pathogen virulence has been elusive. Using chemical proteomics, we found that several virulence factors encoded by Salmonella pathogenicity island-1 (SPI-1) are acylated by SCFAs. Notably, a transcriptional regulator of SPI-1, HilA, was acylated on several key lysine residues. Subsequent incorporation of stable butyryl-lysine analogs using CRISPR-Cas9 gene editing and unnatural amino acid mutagenesis revealed that site-specific modification of HilA impacts its genomic occupancy, expression of SPI-1 genes and attenuates Salmonella enterica serovar Typhimurium invasion of epithelial cells, as well as dissemination in vivo. Moreover, a multiple-site HilA lysine acylation mutant strain of S. Typhimurium was resistant to butyrate inhibition ex vivo and microbiota attenuation in vivo. Our results suggest that prominent microbiota-derived metabolites may directly acylate virulence factors to inhibit microbial pathogenesis in vivo.}, }
@article {pmid31685631, year = {2019}, author = {Coale, TH and Moosburner, M and Horák, A and Oborník, M and Barbeau, KA and Allen, AE}, title = {Reduction-dependent siderophore assimilation in a model pennate diatom.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {47}, pages = {23609-23617}, pmid = {31685631}, issn = {1091-6490}, mesh = {Bacterial Outer Membrane Proteins/*metabolism ; Biological Availability ; Biological Transport ; CRISPR-Cas Systems ; Climate Change ; Diatoms/genetics/growth &amp; development/*metabolism ; FMN Reductase/genetics/*metabolism ; Gallium/metabolism ; Gene Knockout Techniques ; Iron/*metabolism ; Membrane Transport Proteins/genetics/*metabolism ; Microbiota ; Oxidation-Reduction ; Phylogeny ; Receptors, Cell Surface/*metabolism ; Recombinant Fusion Proteins/metabolism ; Seawater/chemistry ; Siderophores/*metabolism ; Species Specificity ; }, abstract = {Iron uptake by diatoms is a biochemical process with global biogeochemical implications. In large regions of the surface ocean diatoms are both responsible for the majority of primary production and frequently experiencing iron limitation of growth. The strategies used by these phytoplankton to extract iron from seawater constrain carbon flux into higher trophic levels and sequestration into sediments. In this study we use reverse genetic techniques to target putative iron-acquisition genes in the model pennate diatom Phaeodactylum tricornutum We describe components of a reduction-dependent siderophore acquisition pathway that relies on a bacterial-derived receptor protein and provides a viable alternative to inorganic iron uptake under certain conditions. This form of iron uptake entails a close association between diatoms and siderophore-producing organisms during low-iron conditions. Homologs of these proteins are found distributed across diatom lineages, suggesting the significance of siderophore utilization by diatoms in the marine environment. Evaluation of specific proteins enables us to confirm independent iron-acquisition pathways in diatoms and characterize their preferred substrates. These findings refine our mechanistic understanding of the multiple iron-uptake systems used by diatoms and help us better predict the influence of iron speciation on taxa-specific iron bioavailability.}, }
@article {pmid31685617, year = {2019}, author = {Xu, M and Kiselar, J and Whited, TL and Hernandez-Sanchez, W and Taylor, DJ}, title = {POT1-TPP1 differentially regulates telomerase via POT1 His266 and as a function of single-stranded telomere DNA length.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {47}, pages = {23527-23533}, pmid = {31685617}, issn = {1091-6490}, support = {P30 EB009998/EB/NIBIB NIH HHS/United States ; P30 CA091842/CA/NCI NIH HHS/United States ; R01 GM133841/GM/NIGMS NIH HHS/United States ; R01 CA240993/CA/NCI NIH HHS/United States ; R01 GM126218/GM/NIGMS NIH HHS/United States ; }, mesh = {Amino Acid Substitution ; CRISPR-Cas Systems ; DNA, Single-Stranded/*metabolism ; HCT116 Cells ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell/genetics/metabolism ; *Mutation, Missense ; *Point Mutation ; *Polymorphism, Single Nucleotide ; Protein Binding ; Recombinant Proteins/metabolism ; Telomere/*metabolism ; Telomere Homeostasis/*physiology ; Telomere-Binding Proteins/genetics/*physiology ; }, abstract = {Telomeres cap the ends of linear chromosomes and terminate in a single-stranded DNA (ssDNA) overhang recognized by POT1-TPP1 heterodimers to help regulate telomere length homeostasis. Here hydroxyl radical footprinting coupled with mass spectrometry was employed to probe protein-protein interactions and conformational changes involved in the assembly of telomere ssDNA substrates of differing lengths bound by POT1-TPP1 heterodimers. Our data identified environmental changes surrounding residue histidine 266 of POT1 that were dependent on telomere ssDNA substrate length. We further determined that the chronic lymphocytic leukemia-associated H266L substitution significantly reduced POT1-TPP1 binding to short ssDNA substrates; however, it only moderately impaired the heterodimer binding to long ssDNA substrates containing multiple protein binding sites. Additionally, we identified a telomerase inhibitory role when several native POT1-TPP1 proteins coat physiologically relevant lengths of telomere ssDNA. This POT1-TPP1 complex-mediated inhibition of telomerase is abrogated in the context of the POT1 H266L mutation, which leads to telomere overextension in a malignant cellular environment.}, }
@article {pmid31685615, year = {2019}, author = {Sagai, T and Amano, T and Maeno, A and Ajima, R and Shiroishi, T}, title = {SHH signaling mediated by a prechordal and brain enhancer controls forebrain organization.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {47}, pages = {23636-23642}, pmid = {31685615}, issn = {1091-6490}, mesh = {Animals ; CRISPR-Cas Systems ; *Enhancer Elements, Genetic ; Eye Proteins/physiology ; *Gene Expression Regulation, Developmental ; Gene Knockout Techniques ; Genes, Reporter ; Hedgehog Proteins/biosynthesis/genetics/*physiology ; Holoprosencephaly/genetics ; Homeodomain Proteins/physiology ; Hypothalamus/abnormalities/embryology/metabolism ; Lac Operon ; Mesencephalon/embryology/metabolism ; Mesoderm/metabolism ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins/biosynthesis/deficiency/genetics/*physiology ; Prosencephalon/abnormalities/*embryology/metabolism ; Signal Transduction ; Transgenes ; }, abstract = {Sonic hedgehog (SHH) signaling plays a pivotal role in 2 different phases during brain development. Early SHH signaling derived from the prechordal plate (PrCP) triggers secondary Shh induction in the forebrain, which overlies the PrCP, and the induced SHH signaling, in turn, directs late neuronal differentiation of the forebrain. Consequently, Shh regulation in the PrCP is crucial for initiation of forebrain development. However, no enhancer that regulates prechordal Shh expression has yet been found. Here, we identified a prechordal enhancer, named SBE7, in the vicinity of a cluster of known forebrain enhancers for Shh This enhancer also directs Shh expression in the ventral midline of the forebrain, which receives the prechordal SHH signal. Thus, the identified enhancer acts not only for the initiation of Shh regulation in the PrCP but also for subsequent Shh induction in the forebrain. Indeed, removal of the enhancer from the mouse genome markedly down-regulated the expression of Shh in the rostral domains of the axial mesoderm and in the ventral midline of the forebrain and hypothalamus in the mouse embryo, and caused a craniofacial abnormality similar to human holoprosencephaly (HPE). These findings demonstrate that SHH signaling mediated by the newly identified enhancer is essential for development and growth of the ventral midline of the forebrain and hypothalamus. Understanding of the Shh regulation governed by this prechordal and brain enhancer provides an insight into the mechanism underlying craniofacial morphogenesis and the etiology of HPE.}, }
@article {pmid31649173, year = {2019}, author = {Cohen, J}, title = {Prime editing promises to be a cut above CRISPR.}, journal = {Science (New York, N.Y.)}, volume = {366}, number = {6464}, pages = {406}, doi = {10.1126/science.366.6464.406}, pmid = {31649173}, issn = {1095-9203}, mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Genetic Engineering/*methods ; RNA, Guide/genetics ; }, }
@article {pmid31467430, year = {2020}, author = {Kweon, J and Jang, AH and Shin, HR and See, JE and Lee, W and Lee, JW and Chang, S and Kim, K and Kim, Y}, title = {A CRISPR-based base-editing screen for the functional assessment of BRCA1 variants.}, journal = {Oncogene}, volume = {39}, number = {1}, pages = {30-35}, pmid = {31467430}, issn = {1476-5594}, mesh = {BRCA1 Protein/*genetics ; Breast Neoplasms/*genetics/pathology ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Survival/genetics ; Cytosine/chemistry ; DNA Repair/genetics ; Exons/genetics ; Female ; Gene Editing ; Genetic Testing ; Genomic Instability/genetics ; High-Throughput Screening Assays ; Humans ; Loss of Function Mutation/genetics ; Ovarian Neoplasms/*genetics/pathology ; }, abstract = {Genetic mutations in BRCA1, which is crucial for the process of DNA repair and maintenance of genomic integrity, are known to increase markedly the risk of breast and ovarian cancers. Clinical genetic testing has been used to identify new BRCA1 variants; however, functional assessment and determination of their pathogenicity still poses challenges for clinical management. Here, we describe that CRISPR-mediated cytosine base editor, known as BE3, can be used for the functional analysis of BRCA1 variants. We performed CRISPR-mediated base-editing screening using 745 gRNAs targeting all exons in BRCA1 to identify loss-of-function variants and identified variants whose function has heretofore remained unknown, such as c.-97C>T, c.154C>T, c.3847C>T, c.5056C>T, and c.4986+5G>A. Our results show that CRISPR-mediated base editor is a powerful tool for the reclassification of variants of uncertain significance (VUSs) in BRCA1.}, }
@article {pmid31104263, year = {2019}, author = {Wang, L and Li, J}, title = {Expansion of the mutant monkey through cloning.}, journal = {Science China. Life sciences}, volume = {62}, number = {6}, pages = {865-867}, doi = {10.1007/s11427-019-9502-2}, pmid = {31104263}, issn = {1869-1889}, mesh = {ARNTL Transcription Factors/genetics ; Animals ; CRISPR-Cas Systems/genetics ; *Cloning, Molecular ; Gene Editing ; Haplorhini ; *Models, Animal ; Mutation ; Phenotype ; }, }
@article {pmid31059574, year = {2019}, author = {Sobh, A and Loguinov, A and Stornetta, A and Balbo, S and Tagmount, A and Zhang, L and Vulpe, CD}, title = {Genome-Wide CRISPR Screening Identifies the Tumor Suppressor Candidate OVCA2 As a Determinant of Tolerance to Acetaldehyde.}, journal = {Toxicological sciences : an official journal of the Society of Toxicology}, volume = {169}, number = {1}, pages = {235-245}, pmid = {31059574}, issn = {1096-0929}, support = {P42 ES004705/ES/NIEHS NIH HHS/United States ; }, mesh = {Acetaldehyde/*toxicity ; *CRISPR-Cas Systems ; Cell Survival/drug effects ; Cell Transformation, Neoplastic/*chemically induced/*genetics/metabolism/pathology ; DNA Adducts/genetics/metabolism ; Dose-Response Relationship, Drug ; Gene Expression Regulation, Neoplastic ; Genome-Wide Association Study ; HEK293 Cells ; Humans ; K562 Cells ; Neoplasms/*chemically induced/*genetics/metabolism/pathology ; Proteins/*genetics/metabolism ; Risk Assessment ; Tumor Suppressor Proteins/*genetics/metabolism ; }, abstract = {Acetaldehyde, a metabolite of ethanol, is a cellular toxicant and a human carcinogen. A genome-wide CRISPR-based loss-of-function screen in erythroleukemic K562 cells revealed candidate genetic contributors affecting acetaldehyde cytotoxicity. Secondary screening exposing cells to a lower acetaldehyde dose simultaneously validated multiple candidate genes whose loss results in increased sensitivity to acetaldehyde. Disruption of genes encoding components of various DNA repair pathways increased cellular sensitivity to acetaldehyde. Unexpectedly, the tumor suppressor gene OVCA2, whose function is unknown, was identified in our screen as a determinant of acetaldehyde tolerance. Disruption of the OVCA2 gene resulted in increased acetaldehyde sensitivity and higher accumulation of the acetaldehyde-derived DNA adduct N2-ethylidene-dG. Together these results are consistent with a role for OVCA2 in adduct removal and/or DNA repair.}, }
@article {pmid31044159, year = {2019}, author = {Xu, Q and Li, R and Weng, L and Sun, Y and Li, M and Xiao, H}, title = {Domain-specific expression of meristematic genes is defined by the LITTLE ZIPPER protein DTM in tomato.}, journal = {Communications biology}, volume = {2}, number = {}, pages = {134}, pmid = {31044159}, issn = {2399-3642}, mesh = {Amino Acid Sequence ; Amino Acid Substitution ; Arabidopsis Proteins/chemistry ; CRISPR-Cas Systems ; Crosses, Genetic ; DNA, Plant/genetics ; Feedback, Physiological ; *Gene Expression Regulation, Plant/genetics ; *Genes, Plant ; Homeodomain Proteins/physiology ; Lycopersicon esculentum/*genetics ; Meristem/*metabolism ; *Mutation, Missense ; Plant Proteins/genetics/*physiology ; Plant Shoots/growth &amp; development/metabolism/ultrastructure ; Protein Binding ; Protein Multimerization ; Seedlings/growth &amp; development ; Sequence Alignment ; Sequence Homology, Amino Acid ; Transcription Factors/physiology ; }, abstract = {Shoot meristems, which harbor a small population of stem cells, are responsible for generating new above-ground organs in plants. The proliferation and differentiation of these stem cells is regulated by a genetic pathway involving two key meristematic genes: CLAVATA3 (CLV3) and WUSCHEL (WUS). However, it is not well understood how CLV3 and WUS expression domains in the shoot meristems are specified and maintained during post-embryogenic development. Here, we show that a tomato mutant with fasciated stems, flowers and fruits, due to impaired stem cell activity, is defective in a LITTLE ZIPPER gene denoted as DEFECTIVE TOMATO MERISTEM (DTM). DTM forms a negative feedback loop with class III homeodomain-leucine zipper (HD-ZIP III) transcription factors to confine CLV3 and WUS expression to specific domains of the shoot meristems. Our findings reveal a new layer of complexity in the regulation of plant stem cell homeostasis.}, }
@article {pmid31023625, year = {2019}, author = {Good, AL and Cannon, CE and Haemmerle, MW and Yang, J and Stanescu, DE and Doliba, NM and Birnbaum, MJ and Stoffers, DA}, title = {JUND regulates pancreatic β cell survival during metabolic stress.}, journal = {Molecular metabolism}, volume = {25}, number = {}, pages = {95-106}, pmid = {31023625}, issn = {2212-8778}, support = {K12 DK094723/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Caspase 3/metabolism ; Caspase 7/metabolism ; Cell Line ; Diabetes Mellitus, Type 2/metabolism ; Fatty Acids ; Gene Expression Regulation ; Glucose/metabolism ; Homeodomain Proteins/genetics ; Humans ; Insulin-Secreting Cells/*metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; Proto-Oncogene Proteins c-jun/*metabolism ; RNA, Messenger/metabolism ; Stress, Physiological/*physiology ; Trans-Activators/genetics ; Transcription Factors ; }, abstract = {OBJECTIVE: In type 2 diabetes (T2D), oxidative stress contributes to the dysfunction and loss of pancreatic β cells. A highly conserved feature of the cellular response to stress is the regulation of mRNA translation; however, the genes regulated at the level of translation are often overlooked due to the convenience of RNA sequencing technologies. Our goal is to investigate translational regulation in β cells as a means to uncover novel factors and pathways pertinent to cellular adaptation and survival during T2D-associated conditions.<br><br>METHODS: Translating ribosome affinity purification (TRAP) followed by RNA-seq or RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of shRNAs to primary mouse islets or CRISPR-Cas9 to Min6 cells. Oxidative stress and apoptosis were measured in primary islets using cell-permeable dyes with fluorescence readouts of oxidation and activated cleaved caspase-3 and-7, respectively. Gene expression was assessed by RNA-seq, RT-qPCR, and western blot. ChIP-qPCR was used to determine chromatin enrichment.<br><br>RESULTS: TRAP-seq in a PDX1-deficiency model of β cell dysfunction uncovered a cohort of genes regulated at the level of mRNA translation, including the transcription factor JUND. Using a panel of diabetes-associated stressors, JUND was found to be upregulated in mouse islets cultured with high concentrations of glucose and free fatty acid, but not after treatment with hydrogen peroxide or thapsigargin. This induction of JUND could be attributed to increased mRNA translation. JUND was also upregulated in islets from diabetic db/db mice and in human islets treated with high glucose and free fatty acid. Depletion of JUND in primary islets reduced oxidative stress and apoptosis in β cells during metabolic stress. Transcriptome assessment identified a cohort of genes, including pro-oxidant and pro-inflammatory genes, regulated by JUND that are commonly dysregulated in models of β cell dysfunction, consistent with a maladaptive role for JUND in islets.<br><br>CONCLUSIONS: A translation-centric approach uncovered JUND as a stress-responsive factor in β cells that contributes to redox imbalance and apoptosis during pathophysiologically relevant stress.}, }
@article {pmid30905739, year = {2019}, author = {Yang, J and Rajan, SS and Friedrich, MJ and Lan, G and Zou, X and Ponstingl, H and Garyfallos, DA and Liu, P and Bradley, A and Metzakopian, E}, title = {Genome-Scale CRISPRa Screen Identifies Novel Factors for Cellular Reprogramming.}, journal = {Stem cell reports}, volume = {12}, number = {4}, pages = {757-771}, pmid = {30905739}, issn = {2213-6711}, support = {/WT_/Wellcome Trust/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; WT098051/WT_/Wellcome Trust/United Kingdom ; }, mesh = {Animals ; Biomarkers ; CRISPR-Cas Systems ; Cell Line ; Cellular Reprogramming/*genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Dosage ; *Genome-Wide Association Study ; Germ Layers/cytology/metabolism ; Humans ; Induced Pluripotent Stem Cells ; Mice ; Octamer Transcription Factor-3/genetics/metabolism ; Pluripotent Stem Cells/cytology/metabolism ; Transcription Factors/genetics/metabolism ; }, abstract = {Primed epiblast stem cells (EpiSCs) can be reverted to a pluripotent embryonic stem cell (ESC)-like state by expression of single reprogramming factor. We used CRISPR activation to perform a genome-scale, reprogramming screen in EpiSCs and identified 142 candidate genes. Our screen validated a total of 50 genes, previously not known to contribute to reprogramming, of which we chose Sall1 for further investigation. We show that Sall1 augments reprogramming of mouse EpiSCs and embryonic fibroblasts and that these induced pluripotent stem cells are indeed fully pluripotent including formation of chimeric mice. We also demonstrate that Sall1 synergizes with Nanog in reprogramming and that overexpression in ESCs delays their conversion back to EpiSCs. Lastly, using RNA sequencing, we identify and validate Klf5 and Fam189a2 as new downstream targets of Sall1 and Nanog. In summary, our work demonstrates the power of using CRISPR technology in understanding molecular mechanisms that mediate complex cellular processes such as reprogramming.}, }
@article {pmid30860482, year = {2019}, author = {Hong, AL and Tseng, YY and Wala, JA and Kim, WJ and Kynnap, BD and Doshi, MB and Kugener, G and Sandoval, GJ and Howard, TP and Li, J and Yang, X and Tillgren, M and Ghandi, M and Sayeed, A and Deasy, R and Ward, A and McSteen, B and Labella, KM and Keskula, P and Tracy, A and Connor, C and Clinton, CM and Church, AJ and Crompton, BD and Janeway, KA and Van Hare, B and Sandak, D and Gjoerup, O and Bandopadhayay, P and Clemons, PA and Schreiber, SL and Root, DE and Gokhale, PC and Chi, SN and Mullen, EA and Roberts, CW and Kadoch, C and Beroukhim, R and Ligon, KL and Boehm, JS and Hahn, WC}, title = {Renal medullary carcinomas depend upon SMARCB1 loss and are sensitive to proteasome inhibition.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {30860482}, issn = {2050-084X}, support = {132943-MRSG-18-202-01-TBG//American Cancer Society/International ; T32 CA136432/CA/NCI NIH HHS/United States ; T32 GM007226/GM/NIGMS NIH HHS/United States ; K12 HD052896/HD/NICHD NIH HHS/United States ; P50 CA101942/CA/NCI NIH HHS/United States ; 328545//CureSearch for Children's Cancer/International ; T32 GM007753/GM/NIGMS NIH HHS/United States ; Young Investigator Award//Alex's Lemonade Stand Foundation for Childhood Cancer/International ; W81XWH-15-1-0659//U.S. Department of Defense/International ; U01 CA176058/CA/NCI NIH HHS/United States ; K08 CA188073/CA/NCI NIH HHS/United States ; Wong Family Award//Dana-Farber Cancer Institute/International ; 14-40-31-HONG//American Association for Cancer Research/International ; OFD BTREC CDA//Boston Children's Hospital/International ; U01 CA217848/CA/NCI NIH HHS/United States ; }, mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Carcinoma, Medullary/drug therapy/*genetics ; Cell Cycle ; Cell Line, Tumor ; Exome ; Female ; Humans ; In Situ Hybridization, Fluorescence ; Kidney/metabolism ; Kidney Neoplasms/drug therapy/*genetics ; Mice ; Mice, Nude ; Mutation ; Neoplasm Transplantation ; Proteasome Endopeptidase Complex/*genetics ; Proteasome Inhibitors/*pharmacology ; RNA Interference ; SMARCB1 Protein/*genetics ; Sequence Analysis, RNA ; Ubiquitin/chemistry ; Whole Genome Sequencing ; }, abstract = {Renal medullary carcinoma (RMC) is a rare and deadly kidney cancer in patients of African descent with sickle cell trait. We have developed faithful patient-derived RMC models and using whole-genome sequencing, we identified loss-of-function intronic fusion events in one SMARCB1 allele with concurrent loss of the other allele. Biochemical and functional characterization of these models revealed that RMC requires the loss of SMARCB1 for survival. Through integration of RNAi and CRISPR-Cas9 loss-of-function genetic screens and a small-molecule screen, we found that the ubiquitin-proteasome system (UPS) was essential in RMC. Inhibition of the UPS caused a G2/M arrest due to constitutive accumulation of cyclin B1. These observations extend across cancers that harbor SMARCB1 loss, which also require expression of the E2 ubiquitin-conjugating enzyme, UBE2C. Our studies identify a synthetic lethal relationship between SMARCB1-deficient cancers and reliance on the UPS which provides the foundation for a mechanism-informed clinical trial with proteasome inhibitors.}, }
@article {pmid30815697, year = {2019}, author = {Sobh, A and Loguinov, A and Yazici, GN and Zeidan, RS and Tagmount, A and Hejazi, NS and Hubbard, AE and Zhang, L and Vulpe, CD}, title = {Functional Profiling Identifies Determinants of Arsenic Trioxide Cellular Toxicity.}, journal = {Toxicological sciences : an official journal of the Society of Toxicology}, volume = {169}, number = {1}, pages = {108-121}, pmid = {30815697}, issn = {1096-0929}, support = {P42 ES004705/ES/NIEHS NIH HHS/United States ; }, mesh = {Antineoplastic Agents/*pharmacology ; Arsenic Trioxide/*pharmacology ; CRISPR-Cas Systems ; Cell Survival/drug effects ; Dose-Response Relationship, Drug ; Gene Editing ; *Gene Expression Profiling ; Gene Expression Regulation, Leukemic ; HEK293 Cells ; Humans ; K562 Cells ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy/genetics/metabolism/pathology ; Signal Transduction ; Sodium Selenite/pharmacology ; Time Factors ; Transcriptome ; }, abstract = {Arsenic exposure is a worldwide health concern associated with an increased risk of skin, lung, and bladder cancer but arsenic trioxide (AsIII) is also an effective chemotherapeutic agent. The current use of AsIII in chemotherapy is limited to acute promyelocytic leukemia (APL). However, AsIII was suggested as a potential therapy for other cancer types including chronic myeloid leukemia (CML), especially when combined with other drugs. Here, we carried out a genome-wide CRISPR-based approach to identify modulators of AsIII toxicity in K562, a human CML cell line. We found that disruption of KEAP1, the inhibitory partner of the key antioxidant transcription factor Nrf2, or TXNDC17, a thioredoxin-like protein, markedly increased AsIII tolerance. Loss of the water channel AQP3, the zinc transporter ZNT1 and its regulator MTF1 also enhanced tolerance to AsIII whereas loss of the multidrug resistance protein ABCC1 increased sensitivity to AsIII. Remarkably, disruption of any of multiple genes, EEFSEC, SECISBP2, SEPHS2, SEPSECS, and PSTK, encoding proteins involved in selenocysteine metabolism increased resistance to AsIII. Our data suggest a model in which an intracellular interaction between selenium and AsIII may impact intracellular AsIII levels and toxicity. Together this work revealed a suite of cellular components/processes which modulate the toxicity of AsIII in CML cells. Targeting such processes simultaneously with AsIII treatment could potentiate AsIII in CML therapy.}, }
@article {pmid30666960, year = {2019}, author = {Champer, J and Chung, J and Lee, YL and Liu, C and Yang, E and Wen, Z and Clark, AG and Messer, PW}, title = {Molecular safeguarding of CRISPR gene drive experiments.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {30666960}, issn = {2050-084X}, support = {F32 AI138476/AI/NIAID NIH HHS/United States ; R21AI130635/NH/NIH HHS/United States ; F32AI138476/NH/NIH HHS/United States ; R01 GM127418/GM/NIGMS NIH HHS/United States ; R21 AI130635/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Drosophila melanogaster/*genetics ; Gene Drive Technology/*methods ; Gene Editing/methods ; Genetics, Population ; Genotype ; Germ Cells ; Phenotype ; RNA, Guide/*genetics/metabolism ; }, abstract = {CRISPR-based homing gene drives have sparked both enthusiasm and deep concerns due to their potential for genetically altering entire species. This raises the question about our ability to prevent the unintended spread of such drives from the laboratory into a natural population. Here, we experimentally demonstrate the suitability of synthetic target site drives as well as split drives as flexible safeguarding strategies for gene drive experiments by showing that their performance closely resembles that of standard homing drives in Drosophila melanogaster. Using our split drive system, we further find that maternal deposition of both Cas9 and gRNA is required to form resistance alleles in the early embryo and that maternally-deposited Cas9 alone can power germline drive conversion in individuals that lack a genomic source of Cas9.}, }
@article {pmid30644359, year = {2019}, author = {Arunsan, P and Ittiprasert, W and Smout, MJ and Cochran, CJ and Mann, VH and Chaiyadet, S and Karinshak, SE and Sripa, B and Young, ND and Sotillo, J and Loukas, A and Brindley, PJ and Laha, T}, title = {Programmed knockout mutation of liver fluke granulin attenuates virulence of infection-induced hepatobiliary morbidity.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {30644359}, issn = {2050-084X}, support = {PHD/0111/2557//Thailand Research Fund/International ; APP1109829//National Health and Medical Research Council/International ; APP1085309//National Health and Medical Research Council/International ; R01CA164719/CA/NCI NIH HHS/United States ; APP1117504//National Health and Medical Research Council/International ; 107475/Z/15/Z//Wellcome/International ; PHD/0111/2557//Royal Golden Jubilee PhD Program, Thailand/International ; }, mesh = {Animals ; Bile Ducts, Intrahepatic/*parasitology/*pathology ; CRISPR-Cas Systems/genetics ; Carcinogenesis/pathology ; Cell Line ; Cell Proliferation ; Chronic Disease ; Cricetinae ; Fibrosis ; Gene Editing ; Gene Expression Regulation ; *Gene Knockout Techniques ; Genome ; Granulins/*genetics/metabolism ; Humans ; Hyperplasia ; Mutation/*genetics ; Opisthorchiasis/genetics/parasitology/pathology ; Opisthorchis/*pathogenicity ; Wound Healing ; }, abstract = {Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2012) for cholangiocarcinoma (CCA) in the Lower Mekong River Basin countries including Thailand, Lao PDR, Vietnam and Cambodia. We exploited this link to explore the role of the secreted growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions by undertaking programmed CRISPR/Cas9 knockout of the Ov-GRN-1 gene from the liver fluke genome. Deep sequencing of amplicon libraries from genomic DNA of gene-edited parasites revealed Cas9-catalyzed mutations within Ov-GRN-1. Gene editing resulted in rapid depletion of Ov-GRN-1 transcripts and the encoded Ov-GRN-1 protein. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, but the infection resulted in reduced pathology as evidenced by attenuated biliary hyperplasia and fibrosis. Not only does this report pioneer programmed gene-editing in parasitic flatworms, but also the striking, clinically-relevant pathophysiological phenotype confirms the role for Ov-GRN-1 in virulence morbidity during opisthorchiasis.}, }
@article {pmid30644357, year = {2019}, author = {Ittiprasert, W and Mann, VH and Karinshak, SE and Coghlan, A and Rinaldi, G and Sankaranarayanan, G and Chaidee, A and Tanno, T and Kumkhaek, C and Prangtaworn, P and Mentink-Kane, MM and Cochran, CJ and Driguez, P and Holroyd, N and Tracey, A and Rodpai, R and Everts, B and Hokke, CH and Hoffmann, KF and Berriman, M and Brindley, PJ}, title = {Programmed genome editing of the omega-1 ribonuclease of the blood fluke, Schistosoma mansoni.}, journal = {eLife}, volume = {8}, number = {}, pages = {}, pmid = {30644357}, issn = {2050-084X}, support = {R21AI109532//National Institute of Allergy and Infectious Diseases/International ; PHD/0047/2556//Royal Golden Jubilee Ph.D Program, Thailand/International ; 107475/Z/15/Z//Wellcome/International ; WT 098051//Wellcome/International ; PHD/0011/2555//Thailand Research Fund/International ; HHSN272201000005C/AI/NIAID NIH HHS/United States ; HHSN272201000005I/AI/NIAID NIH HHS/United States ; PHD/0047/2556//Thailand Research Fund/International ; PHD/00531/2556//Thailand Research Fund/International ; }, mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Line ; Chromosomes/genetics ; DNA Repair/genetics ; Exons/genetics ; *Gene Editing ; Gene Expression Regulation ; Genetic Loci ; Granuloma/pathology ; Homologous Recombination/genetics ; Humans ; Inflammation/pathology ; Lung/parasitology/pathology ; Mice ; Mutation/genetics ; Ovum/enzymology ; RNA, Messenger/genetics/metabolism ; Ribonucleases/*genetics ; Schistosoma mansoni/*enzymology/*genetics ; Th2 Cells/immunology ; Transgenes ; }, abstract = {CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.}, }
@article {pmid30602586, year = {2019}, author = {Braun, M and Meacham, D}, title = {The trust game: CRISPR for human germline editing unsettles scientists and society.}, journal = {EMBO reports}, volume = {20}, number = {2}, pages = {}, pmid = {30602586}, issn = {1469-3178}, support = {BB/L01386X/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Ethics, Research ; *Gene Editing/ethics ; *Germ Cells ; Health Personnel ; Humans ; Scientific Misconduct ; Societies, Scientific ; }, }
@article {pmid30429198, year = {2019}, author = {Li, L and Li, W and Chen, N and Zhao, H and Xu, G and Zhao, Y and Pan, X and Zhang, X and Zhou, L and Yu, D and Li, A and Hu, JF and Cui, J}, title = {FLI1 Exonic Circular RNAs as a Novel Oncogenic Driver to Promote Tumor Metastasis in Small Cell Lung Cancer.}, journal = {Clinical cancer research : an official journal of the American Association for Cancer Research}, volume = {25}, number = {4}, pages = {1302-1317}, doi = {10.1158/1078-0432.CCR-18-1447}, pmid = {30429198}, issn = {1078-0432}, mesh = {A549 Cells ; Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Carcinoma, Non-Small-Cell Lung/blood/genetics/pathology ; Cell Cycle/genetics ; Cell Movement/genetics ; Cell Proliferation/genetics ; Exons/genetics ; Exosomes/genetics ; Heterografts ; Humans ; Mice ; MicroRNAs/*genetics ; Neoplasm Metastasis ; Proto-Oncogene Protein c-fli-1/antagonists &amp; inhibitors/*genetics ; RNA, Circular/*genetics/isolation &amp; purification ; RNA, Small Interfering/genetics ; Small Cell Lung Carcinoma/blood/*genetics/pathology ; rho-Associated Kinases/*genetics ; }, abstract = {PURPOSE: The aberrantly upregulated Friend leukemia virus integration 1 (FLI1) is closely correlated with the malignant phenotype of small cell lung cancer (SCLC). It is interesting to note that the CRISPR gene knockout by Cas9 gRNAs that target the FLI1 coding region and the posttranscriptional knockdown by shRNAs that target the 3' region of FLI1 mRNA yielded distinct antimetastasis effects in SCLC cells. This study attempts to examine if FLI1 exonic circular RNAs (FECR) function as a new malignant driver that determines the metastatic phenotype in SCLC.<br><br>EXPERIMENTAL DESIGN: The clinical relevance of FECRs was examined in 56 primary SCLC tissues and 50 non-small cell lung cancer (NSCLC) tissues. The prognostic value of FECRs was examined by measuring serum exosomal FECRs in a longitudinal cohort of patients with SCLC. The oncogenic activity of FECRs was investigated in both SCLC cell lines and animal xenograft studies. Finally, we explored the molecular mechanisms underlying these noncoding RNAs as a malignant driver.<br><br>RESULTS: Therapeutic comparison of CRISPR Cas9 knockout and shRNA knockdown of FLI1 identified FECRs as a new noncanonical malignant driver in SCLC. Using RNA FISH and quantitative PCR, we found that FECR1 (exons 4-2-3) and FECR2 (exons 5-2-3-4) were aberrantly upregulated in SCLC tissues (P < 0.0001), and was positively associated with lymph node metastasis (P < 0.01). Notably, serum exosomal FECR1 was associated with poor survival (P = 0.038) and clinical response to chemotherapy. Silencing of FECRs significantly inhibited the migration in two highly aggressive SCLC cell lines and reduced tumor metastasis in vivo. Mechanistically, we uncovered that FECRs sequestered and subsequently inactivated tumor suppressor miR584-3p, leading to the activation of the Rho Associated Coiled-Coil Containing Protein Kinase 1 gene (ROCK1).<br><br>CONCLUSIONS: This study identifies FLI1 exonic circular RNAs as a new oncogenic driver that promotes tumor metastasis through the miR584-ROCK1 pathway. Importantly, serum exosomal FECR1 may serve as a promising biomarker to track disease progression of SCLC.}, }
@article {pmid32303549, year = {2020}, author = {Philippe, C and Levesque, S and Dion, MB and Tremblay, DM and Horvath, P and Lüth, N and Cambillau, C and Franz, C and Neve, H and Fremaux, C and Heller, KJ and Moineau, S}, title = {Genomic and morphological characterization of a novel genus of phages infecting Streptococcus thermophilus.}, journal = {Applied and environmental microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1128/AEM.00227-20}, pmid = {32303549}, issn = {1098-5336}, abstract = {Streptococcus thermophilus is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes worldwide as they can inactivate the added starter bacterial cells, leading to low quality fermented dairy products. To date, four genetically distinct groups of phages infecting S. thermophilus have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of S. thermophilus The genomes of phages P738 and D4446 were sequenced and found to contain 34,037 and 33,656 base pairs (bp) as well as 48 and 46 open reading frames, respectively. Comparative genomic analyses revealed that the two phages are highly related to each other but display very limited similarities to other S. thermophilus phages. In fact, these two novel S. thermophilus phages share similarities with streptococcal phages of non-dairy origin, suggesting that they emerged recently in the dairy environment.Importance Despite decades of research and adapted anti-phage strategies such as CRISPR-Cas systems, virulent phages are still a persistent risk for the milk fermentation industry worldwide as they can cause manufacture failures and alter product quality. Phages P738 and D4446 are novel virulent phages that infect the food-grade Gram-positive bacterial species Streptococcus thermophilus These two related viruses represent a fifth group of S. thermophilus phages as they are significantly distinct from other known S. thermophilus phages. Both phages share similarities with phages infecting pathogenic streptococci, suggesting their recent emergence and probable co-existence in dairy environments. These findings highlight the necessity of phage surveillance programs as the viral population evolves in response to the application of anti-phage strategies.}, }
@article {pmid32300337, year = {2020}, author = {Xu, A and Wang, D and Ding, Y and Zheng, Y and Wang, B and Wei, Q and Wang, S and Yang, L and Ma, LZ}, title = {Integrated Comparative Genomic Analysis and Phenotypic Profiling of Pseudomonas aeruginosa Isolates From Crude Oil.}, journal = {Frontiers in microbiology}, volume = {11}, number = {}, pages = {519}, doi = {10.3389/fmicb.2020.00519}, pmid = {32300337}, issn = {1664-302X}, abstract = {Pseudomonas aeruginosa is an environmental microorganism that can thrive in diverse ecological niches including plants, animals, water, soil, and crude oil. It also one of the microorganism widely used in tertiary recovery of crude oil and bioremediation. However, the genomic information regarding the mechanisms of survival and adapation of this bacterium in crude oil is still limited. In this study, three Pseudomonads strains (named as IMP66, IMP67, and IMP68) isolated from crude oil were taken for whole-genome sequencing by using a hybridized PacBio and Illumina approach. The phylogeny analysis showed that the three strains were all P. aeruginosa species and clustered in clade 1, the group with PAO1 as a representitive. Subsequent comparative genomic analysis revealed a high degree of individual genomic plasticity, with a probable alkane degradation genomic island, one type I-F CRISPR-Cas system and several prophages integrated into their genomes. Nine genes encoding alkane hydroxylases (AHs) homologs were found in each strain, which might enable these strains to degrade alkane in crude oil. P. aeruginosa can produce rhamnolipids (RLs) biosurfactant to emulsify oil, which enables their survival in crude oil enviroments. Our previous report showed that IMP67 and IMP68 were high RLs producers, while IMP66 produced little RLs. Genomic analysis suggested that their RLs yield was not likely due to differences at genetic level. We then further analyzed the quorum sensing (QS) signal molecules that regulate RLs synthesis. IMP67 and IMP68 produced more N-acyl-homoserine lactones (AHLs) signal molecules than that of PAO1 and IMP66, which could explain their high RLs yield. This study provides evidence for adaptation of P. aeruginosa in crude oil and proposes the potential application of IMP67 and IMP68 in microbial-enhanced oil recovery and bioremediation.}, }
@article {pmid31727803, year = {2019}, author = {Doudna, J}, title = {CRISPR's unwanted anniversary.}, journal = {Science (New York, N.Y.)}, volume = {366}, number = {6467}, pages = {777}, doi = {10.1126/science.aba1751}, pmid = {31727803}, issn = {1095-9203}, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genome, Human ; Humans ; }, }
@article {pmid31385888, year = {2019}, author = {Li, P and Zhang, W and Smith, LJ and Ayares, D and Cooper, DKC and Ekser, B}, title = {The potential role of 3D-bioprinting in xenotransplantation.}, journal = {Current opinion in organ transplantation}, volume = {24}, number = {5}, pages = {547-554}, pmid = {31385888}, issn = {1531-7013}, support = {S10 OD023595/OD/NIH HHS/United States ; U19 AI090959/AI/NIAID NIH HHS/United States ; UL1 TR001108/TR/NCATS NIH HHS/United States ; }, mesh = {Animals ; Animals, Genetically Modified ; Bioprinting/*methods ; CRISPR-Cas Systems ; Genetic Engineering ; Humans ; *Printing, Three-Dimensional ; Swine ; Transplantation, Heterologous/*methods ; }, abstract = {PURPOSE OF REVIEW: To review the impact of a new technology, 3D-bioprinting, in xenotransplantation research.<br><br>RECENT FINDINGS: Genetically engineered pigs, beginning with human (h) CD55-transgenic and Gal-knockout pigs, have improved the outcomes of xenotransplantation research. Today, there are more than 30 different genetically engineered pigs either expressing human gene(s) or lacking pig gene(s). CRIPSR/cas9 technology has facilitated the production of multigene pigs (up to nine genes in a single pig), which lack multiple pig xenoantigens, and express human transgenes, such as hCD46, hCD55, hThrombomodulin, hCD39, etc. Although recent studies in nonhuman primates (NHPs) have demonstrated prolonged survival after life-supporting pig kidney, heart, and islet xenotransplantation, researchers have difficulty determining the best genetic combination to test in NHPs because of a potential greater than 100 000 genetic combinations. 3D-bioprinting of genetically engineered pig cells: is superior to 2D in-vitro testing, enables organ-specific testing, helps to understand differences in immunogenicity between organs, and is faster and cheaper than testing in NHPs. Moreover, 3D-bioprinted cells can be continuously perfused in a bioreactor, controlling for all variables, except the studied variable.<br><br>SUMMARY: 3D-bioprinting can help in the study of the impact of specific genes (human or pig) in xenotransplantation in a rapid, inexpensive, and reliable way.}, }
@article {pmid32298126, year = {2020}, author = {Liu, B and Ejaz, W and Gong, S and Kurbanov, M and Canakci, M and Anson, F and Thayumanavan, S}, title = {Engineered Interactions with Mesoporous Silica Facilitate Intracellular Delivery of Proteins and Gene Editing.}, journal = {Nano letters}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.nanolett.0c01387}, pmid = {32298126}, issn = {1530-6992}, abstract = {Intracellular delivery of functional proteins is a promising, but challenging, strategy for many therapeutic applications. Here, we report a new methodology that overcomes drawbacks of traditional mesoporous silica (MSi) particles for protein delivery. We hypothesize that engineering enhancement in interactions between proteins and delivery vehicles can facilitate efficient encapsulation and intracellular delivery. In this strategy, surface lysines in proteins were modified with a self-immolative linker containing a terminal boronic acid for stimulus-induced reversibility in functionalization. The boronic acid moiety serves to efficiently interact with amine-functionalized MSi through dative and electrostatic interactions. We show that proteins of different sizes and isoelectric points (pIs) can be quantitatively encapsulated into MSi, even at low protein concentrations. We also show that the proteins can be efficiently delivered into cells with retention of activity. Utility of this approach is further demonstrated with gene editing in cells, through the delivery of a CRISPR/Cas 9 complex.}, }
@article {pmid32296011, year = {2020}, author = {Li, H and Yang, Y and Hong, W and Huang, M and Wu, M and Zhao, X}, title = {Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects.}, journal = {Signal transduction and targeted therapy}, volume = {5}, number = {1}, pages = {1}, doi = {10.1038/s41392-019-0089-y}, pmid = {32296011}, issn = {2059-3635}, support = {No. 81602492//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, abstract = {Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.}, }
@article {pmid31337983, year = {2019}, author = {Yamagishi, A and Susaki, M and Takano, Y and Mizusawa, M and Mishima, M and Iijima, M and Kuroda, S and Okada, T and Nakamura, C}, title = {The Structural Function of Nestin in Cell Body Softening is Correlated with Cancer Cell Metastasis.}, journal = {International journal of biological sciences}, volume = {15}, number = {7}, pages = {1546-1556}, pmid = {31337983}, issn = {1449-2288}, mesh = {Actins/*chemistry ; Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Chemotaxis ; Cytoskeleton/chemistry ; Female ; Humans ; Mice ; Mice, Inbred BALB C ; Microscopy, Atomic Force ; Neoplasm Invasiveness ; Neoplasm Metastasis/*pathology ; Nestin/chemistry/*physiology ; Protein Domains ; Sequence Analysis, RNA ; Stress, Mechanical ; Vimentin/*chemistry ; }, abstract = {Intermediate filaments play significant roles in governing cell stiffness and invasive ability. Nestin is a type VI intermediate filament protein that is highly expressed in several high-metastatic cancer cells. Although inhibition of nestin expression was shown to reduce the metastatic capacity of tumor cells, the relationship between this protein and the mechanism of cancer cell metastasis remains unclear. Here, we show that nestin softens the cell body of the highly metastatic mouse breast cancer cell line FP10SC2, thereby enhancing the metastasis capacity. Proximity ligation assay demonstrated increased binding between actin and vimentin in nestin knockout cells. Because nestin copolymerizes with vimentin and nestin has an extremely long tail domain in its C-terminal region, we hypothesized that the tail domain functions as a steric inhibitor of the vimentin-actin interaction and suppresses association of vimentin filaments with the cortical actin cytoskeleton, leading to reduced cell stiffness. To demonstrate this function, we mechanically pulled vimentin filaments in living cells using a nanoneedle modified with vimentin-specific antibodies under manipulation by atomic force microscopy (AFM). The tensile test revealed that mobility of vimentin filaments was increased by nestin expression in FP10SC2 cells.}, }
@article {pmid31161346, year = {2019}, author = {Balboa, D and Prasad, RB and Groop, L and Otonkoski, T}, title = {Genome editing of human pancreatic beta cell models: problems, possibilities and outlook.}, journal = {Diabetologia}, volume = {62}, number = {8}, pages = {1329-1336}, pmid = {31161346}, issn = {1432-0428}, mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Diabetes Mellitus/*genetics/*therapy ; *Gene Editing/methods/trends ; Gene Silencing ; Genetic Variation ; *Genome, Human ; Genotype ; Humans ; Insulin-Secreting Cells/*metabolism ; Pluripotent Stem Cells ; Polymorphism, Single Nucleotide ; Risk ; }, abstract = {Understanding the molecular mechanisms behind beta cell dysfunction is essential for the development of effective and specific approaches for diabetes care and prevention. Physiological human beta cell models are needed for this work. We review the possibilities and limitations of currently available human beta cell models and how they can be dramatically enhanced using genome-editing technologies. In addition to the gold standard, primary isolated islets, other models now include immortalised human beta cell lines and pluripotent stem cell-derived islet-like cells. The scarcity of human primary islet samples limits their use, but valuable gene expression and functional data from large collections of human islets have been made available to the scientific community. The possibilities for studying beta cell physiology using immortalised human beta cell lines and stem cell-derived islets are rapidly evolving. However, the functional immaturity of these cells is still a significant limitation. CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) has enabled precise engineering of specific genetic variants, targeted transcriptional modulation and genome-wide genetic screening. These approaches can now be exploited to gain understanding of the mechanisms behind coding and non-coding diabetes-associated genetic variants, allowing more precise evaluation of their contribution to diabetes pathogenesis. Despite all the progress, genome editing in primary pancreatic islets remains difficult to achieve, an important limitation requiring further technological development.}, }
@article {pmid31110156, year = {2019}, author = {Goswami, MT and VanDenBerg, KR and Han, S and Wang, LL and Singh, B and Weiss, T and Barlow, M and Kamberov, S and Wilder-Romans, K and Rhodes, DR and Feng, FY and Tomlins, SA}, title = {Identification of TP53RK-Binding Protein (TPRKB) Dependency in TP53-Deficient Cancers.}, journal = {Molecular cancer research : MCR}, volume = {17}, number = {8}, pages = {1652-1664}, pmid = {31110156}, issn = {1557-3125}, support = {R01 CA183857/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; *Apoptosis ; Apoptosis Regulatory Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Cell Cycle ; *Cell Proliferation ; Colonic Neoplasms/genetics/metabolism/*pathology ; Female ; Humans ; Intracellular Signaling Peptides and Proteins/genetics/*metabolism ; Mice ; Mice, Nude ; *Mutation ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53/antagonists &amp; inhibitors/genetics/*metabolism ; Xenograft Model Antitumor Assays ; }, abstract = {Tumor protein 53 (TP53; p53) is the most frequently altered gene in human cancer. Identification of vulnerabilities imposed by TP53 alterations may enable effective therapeutic approaches. Through analyzing short hairpin RNA (shRNA) screening data, we identified TP53RK-Binding Protein (TPRKB), a poorly characterized member of the tRNA-modifying EKC/KEOPS complex, as the most significant vulnerability in TP53-mutated cancer cell lines. In vitro and in vivo, across multiple benign-immortalized and cancer cell lines, we confirmed that TPRKB knockdown in TP53-deficient cells significantly inhibited proliferation, with minimal effect in TP53 wild-type cells. TP53 reintroduction into TP53-null cells resulted in loss of TPRKB sensitivity, confirming the importance of TP53 status in this context. In addition, cell lines with mutant TP53 or amplified MDM2 (E3-ubiquitin ligase for TP53) also showed high sensitivity to TPRKB knockdown, consistent with TPRKB dependence in a wide array of TP53-altered cancers. Depletion of other EKC/KEOPS complex members exhibited TP53-independent effects, supporting complex-independent functions of TPRKB. Finally, we found that TP53 indirectly mediates TPRKB degradation, which was rescued by coexpression of PRPK, an interacting member of the EKC/KEOPS complex, or proteasome inhibition. Together, these results identify a unique and specific requirement of TPRKB in a variety of TP53-deficient cancers. IMPLICATIONS: Cancer cells with genomic alterations in TP53 are dependent on TPRKB.}, }
@article {pmid32294089, year = {2020}, author = {Fajardo-Ortiz, D and Shattuck, A and Hornbostel, S}, title = {Mapping the coevolution, leadership and financing of research on viral vectors, RNAi, CRISPR/Cas9 and other genomic editing technologies.}, journal = {PloS one}, volume = {15}, number = {4}, pages = {e0227593}, doi = {10.1371/journal.pone.0227593}, pmid = {32294089}, issn = {1932-6203}, abstract = {Genomic editing technologies are developing rapidly, promising significant developments for biomedicine, agriculture and other fields. In the present investigation, we analyzed and compared the process of innovation for six genomic technologies: viral vectors, RNAi, TALENs, meganucleases, ZFNs and CRISPR/Cas including the profile of the main research institutions and their funders, to understand how innovation evolved and what institutions influenced research trajectories. A Web of Science search of papers on viral vectors RNAi, CRISPR/Cas, TALENs, ZFNs and meganucleases was used to build a citation network of 16,746 papers. An analysis of network clustering combined with text mining was performed. For viral vectors, a long-term process of incremental innovation was identified, which was largely publicly funded in the United States and the European Union. The trajectory of RNAi research included clusters related to the study of RNAi as a biological phenomenon and its use in functional genomics, biomedicine and pest control. A British philanthropic organization and a US pharmaceutical company played a key role in the development of basic RNAi research and clinical application respectively, in addition to government and academic institutions. In the case of CRISPR/Cas research, basic science discoveries led to the technical improvements, and these two in turn provided the information required for the development of biomedical, agricultural, livestock and industrial applications. The trajectory of CRISPR/Cas research exhibits a geopolitical division of the investigation efforts between the US, as the main producer and funder of basic research and technical improvements, and Chinese research institutions increasingly leading applied research. Our results reflect a change in the model for financing science, with reduced public financing for basic science and applied research on publicly funded technological developments in the US, and the emergence of China as a scientific superpower, with implications for the development of applications of genomic technologies.}, }
@article {pmid32293244, year = {2020}, author = {Silveira, MC and Rocha-de-Souza, CM and Albano, RM and de Oliveira Santos, IC and Carvalho-Assef, APD}, title = {Exploring the success of Brazilian endemic clone Pseudomonas aeruginosa ST277 and its association with the CRISPR-Cas system type I-C.}, journal = {BMC genomics}, volume = {21}, number = {1}, pages = {255}, doi = {10.1186/s12864-020-6650-9}, pmid = {32293244}, issn = {1471-2164}, abstract = {BACKGROUND: The Brazilian endemic clone Pseudomonas aeruginosa ST277 carries important antibiotic resistance determinants, highlighting the gene coding for SPM-1 carbapenemase. However, the resistance and persistence of this clone is apparently restricted to the Brazilian territory. To understand the differences between Brazilian strains from those isolated in other countries, we performed a phylogenetic analysis of 47 P. aeruginosa ST277 genomes as well as analyzed the virulence and resistance gene profiles. Furthermore, we evaluated the distribution of genomic islands and assessed in detail the characteristics of the CRISPR-Cas immunity system in these isolates.<br><br>RESULTS: The Brazilian genomes presented a typical set of resistance and virulence determinants, genomic islands and a high frequency of the CRISPR-Cas system type I-C. Even though the ST277 genomes are closely related, the phylogenetic analysis showed that the Brazilian strains share a great number of exclusively SNPs when compared to other ST277 genomes. We also observed a standard CRISPR spacers content for P. aeruginosa ST277, confirming a strong link between sequence type and spacer acquisition. Most CRISPR spacer targets were phage sequences.<br><br>CONCLUSIONS: Based on our findings, P. aeruginosa ST277 strains circulating in Brazil characteristically acquired In163 and PAGI-25, which can distinguish them from strains that do not accumulate resistance mechanisms and can be found on the Asian, European and North American continents. The distinctive genetic elements accumulated in Brazilian samples can contribute to the resistance, pathogenicity and transmission success that characterize the ST277 in this country.}, }
@article {pmid32292777, year = {2020}, author = {Cen, YK and Lin, JG and Wang, YL and Wang, JY and Liu, ZQ and Zheng, YG}, title = {The Gibberellin Producer Fusarium fujikuroi: Methods and Technologies in the Current Toolkit.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {8}, number = {}, pages = {232}, doi = {10.3389/fbioe.2020.00232}, pmid = {32292777}, issn = {2296-4185}, abstract = {In recent years, there has been a noticeable increase in research interests on the Fusarium species, which includes prevalent plant pathogens and human pathogens, common microbial food contaminants and industrial microbes. Taken the advantage of gibberellin synthesis, Fusarium fujikuroi succeed in being a prevalent plant pathogen. At the meanwhile, F. fujikuroi was utilized for industrial production of gibberellins, a group of extensively applied phytohormone. F. fujikuroi has been known for its outstanding performance in gibberellin production for almost 100 years. Research activities relate to this species has lasted for a very long period. The slow development in biological investigation of F. fujikuroi is largely due to the lack of efficient research technologies and molecular tools. During the past decade, technologies to analyze the molecular basis of host-pathogen interactions and metabolic regulations have been developed rapidly, especially on the aspects of genetic manipulation. At the meanwhile, the industrial fermentation technologies kept sustained development. In this article, we reviewed the currently available research tools/methods for F. fujikuroi research, focusing on the topics about genetic engineering and gibberellin production.}, }
@article {pmid32292501, year = {2020}, author = {Wu, SS and Li, QC and Yin, CQ and Xue, W and Song, CQ}, title = {Advances in CRISPR/Cas-based Gene Therapy in Human Genetic Diseases.}, journal = {Theranostics}, volume = {10}, number = {10}, pages = {4374-4382}, doi = {10.7150/thno.43360}, pmid = {32292501}, issn = {1838-7640}, abstract = {CRISPR/Cas genome editing is a simple, cost effective, and highly specific technique for introducing genetic variations. In mammalian cells, CRISPR/Cas can facilitate non-homologous end joining, homology- directed repair, and single-base exchanges. Cas9/Cas12a nuclease, dCas9 transcriptional regulators, base editors, PRIME editors and RNA editing tools are widely used in basic research. Currently, a variety of CRISPR/Cas-based therapeutics are being investigated in clinical trials. Among many new findings that have advanced the field, we highlight a few recent advances that are relevant to CRISPR/Cas-based gene therapies for monogenic human genetic diseases.}, }
@article {pmid32288252, year = {2018}, author = {Koo, B and Kim, DE and Kweon, J and Jin, CE and Kim, SH and Kim, Y and Shin, Y}, title = {CRISPR/dCas9-mediated biosensor for detection of tick-borne diseases.}, journal = {Sensors and actuators. B, Chemical}, volume = {273}, number = {}, pages = {316-321}, doi = {10.1016/j.snb.2018.06.069}, pmid = {32288252}, issn = {0925-4005}, abstract = {Rapid and highly sensitive detection of biomolecules is greatly needed for pathogen diagnosis in clinical samples, but the method needs to be significantly improved in terms of sensitivity and specificity for actual use in clinical settings. Here, we report the development of an improved molecular diagnostics tool that utilizes CRISPR/dCas9-mediated biosensor that couples a nuclease inactivated Cas9 (dCas9) and single microring resonator biosensor, enables label-free and real-time detection of pathogenic DNA and RNA. We addressed the clinical utility of this CRISPR/dCas9-mediated biosensor in tick-borne illnesses including scrub typhus (ST) and severe fever with thrombocytopenia syndrome (SFTS), whose clinical presentations are too similar to be easily differentiated. By using CRISPR/dCas9-mediated biosensor, we achieved single molecule sensitivity for the detection of ST (0.54 aM) and SFTS (0.63 aM); this detection sensitivity is 100 times more sensitive than that of RT-PCR assay. Finally, CRISPR/dCas9-mediated biosensor was able to clearly distinguish between ST and SFTS in serum samples within 20 min. We believe that CRISPR/dCas9-mediated biosensor will be useful for rapid and accurate molecular diagnostic tool that is suitable for immediate clinical applications.}, }
@article {pmid32015438, year = {2020}, author = {Gan, W and Dai, X and Dai, X and Xie, J and Yin, S and Zhu, J and Wang, C and Liu, Y and Guo, J and Wang, M and Liu, J and Hu, J and Quinton, RJ and Ganem, NJ and Liu, P and Asara, JM and Pandolfi, PP and Yang, Y and He, Z and Gao, G and Wei, W}, title = {LATS suppresses mTORC1 activity to directly coordinate Hippo and mTORC1 pathways in growth control.}, journal = {Nature cell biology}, volume = {22}, number = {2}, pages = {246-256}, pmid = {32015438}, issn = {1476-4679}, support = {R01 CA200651/CA/NCI NIH HHS/United States ; R00 CA181342/CA/NCI NIH HHS/United States ; R01 GM094777/GM/NIGMS NIH HHS/United States ; P30 EY012196/EY/NEI NIH HHS/United States ; R00 CA207867/CA/NCI NIH HHS/United States ; R01 CA222571/CA/NCI NIH HHS/United States ; R01 CA177910/CA/NCI NIH HHS/United States ; P30 CA006516/CA/NCI NIH HHS/United States ; P01 CA120964/CA/NCI NIH HHS/United States ; R01 CA200573/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Colonic Neoplasms/genetics/metabolism/pathology ; Female ; Gene Editing ; *Gene Expression Regulation, Developmental ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Heterografts ; Humans ; Liver/abnormalities/metabolism ; MCF-7 Cells ; Mechanistic Target of Rapamycin Complex 1/*genetics/metabolism ; Mice ; Mice, Nude ; Mice, Transgenic ; Myocardium/metabolism/pathology ; Neurofibromin 2/deficiency/genetics ; Organ Size ; Protein-Serine-Threonine Kinases/deficiency/*genetics/metabolism ; Ras Homolog Enriched in Brain Protein/*genetics/metabolism ; Regulatory-Associated Protein of mTOR/*genetics/metabolism ; Signal Transduction ; Tumor Suppressor Proteins/deficiency/*genetics ; }, abstract = {The Hippo and mammalian target of rapamycin complex 1 (mTORC1) pathways are the two predominant growth-control pathways that dictate proper organ development. We therefore explored potential crosstalk between these two functionally relevant pathways to coordinate their growth-control functions. We found that the LATS1 and LATS2 kinases, the core components of the Hippo pathway, phosphorylate S606 of Raptor, an essential component of mTORC1, to attenuate mTORC1 activation by impairing the interaction of Raptor with Rheb. The phosphomimetic Raptor-S606D knock-in mutant led to a reduction in cell size and proliferation. Compared with Raptor+/+ mice, RaptorD/D knock-in mice exhibited smaller livers and hearts, and a significant inhibition of elevation in mTORC1 signalling induced by Nf2 or Lats1 and Lats2 loss. Thus, our study reveals a direct link between the Hippo and mTORC1 pathways to fine-tune organ growth.}, }
@article {pmid32015436, year = {2020}, author = {Barucci, G and Cornes, E and Singh, M and Li, B and Ugolini, M and Samolygo, A and Didier, C and Dingli, F and Loew, D and Quarato, P and Cecere, G}, title = {Small-RNA-mediated transgenerational silencing of histone genes impairs fertility in piRNA mutants.}, journal = {Nature cell biology}, volume = {22}, number = {2}, pages = {235-245}, doi = {10.1038/s41556-020-0462-7}, pmid = {32015436}, issn = {1476-4679}, support = {P40 OD010440/OD/NIH HHS/United States ; }, mesh = {Animals ; Animals, Genetically Modified ; Argonaute Proteins/deficiency/*genetics/metabolism ; Biological Evolution ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics/metabolism ; Caenorhabditis elegans Proteins/*genetics/metabolism ; Fertility/genetics ; Gene Editing ; *Gene Silencing ; Histones/*genetics/metabolism ; Inheritance Patterns ; Mutation ; RNA, Antisense/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; RNA, Small Interfering/*genetics/metabolism ; Repetitive Sequences, Nucleic Acid ; }, abstract = {PIWI-interacting RNAs (piRNAs) promote fertility in many animals. However, whether this is due to their conserved role in repressing repetitive elements (REs) remains unclear. Here, we show that the progressive loss of fertility in Caenorhabditis elegans lacking piRNAs is not caused by derepression of REs or other piRNA targets but, rather, is mediated by epigenetic silencing of all of the replicative histone genes. In the absence of piRNAs, downstream components of the piRNA pathway relocalize from germ granules and piRNA targets to histone mRNAs to synthesize antisense small RNAs (sRNAs) and induce transgenerational silencing. Removal of the downstream components of the piRNA pathway restores histone mRNA expression and fertility in piRNA mutants, and the inheritance of histone sRNAs in wild-type worms adversely affects their fertility for multiple generations. We conclude that sRNA-mediated silencing of histone genes impairs the fertility of piRNA mutants and may serve to maintain piRNAs across evolution.}, }
@article {pmid31817535, year = {2019}, author = {Kim, HK and Ham, KA and Lee, SW and Choi, HS and Kim, HS and Kim, HK and Shin, HS and Seo, KY and Cho, Y and Nam, KT and Kim, IB and Joe, YA}, title = {Biallelic Deletion of Pxdn in Mice Leads to Anophthalmia and Severe Eye Malformation.}, journal = {International journal of molecular sciences}, volume = {20}, number = {24}, pages = {}, pmid = {31817535}, issn = {1422-0067}, mesh = {Animals ; *Anophthalmos/genetics/metabolism/pathology ; CRISPR-Cas Systems ; Collagen Type IV/genetics/metabolism ; Eye/*growth &amp; development/pathology ; *Gene Deletion ; Mice ; Mice, Knockout ; Peroxidases/*deficiency/metabolism ; Vision, Ocular/genetics ; }, abstract = {Peroxidasin (PXDN) is a unique peroxidase containing extracellular matrix motifs and stabilizes collagen IV networks by forming sulfilimine crosslinks. PXDN gene knockout in Caenorhabditis elegans (C. elegans) and Drosophila results in the demise at the embryonic and larval stages. PXDN mutations lead to severe eye disorders, including microphthalmia, cataract, glaucoma, and anterior segment dysgenesis in humans and mice. To investigate how PXDN loss of function affects organ development, we generated Pxdn knockout mice by deletion of exon 1 and its 5' upstream sequences of the Pxdn gene using the CRISPR/Cas9 system. Loss of both PXDN expression and collagen IV sulfilimine cross-links was detected only in the homozygous mice, which showed completely or almost closed eyelids with small eyes, having no apparent external morphological defects in other organs. In histological analysis of eye tissues, the homozygous mice had extreme defects in eye development, including no eyeballs or drastically disorganized eye structures, whereas the heterozygous mice showed normal eye structure. Visual function tests also revealed no obvious functional abnormalities in the eyes between heterozygous mice and wild-type mice. Thus, these results suggest that PXDN activity is essential in eye development, and also indicate that a single allele of Pxdn gene is sufficient for eye-structure formation and normal visual function.}, }
@article {pmid31754021, year = {2019}, author = {Erwood, S and Brewer, RA and Bily, TMI and Maino, E and Zhou, L and Cohn, RD and Ivakine, EA}, title = {Modeling Niemann-Pick disease type C in a human haploid cell line allows for patient variant characterization and clinical interpretation.}, journal = {Genome research}, volume = {29}, number = {12}, pages = {2010-2019}, pmid = {31754021}, issn = {1549-5469}, mesh = {*CRISPR-Cas Systems ; Cell Line ; *Gene Editing ; *Genome, Human ; *Haploidy ; Humans ; *Models, Genetic ; Niemann-Pick Disease, Type C/*genetics ; *Whole Genome Sequencing ; }, abstract = {The accurate clinical interpretation of human sequence variation is foundational to personalized medicine. This remains a pressing challenge, however, as genome sequencing becomes routine and new functionally undefined variants rapidly accumulate. Here, we describe a platform for the rapid generation, characterization, and interpretation of genomic variants in haploid cells focusing on Niemann-Pick disease type C (NPC) as an example. NPC is a fatal neurodegenerative disorder characterized by a lysosomal accumulation of unesterified cholesterol and glycolipids. In 95% of cases, NPC is caused by mutations in the NPC1 gene, for which more than 200 unique disease-causing variants have been reported to date. Furthermore, the majority of patients with NPC are compound heterozygotes that often carry at least one private mutation, presenting a challenge for the characterization and classification of individual variants. Here, we have developed the first haploid cell model of NPC. This haploid cell model recapitulates the primary biochemical and molecular phenotypes typically found in patient-derived fibroblasts, illustrating its utility in modeling NPC. Additionally, we show the power of CRISPR/Cas9-mediated base editing in quickly and efficiently generating haploid cell models of individual patient variants in NPC. These models provide a platform for understanding the disease mechanisms underlying individual NPC1 variants while allowing for definitive clinical variant interpretation for NPC.}, }
@article {pmid31508509, year = {2019}, author = {Fang, P and De Souza, C and Minn, K and Chien, J}, title = {Genome-scale CRISPR knockout screen identifies TIGAR as a modifier of PARP inhibitor sensitivity.}, journal = {Communications biology}, volume = {2}, number = {}, pages = {335}, pmid = {31508509}, issn = {2399-3642}, support = {P30 CA168524/CA/NCI NIH HHS/United States ; }, mesh = {Antineoplastic Agents/pharmacology ; Apoptosis Regulatory Proteins/*genetics ; Biomarkers ; *CRISPR-Cas Systems ; Cellular Senescence ; DNA Damage ; Drug Resistance, Neoplasm/*genetics ; Gene Knockdown Techniques ; Genome-Wide Association Study ; Humans ; Phosphoric Monoester Hydrolases/*genetics ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology ; Signal Transduction ; }, abstract = {Treatment of cancer with poly (ADP-ribose) polymerase (PARP) inhibitors is currently limited to cells defective in the homologous recombination (HR) pathway. Identification of genetic targets that induce or mimic HR deficiencies will extend the clinical utility of PARP inhibitors. Here we perform a CRISPR/Cas9-based genome-scale loss-of-function screen, using the sensitivity of PARP inhibitor olaparib as a surrogate. We identify C12orf5, encoding TP53 induced glycolysis and apoptosis regulator (TIGAR), as a modifier of PARP inhibitor response. We show that TIGAR is amplified in several cancer types, and higher expression of TIGAR associates with poor overall survival in ovarian cancer. TIGAR knockdown enhances sensitivity to olaparib in cancer cells via downregulation of BRCA1 and the Fanconi anemia pathway and increases senescence of these cells by affecting metabolic pathways and increasing the cytotoxic effects of olaparib. Our results indicate TIGAR should be explored as a therapeutic target for treating cancer and extending the use of PARP inhibitors.}, }
@article {pmid31365053, year = {2019}, author = {Lee, MCS and Lindner, SE and Lopez-Rubio, JJ and Llinás, M}, title = {Cutting back malaria: CRISPR/Cas9 genome editing of Plasmodium.}, journal = {Briefings in functional genomics}, volume = {18}, number = {5}, pages = {281-289}, pmid = {31365053}, issn = {2041-2657}, support = {/WT_/Wellcome Trust/United Kingdom ; R01 AI125565/AI/NIAID NIH HHS/United States ; R21 AI130692/AI/NIAID NIH HHS/United States ; 206194/WT_/Wellcome Trust/United Kingdom ; }, mesh = {CRISPR-Associated Protein 9/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Gene Expression Regulation ; Plasmodium/*genetics ; RNA, Guide/chemistry/genetics ; }, abstract = {CRISPR/Cas9 approaches are revolutionizing our ability to perform functional genomics across a wide range of organisms, including the Plasmodium parasites that cause malaria. The ability to deliver single point mutations, epitope tags and gene deletions at increased speed and scale is enabling our understanding of the biology of these complex parasites, and pointing to potential new therapeutic targets. In this review, we describe some of the biological and technical considerations for designing CRISPR-based experiments, and discuss potential future developments that broaden the applications for CRISPR/Cas9 interrogation of the malaria parasite genome.}, }
@article {pmid31105049, year = {2019}, author = {Park, CY and Sung, JJ and Cho, SR and Kim, J and Kim, DW}, title = {Universal Correction of Blood Coagulation Factor VIII in Patient-Derived Induced Pluripotent Stem Cells Using CRISPR/Cas9.}, journal = {Stem cell reports}, volume = {12}, number = {6}, pages = {1242-1249}, pmid = {31105049}, issn = {2213-6711}, mesh = {*CRISPR-Cas Systems ; *Factor VIII/biosynthesis/genetics ; *Gene Editing ; *Hemophilia A/genetics/metabolism/pathology/therapy ; Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *Mutation ; }, abstract = {Hemophilia A (HA) is caused by genetic mutations in the blood coagulation factor VIII (FVIII) gene. Genome-editing approaches can be used to target the mutated site itself in patient-derived induced pluripotent stem cells (iPSCs). However, these approaches can be hampered by difficulty in preparing thousands of editing platforms for each corresponding variant found in HA patients. Here, we report a universal approach to correct the various mutations in HA patient iPSCs by the targeted insertion of the FVIII gene into the human H11 site via CRISPR/Cas9. We derived corrected clones from two types of patient iPSCs with frequencies of up to 64% and 66%, respectively, without detectable unwanted off-target mutations. Moreover, we demonstrated that endothelial cells differentiated from the corrected iPSCs successfully secreted functional protein. This strategy may provide a universal therapeutic method for correcting all genetic variants found in HA patients.}, }
@article {pmid30865899, year = {2019}, author = {Fang, Z and Weng, C and Li, H and Tao, R and Mai, W and Liu, X and Lu, L and Lai, S and Duan, Q and Alvarez, C and Arvan, P and Wynshaw-Boris, A and Li, Y and Pei, Y and Jin, F and Li, Y}, title = {Single-Cell Heterogeneity Analysis and CRISPR Screen Identify Key β-Cell-Specific Disease Genes.}, journal = {Cell reports}, volume = {26}, number = {11}, pages = {3132-3144.e7}, pmid = {30865899}, issn = {2211-1247}, support = {P30 DK020572/DK/NIDDK NIH HHS/United States ; R01 DK113185/DK/NIDDK NIH HHS/United States ; R01 HG009658/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics/metabolism ; Cells, Cultured ; Chromosomal Proteins, Non-Histone/genetics/metabolism ; Diabetes Mellitus, Type 2/*genetics/metabolism ; Gene Expression Profiling ; *Genetic Heterogeneity ; HEK293 Cells ; Histone Acetyltransferases/genetics/metabolism ; Humans ; Insulin/genetics/metabolism ; Insulin-Secreting Cells/*metabolism ; Mice ; Single-Cell Analysis ; *Transcriptome ; }, abstract = {Identification of human disease signature genes typically requires samples from many donors to achieve statistical significance. Here, we show that single-cell heterogeneity analysis may overcome this hurdle by significantly improving the test sensitivity. We analyzed the transcriptome of 39,905 single islets cells from 9 donors and observed distinct β cell heterogeneity trajectories associated with obesity or type 2 diabetes (T2D). We therefore developed RePACT, a sensitive single-cell analysis algorithm to identify both common and specific signature genes for obesity and T2D. We mapped both β-cell-specific genes and disease signature genes to the insulin regulatory network identified from a genome-wide CRISPR screen. Our integrative analysis discovered the previously unrecognized roles of the cohesin loading complex and the NuA4/Tip60 histone acetyltransferase complex in regulating insulin transcription and release. Our study demonstrated the power of combining single-cell heterogeneity analysis and functional genomics to dissect the etiology of complex diseases.}, }
@article {pmid32289196, year = {2020}, author = {Yu, Y and Wang, X and Sun, H and Liang, Q and Wang, W and Zhang, C and Bian, X and Cao, Q and Li, Q and Xie, Y and Ma, D and Li, Z and Sun, J}, title = {Improving CRISPR-Cas-mediated RNA targeting and gene editing using SPLCV replicon-based expression vectors in Nicotiana benthamiana.}, journal = {Plant biotechnology journal}, volume = {}, number = {}, pages = {}, doi = {10.1111/pbi.13384}, pmid = {32289196}, issn = {1467-7652}, abstract = {In this study, we developed Sweet Potato Leaf Curl Virus (SPLCV) replicon-based expression vectors and tested the efficiency of these vectors in CRISPR-Cas-mediated RNA targeting and gene editing by using Nicotiana benthamiana as model plant. Our results showed that the SPLCV-based vectors remarkably enhance the LwaCas13a-mediated RNA targeting activity nd SpCas9/LbCas12a-mediated gene editing efficiency in Nicotiana benthamiana. Our work expands the existing list of geminiviral replicon-based vectors in plants. The SPLCV-based vectors offer an opportunity to enhance the efficiency of CRISPR-Cas-mediated gene knockdown, knockout, and knockin in plants, especially for its host sweetpotato.}, }
@article {pmid32287058, year = {2020}, author = {Ye, L and Wang, J and Teque, F and Xie, F and Tan, Y and Kan, YW and Levy, JA}, title = {Generation of HIV-1 infected patient's gene-edited iPSC using feeder free culture conditions.}, journal = {AIDS (London, England)}, volume = {}, number = {}, pages = {}, doi = {10.1097/QAD.0000000000002535}, pmid = {32287058}, issn = {1473-5571}, abstract = {: The discovery of induced pluripotent stem cells (iPSC) has brought promise to regenerative medicine since it breaks the ethical barrier of using embryonic stem cells. Such cell culture-derived patient specific autologous stem cells are needed for transplantation. Here we report deriving HIV-1 infected patient's iPSC lines under transgene- free methods and under feeder- free and xeno-free culture conditions to meet the requirement for clinical application. We have reprogrammed patients' peripheral blood mononuclear cells with EBNA1/OriP episomal vectors, or a defective and persistent Sendai virus vector (SeVdp) to ensure a non-integrating iPSC generation. Both single picked and pooled iPSC lines demonstrated high pluripotency and were able to differentiate into various lineage cells in vivo. The established cell lines could be modified by genetic editing using the TALENs or CRISPR/Cas 9 technology to have a bi-allelic CCR5 delta32 mutations seamlessly. All generated iPSC lines and modified cell lines had no evidence of HIV integration and maintained normal karyotype after expansion. Our study provides a reproducible simple procedure for generating therapeutic grade iPSC from HIV-infected subjects and for engineering these cells to possess a naturally occurring genotype for resistance to HIV-1 infection when differentiated into immune cells.}, }
@article {pmid32286628, year = {2020}, author = {Eitzinger, S and Asif, A and Watters, KE and Iavarone, AT and Knott, GJ and Doudna, JA and Minhas, FUAA}, title = {Machine learning predicts new anti-CRISPR proteins.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkaa219}, pmid = {32286628}, issn = {1362-4962}, abstract = {The increasing use of CRISPR-Cas9 in medicine, agriculture, and synthetic biology has accelerated the drive to discover new CRISPR-Cas inhibitors as potential mechanisms of control for gene editing applications. Many anti-CRISPRs have been found that inhibit the CRISPR-Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method to aid direct identification of new potential anti-CRISPRs using only protein sequence information. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking. We then applied AcRanker to predict candidate anti-CRISPRs from predicted prophage regions within self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA20 (ML1) and AcrIIA21 (ML8). We show that AcrIIA20 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA21 inhibits SpyCas9, Streptococcus aureus Cas9 (SauCas9) and SinCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.}, }
@article {pmid32284602, year = {2020}, author = {Mathony, J and Harteveld, Z and Schmelas, C and Upmeier Zu Belzen, J and Aschenbrenner, S and Sun, W and Hoffmann, MD and Stengl, C and Scheck, A and Georgeon, S and Rosset, S and Wang, Y and Grimm, D and Eils, R and Correia, BE and Niopek, D}, title = {Computational design of anti-CRISPR proteins with improved inhibition potency.}, journal = {Nature chemical biology}, volume = {}, number = {}, pages = {}, doi = {10.1038/s41589-020-0518-9}, pmid = {32284602}, issn = {1552-4469}, abstract = {Anti-CRISPR (Acr) proteins are powerful tools to control CRISPR-Cas technologies. However, the available Acr repertoire is limited to naturally occurring variants. Here, we applied structure-based design on AcrIIC1, a broad-spectrum CRISPR-Cas9 inhibitor, to improve its efficacy on different targets. We first show that inserting exogenous protein domains into a selected AcrIIC1 surface site dramatically enhances inhibition of Neisseria meningitidis (Nme)Cas9. Then, applying structure-guided design to the Cas9-binding surface, we converted AcrIIC1 into AcrIIC1X, a potent inhibitor of the Staphylococcus aureus (Sau)Cas9, an orthologue widely applied for in vivo genome editing. Finally, to demonstrate the utility of AcrIIC1X for genome engineering applications, we implemented a hepatocyte-specific SauCas9 ON-switch by placing AcrIIC1X expression under regulation of microRNA-122. Our work introduces designer Acrs as important biotechnological tools and provides an innovative strategy to safeguard CRISPR technologies.}, }
@article {pmid32284587, year = {2020}, author = {Hanna, RE and Doench, JG}, title = {Design and analysis of CRISPR-Cas experiments.}, journal = {Nature biotechnology}, volume = {}, number = {}, pages = {}, doi = {10.1038/s41587-020-0490-7}, pmid = {32284587}, issn = {1546-1696}, abstract = {A large and ever-expanding set of CRISPR-Cas systems now enables the rapid and flexible manipulation of genomes in both targeted and large-scale experiments. Numerous software tools and analytical methods have been developed for the design and analysis of CRISPR-Cas experiments, including resources to design optimal guide RNAs for various modes of manipulation and to analyze the results of such experiments. A major recent focus has been the development of comprehensive tools for use on data from large-scale CRISPR-based genetic screens. As this field continues to progress, a clear ongoing challenge is not only to innovate, but to actively maintain and improve existing tools so that researchers across disciplines can rely on a stable set of excellent computational resources for CRISPR-Cas experiments.}, }
@article {pmid32284320, year = {2020}, author = {Ratner, HK and Weiss, DS}, title = {F. novicida CRISPR-Cas systems can functionally complement each other in DNA defense while providing target flexibility.}, journal = {Journal of bacteriology}, volume = {}, number = {}, pages = {}, doi = {10.1128/JB.00670-19}, pmid = {32284320}, issn = {1098-5530}, abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that facilitate protection of bacteria and archaea against infection by external mobile genetic elements. The model pathogen Francisella novicida encodes a CRISPR-Cas12a (FnoCas12a) system and a CRISPR-Cas9 (FnoCas9) system, the latter of which has an additional and non-canonical function in bacterial virulence. Here, we investigated and compared the functional roles of the FnoCas12a and FnoCas9 systems in transformation inhibition and bacterial virulence. Unlike FnoCas9, FnoCas12a was not required for F. novicida virulence. However, both systems were highly effective at plasmid restriction and acted independently of each other. We further identified a critical protospacer adjacent motif (PAM) necessary for transformation inhibition by FnoCas12a, demonstrating a greater flexibility for target identification by FnoCas12a than previously appreciated, and a specificity that is distinct from that of FnoCas9. The effectors of the two systems exhibited different patterns of expression at the mRNA level, suggesting they may confer distinct benefits to the bacterium in diverse environments. These data suggest that due to the differences between the two CRISPR-Cas systems, together they may provide F. novicida with a more comprehensive defense against foreign nucleic acids. Finally, we demonstrated that the FnoCas12a and FnoCas9 machineries could be simultaneously engineered to restrict the same non-native target, thereby expanding the toolset for prokaryotic genome manipulation.Importance CRISPR-Cas9 and CRISPR-Cas12a systems have been widely commandeered for genome engineering. However, they originate in prokaryotes where they function as adaptive immune systems. The details of this activity and relationship between these systems within native host organisms have been minimally explored. The human pathogen Francisella novicida contains both of these systems, with the Cas9 system also exhibiting a second activity, modulating virulence through transcriptional regulation. We compared and contrasted the ability of these two systems to control virulence and restrict DNA within their native host bacterium, highlighting differences and similarities in these two functions. Collectively, our results indicate that these two distinct and reprogrammable endogenous systems provide F. novicida with a more comprehensive defense against mobile genetic elements.}, }
@article {pmid32066938, year = {2020}, author = {Gutierrez-Arcelus, M and Baglaenko, Y and Arora, J and Hannes, S and Luo, Y and Amariuta, T and Teslovich, N and Rao, DA and Ermann, J and Jonsson, AH and ,  and Navarrete, C and Rich, SS and Taylor, KD and Rotter, JI and Gregersen, PK and Esko, T and Brenner, MB and Raychaudhuri, S}, title = {Allele-specific expression changes dynamically during T cell activation in HLA and other autoimmune loci.}, journal = {Nature genetics}, volume = {52}, number = {3}, pages = {247-253}, pmid = {32066938}, issn = {1546-1718}, support = {U01 GM092691/GM/NIGMS NIH HHS/United States ; UL1 TR001881/TR/NCATS NIH HHS/United States ; R01 HL120393/HL/NHLBI NIH HHS/United States ; U19 AI111224/AI/NIAID NIH HHS/United States ; R01 AR063759/AR/NIAMS NIH HHS/United States ; T32 HG002295/HG/NHGRI NIH HHS/United States ; HHSN268201000001I/HL/NHLBI NIH HHS/United States ; U01 HG009379/HG/NHGRI NIH HHS/United States ; UH2 AR067677/AR/NIAMS NIH HHS/United States ; }, mesh = {Alleles ; Autoimmunity/*genetics ; CD4-Positive T-Lymphocytes ; CRISPR-Cas Systems ; Cell Line ; Gene Expression Regulation ; Genetic Loci ; *Genetic Variation ; Genotyping Techniques ; HLA Antigens/*genetics/metabolism ; HLA-DQ beta-Chains/*genetics/metabolism ; Humans ; Immunity, Cellular ; Lymphocyte Activation/*genetics ; Promoter Regions, Genetic/*genetics ; T-Lymphocytes, Regulatory ; }, abstract = {Genetic studies have revealed that autoimmune susceptibility variants are over-represented in memory CD4+ T cell regulatory elements1-3. Understanding how genetic variation affects gene expression in different T cell physiological states is essential for deciphering genetic mechanisms of autoimmunity4,5. Here, we characterized the dynamics of genetic regulatory effects at eight time points during memory CD4+ T cell activation with high-depth RNA-seq in healthy individuals. We discovered widespread, dynamic allele-specific expression across the genome, where the balance of alleles changes over time. These genes were enriched fourfold within autoimmune loci. We found pervasive dynamic regulatory effects within six HLA genes. HLA-DQB1 alleles had one of three distinct transcriptional regulatory programs. Using CRISPR-Cas9 genomic editing we demonstrated that a promoter variant is causal for T cell-specific control of HLA-DQB1 expression. Our study shows that genetic variation in cis-regulatory elements affects gene expression in a manner dependent on lymphocyte activation status, contributing to the interindividual complexity of immune responses.}, }
@article {pmid31428700, year = {2019}, author = {Johnston, AD and Simões-Pires, CA and Suzuki, M and Greally, JM}, title = {High-efficiency genomic editing in Epstein-Barr virus-transformed lymphoblastoid B cells using a single-stranded donor oligonucleotide strategy.}, journal = {Communications biology}, volume = {2}, number = {}, pages = {312}, pmid = {31428700}, issn = {2399-3642}, support = {T32 GM007288/GM/NIGMS NIH HHS/United States ; }, mesh = {B-Lymphocytes/*metabolism/*virology ; CRISPR-Cas Systems/genetics ; Cell Line, Transformed ; Child ; Clone Cells ; *Gene Editing ; Gene Rearrangement/genetics ; Genetic Loci ; Herpesvirus 4, Human/*metabolism ; Humans ; Oligonucleotides/*metabolism ; }, abstract = {While human lymphoblastoid cell lines represent a valuable resource for population genetic studies, they have usually been regarded as difficult for CRISPR-mediated genomic editing because of very inefficient DNA transfection and retroviral or lentiviral transduction in these cells, which becomes a substantial problem when multiple constructs need to be co-expressed. Here we describe a protocol using a single-stranded donor oligonucleotide strategy for 'scarless' editing in lymphoblastoid cells, yielding 12/60 (20%) of clones with homology-directed recombination, when rates of <5-10% are frequently typical for many other cell types. The protocol does not require the use of lentiviruses or stable transfection, permitting lymphoblastoid cell lines to be used for CRISPR-mediated genomic targeting and screening in population genetic studies.}, }
@article {pmid31216436, year = {2019}, author = {Schwartz, C and Cheng, JF and Evans, R and Schwartz, CA and Wagner, JM and Anglin, S and Beitz, A and Pan, W and Lonardi, S and Blenner, M and Alper, HS and Yoshikuni, Y and Wheeldon, I}, title = {Validating genome-wide CRISPR-Cas9 function improves screening in the oleaginous yeast Yarrowia lipolytica.}, journal = {Metabolic engineering}, volume = {55}, number = {}, pages = {102-110}, doi = {10.1016/j.ymben.2019.06.007}, pmid = {31216436}, issn = {1096-7184}, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Gene Library ; *Genes, Fungal ; Yarrowia/*genetics ; }, abstract = {Genome-wide mutational screens are central to understanding the genetic underpinnings of evolved and engineered phenotypes. The widespread adoption of CRISPR-Cas9 genome editing has enabled such screens in many organisms, but identifying functional sgRNAs still remains a challenge. Here, we developed a methodology to quantify the cutting efficiency of each sgRNA in a genome-scale library, and in doing so improve screens in the biotechnologically important yeast Yarrowia lipolytica. Screening in the presence and absence of native DNA repair enabled high-throughput quantification of sgRNA function leading to the identification of high efficiency sgRNAs that cover 94% of genes. Library validation enhanced the classification of essential genes by identifying inactive guides that create false negatives and mask the effects of successful disruptions. Quantification of guide effectiveness also creates a dataset from which determinants of CRISPR-Cas9 can be identified. Finally, application of the library identified novel mutations for metabolic engineering of high lipid accumulation.}, }
@article {pmid31053612, year = {2019}, author = {Kotov, JA and Kotov, DI and Linehan, JL and Bardwell, VJ and Gearhart, MD and Jenkins, MK}, title = {BCL6 corepressor contributes to Th17 cell formation by inhibiting Th17 fate suppressors.}, journal = {The Journal of experimental medicine}, volume = {216}, number = {6}, pages = {1450-1464}, pmid = {31053612}, issn = {1540-9538}, support = {T32 AI083196/AI/NIAID NIH HHS/United States ; T32 AI007313/AI/NIAID NIH HHS/United States ; R01 HD084459/HD/NICHD NIH HHS/United States ; R01 AI103760/AI/NIAID NIH HHS/United States ; R01 AI039614/AI/NIAID NIH HHS/United States ; F31 AI133716/AI/NIAID NIH HHS/United States ; R37 AI027998/AI/NIAID NIH HHS/United States ; R01 AI027998/AI/NIAID NIH HHS/United States ; R01 CA071540/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; *Cell Lineage ; Co-Repressor Proteins/*metabolism ; Cytokines/metabolism ; F-Box Proteins/metabolism ; Female ; Gene Expression Regulation ; Jumonji Domain-Containing Histone Demethylases/metabolism ; Lymphocyte Subsets/metabolism ; Male ; Mice, Inbred C57BL ; Proto-Oncogene Proteins c-bcl-6/*metabolism ; Receptors, Chemokine/metabolism ; Repressor Proteins/*metabolism ; Signal Transduction ; Streptococcus pyogenes/physiology ; Th17 Cells/*cytology/*metabolism ; }, abstract = {CD4+ T helper 17 (Th17) cells protect vertebrate hosts from extracellular pathogens at mucosal surfaces. Th17 cells form from naive precursors when signals from the T cell antigen receptor (TCR) and certain cytokine receptors induce the expression of the RORγt transcription factor, which activates a set of Th17-specific genes. Using T cell-specific loss-of-function experiments, we find that two components of the Polycomb repressive complex 1.1 (PRC1.1), BCL6 corepressor (BCOR) and KDM2B, which helps target the complex to unmethylated CpG DNA islands, are required for optimal Th17 cell formation in mice after Streptococcus pyogenes infection. Genome-wide expression and BCOR chromatin immunoprecipitation studies revealed that BCOR directly represses Lef1, Runx2, and Dusp4, whose products inhibit Th17 differentiation. Together, the results suggest that the PRC1.1 components BCOR and KDM2B work together to enhance Th17 cell formation by repressing Th17 fate suppressors.}, }
@article {pmid30975893, year = {2019}, author = {Hartweger, H and McGuire, AT and Horning, M and Taylor, JJ and Dosenovic, P and Yost, D and Gazumyan, A and Seaman, MS and Stamatatos, L and Jankovic, M and Nussenzweig, MC}, title = {HIV-specific humoral immune responses by CRISPR/Cas9-edited B cells.}, journal = {The Journal of experimental medicine}, volume = {216}, number = {6}, pages = {1301-1310}, pmid = {30975893}, issn = {1540-9538}, support = {R01 AI129795/AI/NIAID NIH HHS/United States ; UM1 AI100663/AI/NIAID NIH HHS/United States ; }, mesh = {Adoptive Transfer ; Animals ; Antibodies, Neutralizing/immunology ; B-Lymphocytes/*immunology ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; Gene Editing ; HIV Antibodies/immunology ; HIV Infections/*immunology ; Humans ; INDEL Mutation/genetics ; *Immunity, Humoral ; Mice ; Protein Engineering ; }, abstract = {A small number of HIV-1-infected individuals develop broadly neutralizing antibodies to the virus (bNAbs). These antibodies are protective against infection in animal models. However, they only emerge 1-3 yr after infection, and show a number of highly unusual features including exceedingly high levels of somatic mutations. It is therefore not surprising that elicitation of protective immunity to HIV-1 has not yet been possible. Here we show that mature, primary mouse and human B cells can be edited in vitro using CRISPR/Cas9 to express mature bNAbs from the endogenous Igh locus. Moreover, edited B cells retain the ability to participate in humoral immune responses. Immunization with cognate antigen in wild-type mouse recipients of edited B cells elicits bNAb titers that neutralize HIV-1 at levels associated with protection against infection. This approach enables humoral immune responses that may be difficult to elicit by traditional immunization.}, }
@article {pmid30636321, year = {2019}, author = {Sung, LY and Wu, MY and Lin, MW and Hsu, MN and Truong, VA and Shen, CC and Tu, Y and Hwang, KY and Tu, AP and Chang, YH and Hu, YC}, title = {Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli.}, journal = {Biotechnology and bioengineering}, volume = {116}, number = {5}, pages = {1066-1079}, doi = {10.1002/bit.26915}, pmid = {30636321}, issn = {1097-0290}, mesh = {*CRISPR-Cas Systems ; Escherichia coli/*genetics ; Francisella/genetics ; *Gene Knockout Techniques ; *Genetic Engineering ; *Genome, Bacterial ; Staphylococcus aureus/genetics ; Streptococcus pyogenes/genetics ; }, abstract = {CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9-based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single-guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock-in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.}, }
@article {pmid30636320, year = {2019}, author = {Westbrook, A and Tang, X and Marshall, R and Maxwell, CS and Chappell, J and Agrawal, DK and Dunlop, MJ and Noireaux, V and Beisel, CL and Lucks, J and Franco, E}, title = {Distinct timescales of RNA regulators enable the construction of a genetic pulse generator.}, journal = {Biotechnology and bioengineering}, volume = {116}, number = {5}, pages = {1139-1151}, doi = {10.1002/bit.26918}, pmid = {30636320}, issn = {1097-0290}, mesh = {*CRISPR-Cas Systems ; Cell-Free System/chemistry/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Models, Chemical ; RNA/*chemistry/metabolism ; *Transcription, Genetic ; *Transcriptional Activation ; }, abstract = {To build complex genetic networks with predictable behaviors, synthetic biologists use libraries of modular parts that can be characterized in isolation and assembled together to create programmable higher-order functions. Characterization experiments and computational models for gene regulatory parts operating in isolation are routinely used to predict the dynamics of interconnected parts and guide the construction of new synthetic devices. Here, we individually characterize two modes of RNA-based transcriptional regulation, using small transcription activating RNAs (STARs) and clustered regularly interspaced short palindromic repeats interference (CRISPRi), and show how their distinct regulatory timescales can be used to engineer a composed feedforward loop that creates a pulse of gene expression. We use a cell-free transcription-translation system (TXTL) to rapidly characterize the system, and we apply Bayesian inference to extract kinetic parameters for an ordinary differential equation-based mechanistic model. We then demonstrate in simulation and verify with TXTL experiments that the simultaneous regulation of a single gene target with STARs and CRISPRi leads to a pulse of gene expression. Our results suggest the modularity of the two regulators in an integrated genetic circuit, and we anticipate that construction and modeling frameworks that can leverage this modularity will become increasingly important as synthetic circuits increase in complexity.}, }
@article {pmid30063275, year = {2019}, author = {Chang, L and Dykes, EJ and Li, J and Moreno, SNJ and Hortua Triana, MA}, title = {Characterization of Two EF-hand Domain-containing Proteins from Toxoplasma gondii.}, journal = {The Journal of eukaryotic microbiology}, volume = {66}, number = {2}, pages = {343-353}, pmid = {30063275}, issn = {1550-7408}, support = {R21 AI110027/AI/NIAID NIH HHS/United States ; R01 AI128356/AI/NIAID NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Calcium/*metabolism ; Calcium-Binding Proteins/genetics/metabolism ; Protozoan Proteins/*genetics/metabolism ; Toxoplasma/*genetics/growth &amp; development/metabolism ; }, abstract = {The universal role of calcium (Ca2+) as a second messenger in cells depends on a large number of Ca2+ -binding proteins (CBP), which are able to bind Ca2+ through specific domains. Many CBPs share a type of Ca2+ -binding domain known as the EF-hand. The EF-hand motif has been well studied and consists of a helix-loop-helix structural domain with specific amino acids in the loop region that interact with Ca2+ . In Toxoplasma gondii a large number of genes (approximately 68) are predicted to have at least one EF-hand motif. The majority of these genes have not been characterized. We report the characterization of two EF-hand motif-containing proteins, TgGT1_216620 and TgGT1_280480, which localize to the plasma membrane and to the rhoptry bulb, respectively. Genetic disruption of these genes by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) resulted in mutant parasite clones (Δtg216620 and Δtg280480) that grew at a slower rate than control cells. Ca2+ measurements showed that Δtg216620 cells did not respond to extracellular Ca2+ as the parental controls while Δtg280480 cells appeared to respond as the parental cells. Our hypothesis is that TgGT1_216620 is important for Ca2+ influx while TgGT1_280480 may be playing a different role in the rhoptries.}, }
@article {pmid32281426, year = {2020}, author = {Aghamiri, S and Talaei, S and Roshanzamiri, S and Zandsalimi, F and Fazeli, E and Aliyu, M and Kheiry Avarvand, O and Ebrahimi, Z and Keshavarz-Fathi, M and Ghanbarian, H}, title = {Delivery of genome editing tools: A promising strategy for HPV-related cervical malignancy therapy.}, journal = {Expert opinion on drug delivery}, volume = {}, number = {}, pages = {1-14}, doi = {10.1080/17425247.2020.1747429}, pmid = {32281426}, issn = {1744-7593}, abstract = {Introduction: Persistent high-risk human papillomavirus infection is the main cause of various types of cancer especially cervical cancer. The E6 and E7 oncoproteins of HPV play critical roles in promoting carcinogenesis and cancer cell growth. As a result, E6 and E7 oncogenes are considered as promising therapeutic targets for cervical cancer. Recently, the development of genome-editing technologies including transcription activator-like effector nucleases (TALEN), meganucleases (MNs), zinc finger nucleases (ZFN), and more importantly clustered regularly interspaced short palindromic repeat-CRISPR-associated protein (CRISPR-Cas) has sparked a revolution in the cervical cancer-targeted therapy. However, due to immunogenicity, off-target effect, renal clearance, guide RNA (gRNA) nuclease degradation, and difficult direct transportation into the cytoplasm and nucleus, the safe and effective delivery is considered as the Achilles' heel of this robust strategy.Areas covered: In this review, we discuss cutting-edge available strategies for in vivo delivery of genome-editing technologies for HPV-induced cervical cancer therapy. Moreover, the combination of genome-editing tools and other therapies has been fully discussed.Expert opinion: The combination of nanoparticle-based delivery systems and genome-editing tools is a promising powerful strategy for cervical cancer therapy. The most significant limitations of this strategy that need to be focused on are low efficiency and off-target events.}, }
@article {pmid32123335, year = {2020}, author = {Artegiani, B and Hendriks, D and Beumer, J and Kok, R and Zheng, X and Joore, I and Chuva de Sousa Lopes, S and van Zon, J and Tans, S and Clevers, H}, title = {Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing.}, journal = {Nature cell biology}, volume = {22}, number = {3}, pages = {321-331}, doi = {10.1038/s41556-020-0472-5}, pmid = {32123335}, issn = {1476-4679}, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Gene Knock-In Techniques/*methods ; Hepatocytes/cytology/ultrastructure ; Humans ; Intestines/cytology ; Liver/cytology ; Organoids/*cytology/ultrastructure ; Spindle Apparatus/ultrastructure ; Tumor Suppressor Protein p53/physiology ; }, abstract = {CRISPR-Cas9 technology has revolutionized genome editing and is applicable to the organoid field. However, precise integration of exogenous DNA sequences into human organoids is lacking robust knock-in approaches. Here, we describe CRISPR-Cas9-mediated homology-independent organoid transgenesis (CRISPR-HOT), which enables efficient generation of knock-in human organoids representing different tissues. CRISPR-HOT avoids extensive cloning and outperforms homology directed repair (HDR) in achieving precise integration of exogenous DNA sequences into desired loci, without the necessity to inactivate TP53 in untransformed cells, which was previously used to increase HDR-mediated knock-in. CRISPR-HOT was used to fluorescently tag and visualize subcellular structural molecules and to generate reporter lines for rare intestinal cell types. A double reporter-in which the mitotic spindle was labelled by endogenously tagged tubulin and the cell membrane by endogenously tagged E-cadherin-uncovered modes of human hepatocyte division. Combining tubulin tagging with TP53 knock-out revealed that TP53 is involved in controlling hepatocyte ploidy and mitotic spindle fidelity. CRISPR-HOT simplifies genome editing in human organoids.}, }
@article {pmid32066906, year = {2020}, author = {Li, X and Qian, X and Wang, B and Xia, Y and Zheng, Y and Du, L and Xu, D and Xing, D and DePinho, RA and Lu, Z}, title = {Programmable base editing of mutated TERT promoter inhibits brain tumour growth.}, journal = {Nature cell biology}, volume = {22}, number = {3}, pages = {282-288}, doi = {10.1038/s41556-020-0471-6}, pmid = {32066906}, issn = {1476-4679}, support = {R01 CA084628/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; Brain Neoplasms/*genetics/metabolism/pathology ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Cellular Senescence ; *Gene Editing ; Glioblastoma/*genetics/metabolism/pathology ; Humans ; Male ; Mice, Nude ; Middle Aged ; Mutation ; Promoter Regions, Genetic ; Telomerase/*genetics/metabolism ; Telomere Shortening ; Transcription Factors/metabolism ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR interference and programmable base editing have transformed the manipulation of eukaryotic genomes for potential therapeutic applications1-4. Here, we exploited CRISPR interference and programmable base editing to determine their potential in editing a TERT gene promoter-activating mutation, which occurs in many diverse cancer types, particularly glioblastoma5-8. Correction of the -124C>T TERT promoter mutation to -124C was achieved using a single guide RNA (sgRNA)-guided and catalytically impaired Campylobacter jejuni CRISPR-associated protein 9-fused adenine base editor (CjABE). This modification blocked the binding of members of the E26 transcription factor family to the TERT promoter, reduced TERT transcription and TERT protein expression, and induced cancer-cell senescence and proliferative arrest. Local injection of adeno-associated viruses expressing sgRNA-guided CjABE inhibited the growth of gliomas harbouring TERT-promoter mutations. These preclinical proof-of-concept studies establish the feasibility of gene editing as a therapeutic approach for cancer and validate activated TERT-promoter mutations as a cancer-specific therapeutic target.}, }
@article {pmid32001684, year = {2020}, author = {Williams, JB and Li, S and Higgs, EF and Cabanov, A and Wang, X and Huang, H and Gajewski, TF}, title = {Tumor heterogeneity and clonal cooperation influence the immune selection of IFN-γ-signaling mutant cancer cells.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {602}, pmid = {32001684}, issn = {2041-1723}, support = {T32 AI007090/AI/NIAID NIH HHS/United States ; T32 HD007009/HD/NICHD NIH HHS/United States ; }, mesh = {Animals ; B7-H1 Antigen/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Proliferation ; Clone Cells ; Cytotoxicity, Immunologic ; *Genetic Heterogeneity ; Humans ; Immunomodulation ; Interferon-gamma/*metabolism ; Lymphocytes, Tumor-Infiltrating/immunology ; Mice, Inbred C57BL ; Mice, Transgenic ; Mutation/*genetics ; Neoplasms/*genetics/*pathology ; *Signal Transduction ; T-Lymphocytes/immunology ; }, abstract = {PD-1/PD-L1 blockade can promote robust tumor regression yet secondary resistance often occurs as immune selective pressure drives outgrowth of resistant tumor clones. Here using a genome-wide CRISPR screen in B16.SIY melanoma cells, we confirm Ifngr2 and Jak1 as important genes conferring sensitivity to T cell-mediated killing in vitro. However, when implanted into mice, these Ifngr2- and Jak1-deficient tumors paradoxically are better controlled immunologically. This phenotype maps to defective PD-L1 upregulation on mutant tumor cells, which improves anti-tumor efficacy of CD8+ T cells. To reconcile these observations with clinical reports of anti-PD-1 resistance linked to emergence of IFN-γ signaling mutants, we show that when mixed with wild-type tumor cells, IFN-γ-insensitive tumor cells indeed grow out, which depends upon PD-L1 expression by wild-type cells. Our results illustrate the complexity of functions for IFN-γ in anti-tumor immunity and demonstrate that intratumor heterogeneity and clonal cooperation can contribute to immunotherapy resistance.}, }
@article {pmid31980609, year = {2020}, author = {Li, K and Liu, Y and Cao, H and Zhang, Y and Gu, Z and Liu, X and Yu, A and Kaphle, P and Dickerson, KE and Ni, M and Xu, J}, title = {Interrogation of enhancer function by enhancer-targeting CRISPR epigenetic editing.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {485}, pmid = {31980609}, issn = {2041-1723}, support = {R01 CA230631/CA/NCI NIH HHS/United States ; R01 DK111430/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Enhancer Elements, Genetic ; *Epigenesis, Genetic ; Female ; Gene Editing/*methods ; HEK293 Cells ; Hematopoiesis/genetics ; Humans ; Jurkat Cells ; K562 Cells ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Mice, Transgenic ; Neoplasms/genetics ; RNA, Guide/genetics ; T-Cell Acute Lymphocytic Leukemia Protein 1/genetics ; }, abstract = {Tissue-specific gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), yet functional analysis of gene-distal CREs such as enhancers remains challenging. Here we describe CRISPR/dCas9-based enhancer-targeting epigenetic editing systems, enCRISPRa and enCRISPRi, for efficient analysis of enhancer function in situ and in vivo. Using dual effectors capable of re-writing enhancer-associated chromatin modifications, we show that enCRISPRa and enCRISPRi modulate gene transcription by remodeling local epigenetic landscapes at sgRNA-targeted enhancers and associated genes. Comparing with existing methods, the improved systems display more robust perturbations of enhancer activity and gene transcription with minimal off-targets. Allele-specific targeting of enCRISPRa to oncogenic TAL1 super-enhancer modulates TAL1 expression and cancer progression in xenotransplants. Single or multi-loci perturbations of lineage-specific enhancers using an enCRISPRi knock-in mouse establish in vivo evidence for lineage-restricted essentiality of developmental enhancers during hematopoiesis. Hence, enhancer-targeting CRISPR epigenetic editing provides opportunities for interrogating enhancer function in native biological contexts.}, }
@article {pmid31980606, year = {2020}, author = {Nishiguchi, KM and Fujita, K and Miya, F and Katayama, S and Nakazawa, T}, title = {Single AAV-mediated mutation replacement genome editing in limited number of photoreceptors restores vision in mice.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {482}, pmid = {31980606}, issn = {2041-1723}, mesh = {Animals ; CRISPR-Cas Systems ; Cyclic Nucleotide Phosphodiesterases, Type 6/deficiency/genetics ; DNA End-Joining Repair ; Dependovirus/*genetics ; Eye Proteins/genetics ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mutation ; Photoreceptor Cells, Vertebrate/*physiology ; Retinal Dystrophies/*genetics/physiopathology/*therapy ; Targeted Gene Repair/methods ; Transducin/deficiency/genetics ; Visual Acuity/genetics/physiology ; }, abstract = {Supplementing wildtype copies of functionally defective genes with adeno-associated virus (AAV) is a strategy being explored clinically for various retinal dystrophies. However, the low cargo limit of this vector allows its use in only a fraction of patients with mutations in relatively small pathogenic genes. To overcome this issue, we developed a single AAV platform that allows local replacement of a mutated sequence with its wildtype counterpart, based on combined CRISPR-Cas9 and micro-homology-mediated end-joining (MMEJ). In blind mice, the mutation replacement rescued approximately 10% of photoreceptors, resulting in an improvement in light sensitivity and an increase in visual acuity. These effects were comparable to restoration mediated by gene supplementation, which targets a greater number of photoreceptors. This strategy may be applied for the treatment of inherited disorders caused by mutations in larger genes, for which conventional gene supplementation therapy is not currently feasible.}, }
@article {pmid31978056, year = {2020}, author = {Emmer, BT and Lascuna, PJ and Tang, VT and Kotnik, EN and Saunders, TL and Khoriaty, R and Ginsburg, D}, title = {Murine Surf4 is essential for early embryonic development.}, journal = {PloS one}, volume = {15}, number = {1}, pages = {e0227450}, pmid = {31978056}, issn = {1932-6203}, support = {R35 HL135793/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; K08 HL128794/HL/NHLBI NIH HHS/United States ; T32 HL007853/HL/NHLBI NIH HHS/United States ; K08 HL148552/HL/NHLBI NIH HHS/United States ; R01 HL148333/HL/NHLBI NIH HHS/United States ; KL2 TR002241/TR/NCATS NIH HHS/United States ; }, mesh = {Alleles ; Animals ; Apolipoproteins B/blood/metabolism ; CRISPR-Cas Systems/genetics ; Cholesterol/blood ; *Embryonic Development ; Gene Editing ; Heterozygote ; Membrane Proteins/*genetics/metabolism ; Mice ; Mice, Knockout ; Proprotein Convertase 9/blood/metabolism ; }, abstract = {Newly synthesized proteins co-translationally inserted into the endoplasmic reticulum (ER) lumen may be recruited into anterograde transport vesicles by their association with specific cargo receptors. We recently identified a role for the cargo receptor SURF4 in facilitating the secretion of PCSK9 in cultured cells. To examine the function of SURF4 in vivo, we used CRISPR/Cas9-mediated gene editing to generate mice with germline loss-of-function mutations in Surf4. Heterozygous Surf4+/- mice exhibit grossly normal appearance, behavior, body weight, fecundity, and organ development, with no significant alterations in circulating plasma levels of PCSK9, apolipoprotein B, or total cholesterol, and a detectable accumulation of intrahepatic apoliprotein B. Homozygous Surf4-/- mice exhibit embryonic lethality, with complete loss of all Surf4-/- offspring between embryonic days 3.5 and 9.5. In contrast to the milder murine phenotypes associated with deficiency of known SURF4 cargoes, the embryonic lethality of Surf4-/- mice implies the existence of additional SURF4 cargoes or functions that are essential for murine early embryonic development.}, }
@article {pmid31959982, year = {2020}, author = {Crowther, MD and Dolton, G and Legut, M and Caillaud, ME and Lloyd, A and Attaf, M and Galloway, SAE and Rius, C and Farrell, CP and Szomolay, B and Ager, A and Parker, AL and Fuller, A and Donia, M and McCluskey, J and Rossjohn, J and Svane, IM and Phillips, JD and Sewell, AK}, title = {Genome-wide CRISPR-Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1.}, journal = {Nature immunology}, volume = {21}, number = {2}, pages = {178-185}, doi = {10.1038/s41590-019-0578-8}, pmid = {31959982}, issn = {1529-2916}, support = {/WT_/Wellcome Trust/United Kingdom ; 100327/WT_/Wellcome Trust/United Kingdom ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cytotoxicity, Immunologic/*immunology ; Genome-Wide Association Study ; Histocompatibility Antigens Class I/*immunology ; Humans ; Immunotherapy/methods ; Lymphocyte Activation/immunology ; Mice ; Minor Histocompatibility Antigens/*immunology ; Neoplasms/*immunology ; Receptors, Antigen, T-Cell/*immunology ; T-Lymphocyte Subsets/*immunology ; }, abstract = {Human leukocyte antigen (HLA)-independent, T cell-mediated targeting of cancer cells would allow immune destruction of malignancies in all individuals. Here, we use genome-wide CRISPR-Cas9 screening to establish that a T cell receptor (TCR) recognized and killed most human cancer types via the monomorphic MHC class I-related protein, MR1, while remaining inert to noncancerous cells. Unlike mucosal-associated invariant T cells, recognition of target cells by the TCR was independent of bacterial loading. Furthermore, concentration-dependent addition of vitamin B-related metabolite ligands of MR1 reduced TCR recognition of cancer cells, suggesting that recognition occurred via sensing of the cancer metabolome. An MR1-restricted T cell clone mediated in vivo regression of leukemia and conferred enhanced survival of NSG mice. TCR transfer to T cells of patients enabled killing of autologous and nonautologous melanoma. These findings offer opportunities for HLA-independent, pan-cancer, pan-population immunotherapies.}, }
@article {pmid31942057, year = {2020}, author = {Dolgin, E}, title = {The kill-switch for CRISPR that could make gene-editing safer.}, journal = {Nature}, volume = {577}, number = {7790}, pages = {308-310}, doi = {10.1038/d41586-020-00053-0}, pmid = {31942057}, issn = {1476-4687}, mesh = {Animals ; Bacteria/genetics/immunology/virology ; Bacteriophages/immunology/physiology ; Biohazard Release/*prevention &amp; control ; Biosensing Techniques ; Biotechnology/*methods ; CRISPR-Associated Protein 9/antagonists &amp; inhibitors/metabolism ; *CRISPR-Cas Systems ; Culicidae/genetics ; Female ; Gene Drive Technology/*methods/standards ; Gene Editing/*methods/*standards ; Humans ; Male ; Optogenetics ; }, }
@article {pmid31771289, year = {2019}, author = {Santos, R and Amaral, O}, title = {Advances in Sphingolipidoses: CRISPR-Cas9 Editing as an Option for Modelling and Therapy.}, journal = {International journal of molecular sciences}, volume = {20}, number = {23}, pages = {}, pmid = {31771289}, issn = {1422-0067}, support = {PTDC/BIM-MEC/4762/2014//Fundação para a Ciência e a Tecnologia/ ; }, mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Disease Models, Animal ; Enzyme Replacement Therapy ; Gaucher Disease/genetics/therapy ; Gene Editing/*methods ; Humans ; Sphingolipidoses/genetics/*therapy ; beta-Glucosidase/genetics ; }, abstract = {Sphingolipidoses are inherited genetic diseases characterized by the accumulation of glycosphingolipids. Sphingolipidoses (SP), which usually involve the loss of sphingolipid hydrolase function, are of lysosomal origin, and represent an important group of rare diseases among lysosomal storage disorders. Initial treatments consisted of enzyme replacement therapy, but, in recent decades, various therapeutic approaches have been developed. However, these commonly used treatments for SP fail to be fully effective and do not penetrate the blood-brain barrier. New approaches, such as genome editing, have great potential for both the treatment and study of sphingolipidoses. Here, we review the most recent advances in the treatment and modelling of SP through the application of CRISPR-Cas9 genome editing. CRISPR-Cas9 is currently the most widely used method for genome editing. This technique is versatile; it can be used for altering the regulation of genes involved in sphingolipid degradation and synthesis pathways, interrogating gene function, generating knock out models, or knocking in mutations. CRISPR-Cas9 genome editing is being used as an approach to disease treatment, but more frequently it is utilized to create models of disease. New CRISPR-Cas9-based tools of gene editing with diminished off-targeting effects are evolving and seem to be more promising for the correction of individual mutations. Emerging Prime results and CRISPR-Cas9 difficulties are also discussed.}, }
@article {pmid31740817, year = {2020}, author = {Camsund, D and Lawson, MJ and Larsson, J and Jones, D and Zikrin, S and Fange, D and Elf, J}, title = {Time-resolved imaging-based CRISPRi screening.}, journal = {Nature methods}, volume = {17}, number = {1}, pages = {86-92}, doi = {10.1038/s41592-019-0629-y}, pmid = {31740817}, issn = {1548-7105}, mesh = {*CRISPR-Cas Systems ; Cell Cycle ; DNA Replication ; Escherichia coli/genetics/growth &amp; development/*metabolism ; Escherichia coli Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Gene Expression Regulation, Bacterial ; Gene Library ; Genotype ; Image Processing, Computer-Assisted/*methods ; Metabolic Engineering/*methods ; Microfluidic Analytical Techniques/*methods ; Microscopy/*methods ; Phenotype ; }, abstract = {Our ability to connect genotypic variation to biologically important phenotypes has been seriously limited by the gap between live-cell microscopy and library-scale genomic engineering. Here, we show how in situ genotyping of a library of strains after time-lapse imaging in a microfluidic device overcomes this problem. We determine how 235 different CRISPR interference knockdowns impact the coordination of the replication and division cycles of Escherichia coli by monitoring the location of replication forks throughout on average >500 cell cycles per knockdown. Subsequent in situ genotyping allows us to map each phenotype distribution to a specific genetic perturbation to determine which genes are important for cell cycle control. The single-cell time-resolved assay allows us to determine the distribution of single-cell growth rates, cell division sizes and replication initiation volumes. The technology presented in this study enables genome-scale screens of most live-cell microscopy assays.}, }
@article {pmid31666694, year = {2019}, author = {Flavahan, WA and Drier, Y and Johnstone, SE and Hemming, ML and Tarjan, DR and Hegazi, E and Shareef, SJ and Javed, NM and Raut, CP and Eschle, BK and Gokhale, PC and Hornick, JL and Sicinska, ET and Demetri, GD and Bernstein, BE}, title = {Altered chromosomal topology drives oncogenic programs in SDH-deficient GISTs.}, journal = {Nature}, volume = {575}, number = {7781}, pages = {229-233}, doi = {10.1038/s41586-019-1668-3}, pmid = {31666694}, issn = {1476-4687}, support = {DP1 CA216873/CA/NCI NIH HHS/United States ; F32 CA203153/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carcinogenesis/*genetics ; *Chromosome Aberrations ; DNA Methylation ; Enhancer Elements, Genetic/genetics ; Epigenesis, Genetic ; Fibroblast Growth Factor 4/genetics ; Gastrointestinal Stromal Tumors/enzymology/*genetics/*pathology ; Humans ; Mice ; Mutation ; Oncogenes/*genetics ; Proto-Oncogene Proteins c-kit/antagonists &amp; inhibitors ; Receptors, Fibroblast Growth Factor/antagonists &amp; inhibitors ; Succinate Dehydrogenase/*deficiency/genetics ; }, abstract = {Epigenetic aberrations are widespread in cancer, yet the underlying mechanisms and causality remain poorly understood1-3. A subset of gastrointestinal stromal tumours (GISTs) lack canonical kinase mutations but instead have succinate dehydrogenase (SDH) deficiency and global DNA hyper-methylation4,5. Here, we associate this hyper-methylation with changes in genome topology that activate oncogenic programs. To investigate epigenetic alterations systematically, we mapped DNA methylation, CTCF insulators, enhancers, and chromosome topology in KIT-mutant, PDGFRA-mutant and SDH-deficient GISTs. Although these respective subtypes shared similar enhancer landscapes, we identified hundreds of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. We focused on a disrupted insulator that normally partitions a core GIST super-enhancer from the FGF4 oncogene. Recurrent loss of this insulator alters locus topology in SDH-deficient GISTs, allowing aberrant physical interaction between enhancer and oncogene. CRISPR-mediated excision of the corresponding CTCF motifs in an SDH-intact GIST model disrupted the boundary between enhancer and oncogene, and strongly upregulated FGF4 expression. We also identified a second recurrent insulator loss event near the KIT oncogene, which is also highly expressed across SDH-deficient GISTs. Finally, we established a patient-derived xenograft (PDX) from an SDH-deficient GIST that faithfully maintains the epigenetics of the parental tumour, including hypermethylation and insulator defects. This PDX model is highly sensitive to FGF receptor (FGFR) inhibition, and more so to combined FGFR and KIT inhibition, validating the functional significance of the underlying epigenetic lesions. Our study reveals how epigenetic alterations can drive oncogenic programs in the absence of canonical kinase mutations, with implications for mechanistic targeting of aberrant pathways in cancers.}, }
@article {pmid31630543, year = {2019}, author = {Arkhipov, SN and Potter, DL and Geurts, AM and Pavlov, TS}, title = {Knockout of P2rx7 purinergic receptor attenuates cyst growth in a rat model of ARPKD.}, journal = {American journal of physiology. Renal physiology}, volume = {317}, number = {6}, pages = {F1649-F1655}, pmid = {31630543}, issn = {1522-1466}, support = {P30 DK090868/DK/NIDDK NIH HHS/United States ; R00 DK105160/DK/NIDDK NIH HHS/United States ; R00 HL116603/HL/NHLBI NIH HHS/United States ; R24 HL114474/HL/NHLBI NIH HHS/United States ; }, mesh = {Adenosine Triphosphate/urine ; Animals ; CRISPR-Cas Systems ; Connexins/biosynthesis/genetics ; Cysts/genetics/*pathology ; Epithelial Sodium Channels/metabolism ; Female ; Gene Knockout Techniques ; Kidney Diseases/genetics/*pathology ; Mutagenesis, Insertional ; Nerve Tissue Proteins/biosynthesis/genetics ; Polycystic Kidney, Autosomal Recessive/genetics/*metabolism ; Pregnancy ; Rats ; Receptors, Purinergic P2X7/genetics/*metabolism ; Sodium/metabolism ; }, abstract = {The severity of polycystic kidney diseases (PKD) depends on the counterbalancing of genetic predisposition and environmental factors exerting permissive or protective influence on cyst development. One poorly characterized phenomenon in the cystic epithelium is abnormal purinergic signaling. Earlier experimental studies revealed the high importance of the ionotropic P2X receptors (particularly, P2X7) in the pathophysiology of the cyst wall. To study mechanisms of P2X7 involvement in cyst growth and aspects of targeting these receptors in PKD treatment we performed a CRISPR/SpCas9-mediated global knockout of the P2rx7 gene in PCK rats, a model of autosomal recessive PKD (ARPKD). A single base insertion in exon 2 of the P2rx7 gene in the renal tissues of homozygous mutant animals leads to lack of P2X7 protein that did not affect their viability or renal excretory function. However, PCK.P2rx7 rats demonstrated slower cyst growth (but not formation of new cysts) compared with heterozygous and PCK.P2rx7+ littermates. P2X7 receptors are known to activate pannexin-1, a plasma channel capable of releasing ATP, and we found here that pannexin-1 expression in the cystic epithelium is significantly higher than in nondilated tubules. P2X7 deficiency reduces renal pannexin-1 protein expression and daily urinary ATP excretion. Patch-clamp analysis revealed that lack of P2X7 increases epithelial sodium channel activity in renal tissues and restores impaired channel activity in cysts. Interpretation of our current data in the context of earlier studies strongly suggests that P2X7 contributes to cyst growth by increasing pannexin-1-dependent pathogenic ATP release into the lumen and reduction of sodium reabsorption across the cyst walls.}, }
@article {pmid31523199, year = {2019}, author = {Wang, H and Shen, L and Chen, J and Liu, X and Tan, T and Hu, Y and Bai, X and Li, Y and Tian, K and Li, N and Hu, X}, title = {Deletion of CD163 Exon 7 Confers Resistance to Highly Pathogenic Porcine Reproductive and Respiratory Viruses on Pigs.}, journal = {International journal of biological sciences}, volume = {15}, number = {9}, pages = {1993-2005}, pmid = {31523199}, issn = {1449-2288}, mesh = {Animals ; Antigens, CD/*genetics ; Antigens, Differentiation, Myelomonocytic/*genetics ; CRISPR-Cas Systems/genetics ; Exons/*genetics ; Nuclear Transfer Techniques ; Porcine Reproductive and Respiratory Syndrome/*genetics/*prevention &amp; control ; Porcine respiratory and reproductive syndrome virus/*pathogenicity ; Receptors, Cell Surface/*genetics ; Swine ; }, abstract = {Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is a severe infectious disease in the swine industry. PRRSV infection is mediated by porcine CD163 (pCD163). Scavenger receptor cysteine-rich domain 5 coded by exon 7 of pCD163 is essential for PRRSV infection. In this study, we generated CD163 exon 7 deleted (CD163E7D) pigs using CRISPR/Cas9 mediated homologous recombination and somatic cell nuclear transfer (SCNT). The deletion of exon 7 had no adverse effects on CD163-associated functions. Pigs were further challenged with a highly pathogenic PRRSV (HP-PRRSV) strain. The CD163E7D pigs exhibited mild clinical symptoms and had decreased viral loads in blood. All CD163E7D pigs survived the viral challenge, while all the WT pigs displayed severe symptoms, and 2 out of 6 WT pigs died during the challenge. Our results demonstrated that CD163 exon 7 deletion confers resistance to HP-PRRSV infection without impairing the biological functions of CD163.}, }
@article {pmid30932232, year = {2019}, author = {Denner, J and Scobie, L}, title = {Are there better assays to evaluate the risk of transmission of porcine endogenous retroviruses (PERVs) to human cells?.}, journal = {Xenotransplantation}, volume = {26}, number = {4}, pages = {e12510}, doi = {10.1111/xen.12510}, pmid = {30932232}, issn = {1399-3089}, mesh = {Animals ; Biological Assay/economics/*methods ; CRISPR-Cas Systems ; Endogenous Retroviruses/genetics/*physiology ; Genome, Viral ; HEK293 Cells/virology ; Heterografts/*virology ; *Host Specificity ; Humans ; RNA Splicing ; RNA, Messenger/*analysis/biosynthesis/genetics ; RNA, Viral/*analysis/biosynthesis/genetics ; Retroviridae Infections/prevention &amp; control/*transmission ; Retroviridae Proteins/genetics/physiology ; Risk ; Swine/*virology ; *Viral Tropism ; Virus Replication ; }, }
@article {pmid30880567, year = {2019}, author = {Deryabin, P and Griukova, A and Shatrova, A and Petukhov, A and Nikolsky, N and Borodkina, A}, title = {Optimization of lentiviral transduction parameters and its application for CRISPR-based secretome modification of human endometrial mesenchymal stem cells.}, journal = {Cell cycle (Georgetown, Tex.)}, volume = {18}, number = {6-7}, pages = {742-758}, pmid = {30880567}, issn = {1551-4005}, mesh = {CRISPR-Cas Systems/*genetics ; Cells, Cultured ; Cellular Senescence/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endometrium/*physiology ; Female ; Gene Editing/methods ; Genetic Therapy ; Genome/genetics ; HEK293 Cells ; Humans ; Lentivirus/*genetics ; Mesenchymal Stem Cells/*physiology ; Regenerative Medicine/methods ; }, abstract = {Mesenchymal stem cells (MSCs) hold a great promise for successful development of regenerative medicine. Among the plenty of uncovered MSCs sources, desquamated endometrium collected from the menstrual blood probably remains the most accessible. Though numerous studies have been published on human endometrium-derived mesenchymal stem cells (hMESCs) properties in the past years, there are only a few data regarding their genetic modulation. Moreover, there is a lack of information about the fate of the transduced hMESCs. The present study aimed to optimize hMESCs transduction parameters and apply Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology for genome and secretome modification. The fate of hMESCs transduced either in presence of polybrene (Pb) or protamine sulfate (Ps) was assessed by alterations in CD expression profile, growth rate, cell size, migration capability, osteogenic, adipogenic, and decidual differential potentials. Here, we postulated that the use of Ps for hMESCs genetic manipulations is preferable, as it has no impact on the stem-cell properties, whereas Pb application is undesirable, as it induces cellular senescence. Plasminogen activator inhibitor-1 was selected for further targeted hMESCs genome and secretome modification using CRISPR/Cas9 systems. The obtained data provide optimized transduction scheme for hMESCs and verification of its effectiveness by successful hMESCs genome editing via CRISPR/Cas9 technology.}, }
@article {pmid30773093, year = {2019}, author = {Menon, VR and Ananthapadmanabhan, V and Swanson, S and Saini, S and Sesay, F and Yakovlev, V and Florens, L and DeCaprio, JA and Washburn, MP and Dozmorov, M and Litovchick, L}, title = {DYRK1A regulates the recruitment of 53BP1 to the sites of DNA damage in part through interaction with RNF169.}, journal = {Cell cycle (Georgetown, Tex.)}, volume = {18}, number = {5}, pages = {531-551}, pmid = {30773093}, issn = {1551-4005}, support = {P01 CA203655/CA/NCI NIH HHS/United States ; P30 CA016059/CA/NCI NIH HHS/United States ; R01 CA188571/CA/NCI NIH HHS/United States ; R25 GM090084/GM/NIGMS NIH HHS/United States ; R01 CA063113/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Cycle Checkpoints/radiation effects ; Cell Line, Tumor ; *DNA Damage/radiation effects ; DNA Repair ; Gamma Rays ; Gene Editing ; Humans ; Metabolic Networks and Pathways ; Mice ; Phosphorylation ; Protein Binding ; Protein-Serine-Threonine Kinases/deficiency/genetics/*metabolism ; Protein-Tyrosine Kinases/deficiency/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering/metabolism ; Tumor Suppressor p53-Binding Protein 1/*metabolism ; Ubiquitin-Protein Ligases/antagonists &amp; inhibitors/genetics/*metabolism ; }, abstract = {Human Dual-specificity tyrosine (Y) Regulated Kinase 1A (DYRK1A) is encoded by a dosage dependent gene whereby either trisomy or haploinsufficiency result in developmental abnormalities. However, the function and regulation of this important protein kinase are not fully understood. Here, we report proteomic analysis of DYRK1A in human cells that revealed a novel role of DYRK1A in DNA double-strand breaks (DSBs) repair, mediated in part by its interaction with the ubiquitin-binding protein RNF169 that accumulates at the DSB sites and promotes homologous recombination repair (HRR) by displacing 53BP1, a key mediator of non-homologous end joining (NHEJ). We found that overexpression of active, but not the kinase inactive DYRK1A in U-2 OS cells inhibits accumulation of 53BP1 at the DSB sites in the RNF169-dependent manner. DYRK1A phosphorylates RNF169 at two sites that influence its ability to displace 53BP1 from the DSBs. Although DYRK1A is not required for the recruitment of RNF169 to the DSB sites and 53BP1 displacement, inhibition of DYRK1A or mutation of the DYRK1A phosphorylation sites in RNF169 decreases its ability to block accumulation of 53BP1 at the DSB sites. Interestingly, CRISPR-Cas9 knockout of DYRK1A in human and mouse cells also diminished the 53BP1 DSB recruitment in a manner that did not require RNF169, suggesting that dosage of DYRK1A can influence the DNA repair processes through both RNF169-dependent and independent mechanisms. Human U-2 OS cells devoid of DYRK1A display an increased HRR efficiency and resistance to DNA damage, therefore our findings implicate DYRK1A in the DNA repair processes.}, }
@article {pmid30565897, year = {2019}, author = {Li, Y and Li, AC and Xu, Q}, title = {Intracellular Delivery of His-Tagged Genome-Editing Proteins Enabled by Nitrilotriacetic Acid-Containing Lipidoid Nanoparticles.}, journal = {Advanced healthcare materials}, volume = {8}, number = {6}, pages = {e1800996}, pmid = {30565897}, issn = {2192-2659}, support = {R21 EB024041/EB/NIBIB NIH HHS/United States ; R01 EB027170/EB/NIBIB NIH HHS/United States ; UG3 TR002636/TR/NCATS NIH HHS/United States ; DMR1452122//National Science Foundation/International ; }, mesh = {CRISPR-Cas Systems/genetics ; Cell Line ; Cell Survival/drug effects ; Gene Editing/*methods ; Green Fluorescent Proteins/genetics/metabolism ; Histidine/genetics/metabolism ; Humans ; Lipids/*chemistry ; Nanoparticles/*chemistry/toxicity ; Nitrilotriacetic Acid/*chemistry ; Oligopeptides/genetics/metabolism ; Transfection ; }, abstract = {Protein- and peptide-based therapeutics with high tolerance and specificity along with low off-target effects and genetic risks have attracted tremendous attention over the last three decades. Herein, a new type of noncationic lipidoid nanoparticle (LNP) is reported for His-tagged protein delivery. Active lipidoids are synthesized by conjugating a nitrilotriacetic acid group with hydrophobic tails (EC16, O16B, and O17O) and nanoparticles are formulated in the presence of nickel ions and helper lipids (cholesterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). It is demonstrated that the newly developed LNPs are capable of delivering various His-tagged proteins including green fluorescent protein (GFP), (-30)GFP-Cre recombinase, and CRISPR/Cas9 ribonucleoprotein into mammalian cells.}, }
@article {pmid30032190, year = {2018}, author = {Hirayama, T and Lei, GJ and Yamaji, N and Nakagawa, N and Ma, JF}, title = {The Putative Peptide Gene FEP1 Regulates Iron Deficiency Response in Arabidopsis.}, journal = {Plant &amp; cell physiology}, volume = {59}, number = {9}, pages = {1739-1752}, doi = {10.1093/pcp/pcy145}, pmid = {30032190}, issn = {1471-9053}, mesh = {Arabidopsis/*metabolism ; Arabidopsis Proteins/genetics/*metabolism ; Base Sequence ; Biological Transport ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant/*drug effects ; Iron/metabolism/*pharmacology ; Mutation ; Plant Roots/metabolism ; Plant Shoots/metabolism ; Plants, Genetically Modified ; RNA, Plant ; Up-Regulation ; }, abstract = {Iron is an essential element for all organisms, and plants have developed sophisticated systems to acquire iron and maintain iron homeostasis. We found that an Arabidopsis thaliana ABA-hypersensitive mutant, aba hypersensitive germination2-1 (ahg2-1), that is known to be defective in mitochondrial mRNA regulation, had increased expression of iron deficiency response genes. The ahg2-1 mutant had lower heme levels than the wild type. Transcriptome data further revealed that novel genes encoding short polypeptides were highly expressed in this mutant. The expression of one of these genes, which we named FE-UPTAKE-INDUCING PEPTIDE 1 (FEP1), was induced under iron-deficient conditions and was observed in the vascular tissues of the leaves and roots, as well as in leaf mesophyll cells. Notably, deletion or insertion mutations of FEP1 exhibited impaired iron accumulation in shoots but normal iron levels in roots. Artificially induced expression of FEP1 was sufficient to induce iron deficiency response genes, such as basic HELIX-LOOP-HELIX 38 (bHLH38), bHLH39, IRON-REGULATED TRANSPORTER1 (IRT1) and FERRIC REDUCTION OXIDASE2 (FRO2), and led to iron accumulation in planta. Further analysis confirmed that the encoded peptide, but not the FEP1 RNA, was responsible for this activity. Remarkably, the activation of bHLH39 by FEP1 was independent of FER-LIKE IRON DEFICIENCY INDUCED (FIT), a key transcription factor in the iron deficiency response. Taken together, our results indicate that FEP1 functions in iron homeostasis through a previously undescribed regulatory mechanism for iron acquisition in Arabidopsis.}, }
@article {pmid32279281, year = {2020}, author = {Zhang, Y and Zhang, Q and Chen, QJ}, title = {Agrobacterium-mediated delivery of CRISPR/Cas reagents for genome editing in plants enters an era of ternary vector systems.}, journal = {Science China. Life sciences}, volume = {}, number = {}, pages = {}, doi = {10.1007/s11427-020-1685-9}, pmid = {32279281}, issn = {1869-1889}, abstract = {Lack of appropriate methods for delivery of genome-editing reagents is a major barrier to CRISPR/Cas-mediated genome editing in plants. Agrobacterium-mediated genetic transformation (AMGT) is the preferred method of CRISPR/Cas reagent delivery, and researchers have recently made great improvements to this process. In this article, we review the development of AMGT and AMGT-based delivery of CRISPR/Cas reagents. We give an overview of the development of AMGT vectors including binary vector, superbinary vector, dual binary vector, and ternary vector systems. We also review the progress in Agrobacterium genomics and Agrobacterium genetic engineering for optimal strains. We focus in particular on the ternary vector system and the resources we developed. In summary, it is our opinion that Agrobacterium-mediated CRISPR/Cas genome editing in plants is entering an era of ternary vector systems, which are often integrated with morphogenic regulators. The new vectors described in this article are available from Addgene and/or MolecularCloud for sharing with academic investigators for noncommercial research.}, }
@article {pmid32278307, year = {2020}, author = {Yang, YC and Chen, YH and Kao, JH and Ching, C and Liu, IJ and Wang, CC and Tsai, CH and Wu, FY and Liu, CJ and Chen, PJ and Chen, DS and Yang, HC}, title = {Permanent Inactivation of HBV Genomes by CRISPR/Cas9-Mediated Non-cleavage Base Editing.}, journal = {Molecular therapy. Nucleic acids}, volume = {20}, number = {}, pages = {480-490}, doi = {10.1016/j.omtn.2020.03.005}, pmid = {32278307}, issn = {2162-2531}, abstract = {Current antiviral therapy fails to cure chronic hepatitis B virus (HBV) infection because of persistent covalently closed circular DNA (cccDNA). CRISPR/Cas9-mediated specific cleavage of cccDNA is a potentially curative strategy for chronic hepatitis B (CHB). However, the CRISPR/Cas system inevitably targets integrated HBV DNA and induces double-strand breaks (DSBs) of host genome, bearing the risk of genomic rearrangement and damage. Herein, we examined the utility of recently developed CRISPR/Cas-mediated &quot;base editors&quot; (BEs) in inactivating HBV gene expression without cleavage of DNA. Candidate target sites of the SpCas9-derived BE and its variants in HBV genomes were screened for generating nonsense mutations of viral genes with individual guide RNAs (gRNAs). SpCas9-BE with certain gRNAs effectively base-edited polymerase and surface genes and reduced HBV gene expression in cells harboring integrated HBV genomes, but induced very few insertions or deletions (indels). Interestingly, some point mutations introduced by base editing resulted in simultaneous suppression of both polymerase and surface genes. Finally, the episomal cccDNA was successfully edited by SpCas9-BE for suppression of viral gene expression in an in vitro HBV infection system. In conclusion, Cas9-mediated base editing is a potential strategy to cure CHB by permanent inactivation of integrated HBV DNA and cccDNA without DSBs of the host genome.}, }
@article {pmid32277462, year = {2020}, author = {Bhandawat, A and Sharma, V and Rishi, V and K Roy, J}, title = {Biolistic Delivery of Programmable Nuclease (CRISPR/Cas9) in Bread Wheat.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2124}, number = {}, pages = {309-329}, doi = {10.1007/978-1-0716-0356-7_17}, pmid = {32277462}, issn = {1940-6029}, abstract = {The discovery of site-specific programmable nucleases has led to a major breakthrough in the area of genome editing. In the past few years, CRISPR/Cas system has been utilized for genome editing of a large number of crops including cereals like wheat, rice, maize, and barley. In terms of consumption, wheat is second only to rice as the most important crop of the world. In the present chapter, we describe biolistic delivery method of ribonucleoprotein (RNP) complexes of programmable nuclease (CRISPR/Cas9) for targeted genome editing and selection-free screening of transformants in wheat. The method not only overcomes the problem of random integration into the genome but also reduces the off-targets. Besides the step-by-step protocol, plausible challenges and ways to overcome them are also discussed. By using the described method of biolistic delivery of CRISPR/Cas9 in plant systems, genome-edited plants can be identified within 11 weeks.}, }
@article {pmid32277460, year = {2020}, author = {Imani, J and Kogel, KH}, title = {Plant Transformation Techniques: Agrobacterium- and Microparticle-Mediated Gene Transfer in Cereal Plants.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2124}, number = {}, pages = {281-294}, doi = {10.1007/978-1-0716-0356-7_15}, pmid = {32277460}, issn = {1940-6029}, abstract = {Biotechnological methods for targeted gene transfers into plants are key for successful breeding in the twenty-first century and thus essential for the survival of humanity. Two decades ago, genetic transformation of crop plants was not routine, and it was all but impossible with important cereals such as barley and wheat. The recent focus on crop plant genomics-yet based on the Arabidopsis toolbox-boosted the research for more efficient plant transformation protocols, thereby considerably widened the number of genetically tractable crops. Moreover, modern genome editing methods such as the CRISPR/Cas technique are game changers in plant breeding, though heavily dependent on technical optimization of plant transformation. Basically, there are two successful ways of introducing DNA into plant cells: one is making use of a living DNA vector, namely, microbes such as the soil bacterium Agrobacterium tumefaciens that infects plants and naturally transfers and subsequently integrates DNA into the plant genome. The other method uses a direct physical transfer of DNA by means of microinjection, microprojectile bombardment, or polymers such as polyethylene glycol. Both ways subsequently require sophisticated strategies for selecting and multiplying the transformed cells under tissue culture conditions to develop into a fully functional plant with the new desirable characteristics. Here we discuss practical and theoretical aspects of cereal crop plant transformation by Agrobacterium-mediated transformation and microparticle bombardment. Using immature embryos as explants, the efficiency of cereal transformation is compelling, reaching today up to 80% transformation efficiency.}, }
@article {pmid32275780, year = {2020}, author = {Nieves-Cordones, M and Lara, A and Silva, M and Amo, J and Rodriguez-Sepulveda, P and Rivero, RM and Martínez, V and Botella, MA and Rubio, F}, title = {Root high-affinity K+ and Cs+ uptake and plant fertility in tomato plants are dependent on the activity of the high-affinity K+ transporter SlHAK5.}, journal = {Plant, cell &amp; environment}, volume = {}, number = {}, pages = {}, doi = {10.1111/pce.13769}, pmid = {32275780}, issn = {1365-3040}, abstract = {Root K+ acquisition is a key process for plant growth and development, extensively studied in the model plant Arabidopsis thaliana. Because important differences may exist among species, translational research supported by specific studies in crops such as tomato are needed. Here we present a reverse genetics study to demonstrate the role of the SlHAK5 K+ transporter in tomato K+ nutrition, Cs+ accumulation and its fertility. slhak5 KO lines, generated by CRISPR-Cas edition, were characterized in growth experiments, Rb+ and Cs+ uptake tests and root cells K+ -induced plasma membrane depolarizations. Pollen viability and its K+ accumulation capacity was estimated by using the K+ -sensitive dye Ion Potassium Green 4. SlHAK5 is the major system for high-affinity root K+ uptake required for plant growth at low K+ , even in the presence of salinity. It also constitutes a pathway for Cs+ entry in tomato plants with a strong impact on fruit Cs+ accumulation. SlHAK5 also contributes to pollen K+ uptake and viability and its absence produces almost seedless fruits. Knowledge gained into SlHAK5 can serve as a model for other crops with fleshy fruits and it can help to generate tools to develop low-Cs+ or seedless fruits crops. This article is protected by copyright. All rights reserved.}, }
@article {pmid32273345, year = {2020}, author = {Ling, L and Mulaka, M and Munro, J and Dass, S and Mather, MW and Riscoe, MK and Llinás, M and Zhou, J and Ke, H}, title = {Genetic ablation of the mitoribosome in the malaria parasite Plasmodium falciparum sensitizes it to antimalarials that target mitochondrial functions.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1074/jbc.RA120.012646}, pmid = {32273345}, issn = {1083-351X}, abstract = {The mitochondrion of malaria parasites contains several clinically validated drug targets. Within Plasmodium spp., the causative agents of malaria, the mitochondrial DNA (mtDNA) is only 6 kb long, being the smallest mitochondrial genome among all eukaryotes. The mtDNA encodes only three proteins of the mitochondrial electron transport chain and ~27 small, fragmented rRNA genes having lengths of 22-195 nucleotides. The rRNA fragments are thought to form a mitochondrial ribosome (mitoribosome), together with ribosomal proteins imported from the cytosol. The mitoribosome of Plasmodium falciparum is essential for maintenance of the mitochondrial membrane potential and parasite viability. However, the role of the mitoribosome in sustaining the metabolic status of the parasite mitochondrion remains unclear. The small ribosomal subunit in P. falciparum has 14 annotated mitoribosomal proteins, and employing a CRISPR/Cas9-based conditional knockdown tool, here we verified the location and tested the essentiality of three candidates (PfmtRPS12, PfmtRPS17, and PfmtRPS18). Using immuno-EM, we provide evidence that the P. falciparum mitoribosome is closely associated with the mitochondrial inner membrane. Upon knockdown of the mitoribosome, parasites became hypersensitive to inhibitors targeting mitochondrial Complex III (bc1), dihydroorotate dehydrogenase (DHODH), and the F1Fo ATP synthase complex. Furthermore, the mitoribosome knockdown blocked the pyrimidine biosynthesis pathway and reduced the cellular pool of pyrimidine nucleotides. These results suggest that disruption of the P. falciparum mitoribosome compromises the metabolic capacity of the mitochondrion, rendering the parasite hypersensitive to a panel of inhibitors that target mitochondrial functions.}, }
@article {pmid32272843, year = {2020}, author = {Wang, G and Tian, W and Liu, X and Ren, W and Liu, C}, title = {A new CRISPR-derived microRNA sensing mechanism based on Cas12a self-powered and rolling circle transcription (RCT)-unleashed real-time crRNA recruiting.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.0c00680}, pmid = {32272843}, issn = {1520-6882}, abstract = {Current CRISPR-Cas-based nucleic acid sensing methods relying on the pre-assembled Cas-crRNA complexes are gen-erally limited to the detection of protospacer-adjacent motif (PAM)-containing sequences, and nonspecific back-grounds are inevitable. Herein, we propose a new CRISPR-derived microRNA sensing mechanism based on rolling cir-cular transcription (RCT)-unleashed self-recruiting of crRNA by Cas12a (Cas12a-SCR). In Cas12a-SCR, target microRNA can specifically trigger RCT to produce a long single-strand RNA with numerous pre-crRNA repeats, which can be trimmed and recruited by Cas12a actively. This new target-initiated, real-time producing, trimming, and self-assembling manner of Cas12a-crRNA remarkably suppresses the nonspecific background and relieves the stringent re-quirement of PAM site in the target sequence. Thus, the universality of the Cas12a-SCR towards different nucleic acid sequences is greatly expanded.}, }
@article {pmid32152546, year = {2020}, author = {Girardi, E and César-Razquin, A and Lindinger, S and Papakostas, K and Konecka, J and Hemmerich, J and Kickinger, S and Kartnig, F and Gürtl, B and Klavins, K and Sedlyarov, V and Ingles-Prieto, A and Fiume, G and Koren, A and Lardeau, CH and Kumaran Kandasamy, R and Kubicek, S and Ecker, GF and Superti-Furga, G}, title = {A widespread role for SLC transmembrane transporters in resistance to cytotoxic drugs.}, journal = {Nature chemical biology}, volume = {16}, number = {4}, pages = {469-478}, doi = {10.1038/s41589-020-0483-3}, pmid = {32152546}, issn = {1552-4469}, mesh = {Amino Acid Transport Systems, Neutral/genetics/metabolism ; Antineoplastic Agents ; Biochemical Phenomena ; Biological Transport/genetics/physiology ; CRISPR-Cas Systems ; Cation Transport Proteins/genetics/metabolism ; Drug Resistance/*genetics/physiology ; Genetic Testing ; Humans ; Monocarboxylic Acid Transporters/genetics/metabolism ; Monosaccharide Transport Proteins/genetics/metabolism ; Protein Transport/physiology ; Solute Carrier Proteins/*metabolism/physiology ; Symporters/genetics/metabolism ; }, abstract = {Solute carriers (SLCs) are the largest family of transmembrane transporters in humans and are major determinants of cellular metabolism. Several SLCs have been shown to be required for the uptake of chemical compounds into cellular systems, but systematic surveys of transporter-drug relationships in human cells are currently lacking. We performed a series of genetic screens in a haploid human cell line against 60 cytotoxic compounds representative of the chemical space populated by approved drugs. By using an SLC-focused CRISPR-Cas9 library, we identified transporters whose absence induced resistance to the drugs tested. This included dependencies involving the transporters SLC11A2/SLC16A1 for artemisinin derivatives and SLC35A2/SLC38A5 for cisplatin. The functional dependence on SLCs observed for a significant proportion of the screened compounds suggests a widespread role for SLCs in the uptake and cellular activity of cytotoxic drugs and provides an experimentally validated set of SLC-drug associations for a number of clinically relevant compounds.}, }
@article {pmid32123334, year = {2020}, author = {Yang, Q and Oost, KC and Liberali, P}, title = {Engineering human knock-in organoids.}, journal = {Nature cell biology}, volume = {22}, number = {3}, pages = {261-263}, doi = {10.1038/s41556-020-0478-z}, pmid = {32123334}, issn = {1476-4679}, mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; Humans ; *Organoids ; Tissue Engineering ; }, }
@article {pmid31932729, year = {2020}, author = {Jost, M and Santos, DA and Saunders, RA and Horlbeck, MA and Hawkins, JS and Scaria, SM and Norman, TM and Hussmann, JA and Liem, CR and Gross, CA and Weissman, JS}, title = {Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs.}, journal = {Nature biotechnology}, volume = {38}, number = {3}, pages = {355-364}, doi = {10.1038/s41587-019-0387-5}, pmid = {31932729}, issn = {1546-1696}, support = {U01 CA168370/CA/NCI NIH HHS/United States ; U01 CA217882/CA/NCI NIH HHS/United States ; F32 GM116331/GM/NIGMS NIH HHS/United States ; R35 GM118061/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; K99 GM130964/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; Computational Biology/*methods ; Deep Learning ; Gene Editing ; *Gene Expression ; Genomic Library ; HeLa Cells ; Humans ; K562 Cells ; Phenotype ; RNA, Guide/*genetics ; Sequence Analysis, RNA ; Single-Cell Analysis/*methods ; }, abstract = {A lack of tools to precisely control gene expression has limited our ability to evaluate relationships between expression levels and phenotypes. Here, we describe an approach to titrate expression of human genes using CRISPR interference and series of single-guide RNAs (sgRNAs) with systematically modulated activities. We used large-scale measurements across multiple cell models to characterize activities of sgRNAs containing mismatches to their target sites and derived rules governing mismatched sgRNA activity using deep learning. These rules enabled us to synthesize a compact sgRNA library to titrate expression of ~2,400 genes essential for robust cell growth and to construct an in silico sgRNA library spanning the human genome. Staging cells along a continuum of gene expression levels combined with single-cell RNA-seq readout revealed sharp transitions in cellular behaviors at gene-specific expression thresholds. Our work provides a general tool to control gene expression, with applications ranging from tuning biochemical pathways to identifying suppressors for diseases of dysregulated gene expression.}, }
@article {pmid31873222, year = {2020}, author = {Yu, Y and Guo, Y and Tian, Q and Lan, Y and Yeh, H and Zhang, M and Tasan, I and Jain, S and Zhao, H}, title = {An efficient gene knock-in strategy using 5'-modified double-stranded DNA donors with short homology arms.}, journal = {Nature chemical biology}, volume = {16}, number = {4}, pages = {387-390}, doi = {10.1038/s41589-019-0432-1}, pmid = {31873222}, issn = {1552-4469}, support = {U54 DK107965/DK/NIDDK NIH HHS/United States ; UM1 HG009402/HG/NHGRI NIH HHS/United States ; }, mesh = {5' Flanking Region/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA/genetics ; Gene Knock-In Techniques/*methods ; Genome/genetics ; HEK293 Cells ; Humans ; RNA, Guide/genetics ; Sequence Homology, Nucleic Acid ; }, abstract = {Here, we report a rapid CRISPR-Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5'-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65/40% knock-in rates for 0.7/2.5 kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5'-end modification led to up to a fivefold increase in gene knock-in rates at various genomic loci in human cancer and stem cells.}, }
@article {pmid31492696, year = {2019}, author = {Dong, Y and Li, H and Zhao, L and Koopman, P and Zhang, F and Huang, JX}, title = {Genome-Wide Off-Target Analysis in CRISPR-Cas9 Modified Mice and Their Offspring.}, journal = {G3 (Bethesda, Md.)}, volume = {9}, number = {11}, pages = {3645-3651}, pmid = {31492696}, issn = {2160-1836}, mesh = {Animals ; *CRISPR-Cas Systems ; Female ; *Gene Editing ; Gene Targeting ; Genome ; Mice, Inbred C57BL ; Mice, Knockout ; Mutation ; Receptors, Progesterone/genetics ; Whole Genome Sequencing ; }, abstract = {The emergence of the CRISPR-Cas9 system has triggered a technical revolution in mammalian genome editing. Compared to traditional gene-targeting strategies, CRISPR-Cas9 technology offers a more efficient and cost-effective approach for generating genetically modified animal models. However, off-target cleavage in CRISPR-mediated genome editing is a major concern in the analysis of phenotypes as well as the selection of therapeutic targets. Here, we analyzed whole-genome sequencing (WGS) data from two knock-out (KO) mouse strains generated by using the CRISPR-Cas9 system targeting the Mmd and Paqr8 loci. A total of nine individuals were sequenced including two parents, four F1 offspring and three uninjected control mice. Using GATK and bcftools software, we identified two off-target events in the founder mice. The two CRISPR-Cas9-induced off-target events were predictable using Cas-OFFinder and were not passed on to the offspring that we investigated. In addition, our results indicated that the number of CRISPR-Cas9-induced mutations was not statistically distinguishable from the background de novo mutations (DNMs).}, }
@article {pmid31463914, year = {2020}, author = {Mead, TJ}, title = {Alizarin Red and Alcian Blue Preparations to Visualize the Skeleton.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2043}, number = {}, pages = {207-212}, doi = {10.1007/978-1-4939-9698-8_17}, pmid = {31463914}, issn = {1940-6029}, mesh = {Alcian Blue/*chemistry ; Animals ; Animals, Newborn ; Anthraquinones/*chemistry ; Body Patterning ; Bone and Bones/chemistry/*embryology ; CRISPR-Cas Systems ; Embryonic Development ; Mice ; }, abstract = {Understanding proteolytic remodeling of extracellular matrix involves the generation of global or conditional knockout mice by homologous recombination in embryonic stem cells or their manipulation through new advanced technologies such as CRISPR-Cas9. These models provide opportunities to understand the roles of ADAMTS genes in skeletogenesis. Whole-mount skeletal preparations are necessary for assessment of the skeletal phenotype. They allow for facile visualization of skeletal patterning, size and shape of skeletal elements, and skeletal structure. This protocol describes the staining of the murine skeleton using Alcian blue to identify cartilage and alizarin red to identify bone.}, }
@article {pmid31463899, year = {2020}, author = {Peris-Torres, C and Serrano, O and Plaza-Calonge, MDC and Rodríguez-Manzaneque, JC}, title = {Inhibition of ADAMTS1 Expression by Lentiviral CRISPR/Cas9 Gene Editing Technology.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2043}, number = {}, pages = {13-24}, doi = {10.1007/978-1-4939-9698-8_2}, pmid = {31463899}, issn = {1940-6029}, mesh = {ADAMTS1 Protein/*antagonists &amp; inhibitors/genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Sequence Analysis, RNA ; Single-Cell Analysis/*methods ; }, abstract = {The continuous improvement of gene editing tools has allowed a major revolution in biological sciences. Although a variety of gain and loss-of-function approaches have been widely used for the last decades, some limitations arose from non-specific targeting or lack of complete inhibition of the gene of interest. CRISPR/Cas9 editing technology introduced new and significant advantages because it can directly modify the gene of interest and completely blocks its expression.In the context of cancer studies, the heterogeneity of the tumor microenvironment requires comprehensive approaches to unveil the contribution of multiple genes. For example, a deeper understanding of the biology of proteases such as ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) will improve our perspective of complex phenomena affected by extracellular matrix remodeling, including embryonic development, angiogenesis, immune infiltration, metastasis, and tumor plasticity. Here, we present a method using CRISPR/Cas9 technology to inhibit the expression of the representative ADAMTS1 in cancer cells. Following the first steps of gene edition, we pursue further selection of silenced cells and provide a detailed description of sequence analysis and validation assays. This method leads to inactivation of ADAMTS1 in cancer cells, providing a relevant biological tool that will allow subsequent in vivo and in vitro ADAMTS1 functional analysis.}, }
@article {pmid31427540, year = {2019}, author = {Hu, Q and Wolfner, MF}, title = {The Drosophila Trpm channel mediates calcium influx during egg activation.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {38}, pages = {18994-19000}, pmid = {31427540}, issn = {1091-6490}, support = {R21 HD088744/HD/NICHD NIH HHS/United States ; S10 OD018516/OD/NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Calcium/*metabolism ; Calcium Signaling/physiology ; Drosophila Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Drosophila melanogaster/embryology/*metabolism ; *Embryonic Development ; Female ; Fertilization/*physiology ; Male ; Oocytes/cytology/*metabolism ; TRPM Cation Channels/antagonists &amp; inhibitors/genetics/*metabolism ; }, abstract = {Egg activation is the process in which mature oocytes are released from developmental arrest and gain competency for embryonic development. In Drosophila and other arthropods, eggs are activated by mechanical pressure in the female reproductive tract, whereas in most other species, eggs are activated by fertilization. Despite the difference in the trigger, Drosophila shares many conserved features with higher vertebrates in egg activation, including a rise of intracellular calcium in response to the trigger. In Drosophila, this calcium rise is initiated by entry of extracellular calcium due to opening of mechanosensitive ion channels and initiates a wave that passes across the egg prior to initiation of downstream activation events. Here, we combined inhibitor tests, germ-line-specific RNAi knockdown, and germ-line-specific CRISPR/Cas9 knockout to identify the Transient Receptor Potential (TRP) channel subfamily M (Trpm) as a critical channel that mediates the calcium influx and initiates the calcium wave during Drosophila egg activation. We observed a reduction in the proportion of eggs that hatched from trpm germ-line knockout mutant females, although eggs were able to complete some egg activation events including cell cycle resumption. Since a mouse ortholog of Trpm was recently reported also to be involved in calcium influx during egg activation and in further embryonic development, our results suggest that calcium uptake from the environment via TRPM channels is a deeply conserved aspect of egg activation.}, }
@article {pmid31163979, year = {2019}, author = {Jaffré, F and Miller, CL and Schänzer, A and Evans, T and Roberts, AE and Hahn, A and Kontaridis, MI}, title = {Inducible Pluripotent Stem Cell-Derived Cardiomyocytes Reveal Aberrant Extracellular Regulated Kinase 5 and Mitogen-Activated Protein Kinase Kinase 1/2 Signaling Concomitantly Promote Hypertrophic Cardiomyopathy in RAF1-Associated Noonan Syndrome.}, journal = {Circulation}, volume = {140}, number = {3}, pages = {207-224}, pmid = {31163979}, issn = {1524-4539}, support = {R01 HL114775/HL/NHLBI NIH HHS/United States ; R01 HL102368/HL/NHLBI NIH HHS/United States ; R35 HL135778/HL/NHLBI NIH HHS/United States ; R00 HL125912/HL/NHLBI NIH HHS/United States ; R01 HL122238/HL/NHLBI NIH HHS/United States ; }, mesh = {Adolescent ; CRISPR-Cas Systems/physiology ; Cardiomyopathy, Hypertrophic/*genetics/metabolism ; Cells, Cultured ; Child ; Female ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells/*physiology ; MAP Kinase Kinase 1/*genetics/metabolism ; MAP Kinase Kinase 2/*genetics/metabolism ; Male ; Mitogen-Activated Protein Kinase 7/*genetics/metabolism ; Myocytes, Cardiac/physiology ; Noonan Syndrome/*genetics/metabolism ; Proto-Oncogene Proteins c-raf/*genetics/metabolism ; }, abstract = {BACKGROUND: More than 90% of individuals with Noonan syndrome (NS) with mutations clustered in the CR2 domain of RAF1 present with severe and often lethal hypertrophic cardiomyopathy (HCM). The signaling pathways by which NS RAF1 mutations promote HCM remain elusive, and so far, there is no known treatment for NS-associated HCM.<br><br>METHODS: We used patient-derived RAF1S257L/+ and CRISPR-Cas9-generated isogenic control inducible pluripotent stem cell (iPSC)-derived cardiomyocytes to model NS RAF1-associated HCM and to further delineate the molecular mechanisms underlying the disease.<br><br>RESULTS: We show that mutant iPSC-derived cardiomyocytes phenocopy the pathology seen in hearts of patients with NS by exhibiting hypertrophy and structural defects. Through pharmacological and genetic targeting, we identify 2 perturbed concomitant pathways that, together, mediate HCM in RAF1 mutant iPSC-derived cardiomyocytes. Hyperactivation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), but not extracellular regulated kinase 1/2, causes myofibrillar disarray, whereas the enlarged cardiomyocyte phenotype is a direct consequence of increased extracellular regulated kinase 5 (ERK5) signaling, a pathway not previously known to be involved in NS. RNA-sequencing reveals genes with abnormal expression in RAF1 mutant iPSC-derived cardiomyocytes and identifies subsets of genes dysregulated by aberrant MEK1/2 or ERK5 pathways that could contribute to the NS-associated HCM.<br><br>CONCLUSIONS: Taken together, the results of our study identify the molecular mechanisms by which NS RAF1 mutations cause HCM and reveal downstream effectors that could serve as therapeutic targets for treatment of NS and perhaps other, more common, congenital HCM disorders.}, }
@article {pmid32271968, year = {2020}, author = {Zhang, D and Hussain, A and Manghwar, H and Xie, K and Xie, S and Zhao, S and Larkin, RM and Qing, P and Jin, S and Ding, F}, title = {Genome editing with the CRISPR-Cas system: an art, ethics and global regulatory perspective.}, journal = {Plant biotechnology journal}, volume = {}, number = {}, pages = {}, doi = {10.1111/pbi.13383}, pmid = {32271968}, issn = {1467-7652}, abstract = {Over the last three decades, the development of new genome editing techniques, such as ODM, TALENs, ZFNs and the CRISPR-Cas system has led to significant progress in the field of plant and animal breeding. The CRISPR-Cas system is the most versatile genome editing tool discovered in the history of molecular biology because it can be used to alter diverse genomes (e.g., genomes from both plants and animals) including human genomes with unprecedented ease, accuracy and high efficiency. The recent development and scope of CRISPR-Cas system have raised new regulatory challenges around the world due to moral, ethical, safety and technical concerns associated with its applications in pre-clinical and clinical research, biomedicine and agriculture. Here, we review the art, applications and potential risks of CRISPR-Cas system in genome editing. We also highlight the patent and ethical issues of this technology along with regulatory frameworks established by various nations to regulate CRISPR-Cas-modified organisms/products.}, }
@article {pmid32271547, year = {2020}, author = {Cooper, RM and Hasty, DJ}, title = {One-Day Construction of Multiplex Arrays to Harness Natural CRISPR-Cas Systems.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.9b00489}, pmid = {32271547}, issn = {2161-5063}, abstract = {CRISPR-Cas systems are prokaryotic immune systems that have proliferated widely not only in bacteria and archaea, but also much more recently, in human biological research and applications. Much work to date has utilized synthetic sgRNAs along with the CRISPR nuclease Cas9, but the discovery of array-processing nucleases now allows the use of more compact, natural CRISPR arrays in heterologous hosts, in addition to organisms with endogenous systems. Unfortunately, the construction of multiplex natural CRISPR arrays remains technically challenging, expensive, and/or time-consuming. This limitation hampers research involving natural CRISPR arrays in both native and heterologous hosts. To address this problem, we present a method to assemble CRISPR arrays that is simple, rapid, affordable, and highly scalable - we assembled 9-spacer arrays with one day's worth of work. We used this method to harness the endogenous CRISPR-Cas system of the highly competent bacterium Acinetobacter baylyi, showing that while single spacers are not always completely effective at blocking DNA acquisition through natural competence, multiplex natural CRISPR arrays enable both nearly complete DNA exclusion and genome editing, including with multiple targets for both. In addition to demonstrating a CRISPR array assembly method that will benefit a variety of applications, we also find a potential bet-hedging strategy for balancing CRISPR defense vs. DNA acquisition in naturally competent A. baylyi.}, }
@article {pmid32269948, year = {2019}, author = {Wang, JH and Tseng, SY and Tung, KC}, title = {Genomic investigation of emerging zoonotic pathogen Shewanella xiamenensis.}, journal = {Ci ji yi xue za zhi = Tzu-chi medical journal}, volume = {32}, number = {2}, pages = {162-166}, doi = {10.4103/tcmj.tcmj_69_19}, pmid = {32269948}, issn = {1016-3190}, abstract = {Objective: Shewanella xiamenensis is an emerging zoonotic pathogen commonly found in aquatic ecosystem. Clustered regularly interspaced short palindromic repeats (CRISPR) and (CRISPR)-associated gene systems act as adaptive immune system of prokaryotes. Recently, growing evidence suggested their role in bacterial virulence and resistance. Despite its medical importance, little is known about the genomic characteristics of S. xiamenensis.<br><br>Materials and Methods: Strain ZYW6 was isolated from Epinephelus awoara. We sequenced the 16S rRNA gene and blast against the GenBank bacterial database. Antibiotic susceptibility tests and interpretation were performed by automatic VITEK 2 system. We extracted the genomic DNA with QIAGEN Genomic-tip 100/G kit and QIAGEN Genomic DNA Buffer Set. Whole-genome shotgun sequencing was performed using the Illumina MiSeq sequencer. To identify the CRISPR-Cas System in the genome of S. xiamenensis ZYW6, the Integrated Microbial Genomes and Microbiomes and CRISPRFinder were used.<br><br>Results: We characterized the genome of a S. xiamenensis strain. The genome is 4,765,190 bp in length and encodes 4262 open-reading frames. Type I CRISPR-Cas system and serine biosynthesis genes were identified.<br><br>Conclusion: Our results demonstrate the genetic structure of CRISPR-Cas system, l-serine synthesis, and oxacillinase in S. xiamenensis. The report of antibiotics resistance genes in the study might indicate a possible reservoir of antimicrobial drug resistance determinants in food animal, resulting in potential infection source. The findings provide insights into the structure and composition of CRISPR-Cas system in S. xiamenensis and foundation for future biological validation.}, }
@article {pmid32242409, year = {2020}, author = {Bao, Y and Jiang, Y and Xiong, E and Tian, T and Zhang, Z and Lv, J and Li, Y and Zhou, X}, title = {CUT-LAMP: Contamination-Free Loop-Mediated Isothermal Amplification Based on the CRISPR/Cas9 Cleavage.}, journal = {ACS sensors}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssensors.0c00034}, pmid = {32242409}, issn = {2379-3694}, abstract = {Loop-mediated isothermal amplification (LAMP) is a sensitive and widely used gene amplification technique. However, high amplification efficiency and amplification products containing multiple inverted repeats make the LAMP reaction extremely vulnerable to false-positive amplification caused by contamination. Herein, a contamination-free LAMP (CUT-LAMP) assisted by the CRISPR/Cas9 cleavage with superior reliability and durability has been reported. The core of CUT-LAMP is the engineering of the forward or backward inner primer in the target-independent region, which makes the LAMP products contain a protospacer adjacent motif (PAM) site for the CRISPR/Cas9 recognition. For the CUT-LAMP reaction, cross-contamination can be efficiently cleaved by the corresponding Cas9/sgRNA, but the target gene can get rid of digestion due to the lack of a PAM site near the recognition region. CUT-LAMP shows impressive contamination resistance but does not significantly increase procedure complexity; thus, it represents a simple and versatile toolkit facilitating the adoption by open- and closed-tube detection format.}, }
@article {pmid32234883, year = {2020}, author = {Sekino, Y and Hagura, T and Han, X and Babasaki, T and Goto, K and Inoue, S and Hayashi, T and Teishima, J and Shigeta, M and Taniyama, D and Kuraoka, K and Sentani, K and Yasui, W and Matsubara, A}, title = {PTEN Is Involved in Sunitinib and Sorafenib Resistance in Renal Cell Carcinoma.}, journal = {Anticancer research}, volume = {40}, number = {4}, pages = {1943-1951}, doi = {10.21873/anticanres.14149}, pmid = {32234883}, issn = {1791-7530}, mesh = {Biomarkers, Tumor/genetics ; CRISPR-Cas Systems ; Carcinoma, Renal Cell/*drug therapy/genetics/pathology ; Drug Resistance, Neoplasm/drug effects ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockout Techniques ; Humans ; Kaplan-Meier Estimate ; Male ; PTEN Phosphohydrolase/*genetics ; Progression-Free Survival ; Protein Kinase Inhibitors/pharmacology ; Sorafenib/*pharmacology ; Spheroids, Cellular/drug effects ; Sunitinib/*pharmacology ; }, abstract = {BACKGROUND/AIM: Targeted receptor tyrosine kinase inhibitor (TKI) is a standard treatment in advanced renal cell carcinoma (RCC). However, the role of PTEN in TKI resistance remains poorly understood. We aimed to determine the functional role of PTEN knockout and analyse the predictive significance of PTEN expression for TKI treatment in RCC.<br><br>MATERIALS AND METHODS: We developed PTEN knockout cells in RCC cell lines using the CRISPR-Cas9 system and analysed the effect of PTEN knockout on spheroid formation and resistance to sunitinib and sorafenib.<br><br>RESULTS: PTEN knockout promoted spheroid formation and decreased sunitinib/sorafenib sensitivity in RCC cell lines. PTEN immunohistochemistry in 74 metastatic RCCs treated with sunitinib and sorafenib revealed negative PTEN expression in 23% of samples. Kaplan-Meier analysis showed a significant association of negative PTEN expression with poor progression-free survival in metastatic RCC treated with sunitinib and sorafenib (p=0.024) or sunitinib alone (p=0.009).<br><br>CONCLUSION: PTEN may be a biomarker and therapeutic target in patients with metastatic RCC.}, }
@article {pmid32165588, year = {2020}, author = {Loganathan, SK and Schleicher, K and Malik, A and Quevedo, R and Langille, E and Teng, K and Oh, RH and Rathod, B and Tsai, R and Samavarchi-Tehrani, P and Pugh, TJ and Gingras, AC and Schramek, D}, title = {Rare driver mutations in head and neck squamous cell carcinomas converge on NOTCH signaling.}, journal = {Science (New York, N.Y.)}, volume = {367}, number = {6483}, pages = {1264-1269}, doi = {10.1126/science.aax0902}, pmid = {32165588}, issn = {1095-9203}, support = {//Canadian Institute of Health Research/International ; }, mesh = {ADAM10 Protein/genetics ; Amyloid Precursor Protein Secretases/genetics ; Animals ; CRISPR-Cas Systems ; Female ; *Genes, Tumor Suppressor ; *Genetic Predisposition to Disease ; Genetic Testing ; HEK293 Cells ; Head and Neck Neoplasms/*genetics ; Humans ; LIM Domain Proteins/genetics ; Male ; Membrane Proteins/genetics ; Mice ; Mice, Inbred C57BL ; Mice, Mutant Strains ; Mutation ; Receptors, Notch/genetics ; Signal Transduction/genetics ; Squamous Cell Carcinoma of Head and Neck/*genetics ; Tumor Suppressor Proteins/*genetics ; }, abstract = {In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent &quot;long tail&quot; mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, ADAM10 and AJUBA suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. ADAM10 and AJUBA mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.}, }
@article {pmid31953404, year = {2020}, author = {López Del Amo, V and Bishop, AL and Sánchez C, HM and Bennett, JB and Feng, X and Marshall, JM and Bier, E and Gantz, VM}, title = {A transcomplementing gene drive provides a flexible platform for laboratory investigation and potential field deployment.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {352}, pmid = {31953404}, issn = {2041-1723}, support = {DP5 OD023098/OD/NIH HHS/United States ; }, mesh = {Alleles ; Animals ; Animals, Genetically Modified ; Base Sequence ; CRISPR-Cas Systems ; Diptera ; Ecosystem ; Female ; Gene Drive Technology/*methods ; Gene Editing ; Genes, X-Linked ; Genetics, Population/*methods ; Male ; Models, Theoretical ; RNA, Guide/genetics ; Transgenes ; }, abstract = {CRISPR-based gene drives can spread through wild populations by biasing their own transmission above the 50% value predicted by Mendelian inheritance. These technologies offer population-engineering solutions for combating vector-borne diseases, managing crop pests, and supporting ecosystem conservation efforts. Current technologies raise safety concerns for unintended gene propagation. Herein, we address such concerns by splitting the drive components, Cas9 and gRNAs, into separate alleles to form a trans-complementing split-gene-drive (tGD) and demonstrate its ability to promote super-Mendelian inheritance of the separate transgenes. This dual-component configuration allows for combinatorial transgene optimization and increases safety by restricting escape concerns to experimentation windows. We employ the tGD and a small-molecule-controlled version to investigate the biology of component inheritance and resistant allele formation, and to study the effects of maternal inheritance and impaired homology on efficiency. Lastly, mathematical modeling of tGD spread within populations reveals potential advantages for improving current gene-drive technologies for field population modification.}, }
@article {pmid31873217, year = {2020}, author = {Kwon, CT and Heo, J and Lemmon, ZH and Capua, Y and Hutton, SF and Van Eck, J and Park, SJ and Lippman, ZB}, title = {Rapid customization of Solanaceae fruit crops for urban agriculture.}, journal = {Nature biotechnology}, volume = {38}, number = {2}, pages = {182-188}, doi = {10.1038/s41587-019-0361-2}, pmid = {31873217}, issn = {1546-1696}, mesh = {Agriculture/*methods ; Base Sequence ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/*physiology ; Fruit/*physiology ; Gene Editing ; Inflorescence/physiology ; Mutation/genetics ; Phylogeny ; Plant Shoots/physiology ; Solanaceae/*physiology ; }, abstract = {Cultivation of crops in urban environments might reduce the environmental impact of food production1-4. However, lack of available land in cities and a need for rapid crop cycling, to yield quickly and continuously, mean that so far only lettuce and related 'leafy green' vegetables are cultivated in urban farms5. New fruit varieties with architectures and yields suitable for urban farming have proven difficult to breed1,5. We identified a regulator of tomato stem length (SlER) and devised a trait-stacking strategy to combine mutations for condensed shoots, rapid flowering (SP5G) and precocious growth termination (SP). Application of our strategy using one-step CRISPR-Cas9 genome editing restructured vine-like tomato plants into compact, early yielding plants suitable for urban agriculture. Field data confirmed that yields were maintained, and we demonstrated cultivation in indoor farming systems. Targeting the same stem length regulator alone in groundcherry, another Solanaceae plant, also enabled engineering to a compact stature. Our approach can expand the repertoire of crops for urban agriculture.}, }
@article {pmid31571024, year = {2019}, author = {Yang, X and Zhou, H and Zhou, X}, title = {Rock paper scissors: CRISPR/Cas9-mediated interference with geminiviruses in plants.}, journal = {Science China. Life sciences}, volume = {62}, number = {10}, pages = {1389-1391}, doi = {10.1007/s11427-019-9825-4}, pmid = {31571024}, issn = {1869-1889}, mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems/*genetics/physiology ; Disease Resistance/genetics ; Geminiviridae/*metabolism ; Gene Editing/methods ; Gene Expression Regulation/physiology ; Genome, Viral ; Plant Diseases/*prevention &amp; control ; Plants, Genetically Modified/*genetics/metabolism ; }, }
@article {pmid31357516, year = {2019}, author = {Gasparis, S and Przyborowski, M and Kała, M and Nadolska-Orczyk, A}, title = {Knockout of the HvCKX1 or HvCKX3 Gene in Barley (Hordeum vulgare L.) by RNA-Guided Cas9 Nuclease Affects the Regulation of Cytokinin Metabolism and Root Morphology.}, journal = {Cells}, volume = {8}, number = {8}, pages = {}, pmid = {31357516}, issn = {2073-4409}, mesh = {Base Sequence ; CRISPR-Cas Systems ; Cytokinins/*metabolism ; *Gene Editing ; Gene Expression Regulation ; Gene Expression Regulation, Plant ; Hordeum/*physiology ; Humans ; Mutation ; Oxidoreductases/*genetics/metabolism ; Oxidoreductases Acting on CH-NH Group Donors/*genetics/metabolism ; Plant Proteins/genetics/metabolism ; Plant Roots/*physiology ; Plants, Genetically Modified ; RNA, Guide ; }, abstract = {Barley is among four of the most important cereal crops with respect to global production. Increasing barley yields to desired levels can be achieved by the genetic manipulation of cytokinin content. Cytokinins are plant hormones that regulate many developmental processes and have a strong influence on grain yield. Cytokinin homeostasis is regulated by members of several multigene families. CKX genes encode the cytokinin oxidase/dehydrogenase enzyme, which catalyzes the irreversible degradation of cytokinin. Several recent studies have demonstrated that the RNAi-based silencing of CKX genes leads to increased grain yields in some crop species. To assess the possibility of increasing the grain yield of barley by knocking out CKX genes, we used an RNA-guided Cas9 system to generate ckx1 and ckx3 mutant lines with knockout mutations in the HvCKX1 and HvCKX3 genes, respectively. Homozygous, transgene-free mutant lines were subsequently selected and analyzed. A significant decrease in CKX enzyme activity was observed in the spikes of the ckx1 lines, while in the ckx3 lines, the activity remained at a similar level to that in the control plants. Despite these differences, no changes in grain yield were observed in either mutant line. In turn, differences in CKX activity in the roots between the ckx1 and ckx3 mutants were reflected via root morphology. The decreased CKX activity in the ckx1 lines corresponded to greater root length, increased surface area, and greater numbers of root hairs, while the increased CKX activity in the ckx3 mutants gave the opposite results. RNA-seq analysis of the spike and root transcriptomes revealed an altered regulation of genes controlling cytokinin metabolism and signaling, as well as other genes that are important during seed development, such as those that encode nutrient transporters. The observed changes suggest that the knockout of a single CKX gene in barley may be not sufficient for disrupting cytokinin homeostasis or increasing grain yields.}, }
@article {pmid30952800, year = {2019}, author = {Valverde, DP and Yu, S and Boggavarapu, V and Kumar, N and Lees, JA and Walz, T and Reinisch, KM and Melia, TJ}, title = {ATG2 transports lipids to promote autophagosome biogenesis.}, journal = {The Journal of cell biology}, volume = {218}, number = {6}, pages = {1787-1798}, pmid = {30952800}, issn = {1540-8140}, support = {T32 GM007223/GM/NIGMS NIH HHS/United States ; R01 GM114068/GM/NIGMS NIH HHS/United States ; R01 NS063973/NS/NINDS NIH HHS/United States ; R35 GM131715/GM/NIGMS NIH HHS/United States ; R01 GM100930/GM/NIGMS NIH HHS/United States ; R01 GM080616/GM/NIGMS NIH HHS/United States ; }, mesh = {Autophagosomes/*physiology ; *Autophagy ; Autophagy-Related Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; Biological Transport ; CRISPR-Cas Systems ; Endoplasmic Reticulum/*metabolism ; HEK293 Cells ; Humans ; Lipids/*physiology ; Vesicular Transport Proteins/antagonists &amp; inhibitors/genetics/*metabolism ; }, abstract = {During macroautophagic stress, autophagosomes can be produced continuously and in high numbers. Many different organelles have been reported as potential donor membranes for this sustained autophagosome growth, but specific machinery to support the delivery of lipid to the growing autophagosome membrane has remained unknown. Here we show that the autophagy protein, ATG2, without a clear function since its discovery over 20 yr ago, is in fact a lipid-transfer protein likely operating at the ER-autophagosome interface. ATG2A can bind tens of glycerophospholipids at once and transfers lipids robustly in vitro. An N-terminal fragment of ATG2A that supports lipid transfer in vitro is both necessary and fully sufficient to rescue blocked autophagosome biogenesis in ATG2A/ATG2B KO cells, implying that regulation of lipid homeostasis is the major autophagy-dependent activity of this protein and, by extension, that protein-mediated lipid transfer across contact sites is a principal contributor to autophagosome formation.}, }
@article {pmid30951293, year = {2019}, author = {Liu, Y and Ren, CY and Wei, WP and You, D and Yin, BC and Ye, BC}, title = {A CRISPR-Cas9 Strategy for Activating the Saccharopolyspora erythraea Erythromycin Biosynthetic Gene Cluster with Knock-in Bidirectional Promoters.}, journal = {ACS synthetic biology}, volume = {8}, number = {5}, pages = {1134-1143}, doi = {10.1021/acssynbio.9b00024}, pmid = {30951293}, issn = {2161-5063}, mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Erythromycin/*biosynthesis ; Flavoproteins/genetics ; Isocitrate Lyase/genetics ; Multigene Family ; Promoter Regions, Genetic ; RNA, Guide/metabolism ; Saccharopolyspora/*genetics ; }, abstract = {The regulation of biosynthetic pathways is a universal strategy for industrial strains that overproduce metabolites. Erythromycin produced by Saccharopolyspora erythraea has extensive clinical applications. In this study, promoters of the erythromycin biosynthesis gene cluster were tested by reporter mCherry. The SACE_0720 (eryBIV)-SACE_0721 (eryAI) spacer was selected as a target regulatory region, and bidirectional promoters with dual single guide RNAs (sgRNAs) were knocked-in using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 method. qPCR results indicated that knock-in of Pj23119-PkasO, which replaced the native promoter, enabled biosynthetic gene cluster activation, with eryBIV and eryAI expression increased 32 and 79 times, respectively. High performance liquid chromatography results showed that, compared with the wild-type strain, the yield of erythromycin was increased (58.3%) in bidirectional promoter knock-in recombinant strains. On the basis of the activated strain Ab::Pj23119-PkasO, further investigation showed that CRISPR-based interference of sdhA gene affected erythromycin biosynthesis and cell growth. Finally, regulating the culture temperature to optimize the inhibition intensity of sdhA further increased the yield by 15.1%. In summary, this study showed that bidirectional promoter knock-in and CRISPR interference could regulate gene expression in S. erythraea. This strategy has potential application for biosynthetic gene cluster activation and gene regulation in Actinobacteria.}, }
@article {pmid30925042, year = {2019}, author = {Stark, JC and Huang, A and Hsu, KJ and Dubner, RS and Forbrook, J and Marshalla, S and Rodriguez, F and Washington, M and Rybnicky, GA and Nguyen, PQ and Hasselbacher, B and Jabri, R and Kamran, R and Koralewski, V and Wightkin, W and Martinez, T and Jewett, MC}, title = {BioBits Health: Classroom Activities Exploring Engineering, Biology, and Human Health with Fluorescent Readouts.}, journal = {ACS synthetic biology}, volume = {8}, number = {5}, pages = {1001-1009}, doi = {10.1021/acssynbio.8b00381}, pmid = {30925042}, issn = {2161-5063}, mesh = {CRISPR-Cas Systems/genetics ; Cell-Free System ; Drug Resistance, Microbial/genetics ; Gene Editing/methods ; Gene Transfer, Horizontal ; *Genetic Engineering ; Humans ; Optical Imaging ; Synthetic Biology/*education/methods ; }, abstract = {Recent advances in synthetic biology have resulted in biological technologies with the potential to reshape the way we understand and treat human disease. Educating students about the biology and ethics underpinning these technologies is critical to empower them to make informed future policy decisions regarding their use and to inspire the next generation of synthetic biologists. However, hands-on, educational activities that convey emerging synthetic biology topics can be difficult to implement due to the expensive equipment and expertise required to grow living cells. We present BioBits Health, an educational kit containing lab activities and supporting curricula for teaching antibiotic resistance mechanisms and CRISPR-Cas9 gene editing in high school classrooms. This kit links complex biological concepts to visual, fluorescent readouts in user-friendly freeze-dried cell-free reactions. BioBits Health represents a set of educational resources that promises to encourage teaching of cutting-edge, health-related synthetic biology topics in classrooms and other nonlaboratory settings.}, }
@article {pmid30775458, year = {2019}, author = {Ono, R and Yasuhiko, Y and Aisaki, KI and Kitajima, S and Kanno, J and Hirabayashi, Y}, title = {Exosome-mediated horizontal gene transfer occurs in double-strand break repair during genome editing.}, journal = {Communications biology}, volume = {2}, number = {}, pages = {57}, pmid = {30775458}, issn = {2399-3642}, mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; DNA/*genetics/metabolism ; DNA Breaks, Double-Stranded ; *DNA Repair ; Embryo, Mammalian ; Escherichia coli/genetics/metabolism ; Exosomes/*genetics/metabolism ; Gene Editing/*ethics ; *Gene Transfer, Horizontal ; *Genome ; Goats ; High-Throughput Nucleotide Sequencing ; Humans ; Mice ; NIH 3T3 Cells ; Plasmids/chemistry/metabolism ; Retroelements ; Short Interspersed Nucleotide Elements ; }, abstract = {The CRISPR-Cas9 system has been successfully applied in many organisms as a powerful genome-editing tool. Undoubtedly, it will soon be applied to human genome editing, including gene therapy. We have previously reported that unintentional DNA sequences derived from retrotransposons, genomic DNA, mRNA and vectors are captured at double-strand breaks (DSBs) sites when DSBs are introduced by the CRISPR-Cas9 system. Therefore, it is possible that unintentional insertions associated with DSB repair represent a potential risk for human genome editing gene therapies. To address this possibility, comprehensive sequencing of DSB sites was performed. Here, we report that exosome-mediated horizontal gene transfer occurs in DSB repair during genome editing. Exosomes are present in all fluids from living animals, including seawater and breathing mammals, suggesting that exosome-mediated horizontal gene transfer is the driving force behind mammalian genome evolution. The findings of this study highlight an emerging new risk for this leading-edge technology.}, }
@article {pmid30729184, year = {2019}, author = {Tripathi, JN and Ntui, VO and Ron, M and Muiruri, SK and Britt, A and Tripathi, L}, title = {CRISPR/Cas9 editing of endogenous banana streak virus in the B genome of Musa spp. overcomes a major challenge in banana breeding.}, journal = {Communications biology}, volume = {2}, number = {}, pages = {46}, pmid = {30729184}, issn = {2399-3642}, mesh = {Badnavirus/*genetics/pathogenicity ; Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Chimera/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; *Genome, Plant ; *Genome, Viral ; Musa/*genetics/virology ; Mutation ; Plant Breeding/*methods ; Plant Diseases/genetics/virology ; Plants, Genetically Modified ; Plasmids/chemistry/metabolism ; RNA, Guide/genetics/metabolism ; Sequence Alignment ; Stress, Physiological ; }, abstract = {Presence of the integrated endogenous banana streak virus (eBSV) in the B genome of plantain (AAB) is a major challenge for breeding and dissemination of hybrids. As the eBSV activates into infectious viral particles under stress, the progenitor Musa balbisiana and its derivants, having at least one B genome, cannot be used as parents for crop improvement. Here, we report a strategy to inactivate the eBSV by editing the virus sequences. The regenerated genome-edited events of Gonja Manjaya showed mutations in the targeted sites with the potential to prevent proper transcription or/and translational into functional viral proteins. Seventy-five percent of the edited events remained asymptomatic in comparison to the non-edited control plants under water stress conditions, confirming inactivation of eBSV into infectious viral particles. This study paves the way for the improvement of B genome germplasm and its use in breeding programs to produce hybrids that can be globally disseminated.}, }
@article {pmid32265856, year = {2020}, author = {Iranzo, J and Faure, G and Wolf, YI and Koonin, EV}, title = {Game-Theoretical Modeling of Interviral Conflicts Mediated by Mini-CRISPR Arrays.}, journal = {Frontiers in microbiology}, volume = {11}, number = {}, pages = {381}, doi = {10.3389/fmicb.2020.00381}, pmid = {32265856}, issn = {1664-302X}, abstract = {All cellular organisms coevolve with multiple viruses, so that both virus-host and intervirus conflicts are major factors of evolution. Accordingly, hosts evolve multiple, elaborate defense systems and viruses respond by evolving means of antidefense. Although less thoroughly characterized, several dedicated mechanisms of intervirus competition have been described as well. Recently, the genomes of some bacterial and archaeal viruses have been shown to harbor CRISPR mini-arrays that typically contain a single spacer targeting a closely related virus. The involvement of mini-arrays in an intervirus conflict has been experimentally demonstrated for a pair of archaeal viruses. We model the evolution of virus-encoded CRISPR mini-arrays using a game theoretical approach. Analysis of the model reveals multiple equilibria that include mutual targeting, unidirectional targeting, no targeting, cyclic polymorphism, and bistability. The choice between these evolutionary regimes depends on the model parameters including the coinfection frequency, differential productivity of the conflicting viruses, and the fitness cost of mini-arrays. At high coinfection frequencies, the model becomes a version of the Prisoner's dilemma in which defection, i.e., mutual targeting between the competing viruses, is the winning strategy.}, }
@article {pmid32265471, year = {2020}, author = {Menchaca, A and Dos Santos-Neto, PC and Souza-Neves, M and Cuadro, F and Mulet, AP and Tesson, L and Chenouard, V and Guiffès, A and Heslan, JM and Gantier, M and Anegón, I and Crispo, M}, title = {Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair.}, journal = {Scientific reports}, volume = {10}, number = {1}, pages = {5995}, doi = {10.1038/s41598-020-62879-y}, pmid = {32265471}, issn = {2045-2322}, abstract = {Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.}, }
@article {pmid32041517, year = {2020}, author = {Liu, Q and Cheng, X and Liu, G and Li, B and Liu, X}, title = {Deep learning improves the ability of sgRNA off-target propensity prediction.}, journal = {BMC bioinformatics}, volume = {21}, number = {1}, pages = {51}, pmid = {32041517}, issn = {1471-2105}, support = {No.FRF-BR-18-008B//the Fundamental Research Funds for the Central Universities/ ; }, mesh = {*CRISPR-Cas Systems ; *Deep Learning ; Gene Editing ; Humans ; RNA/metabolism ; }, abstract = {BACKGROUND: CRISPR/Cas9 system, as the third-generation genome editing technology, has been widely applied in target gene repair and gene expression regulation. Selection of appropriate sgRNA can improve the on-target knockout efficacy of CRISPR/Cas9 system with high sensitivity and specificity. However, when CRISPR/Cas9 system is operating, unexpected cleavage may occur at some sites, known as off-target. Presently, a number of prediction methods have been developed to predict the off-target propensity of sgRNA at specific DNA fragments. Most of them use artificial feature extraction operations and machine learning techniques to obtain off-target scores. With the rapid expansion of off-target data and the rapid development of deep learning theory, the existing prediction methods can no longer satisfy the prediction accuracy at the clinical level.<br><br>RESULTS: Here, we propose a prediction method named CnnCrispr to predict the off-target propensity of sgRNA at specific DNA fragments. CnnCrispr automatically trains the sequence features of sgRNA-DNA pairs with GloVe model, and embeds the trained word vector matrix into the deep learning model including biLSTM and CNN with five hidden layers. We conducted performance verification on the data set provided by DeepCrispr, and found that the auROC and auPRC in the &quot;leave-one-sgRNA-out&quot; cross validation could reach 0.957 and 0.429 respectively (the Pearson value and spearman value could reach 0.495 and 0.151 respectively under the same settings).<br><br>CONCLUSION: Our results show that CnnCrispr has better classification and regression performance than the existing states-of-art models. The code for CnnCrispr can be freely downloaded from https://github.com/LQYoLH/CnnCrispr.}, }
@article {pmid31980629, year = {2020}, author = {Carpenter, MD and Hu, Q and Bond, AM and Lombroso, SI and Czarnecki, KS and Lim, CJ and Song, H and Wimmer, ME and Pierce, RC and Heller, EA}, title = {Nr4a1 suppresses cocaine-induced behavior via epigenetic regulation of homeostatic target genes.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {504}, pmid = {31980629}, issn = {2041-1723}, support = {T32 GM008076/GM/NIGMS NIH HHS/United States ; DP1 DA044250/DA/NIDA NIH HHS/United States ; R21 DA039038/DA/NIDA NIH HHS/United States ; R01 DA033641/DA/NIDA NIH HHS/United States ; R01 DA015214/DA/NIDA NIH HHS/United States ; }, mesh = {Animals ; Behavior, Animal/*drug effects ; CRISPR-Cas Systems/genetics ; Cocaine/administration &amp; dosage/*pharmacology ; *Epigenesis, Genetic/drug effects ; Female ; Histones/metabolism ; Homeostasis/*drug effects/genetics ; Male ; Mice, Inbred C57BL ; Neurons/drug effects/metabolism ; Nuclear Receptor Subfamily 4, Group A, Member 1/*metabolism ; Phenylacetates/pharmacology ; Promoter Regions, Genetic/genetics ; Protein Processing, Post-Translational ; Synapsins/metabolism ; }, abstract = {Endogenous homeostatic mechanisms can restore normal neuronal function following cocaine-induced neuroadaptations. Such mechanisms may be exploited to develop novel therapies for cocaine addiction, but a molecular target has not yet been identified. Here we profiled mouse gene expression during early and late cocaine abstinence to identify putative regulators of neural homeostasis. Cocaine activated the transcription factor, Nr4a1, and its target gene, Cartpt, a key molecule involved in dopamine metabolism. Sustained activation of Cartpt at late abstinence was coupled with depletion of the repressive histone modification, H3K27me3, and enrichment of activating marks, H3K27ac and H3K4me3. Using both CRISPR-mediated and small molecule Nr4a1 activation, we demonstrated the direct causal role of Nr4a1 in sustained activation of Cartpt and in attenuation of cocaine-evoked behavior. Our findings provide evidence that targeting abstinence-induced homeostatic gene expression is a potential therapeutic target in cocaine addiction.}, }
@article {pmid31959981, year = {2020}, author = {Mori, L and De Libero, G}, title = {'Bohemian Rhapsody' of MR1T cells.}, journal = {Nature immunology}, volume = {21}, number = {2}, pages = {108-110}, doi = {10.1038/s41590-019-0588-6}, pmid = {31959981}, issn = {1529-2916}, mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Early Detection of Cancer ; Histocompatibility Antigens Class I ; *T-Lymphocytes ; }, }
@article {pmid31949143, year = {2020}, author = {Sirvent, S and Vallejo, AF and Davies, J and Clayton, K and Wu, Z and Woo, J and Riddell, J and Chaudhri, VK and Stumpf, P and Nazlamova, LA and Wheway, G and Rose-Zerilli, M and West, J and Pujato, M and Chen, X and Woelk, CH and MacArthur, B and Ardern-Jones, M and Friedmann, PS and Weirauch, MT and Singh, H and Polak, ME}, title = {Genomic programming of IRF4-expressing human Langerhans cells.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {313}, pmid = {31949143}, issn = {2041-1723}, support = {MC_PC_15078/MRC_/Medical Research Council/United Kingdom ; }, mesh = {Antigen Presentation/genetics ; Basic-Leucine Zipper Transcription Factors/metabolism ; CRISPR-Cas Systems ; Cell Movement ; Cytokines/metabolism ; Gene Editing ; Gene Expression Profiling ; *Genomics ; Histocompatibility Antigens Class I ; Histocompatibility Antigens Class II ; Humans ; Interferon Regulatory Factors/*genetics/*metabolism ; Langerhans Cells/immunology/*metabolism ; NF-kappa B/metabolism ; Proto-Oncogene Proteins/metabolism ; Repressor Proteins/metabolism ; Trans-Activators/metabolism ; Transcription, Genetic ; Transcriptional Activation ; Up-Regulation ; }, abstract = {Langerhans cells (LC) can prime tolerogenic as well as immunogenic responses in skin, but the genomic states and transcription factors (TF) regulating these context-specific responses are unclear. Bulk and single-cell transcriptional profiling demonstrates that human migratory LCs are robustly programmed for MHC-I and MHC-II antigen presentation. Chromatin analysis reveals enrichment of ETS-IRF and AP1-IRF composite regulatory elements in antigen-presentation genes, coinciding with expression of the TFs, PU.1, IRF4 and BATF3 but not IRF8. Migration of LCs from the epidermis is accompanied by upregulation of IRF4, antigen processing components and co-stimulatory molecules. TNF stimulation augments LC cross-presentation while attenuating IRF4 expression. CRISPR-mediated editing reveals IRF4 to positively regulate the LC activation programme, but repress NF2EL2 and NF-kB pathway genes that promote responsiveness to oxidative stress and inflammatory cytokines. Thus, IRF4-dependent genomic programming of human migratory LCs appears to enable LC maturation while attenuating excessive inflammatory and immunogenic responses in the epidermis.}, }
@article {pmid31935181, year = {2020}, author = {Ho, J and Zhao, M and Wojcik, S and Taiaroa, G and Butler, M and Poulter, R}, title = {The application of the CRISPR-Cas9 system in Pseudomonas syringae pv. actinidiae.}, journal = {Journal of medical microbiology}, volume = {69}, number = {3}, pages = {478-486}, doi = {10.1099/jmm.0.001124}, pmid = {31935181}, issn = {1473-5644}, mesh = {Actinidia/microbiology ; CRISPR-Cas Systems/genetics/*physiology ; Escherichia coli/physiology ; Fruit/microbiology ; Gene Deletion ; Gene Knockout Techniques ; Gene Targeting ; Genetic Engineering ; Peptide Synthases/genetics ; Plasmids ; Pseudomonas syringae/genetics/*physiology ; Sequence Analysis, DNA ; Streptococcus pyogenes/physiology ; Whole Genome Sequencing ; }, abstract = {Introduction.Pseudomonas syringae pv. actinidiae (Psa) has emerged as a major bacterial pathogen of kiwifruit cultivation throughout the world.Aim. We aim to introduce a CRISPR-Cas9 system, a commonly used genome editing tool, into Psa. The protocols may also be useful in other Pseudomonas species.Methodology. Using standard molecular biology techniques, we modified plasmid pCas9, which carries the CRISPR-Cas9 sequences from Streptococcus pyogenes, for use in Psa. The final plasmid, pJH1, was produced in a series of steps and is maintained with selection in both Escherichia coli and Psa.Results. We have constructed plasmids carrying a CRISPR-Cas9 system based on that of S. pyogenes, which can be maintained, under selection, in Psa. We have shown that the gene targeting capacity of the CRISPR-Cas9 system is active and that the Cas9 protein is able to cleave the targeted sites. The Cas9 was directed to several different sites in the P. syringae genome. Using Cas9 we have generated Psa transformants that no longer carry the native plasmid present in Psa, and other transformants that lack the integrative, conjugative element, Pac_ICE1. Targeting of a specific gene, a chromosomal non-ribosomal peptide synthetase, led to gene knockouts with the transformants having deletions encompassing the target site.Conclusion. We have constructed shuttle plasmids carrying a CRISPR-Cas9 system that are maintained in both E. coli and P. syringae pv. actinidiae. We have used this gene editing system to eliminate features of the accessory genome (plasmids or ICEs) from Psa and to target a single chromosomal gene.}, }
@article {pmid31919444, year = {2020}, author = {}, title = {When the world needs to know.}, journal = {Nature biotechnology}, volume = {38}, number = {1}, pages = {1}, doi = {10.1038/s41587-019-0396-4}, pmid = {31919444}, issn = {1546-1696}, mesh = {CRISPR-Cas Systems/genetics ; *Ethics, Research ; Fertilization in Vitro ; Gene Editing ; Humans ; Publications/ethics ; }, }
@article {pmid31910234, year = {2020}, author = {Alcaraz, E and Vilardell, J and Borgo, C and Sarró, E and Plana, M and Marin, O and Pinna, LA and Bayascas, JR and Meseguer, A and Salvi, M and Itarte, E and Ruzzene, M}, title = {Effects of CK2β subunit down-regulation on Akt signalling in HK-2 renal cells.}, journal = {PloS one}, volume = {15}, number = {1}, pages = {e0227340}, pmid = {31910234}, issn = {1932-6203}, mesh = {CRISPR-Cas Systems/genetics ; Casein Kinase II/*genetics ; Cell Line ; Chromones/pharmacology ; Epithelial-Mesenchymal Transition/genetics ; Gene Expression Regulation, Enzymologic/drug effects ; Gene Knockout Techniques ; Glycogen Synthase Kinase 3 beta/*genetics ; Humans ; Kidney/drug effects/metabolism ; MAP Kinase Signaling System/drug effects ; Morpholines/pharmacology ; Naphthyridines/pharmacology ; Oncogene Protein v-akt/*genetics ; Phosphatidylinositol 3-Kinases/genetics ; Phosphorylation/drug effects ; Protein Isoforms ; Ribosomal Protein S6 Kinases, 90-kDa/genetics ; Signal Transduction/drug effects ; Snail Family Transcription Factors/*genetics ; }, abstract = {The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit β (shCK2β cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways. We found that all CK2 isoforms phosphorylate S129 in vitro, independently of CK2β. However, in HK-2 cells the dependence on CK2β was confirmed by rescue experiments (CK2β re-expression in shCK2β HK-2 cells), suggesting the presence of additional components that drive Akt recognition by CK2 in cells. We also found that CK2β downregulation altered the phosphorylation ratio between the two canonical Akt activation sites (pT308 strongly reduced, pS473 slightly increased) in HK-2 cells. Similar results were found in other cell lines where CK2β was stably knocked out by CRISPR-Cas9 technology. The phosphorylation of rpS6 S235/S236, a downstream effector of Akt, was strongly reduced in shCK2β HK-2 cells, while the phosphorylation of two Akt direct targets, PRAS40 T246 and GSK3β S9, was increased. Differently to what observed in response to CK2β down-regulation, the chemical inhibition of CK2 activity by cell treatment with the specific inhibitor CX-4945 reduced both the Akt canonical sites, pT308 and pS473. In CX-4945-treated cells, the changes in rpS6 pS235/S236 and GSK3β pS9 mirrored those induced by CK2β knock-down (reduction and slight increase, respectively); on the contrary, the effect on PRAS40 pT246 phosphorylation was sharply different, being strongly reduced by CK2 inhibition; this suggests that this Akt target might be dependent on Akt pS473 status in HK-2 cells. Since PI3K/Akt and ERK1/2/p90rsk pathways are known to be interconnected and both modulated by CK2, with GSK3β pS9 representing a convergent point, we investigated if ERK1/2/p90rsk signaling was affected by CK2β knock-down and CX-4945 treatment in HK-2 cells. We found that p90rsk was insensitive to any kind of CK2 targeting; therefore, the observation that, similarly, GSK3β pS9 was not reduced by CK2 blockade suggests that GSK3β phosphorylation is mainly under the control of p90rsk in these cells. However, we found that the PI3K inhibitor LY294002 reduced GSK3β pS9, and concomitantly decreased Snail1 levels (a GSK3β target and Epithelial-to-Mesenchymal transition marker). The effects of LY294002 were observed also in CK2β-downregulated cells, suggesting that reducing GSK3β pS9 could be a strategy to control Snail1 levels in any situation where CK2β is defective, as possibly occurring in cancer cells.}, }
@article {pmid31891587, year = {2019}, author = {Foronda, M and Tarumoto, Y and Schatoff, EM and Leach, BI and Diaz, BJ and Zimmerman, J and Goswami, S and Shusterman, M and Vakoc, CR and Dow, LE}, title = {Tankyrase inhibition sensitizes cells to CDK4 blockade.}, journal = {PloS one}, volume = {14}, number = {12}, pages = {e0226645}, pmid = {31891587}, issn = {1932-6203}, support = {K22 CA181280/CA/NCI NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cellular Senescence ; Colorectal Neoplasms/*enzymology/therapy ; Cyclin-Dependent Kinase 4/*genetics ; Cyclin-Dependent Kinase 6/genetics ; Drug Resistance, Neoplasm/*genetics ; G1 Phase Cell Cycle Checkpoints ; Humans ; Mutation ; Poly(ADP-ribose) Polymerase Inhibitors/*pharmacology/therapeutic use ; Tankyrases/*antagonists &amp; inhibitors ; Wnt Signaling Pathway/drug effects ; }, abstract = {Tankyrase (TNKS) 1/2 are positive regulators of WNT signaling by controlling the activity of the ß-catenin destruction complex. TNKS inhibitors provide an opportunity to suppress hyperactive WNT signaling in tumors, however, they have shown limited anti-proliferative activity as a monotherapy in human cancer cell lines. Here we perform a kinome-focused CRISPR screen to identify potential effective drug combinations with TNKS inhibition. We show that the loss of CDK4, but not CDK6, synergizes with TNKS1/2 blockade to drive G1 cell cycle arrest and senescence. Through precise modelling of cancer-associated mutations using cytidine base editors, we show that this therapeutic approach is absolutely dependent on suppression of canonical WNT signaling by TNKS inhibitors and is effective in cells from multiple epithelial cancer types. Together, our results suggest that combined WNT and CDK4 inhibition might provide a potential therapeutic strategy for difficult-to-treat epithelial tumors.}, }
@article {pmid31862961, year = {2019}, author = {Dempster, JM and Pacini, C and Pantel, S and Behan, FM and Green, T and Krill-Burger, J and Beaver, CM and Younger, ST and Zhivich, V and Najgebauer, H and Allen, F and Gonçalves, E and Shepherd, R and Doench, JG and Yusa, K and Vazquez, F and Parts, L and Boehm, JS and Golub, TR and Hahn, WC and Root, DE and Garnett, MJ and Tsherniak, A and Iorio, F}, title = {Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets.}, journal = {Nature communications}, volume = {10}, number = {1}, pages = {5817}, pmid = {31862961}, issn = {2041-1723}, support = {U01 CA176058/CA/NCI NIH HHS/United States ; }, mesh = {Antineoplastic Agents/pharmacology/therapeutic use ; Biomarkers, Tumor/antagonists &amp; inhibitors/*genetics ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Datasets as Topic ; Drug Screening Assays, Antitumor/*methods ; Gene Expression Profiling ; Genes, Essential/drug effects/genetics ; Genomics/*methods ; Humans ; Molecular Targeted Therapy/methods ; Neoplasms/drug therapy/*genetics ; Oncogenes/drug effects/genetics ; Precision Medicine/methods ; Reproducibility of Results ; Small Molecule Libraries/pharmacology ; }, abstract = {Genome-scale CRISPR-Cas9 viability screens performed in cancer cell lines provide a systematic approach to identify cancer dependencies and new therapeutic targets. As multiple large-scale screens become available, a formal assessment of the reproducibility of these experiments becomes necessary. We analyze data from recently published pan-cancer CRISPR-Cas9 screens performed at the Broad and Sanger Institutes. Despite significant differences in experimental protocols and reagents, we find that the screen results are highly concordant across multiple metrics with both common and specific dependencies jointly identified across the two studies. Furthermore, robust biomarkers of gene dependency found in one data set are recovered in the other. Through further analysis and replication experiments at each institute, we show that batch effects are driven principally by two key experimental parameters: the reagent library and the assay length. These results indicate that the Broad and Sanger CRISPR-Cas9 viability screens yield robust and reproducible findings.}, }
@article {pmid31857575, year = {2019}, author = {Lian, J and Schultz, C and Cao, M and HamediRad, M and Zhao, H}, title = {Multi-functional genome-wide CRISPR system for high throughput genotype-phenotype mapping.}, journal = {Nature communications}, volume = {10}, number = {1}, pages = {5794}, pmid = {31857575}, issn = {2041-1723}, mesh = {Biotechnology/methods ; CRISPR-Cas Systems/*genetics ; Chromosome Mapping/*methods ; Gene Editing/methods ; Gene Expression Regulation, Fungal ; Genome, Fungal/*genetics ; Genomic Library ; Genomics/*methods ; Genotype ; High-Throughput Nucleotide Sequencing ; High-Throughput Screening Assays/*methods ; Phenotype ; Saccharomyces cerevisiae/genetics ; }, abstract = {Genome-scale engineering is an indispensable tool to understand genome functions due to our limited knowledge of cellular networks. Unfortunately, most existing methods for genome-wide genotype-phenotype mapping are limited to a single mode of genomic alteration, i.e. overexpression, repression, or deletion. Here we report a multi-functional genome-wide CRISPR (MAGIC) system to precisely control the expression level of defined genes to desired levels throughout the whole genome. By combining the tri-functional CRISPR system and array-synthesized oligo pools, MAGIC is used to create, to the best of our knowledge, one of the most comprehensive and diversified genomic libraries in yeast ever reported. The power of MAGIC is demonstrated by the identification of previously uncharacterized genetic determinants of complex phenotypes, particularly those having synergistic interactions when perturbed to different expression levels. MAGIC represents a powerful synthetic biology tool to investigate fundamental biological questions as well as engineer complex phenotypes for biotechnological applications.}, }
@article {pmid31848331, year = {2019}, author = {Ujihara, Y and Kanagawa, M and Mohri, S and Takatsu, S and Kobayashi, K and Toda, T and Naruse, K and Katanosaka, Y}, title = {Elimination of fukutin reveals cellular and molecular pathomechanisms in muscular dystrophy-associated heart failure.}, journal = {Nature communications}, volume = {10}, number = {1}, pages = {5754}, pmid = {31848331}, issn = {2041-1723}, mesh = {Adult ; Age Factors ; Animals ; Animals, Newborn ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Disease Models, Animal ; Dystroglycans/metabolism ; Female ; Gene Knockout Techniques ; Glycosylation ; HEK293 Cells ; Heart Failure/*etiology/pathology ; Heart Ventricles/cytology/pathology ; Humans ; Male ; Membrane Proteins/genetics ; Mice ; Mice, Knockout ; Muscle, Skeletal/cytology/*pathology ; Muscular Dystrophies/*complications/genetics/pathology ; Myocardial Contraction/genetics ; Myocytes, Cardiac/cytology/*pathology ; Primary Cell Culture ; Sarcolemma/pathology ; Transferases/*genetics/metabolism ; }, abstract = {Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.}, }
@article {pmid31848330, year = {2019}, author = {Woronik, A and Tunström, K and Perry, MW and Neethiraj, R and Stefanescu, C and Celorio-Mancera, MP and Brattström, O and Hill, J and Lehmann, P and Käkelä, R and Wheat, CW}, title = {A transposable element insertion is associated with an alternative life history strategy.}, journal = {Nature communications}, volume = {10}, number = {1}, pages = {5757}, doi = {10.1038/s41467-019-13596-2}, pmid = {31848330}, issn = {2041-1723}, support = {R00 EY027016/EY/NEI NIH HHS/United States ; }, mesh = {Animals ; Butterflies/*physiology ; CRISPR-Cas Systems/genetics ; Color ; DNA Transposable Elements/*genetics ; Female ; Gene Editing/methods ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; *Genetic Loci ; Homeodomain Proteins/*genetics/metabolism ; *Life History Traits ; Male ; Pigmentation/genetics ; Pigments, Biological/biosynthesis ; Reproduction/genetics ; Sex Factors ; Whole Genome Sequencing ; Wings, Animal/metabolism/ultrastructure ; }, abstract = {Tradeoffs affect resource allocation during development and result in fitness consequences that drive the evolution of life history strategies. Yet despite their importance, we know little about the mechanisms underlying life history tradeoffs. Many species of Colias butterflies exhibit an alternative life history strategy (ALHS) where females divert resources from wing pigment synthesis to reproductive and somatic development. Due to this reallocation, a wing color polymorphism is associated with the ALHS: either yellow/orange or white. Here we map the locus associated with this ALHS in Colias crocea to a transposable element insertion located downstream of the Colias homolog of BarH-1, a homeobox transcription factor. Using CRISPR/Cas9 gene editing, antibody staining, and electron microscopy we find white-specific expression of BarH-1 suppresses the formation of pigment granules in wing scales and gives rise to white wing color. Lipid and transcriptome analyses reveal physiological differences associated with the ALHS. Together, these findings characterize a mechanism for a female-limited ALHS.}, }
@article {pmid31739488, year = {2019}, author = {Lentsch, E and Li, L and Pfeffer, S and Ekici, AB and Taher, L and Pilarsky, C and Grützmann, R}, title = {CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines.}, journal = {International journal of molecular sciences}, volume = {20}, number = {22}, pages = {}, pmid = {31739488}, issn = {1422-0067}, mesh = {Alleles ; Amino Acid Substitution ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing ; Gene Expression Profiling ; *Gene Knockout Techniques ; Gene Targeting ; Humans ; *Mutation ; Pancreatic Neoplasms/*genetics ; Proto-Oncogene Proteins p21(ras)/*genetics ; Signal Transduction ; }, abstract = {In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the KrasG12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells.}, }
@article {pmid31733872, year = {2020}, author = {Xiong, ZQ and Wei, YY and Kong, LH and Song, X and Yi, HX and Ai, LZ}, title = {Short communication: An inducible CRISPR/dCas9 gene repression system in Lactococcus lactis.}, journal = {Journal of dairy science}, volume = {103}, number = {1}, pages = {161-165}, doi = {10.3168/jds.2019-17346}, pmid = {31733872}, issn = {1525-3198}, mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Targeting ; Lactobacillales/enzymology/*genetics ; Lactococcus lactis/enzymology/*genetics ; Nisin/metabolism ; Plasmids/genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide/*genetics ; }, abstract = {Lactococcus lactis, one of the most important probiotic lactic acid bacteria (LAB), is widely used in the dairy industry as a cell factory for recombinant protein production. Currently, a nisin-controlled inducible expression system is used for this purpose and represents the only commercial expression system in LAB. However, the available genetic modification methods are rather limited for modulating gene expression in L. lactis. Here, we developed a 2-plasmid system for gene transcription repression in L. lactis NZ9000 that uses inducible clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9. An inducible promoter Pnisin was used to drive the expression of dCas9 from Streptococcus pyogenes, whereas a strong constitutive promoter P44 drove single guide RNA expression for single or multiple target genes. dCas9 enabled CRISPR interference-mediated silencing of single or multiple target genes with significant reduction of gene expression, up to 99%. In addition, LLNZ_07335, a putative penicillin acylase, was identified as bile salt hydrolase for bile salt resistance in NZ9000 using this system. To our knowledge, this report is the first for a functional gene for bile salt tolerance in L. lactis. Overall, our work introduces a new gene repression tool for various applications in L. lactis or other LAB.}, }
@article {pmid31712774, year = {2020}, author = {Chiarella, AM and Butler, KV and Gryder, BE and Lu, D and Wang, TA and Yu, X and Pomella, S and Khan, J and Jin, J and Hathaway, NA}, title = {Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery.}, journal = {Nature biotechnology}, volume = {38}, number = {1}, pages = {50-55}, pmid = {31712774}, issn = {1546-1696}, support = {R01 GM118653/GM/NIGMS NIH HHS/United States ; R01 HD088626/HD/NICHD NIH HHS/United States ; R01 GM122749/GM/NIGMS NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems/*genetics ; Cell Cycle Proteins/metabolism ; Chromatin/*metabolism ; Epigenesis, Genetic ; *Gene Expression Regulation ; Genome, Human ; HEK293 Cells ; Humans ; RNA, Guide/metabolism ; Time Factors ; Transcription Factors/metabolism ; }, abstract = {Gene expression can be activated or suppressed using CRISPR--Cas9 systems. However, tools that enable dose-dependent activation of gene expression without the use of exogenous transcription regulatory proteins are lacking. Here we describe chemical epigenetic modifiers (CEMs) designed to activate the expression of target genes by recruiting components of the endogenous chromatin-activating machinery, eliminating the need for exogenous transcriptional activators. The system has two parts: catalytically inactive Cas9 (dCas9) in complex with FK506-binding protein (FKBP) and a CEM consisting of FK506 linked to a molecule that interacts with cellular epigenetic machinery. We show that CEMs upregulate gene expression at target endogenous loci up to 20-fold or more depending on the gene. We also demonstrate dose-dependent control of transcriptional activation, function across multiple diverse genes, reversibility of CEM activity and specificity of our best-in-class CEM across the genome.}, }
@article {pmid31703569, year = {2019}, author = {Zheng, G and Zhu, Q and Dong, J and Lin, X and Zhu, C}, title = {Rapid generation and selection of Cas9-engineering TRP53 R172P mice that do not have off-target effects.}, journal = {BMC biotechnology}, volume = {19}, number = {1}, pages = {74}, pmid = {31703569}, issn = {1472-6750}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Fibroblasts/metabolism ; Gene Editing ; Humans ; Male ; Mice ; Mice, Inbred C57BL ; Mutation/genetics ; Tumor Suppressor Protein p53/genetics/*metabolism ; }, abstract = {BACKGROUND: Genetic mutations cause severe human diseases, and suitable animal models to study the regulatory mechanisms involved are required. The CRISPR/Cas9 system is a powerful, highly efficient and easily manipulated tool for genetic modifications. However, utilization of CRISPR/Cas9 to introduce point mutations and the exclusion of off-target effects in mice remain challenging. TP53-R175 is one of the most frequently mutated sites in human cancers, and it plays crucial roles in human diseases, including cancers and diabetes.<br><br>RESULTS: Here, we generated TRP53-R172P mutant mice (C57BL/6 J, corresponding to TP53-R175P in humans) using a single microinjection of the CRISPR/Cas9 system. The optimal parameters comprised gRNA selection, donor designation (silent mutations within gRNA region), the concentration of CRISPR components and the cellular sites of injection. TRP53-R172P conversion was genetically and functionally confirmed. Combination of TA cloning and Sanger sequencing helped identify the correctly targeted mice as well as the off-target effects in the engineered mice, which provide us a strategy to select the on-target mice without off-target effects quickly and efficiently.<br><br>CONCLUSIONS: A single injection of the this optimized CRISPR/Cas9 system can be applied to introduce particular mutations in the genome of mice without off-target effects to model various human diseases.}, }
@article {pmid31697407, year = {2020}, author = {Chan, B}, title = {Improving the justice-based argument for conducting human gene editing research to cure sickle cell disease.}, journal = {Bioethics}, volume = {34}, number = {2}, pages = {200-202}, doi = {10.1111/bioe.12690}, pmid = {31697407}, issn = {1467-8519}, mesh = {*Anemia, Sickle Cell ; CRISPR-Cas Systems ; *Gene Editing ; Humans ; Social Justice ; }, abstract = {In a recent article, Marilyn Baffoe-Bonnie offers three arguments that conducting CRISPR/Cas9 biotechnology research to cure sickle cell disease (SCD) would help address historical and current injustices in SCD research and care. I will grant that the first argument is sound, but show that the second and third arguments suffer from roughly the same defect, which is that they really argue for something else rather than for conducting CRISPR/Cas9 research to cure SCD. I conclude that a better justice-based argument would use only Baffoe-Bonnie's first argument.}, }
@article {pmid31684940, year = {2019}, author = {Arndell, T and Sharma, N and Langridge, P and Baumann, U and Watson-Haigh, NS and Whitford, R}, title = {gRNA validation for wheat genome editing with the CRISPR-Cas9 system.}, journal = {BMC biotechnology}, volume = {19}, number = {1}, pages = {71}, pmid = {31684940}, issn = {1472-6750}, mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genome, Plant/*genetics ; Protoplasts/metabolism ; RNA, Guide/*genetics ; Triticum/*genetics ; }, abstract = {BACKGROUND: The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate.<br><br>RESULTS: The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat.<br><br>CONCLUSIONS: We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.}, }
@article {pmid31670340, year = {2019}, author = {Jain, PK and Lo, JH and Rananaware, S and Downing, M and Panda, A and Tai, M and Raghavan, S and Fleming, HE and Bhatia, SN}, title = {Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes.}, journal = {Nanoscale}, volume = {11}, number = {44}, pages = {21317-21323}, doi = {10.1039/c9nr01786k}, pmid = {31670340}, issn = {2040-3372}, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Gene Transfer Techniques ; HeLa Cells ; Humans ; }, abstract = {There is a critical need for the development of safe and efficient delivery technologies for CRISPR/Cas9 to advance translation of genome editing to the clinic. Non-viral methods that are simple, efficient, and completely based on biologically-derived materials could offer such potential. Here we report a simple and modular tandem peptide-based nanocomplex system with cell-targeting capacity that efficiently combines guide RNA (sgRNA) with Cas9 protein, and facilitates internalization of sgRNA/Cas9 ribonucleoprotein complexes to yield robust genome editing across multiple cell lines.}, }
@article {pmid31622618, year = {2020}, author = {Kawasaki, K and Fujii, M and Sugimoto, S and Ishikawa, K and Matano, M and Ohta, Y and Toshimitsu, K and Takahashi, S and Hosoe, N and Sekine, S and Kanai, T and Sato, T}, title = {Chromosome Engineering of Human Colon-Derived Organoids to Develop a Model of Traditional Serrated Adenoma.}, journal = {Gastroenterology}, volume = {158}, number = {3}, pages = {638-651.e8}, doi = {10.1053/j.gastro.2019.10.009}, pmid = {31622618}, issn = {1528-0012}, mesh = {Adenoma/*genetics/pathology ; Animals ; CRISPR-Cas Systems ; Colonic Neoplasms/*genetics/pathology ; Eukaryotic Initiation Factor-3/genetics ; Gene Fusion ; Genetic Engineering ; Humans ; Intercellular Signaling Peptides and Proteins/*genetics ; Male ; Mice ; Models, Biological ; Neoplasm Transplantation ; Organoids/*pathology ; Proto-Oncogene Proteins B-raf/genetics ; Receptor-Like Protein Tyrosine Phosphatases, Class 2/genetics ; Receptors, G-Protein-Coupled/genetics ; Thrombospondins/*genetics ; Tumor Suppressor Protein p53/genetics ; Wnt Signaling Pathway ; }, abstract = {BACKGROUND &amp; AIMS: Traditional serrated adenomas (TSAs) are rare colorectal polyps with unique histologic features. Fusions in R-spondin genes have been found in TSAs, but it is not clear whether these are sufficient for TSA development, due to the lack of a chromosome engineering platform for human tissues. We studied the effects of fusions in R-spondin genes and other genetic alterations found in TSA using CRISPR-Cas9-mediated chromosome and genetic modification of human colonic organoids.<br><br>METHODS: We introduced chromosome rearrangements that involve R-spondin genes into human colonic organoids, with or without disruption of TP53, using CRISPR-Cas9 (chromosome-engineered organoids). We then knocked a mutation into BRAF encoding the V600E substitution and overexpressed the GREM1 transgene; the organoids were transplanted into colons of NOG mice and growth of xenograft tumors was measured. Colon tissues were collected and analyzed by immunohistochemistry or in situ hybridization. We also established 2 patient-derived TSA organoid lines and characterized their genetic features and phenotypes. We inserted a bicistronic cassette expressing a dimerizer-inducible suicide gene and fluorescent marker downstream of the LGR5 gene in the chromosome-engineered organoids; addition of the dimerizer eradicates LGR5+ cells. Some tumor-bearing mice were given intraperitoneal injections of the dimerizer to remove LGR5-expressing cells.<br><br>RESULTS: Chromosome engineering of organoids required disruption of TP53 or culture in medium containing IGF1 and FGF2. In colons of mice, organoids that expressed BRAFV600E and fusions in R-spondin genes formed flat serrated lesions. Patient-derived TSA organoids grew independent of exogenous R-spondin, and 1 line grew independent of Noggin. Organoids that overexpressed GREM1, in addition to BRAFV600E and fusions in R-spondin genes, formed polypoid tumors in mice that had histologic features similar to TSAs. Xenograft tumors persisted after loss of LGR5-expressing cells.<br><br>CONCLUSIONS: We demonstrated efficient chromosomal engineering of human normal colon organoids. We introduced genetic and chromosome alterations into human colon organoids found in human TSAs; tumors grown from these organoids in mice had histopathology features of TSAs. This model might be used to study progression of human colorectal tumors with RSPO fusion gene and GREM1 overexpression.}, }
@article {pmid31578590, year = {2019}, author = {Fan, Y and Li, J and Wei, W and Fang, H and Duan, Y and Li, N and Zhang, Y and Yu, J and Wang, J}, title = {Ku80 gene knockdown by the CRISPR/Cas9 technique affects the biological functions of human thyroid carcinoma cells.}, journal = {Oncology reports}, volume = {42}, number = {6}, pages = {2486-2498}, pmid = {31578590}, issn = {1791-2431}, mesh = {Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Case-Control Studies ; Cell Line, Tumor ; Cell Proliferation/genetics ; Gene Expression Profiling ; *Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Hashimoto Disease/genetics/pathology ; Humans ; Ku Autoantigen/genetics/*metabolism ; RNA, Messenger/metabolism ; Thyroid Cancer, Papillary/*genetics/pathology ; Thyroid Gland/pathology ; Thyroid Neoplasms/*genetics/pathology ; }, abstract = {In the present study, to evaluate the role of Ku80 in thyroid carcinoma (TC), 86 thyroid tissue samples from patients with a spectrum of thyroid disorders were examined for protein levels of Ku80, nuclear factor‑κB (NF‑κB) and RET/TC by immunohistochemistry. Furthermore, in TC cells, Ku80 mRNA was detected by reverse transcription‑quantitative PCR analysis and silenced using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein 9 (Cas9) technique to assess its role. An antibody array was used to identify Ku80‑related regulatory genes. The protein levels of Ku80 in the TC tissues were significantly higher than those in non‑neoplastic adjacent tissue samples (P<0.01). The activation of NF‑kB and expression of RET/TC in the TC group were significantly increased (P<0.05) and were correlated with the protein expression of Ku80 (P<0.05). In papillary TC cells, the mRNA levels of Ku80 were high; Ku80 knockdown resulted in reductions in proliferation, invasion and colony formation, increased apoptosis, and reduced levels of proteins involved in MAPK signaling, cell proliferation and apoptosis. The high expression of Ku80 in TC was found to be associated with the expression of RET/TC and activation of NF‑κB, and Ku80 knockdown decreased the malignancy of TC cells.}, }
@article {pmid31548381, year = {2019}, author = {Tong, Y and Whitford, CM and Robertsen, HL and Blin, K and Jørgensen, TS and Klitgaard, AK and Gren, T and Jiang, X and Weber, T and Lee, SY}, title = {Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {41}, pages = {20366-20375}, pmid = {31548381}, issn = {1091-6490}, mesh = {*CRISPR-Cas Systems ; DNA, Bacterial/genetics ; *Gene Editing ; Gene Expression Regulation, Bacterial ; Genome, Bacterial ; Genome-Wide Association Study ; Plasmids ; Streptomyces coelicolor/*genetics ; }, abstract = {Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide-resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tü365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.}, }
@article {pmid31527264, year = {2019}, author = {Binger, KJ and Neukam, M and Tattikota, SG and Qadri, F and Puchkov, D and Willmes, DM and Wurmsee, S and Geisberger, S and Dechend, R and Raile, K and Kurth, T and Nguyen, G and Poy, MN and Solimena, M and Muller, DN and Birkenfeld, AL}, title = {Atp6ap2 deletion causes extensive vacuolation that consumes the insulin content of pancreatic β cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {40}, pages = {19983-19988}, pmid = {31527264}, issn = {1091-6490}, mesh = {Animals ; Autophagy ; CRISPR-Cas Systems ; Cytosol/metabolism ; Female ; *Gene Deletion ; Gene Silencing ; Insulin/*metabolism ; Insulin-Secreting Cells/*metabolism ; Insulinoma/metabolism ; Lysosomes/metabolism ; Male ; Mice ; Phenotype ; Promoter Regions, Genetic ; Proton-Translocating ATPases/*genetics ; RNA, Small Interfering/metabolism ; Rats ; Receptors, Cell Surface/*genetics/metabolism ; Receptors, Estrogen/metabolism ; Vacuolar Proton-Translocating ATPases/metabolism ; Vacuoles/metabolism ; }, abstract = {Pancreatic β cells store insulin within secretory granules which undergo exocytosis upon elevation of blood glucose levels. Crinophagy and autophagy are instead responsible to deliver damaged or old granules to acidic lysosomes for intracellular degradation. However, excessive consumption of insulin granules can impair β cell function and cause diabetes. Atp6ap2 is an essential accessory component of the vacuolar ATPase required for lysosomal degradative functions and autophagy. Here, we show that Cre recombinase-mediated conditional deletion of Atp6ap2 in mouse β cells causes a dramatic accumulation of large, multigranular vacuoles in the cytoplasm, with reduction of insulin content and compromised glucose homeostasis. Loss of insulin stores and gigantic vacuoles were also observed in cultured insulinoma INS-1 cells upon CRISPR/Cas9-mediated removal of Atp6ap2. Remarkably, these phenotypic alterations could not be attributed to a deficiency in autophagy or acidification of lysosomes. Together, these data indicate that Atp6ap2 is critical for regulating the stored insulin pool and that a balanced regulation of granule turnover is key to maintaining β cell function and diabetes prevention.}, }
@article {pmid31455729, year = {2019}, author = {Fujihara, Y and Noda, T and Kobayashi, K and Oji, A and Kobayashi, S and Matsumura, T and Larasati, T and Oura, S and Kojima-Kita, K and Yu, Z and Matzuk, MM and Ikawa, M}, title = {Identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {37}, pages = {18498-18506}, pmid = {31455729}, issn = {1091-6490}, support = {P01 HD087157/HD/NICHD NIH HHS/United States ; R01 HD088412/HD/NICHD NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Membrane/metabolism ; Disease Models, Animal ; Fallopian Tubes/physiology ; Female ; Fertility/*genetics ; Humans ; Infertility, Male/*genetics ; Male ; Membrane Proteins/*genetics/metabolism ; Mice ; Mice, Knockout ; Multigene Family/*genetics ; Mutation ; Phosphoric Diester Hydrolases/genetics/metabolism ; Sperm Motility/*genetics ; Spermatozoa/cytology/physiology ; }, abstract = {CRISPR/Cas9-mediated genome editing technology enables researchers to efficiently generate and analyze genetically modified animals. We have taken advantage of this game-changing technology to uncover essential factors for fertility. In this study, we generated knockouts (KOs) of multiple male reproductive organ-specific genes and performed phenotypic screening of these null mutant mice to attempt to identify proteins essential for male fertility. We focused on making large deletions (dels) within 2 gene clusters encoding cystatin (CST) and prostate and testis expressed (PATE) proteins and individual gene mutations in 2 other gene families encoding glycerophosphodiester phosphodiesterase domain (GDPD) containing and lymphocyte antigen 6 (Ly6)/Plaur domain (LYPD) containing proteins. These gene families were chosen because many of the genes demonstrate male reproductive tract-specific expression. Although Gdpd1 and Gdpd4 mutant mice were fertile, disruptions of Cst and Pate gene clusters and Lypd4 resulted in male sterility or severe fertility defects secondary to impaired sperm migration through the oviduct. While absence of the epididymal protein families CST and PATE affect the localization of the sperm membrane protein A disintegrin and metallopeptidase domain 3 (ADAM3), the sperm acrosomal membrane protein LYPD4 regulates sperm fertilizing ability via an ADAM3-independent pathway. Thus, use of CRISPR/Cas9 technologies has allowed us to quickly rule in and rule out proteins required for male fertility and expand our list of male-specific proteins that function in sperm migration through the oviduct.}, }
@article {pmid31451649, year = {2019}, author = {Wang, L and Zhao, L and Zhang, X and Zhang, Q and Jia, Y and Wang, G and Li, S and Tian, D and Li, WH and Yang, S}, title = {Large-scale identification and functional analysis of NLR genes in blast resistance in the Tetep rice genome sequence.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {37}, pages = {18479-18487}, pmid = {31451649}, issn = {1091-6490}, mesh = {CRISPR-Cas Systems/genetics ; Chromosome Mapping ; Cloning, Molecular ; Disease Resistance/*genetics ; Gene Knockout Techniques ; Gene Regulatory Networks/immunology ; Genome, Plant/genetics ; Magnaporthe/*immunology ; NLR Proteins/*genetics/immunology ; Oryza/*genetics/immunology/microbiology ; Phylogeny ; Plant Breeding ; Plant Diseases/genetics/*immunology/microbiology ; Plant Proteins/*genetics/immunology ; Plants, Genetically Modified/genetics/immunology/microbiology ; Sequence Analysis, DNA ; }, abstract = {Tetep is a rice cultivar known for broad-spectrum resistance to blast, a devastating fungal disease. The molecular basis for its broad-spectrum resistance is still poorly understood. Is it because Tetep has many more NLR genes than other cultivars? Or does Tetep possess multiple major NLR genes that can individually confer broad-spectrum resistance to blast? Moreover, are there many interacting NLR pairs in the Tetep genome? We sequenced its genome, obtained a high-quality assembly, and annotated 455 nucleotide-binding site leucine-rich repeat (NLR) genes. We cloned and tested 219 NLR genes as transgenes in 2 susceptible cultivars using 5 to 12 diversified pathogen strains; in many cases, fewer than 12 strains were successfully cultured for testing. Ninety cloned NLRs showed resistance to 1 or more pathogen strains and each strain was recognized by multiple NLRs. However, few NLRs showed resistance to >6 strains, so multiple NLRs are apparently required for Tetep's broad-spectrum resistance to blast. This was further supported by the pedigree analyses, which suggested a correlation between resistance and the number of Tetep-derived NLRs. In developing a method to identify NLR pairs each of which functions as a unit, we found that >20% of the NLRs in the Tetep and 3 other rice genomes are paired. Finally, we designed an extensive set of molecular markers for rapidly introducing clustered and paired NLRs in the Tetep genome for breeding new resistant cultivars. This study increased our understanding of the genetic basis of broad-spectrum blast resistance in rice.}, }
@article {pmid31446002, year = {2019}, author = {Nakagawa, Y and Kaneko, T}, title = {Rapid and efficient production of genome-edited animals by electroporation into oocytes injected with frozen or freeze-dried sperm.}, journal = {Cryobiology}, volume = {90}, number = {}, pages = {71-74}, doi = {10.1016/j.cryobiol.2019.08.004}, pmid = {31446002}, issn = {1090-2392}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cryopreservation/methods ; Electroporation/*methods ; Female ; Freeze Drying/methods ; Gene Editing/*methods ; Genome/genetics ; Male ; Oocytes/*growth &amp; development ; Rats ; Rats, Transgenic ; Semen Preservation/*methods ; Sperm Injections, Intracytoplasmic/*methods ; Spermatozoa/physiology ; }, abstract = {Sperm preservation is a useful technique for maintaining valuable animal strains. Rat sperm could be frozen or freeze-dried in a simple Tris-EDTA solution (TE buffer), and oocytes that were fertilized with these sperm by intracytoplasmic sperm injection (ICSI) developed into offspring. Genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system enables the rapid production of genetically modified rats. The recent innovative method, named the TAKE method, could easily produce genome edited rats by electroporation of endonucleases into embryos. Although various rat strains have been applied for genome editing, genome editing using strains that were preserved as sperm took longer because it required collecting embryos after maturation of animals regenerated from sperm. To reduce the production period, we directly electroporated Cas9 protein and gRNA into oocytes that were injected with frozen or freeze-dried sperm in TE buffer. No effect of electroporation until 30 V to ICSI-embryos derived from frozen or freeze-dried sperm were shown in the development of offspring. Furthermore, the rate of genome editing in offspring was high (56% for frozen and 50% for freeze-dried sperm). These results concluded that the combination of ICSI and the TAKE method was useful for the rapid production of genome-edited animals from sperm that have been preserved as genetic resources.}, }
@article {pmid31433690, year = {2019}, author = {Yuan, F and Zhou, J and Xu, L and Jia, W and Chun, L and Xu, XZS and Liu, J}, title = {GABA receptors differentially regulate life span and health span in C. elegans through distinct downstream mechanisms.}, journal = {American journal of physiology. Cell physiology}, volume = {317}, number = {5}, pages = {C953-C963}, pmid = {31433690}, issn = {1522-1563}, support = {R35 GM126917/GM/NIGMS NIH HHS/United States ; }, mesh = {Aging/*genetics/metabolism ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/physiology ; Caenorhabditis elegans ; Caenorhabditis elegans Proteins/*genetics/metabolism ; Longevity/*physiology ; Oxidative Stress/physiology ; Receptors, GABA/*genetics/metabolism ; Signal Transduction/*physiology ; }, abstract = {GABA, a prominent inhibitory neurotransmitter, is best known to regulate neuronal functions in the nervous system. However, much less is known about the role of GABA signaling in other physiological processes. Interestingly, recent work showed that GABA signaling can regulate life span via a metabotropic GABAB receptor in Caenorhabditis elegans. However, the role of other types of GABA receptors in life span has not been clearly defined. It is also unclear whether GABA signaling regulates health span. Here, using C. elegans as a model, we systematically interrogated the role of various GABA receptors in both life span and health span. We find that mutations in four different GABA receptors extend health span by promoting resistance to stress and pathogen infection and that two such receptor mutants also show extended life span. Different GABA receptors engage distinct transcriptional factors to regulate life span and health span, and even the same receptor regulates life span and health span via different transcription factors. Our results uncover a novel, profound role of GABA signaling in aging in C. elegans, which is mediated by different GABA receptors coupled to distinct downstream effectors.}, }
@article {pmid31422865, year = {2019}, author = {Tian, R and Gachechiladze, MA and Ludwig, CH and Laurie, MT and Hong, JY and Nathaniel, D and Prabhu, AV and Fernandopulle, MS and Patel, R and Abshari, M and Ward, ME and Kampmann, M}, title = {CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons.}, journal = {Neuron}, volume = {104}, number = {2}, pages = {239-255.e12}, pmid = {31422865}, issn = {1097-4199}, support = {U54 NS100717/NS/NINDS NIH HHS/United States ; R01 AG062359/AG/NIA NIH HHS/United States ; R56 AG057528/AG/NIA NIH HHS/United States ; ZIA NS003155-01/ImNIH/Intramural NIH HHS/United States ; F30 AG060722/AG/NIA NIH HHS/United States ; ZIA NS003155-03/ImNIH/Intramural NIH HHS/United States ; DP2 GM119139/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Cell Survival ; Gene Knockdown Techniques/*methods ; Humans ; *Induced Pluripotent Stem Cells ; Microscopy, Confocal ; Neurons/cytology/*metabolism ; RNA-Seq ; Single-Cell Analysis ; }, abstract = {CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type-specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons. VIDEO ABSTRACT.}, }
@article {pmid31416668, year = {2019}, author = {Kwart, D and Gregg, A and Scheckel, C and Murphy, EA and Paquet, D and Duffield, M and Fak, J and Olsen, O and Darnell, RB and Tessier-Lavigne, M}, title = {A Large Panel of Isogenic APP and PSEN1 Mutant Human iPSC Neurons Reveals Shared Endosomal Abnormalities Mediated by APP β-CTFs, Not Aβ.}, journal = {Neuron}, volume = {104}, number = {2}, pages = {256-270.e5}, doi = {10.1016/j.neuron.2019.07.010}, pmid = {31416668}, issn = {1097-4199}, mesh = {Alzheimer Disease/*genetics/pathology ; Amyloid Precursor Protein Secretases ; Amyloid beta-Peptides/*metabolism ; Amyloid beta-Protein Precursor/*genetics ; Aspartic Acid Endopeptidases ; CRISPR-Cas Systems ; Cell Line ; Endocytosis/*genetics ; Endosomes/*metabolism/pathology ; Gene Expression Profiling ; Gene Knock-In Techniques ; Heterozygote ; Homozygote ; Humans ; Induced Pluripotent Stem Cells ; Mutation ; Neurons/*metabolism ; Organelle Size ; Peptide Fragments/*metabolism ; Phenotype ; Presenilin-1/*genetics ; Proteomics ; rab5 GTP-Binding Proteins/metabolism ; }, abstract = {Familial Alzheimer's disease (fAD) results from mutations in the amyloid precursor protein (APP) and presenilin (PSEN1 and PSEN2) genes. Here we leveraged recent advances in induced pluripotent stem cell (iPSC) and CRISPR/Cas9 genome editing technologies to generate a panel of isogenic knockin human iPSC lines carrying APP and/or PSEN1 mutations. Global transcriptomic and translatomic profiling revealed that fAD mutations have overlapping effects on the expression of AD-related and endocytosis-associated genes. Mutant neurons also increased Rab5+ early endosome size. APP and PSEN1 mutations had discordant effects on Aβ production but similar effects on APP β C-terminal fragments (β-CTFs), which accumulate in all mutant neurons. Importantly, endosomal dysfunction correlated with accumulation of β-CTFs, not Aβ, and could be rescued by pharmacological modulation of β-secretase (BACE). These data display the utility of our mutant iPSCs in studying AD-related phenotypes in a non-overexpression human-based system and support mounting evidence that β-CTF may be critical in AD pathogenesis.}, }
@article {pmid31407276, year = {2019}, author = {Ochiai, H}, title = {Real-Time Observation of Localization and Expression (ROLEX) System for Live Imaging of the Transcriptional Activity and Nuclear Position of a Specific Endogenous Gene.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2038}, number = {}, pages = {35-45}, doi = {10.1007/978-1-4939-9674-2_3}, pmid = {31407276}, issn = {1940-6029}, mesh = {Animals ; CRISPR-Cas Systems ; Cell Nucleus/genetics/*metabolism ; Gene Expression Regulation ; *Genetic Loci ; Mice ; *Microscopy, Fluorescence ; Molecular Imaging/*methods ; Mouse Embryonic Stem Cells/*metabolism ; RNA, Messenger/genetics/*metabolism ; Single Molecule Imaging/*methods ; Time Factors ; *Transcription, Genetic ; }, abstract = {Long genomic DNA is folded in a cell-type-specific manner and stored in the cell nucleus. The higher-order structure of genomic DNA is thought to be important for DNA transcription, repair, and replication. Recent advancements in live cell imaging techniques that enable the labeling of specific genomic loci and RNA have made it possible to capture the dynamic relationships between higher-order genomic structure and gene expression. We have established the real-time observation of localization and expression (ROLEX) system for live imaging of the transcriptional state and nuclear position of a specific endogenous gene. In this chapter, I will introduce the detailed protocol of ROLEX imaging in mouse embryonic stem cells.}, }
@article {pmid31395429, year = {2019}, author = {Garcia-Marques, J and Yang, CP and Espinosa-Medina, I and Mok, K and Koyama, M and Lee, T}, title = {Unlimited Genetic Switches for Cell-Type-Specific Manipulation.}, journal = {Neuron}, volume = {104}, number = {2}, pages = {227-238.e7}, doi = {10.1016/j.neuron.2019.07.005}, pmid = {31395429}, issn = {1097-4199}, mesh = {Animals ; *CRISPR-Cas Systems ; *DNA Repair ; Drosophila ; Gene Targeting/*methods ; Genetic Techniques ; RNA, Guide ; Recombinases/genetics ; Zebrafish ; }, abstract = {Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. Here we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.}, }
@article {pmid31394408, year = {2019}, author = {Readler, JM and AlKahlout, AS and Sharma, P and Excoffon, KJDA}, title = {Isoform specific editing of the coxsackievirus and adenovirus receptor.}, journal = {Virology}, volume = {536}, number = {}, pages = {20-26}, pmid = {31394408}, issn = {1096-0341}, support = {R01 AI127816/AI/NIAID NIH HHS/United States ; R15 AI090625/AI/NIAID NIH HHS/United States ; }, mesh = {Adenoviruses, Human/*genetics/metabolism ; Animals ; Base Sequence ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Coxsackie and Adenovirus Receptor-Like Membrane Protein/*genetics/metabolism ; DNA, Complementary/genetics/metabolism ; Dogs ; Doxycycline/pharmacology ; Exons ; Gene Editing/*methods ; *Gene Expression Regulation, Viral ; Humans ; Madin Darby Canine Kidney Cells ; Promoter Regions, Genetic/drug effects ; Protein Isoforms/genetics/metabolism ; RNA, Guide/genetics/metabolism ; }, abstract = {The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAREx7) exhibits basolateral localization, the eight exon-encoded isoform (CAREx8) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms, CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAREx8 cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized, and doxycycline-induction of CAREx8 restored sensitivity to adenovirus. These data reinforce the importance of CAREx8 in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.}, }
@article {pmid31360116, year = {2019}, author = {Li, X and Hao, F and Hu, X and Wang, H and Dai, B and Wang, X and Liang, H and Cang, M and Liu, D}, title = {Generation of Tβ4 knock-in Cashmere goat using CRISPR/Cas9.}, journal = {International journal of biological sciences}, volume = {15}, number = {8}, pages = {1743-1754}, pmid = {31360116}, issn = {1449-2288}, mesh = {Animals ; CRISPR-Cas Systems ; Goats ; Hair Follicle/metabolism ; Receptors, CCR5/genetics/metabolism ; Thymosin/genetics/*metabolism ; }, abstract = {The cashmere goat breed is known to provide excellent quality cashmere. Here, we attempted to breed high-yielding cashmere goats by specifically inserting the Tβ4 gene into the goat CCR5 locus and provided an animal model for future research. We successfully obtained Tβ4 knock-in goat without any screening and fluorescent markers using CRISPR/Cas9 technology. A series of experiments were performed to examine physical conditions and characteristics of the Tβ4 knock-in goat. The goat exhibited an increase in cashmere yield by 74.5% without affecting the fineness and quality. Additionally, RNA-seq analysis indicated that Tβ4 may promote hair growth by affecting processes such as vasoconstriction, angiogenesis, and vascular permeability around secondary hair follicles. Together, our study can significantly improve the breeding of cashmere goat and thereby increase economic efficiency.}, }
@article {pmid31332749, year = {2019}, author = {Khoshnejad, M and Brenner, JS and Parhiz, H and Muzykantov, VR}, title = {CRISPR/Cas9-Mediated Genetic Engineering of Hybridomas for Creation of Antibodies that Allow for Site-Specific Conjugation.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2033}, number = {}, pages = {81-93}, doi = {10.1007/978-1-4939-9654-4_7}, pmid = {31332749}, issn = {1940-6029}, mesh = {Animals ; Antibodies, Monoclonal/*genetics/immunology ; CRISPR-Cas Systems/genetics/immunology ; Gene Editing/*methods ; Genetic Engineering/*methods ; Humans ; Hybridomas/immunology ; Immunoconjugates/*genetics/immunology ; Mice ; Oligopeptides/genetics/immunology ; }, abstract = {Covalent conjugation of chemical moieties to antibodies has numerous applications, including antibody-drug conjugates, antibody conjugation for diagnostics, and more. Most nonspecific chemical conjugation methods ligate onto any of a number of sites on the antibody, leading to multiple conjugated species, many of which perturb antibody function. To solve these problems, we used CRISPR/Cas9-edited hybridomas to introduce a Sortase tag (LPXTG) and a Flag tag at the 3' end of the CH3 heavy chain region of a mouse monoclonal antibody. The Flag tag allows easy purification of the antibody, while the LPXTG is then acted on by the bacterial transpeptidase Sortase to site-specifically add on any of a number of chemical moieties that possess a triglycine repeat. This technique thus allows rapid production of an antibody onto which a wide array of chemical moieties can be site-specifically conjugated.}, }
@article {pmid31213543, year = {2019}, author = {Panganiban, RA and Park, HR and Sun, M and Shumyatcher, M and Himes, BE and Lu, Q}, title = {Genome-wide CRISPR screen identifies suppressors of endoplasmic reticulum stress-induced apoptosis.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {27}, pages = {13384-13393}, pmid = {31213543}, issn = {1091-6490}, support = {P30 ES000002/ES/NIEHS NIH HHS/United States ; R01 ES029097/ES/NIEHS NIH HHS/United States ; R01 HL133433/HL/NHLBI NIH HHS/United States ; }, mesh = {Animals ; *Apoptosis ; *CRISPR-Cas Systems ; Endoplasmic Reticulum/metabolism ; *Endoplasmic Reticulum Stress ; Fibroblasts ; Gene Knockout Techniques ; Genes, Suppressor ; Genome-Wide Association Study ; HEK293 Cells ; Humans ; Mice ; Signal Transduction ; Transcription Factor CHOP/metabolism ; Unfolded Protein Response ; }, abstract = {Sensing misfolded proteins in the endoplasmic reticulum (ER), cells initiate the ER stress response and, when overwhelmed, undergo apoptosis. However, little is known about how cells prevent excessive ER stress response and cell death to restore homeostasis. Here, we report the identification and characterization of cellular suppressors of ER stress-induced apoptosis. Using a genome-wide CRISPR library, we screen for genes whose inactivation further increases ER stress-induced up-regulation of C/EBP homologous protein 10 (CHOP)-the transcription factor central to ER stress-associated apoptosis. Among the top validated hits are two interacting components of the polycomb repressive complex (L3MBTL2 [L(3)Mbt-Like 2] and MGA [MAX gene associated]), and microRNA-124-3 (miR-124-3). CRISPR knockout of these genes increases CHOP expression and sensitizes cells to apoptosis induced by multiple ER stressors, while overexpression confers the opposite effects. L3MBTL2 associates with the CHOP promoter in unstressed cells to repress CHOP induction but dissociates from the promoter in the presence of ER stress, whereas miR-124-3 directly targets the IRE1 branch of the ER stress pathway. Our study reveals distinct mechanisms that suppress ER stress-induced apoptosis and may lead to a better understanding of diseases whose pathogenesis is linked to overactive ER stress response.}, }
@article {pmid31213532, year = {2019}, author = {Boned Del Río, I and Young, LC and Sari, S and Jones, GG and Ringham-Terry, B and Hartig, N and Rejnowicz, E and Lei, W and Bhamra, A and Surinova, S and Rodriguez-Viciana, P}, title = {SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {27}, pages = {13330-13339}, pmid = {31213532}, issn = {1091-6490}, support = {/CRUK_/Cancer Research UK/United Kingdom ; }, mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line, Tumor ; Gene Editing ; Gene Knockout Techniques ; Humans ; Intracellular Signaling Peptides and Proteins/*metabolism ; *MAP Kinase Signaling System ; Phosphorylation ; Protein Multimerization ; raf Kinases/*chemistry/*metabolism ; ras Proteins/metabolism ; }, abstract = {Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved &quot;S259&quot; inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.}, }
@article {pmid31209054, year = {2019}, author = {Lee, J and Ma, J and Lee, K}, title = {Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {27}, pages = {13288-13292}, pmid = {31209054}, issn = {1091-6490}, mesh = {Adenoviridae/genetics ; Animals ; *Blastoderm ; *CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Chimera/genetics ; Coturnix/embryology/*genetics ; Feathers/anatomy &amp; histology ; Female ; Gene Editing/*methods ; Gene Knockdown Techniques/*methods ; Male ; }, abstract = {Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.}, }
@article {pmid31182574, year = {2019}, author = {Goto, N and Fukuda, A and Yamaga, Y and Yoshikawa, T and Maruno, T and Maekawa, H and Inamoto, S and Kawada, K and Sakai, Y and Miyoshi, H and Taketo, MM and Chiba, T and Seno, H}, title = {Lineage tracing and targeting of IL17RB+ tuft cell-like human colorectal cancer stem cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {26}, pages = {12996-13005}, pmid = {31182574}, issn = {1091-6490}, mesh = {Animals ; Biomarkers, Tumor/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Carcinogenesis ; Cell Differentiation ; Cell Lineage ; Colorectal Neoplasms/*pathology ; Gene Knock-In Techniques ; Humans ; Intestinal Mucosa/cytology/pathology ; Mice ; Mice, Transgenic ; Neoplastic Stem Cells/*pathology ; Octamer Transcription Factors/metabolism ; Primary Cell Culture ; Protein-Serine-Threonine Kinases/genetics ; RNA, Small Interfering/metabolism ; Receptors, Interleukin-17/genetics/*metabolism ; Spheroids, Cellular ; Time-Lapse Imaging ; Tumor Cells, Cultured ; Up-Regulation ; Xenograft Model Antitumor Assays ; }, abstract = {Cancer stem cell (CSC)-specific markers may be potential therapeutic targets. We previously identified that Dclk1, a tuft cell marker, marks tumor stem cells (TSCs) in mouse intestinal adenomas. Based on the analysis of mouse Dclk1+ tumor cells, we aimed to identify a CSC-specific cell surface marker in human colorectal cancers (hCRCs) and validate the therapeutic effect of targeting it. IL17RB was distinctively expressed by Dclk1+ mouse intestinal tumor cells. Using Il17rb-CreERT2-IRES-EGFP mice, we show that IL17RB marked intestinal TSCs in an IL13-dependent manner. Tuft cell-like cancer cells were detected in a subset of hCRCs. In these hCRCs, lineage-tracing experiments in CRISPR-Cas9-mediated IL17RB-CreERT2 knockin organoids and xenograft tumors revealed that IL17RB marks CSCs that expand independently of IL-13. We observed up-regulation of POU2F3, a master regulator of tuft cell differentiation, and autonomous tuft cell-like cancer cell differentiation in the hCRCs. Furthermore, long-term ablation of IL17RB-expressing CSCs strongly suppressed the tumor growth in vivo. These findings reveal insights into a CSC-specific marker IL17RB in a subset of hCRCs, and preclinically validate IL17RB+ CSCs as a cancer therapeutic target.}, }
@article {pmid31085639, year = {2019}, author = {Wang, C and Lu, T and Emanuel, G and Babcock, HP and Zhuang, X}, title = {Imaging-based pooled CRISPR screening reveals regulators of lncRNA localization.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {22}, pages = {10842-10851}, pmid = {31085639}, issn = {1091-6490}, support = {R01 MH113094/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Cell Nucleus/chemistry/metabolism ; Gene Editing ; High-Throughput Nucleotide Sequencing/methods ; Humans ; Image Processing, Computer-Assisted/*methods ; In Situ Hybridization, Fluorescence/*methods ; Molecular Probes/chemistry/genetics/metabolism ; RNA, Guide/chemistry/genetics/metabolism ; *RNA, Long Noncoding/chemistry/genetics/metabolism ; RNA-Binding Proteins/chemistry/metabolism ; Single-Cell Analysis/*methods ; }, abstract = {Pooled-library CRISPR screening provides a powerful means to discover genetic factors involved in cellular processes in a high-throughput manner. However, the phenotypes accessible to pooled-library screening are limited. Complex phenotypes, such as cellular morphology and subcellular molecular organization, as well as their dynamics, require imaging-based readout and are currently beyond the reach of pooled-library CRISPR screening. Here we report an all imaging-based pooled-library CRISPR screening approach that combines high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification in individual cells. In this approach, sgRNAs are codelivered to cells with corresponding barcodes placed at the 3' untranslated region of a reporter gene using a lentiviral delivery system with reduced recombination-induced sgRNA-barcode mispairing. Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is used to read out the barcodes and hence identify the sgRNAs with high accuracy. We used this approach to screen 162 sgRNAs targeting 54 RNA-binding proteins for their effects on RNA localization to nuclear compartments and uncovered previously unknown regulatory factors for nuclear RNA localization. Notably, our screen revealed both positive and negative regulators for the nuclear speckle localization of a long noncoding RNA, MALAT1, suggesting a dynamic regulation of lncRNA localization in subcellular compartments.}, }
@article {pmid30954217, year = {2019}, author = {Eom, SY and Hwang, SH and Yeom, H and Lee, M}, title = {An ATG5 knockout promotes paclitaxel resistance in v-Ha-ras-transformed NIH 3T3 cells.}, journal = {Biochemical and biophysical research communications}, volume = {513}, number = {1}, pages = {234-241}, doi = {10.1016/j.bbrc.2019.03.197}, pmid = {30954217}, issn = {1090-2104}, mesh = {Animals ; Antineoplastic Agents, Phytogenic/*pharmacology ; Apoptosis/drug effects ; Autophagy/drug effects ; Autophagy-Related Protein 5/*genetics ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; *Drug Resistance, Neoplasm ; Gene Knockout Techniques ; Genes, ras ; Mice ; NIH 3T3 Cells ; Neoplasms/drug therapy/genetics ; Paclitaxel/*pharmacology ; Tubulin Modulators/pharmacology ; }, abstract = {Autophagy plays a contradictory role in cell survival and death. Here, we investigated changes in paclitaxel sensitivity of cells with an ATG5 gene-knockout (KO), incapable of synthesizing an E3 ubiquitin ligase necessary for autophagy. The ATG5 KO in v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3) was established using the CRISPR/Cas9 system. An LC3 immunoblot and a qRT-PCR assay were used to confirm the KO of functional ATG5. We found that the ATG5 KO led to paclitaxel resistance in Ras-NIH 3T3 cells through an ATP-binding cassette (ABC) transporter-independent mechanism. Flow cytometric analyses revealed that paclitaxel induced a remarkable significant G2/M arrest in parental cells, whereas it was relatively less effective in ATG5 KO cells. Additionally, the proportion of early apoptotic cells significantly decreased in ATG5 KO cells treated with paclitaxel than in parental cells. Interestingly, overexpression of ATG5 N-terminal cleavage product in ATG5 KO cells restored their sensitivity to paclitaxel. Taken together, our results suggest that ATG5 KO cells are resistant to paclitaxel due to the inability to produce tATG5.}, }
@article {pmid30952431, year = {2019}, author = {Ali, M and Mutahir, Z and Riaz, A}, title = {CRISPR/Cas9 engineering of ERK5 identifies its FAK/PYK2 dependent role in adhesion-mediated cell survival.}, journal = {Biochemical and biophysical research communications}, volume = {513}, number = {1}, pages = {179-185}, doi = {10.1016/j.bbrc.2019.03.145}, pmid = {30952431}, issn = {1090-2104}, mesh = {Breast Neoplasms/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Adhesion ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Female ; Focal Adhesion Kinase 1/*metabolism ; Focal Adhesion Kinase 2/*metabolism ; Humans ; Mitogen-Activated Protein Kinase 7/*genetics/metabolism ; }, abstract = {Extracellular signal-regulated kinase 5 (ERK5) is now considered a key regulator of breast cancer cell proliferation, migration and invasion. It is also implicated in growth factor induced anti-apoptotic signaling. But its contribution to adhesion-induced survival signaling is not clear. In the present study, using CRISPR/Cas9 editing, we knocked-out ERK5 expression in several cancer cell lines. Then MDA-MB 231 breast cancer cells lacking ERK5 were used to understand its role in adhesion-mediated cell viability. We demonstrated that ERK5 deficient cells exhibited reduced cell attachment to matrix proteins fibronectin and vitronectin. The adhesion ability of these cells was further reduced upon chemical inhibition of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2) by PF 431396. FAK/PYK2 inhibited ERK5 knock-out cells also showed markedly reduced cell-viability and increased apoptotic signaling. This was evident from the detection of cleaved PARP and caspase 9 in these cells. Thus, our data suggests a FAK/PYK2 regulated pro-survival role of ERK5 in response to cell adhesion.}, }
@article {pmid30914202, year = {2019}, author = {Kawaguchi, K and Sato, T and Kondo, S and Yamamoto-Hino, M and Goto, S}, title = {Stability of the transamidase complex catalyzing GPI anchoring of proteins.}, journal = {Biochemical and biophysical research communications}, volume = {512}, number = {3}, pages = {584-590}, doi = {10.1016/j.bbrc.2019.03.103}, pmid = {30914202}, issn = {1090-2104}, mesh = {Aminoacyltransferases/genetics/*metabolism ; Animals ; CRISPR-Cas Systems ; Drosophila Proteins/genetics/*metabolism ; Drosophila melanogaster/genetics/*metabolism ; Enzyme Stability ; GPI-Linked Proteins/*metabolism ; Protein Subunits/genetics/metabolism ; }, abstract = {Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors some proteins to the plasma membrane. This anchoring is catalyzed by a transamidase complex (TAC) composed of five subunits: PIG-K, GAA1, PIG-U, PIG-T, and PIG-S (Fig. 1A). PIG-K and GAA1 are predicted to catalyze the first and second steps during attachment of proproteins of GPI-anchored proteins (GPI-APs) to GPI. GPI may be delivered by PIG-U, and PIG-T is required for stability of all TAC subunits when overexpressed in cultured cells. However, protein stability of TAC has not been analyzed using loss-of-function mutants for each subunit. Herein, we analyzed the stability of TAC in knockout and/or knockdown mutants for each subunit. PIG-T and PIG-U, or PIG-T and GAA1, were mutually required for stability, and all three subunits were stable without PIG-S or PIG-K. However, these three subunits were essential for the stability of both PIG-S and PIG-K. By contrast, loss of PIG-S reduced the stability of PIG-K and left the other subunits unaffected. Reduction of PIG-K did not impact any of the other subunits. Thus, PIG-T, PIG-U, and GAA1 may form a core complex associated by PIG-S, and these four subunits may stabilize PIG-K, triggering GPI anchoring reactions. Instability of PIG-K in the absence of the other four subunits may ensure that GPI anchoring is catalyzed only by the completely assembled complex.}, }
@article {pmid30905411, year = {2019}, author = {Kasamatsu, A and Uzawa, K and Hayashi, F and Kita, A and Okubo, Y and Saito, T and Kimura, Y and Miyamoto, I and Oka, N and Shiiba, M and Ito, C and Toshimori, K and Miki, T and Yamauchi, M and Tanzawa, H}, title = {Deficiency of lysyl hydroxylase 2 in mice causes systemic endoplasmic reticulum stress leading to early embryonic lethality.}, journal = {Biochemical and biophysical research communications}, volume = {512}, number = {3}, pages = {486-491}, doi = {10.1016/j.bbrc.2019.03.091}, pmid = {30905411}, issn = {1090-2104}, mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Disease Models, Animal ; Embryo Loss/*genetics/pathology ; Embryo, Mammalian/metabolism/*pathology ; *Endoplasmic Reticulum Stress ; Heart/embryology ; Heart Defects, Congenital/*genetics/pathology ; Mice ; Mice, Knockout ; Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/*genetics ; }, abstract = {Lysyl hydroxylase 2 (LH2) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens. This is a critical modification to determine the fate of collagen cross-linking pathway that contributes to the stability of collagen fibrils. Studies have demonstrated that the aberrant LH2 function causes various diseases including osteogenesis imperfecta, fibrosis, and cancer metastasis. However, surprisingly, a LH2-deficient animal model has not been reported. In the current study, to better understand the function of LH2, we generated LH2 gene knockout mice by CRISPR/Cas9 technology. LH2 deficiency was confirmed by genotyping polymerase chain reaction (PCR), reverse transcriptase-PCR, and immunohistochemical analyses. Homozygous LH2 knockout (LH2-/-) embryos failed to develop normally and died at early embryonic stage E10.5 with abnormal common ventricle in a heart, i.e., an insufficient wall, a thin ventricular wall, and loosely packed cells. In the LH2-/- mice, the ER stress-responsive genes, ATF4 and CHOP were significantly up-regulated leading to increased levels of Bax and cleaved caspase-3. These data indicate that LH2 plays an essential role in cardiac development through an ER stress-mediated apoptosis pathway.}, }
@article {pmid30846391, year = {2019}, author = {Kowalski, PS and Rudra, A and Miao, L and Anderson, DG}, title = {Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {27}, number = {4}, pages = {710-728}, pmid = {30846391}, issn = {1525-0024}, mesh = {Animals ; CRISPR-Cas Systems ; Cell-Penetrating Peptides/chemistry ; Dendrimers/chemistry ; Drug Delivery Systems/*methods ; Gene Editing/methods ; Genetic Therapy/*methods ; Humans ; Lipids/chemistry ; Mice ; Nanoparticles/chemistry ; Polymers/chemistry ; RNA, Messenger/genetics/*therapeutic use ; Vaccination/methods ; }, abstract = {mRNA has broad potential as a therapeutic. Current clinical efforts are focused on vaccination, protein replacement therapies, and treatment of genetic diseases. The clinical translation of mRNA therapeutics has been made possible through advances in the design of mRNA manufacturing and intracellular delivery methods. However, broad application of mRNA is still limited by the need for improved delivery systems. In this review, we discuss the challenges for clinical translation of mRNA-based therapeutics, with an emphasis on recent advances in biomaterials and delivery strategies, and we present an overview of the applications of mRNA-based delivery for protein therapy, gene editing, and vaccination.}, }
@article {pmid30834637, year = {2019}, author = {Li, R and Qiu, Z and Wang, X and Gong, P and Xu, Q and Yu, QB and Guan, Y}, title = {Pooled CRISPR/Cas9 reveals redundant roles of plastidial phosphoglycerate kinases in carbon fixation and metabolism.}, journal = {The Plant journal : for cell and molecular biology}, volume = {98}, number = {6}, pages = {1078-1089}, doi = {10.1111/tpj.14303}, pmid = {30834637}, issn = {1365-313X}, mesh = {Arabidopsis/*enzymology/genetics/physiology ; Arabidopsis Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Carbon/*metabolism ; *Carbon Cycle ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytosol/metabolism ; Glycolipids/*metabolism ; Glycolysis ; Isoenzymes ; Mutation ; Phenotype ; Phosphoglycerate Kinase/genetics/*metabolism ; Photosynthesis ; Plastids/enzymology ; }, abstract = {Phosphoglycerate kinase (PGK) is a highly conserved reversible enzyme that participates in both glycolysis and photosynthesis. In Arabidopsis thaliana, one cytosolic PGK (PGKc) and two plastidial PGKs (PGKp) are known. It remains debatable whether the two PGKp isozymes are functionally redundant or specialized in plastidial carbon metabolism and fixation. Here, using a pooled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) strategy, we found that plants with single mutations in pgkp1 or pgkp2 were not significantly affected, whereas a pgkp1pgkp2 double mutation was lethal due to retarded carbon fixation, suggesting that PGKp isozymes play redundant functional roles. Metabolomic analysis demonstrated that the sugar-deficient pgkp1pgkp2 double mutation was partially complemented by exogenous sugar, although respiration intermediates were not rescued. Chloroplast development was defective in pgkp1pgkp2, due to a deficiency in glycolysis-dependent galactoglycerolipid biosynthesis. Ectopic expression of a plastid targeting PGKc did not reverse the pgkp1pgkp2 double-mutant phenotypes. Therefore, PGKp1 and PGKp2 play redundant roles in carbon fixation and metabolism, whereas the molecular function of PGKc is more divergent. Our study demonstrated the functional conservation and divergence of glycolytic enzymes.}, }
@article {pmid30807691, year = {2019}, author = {Ji, CH and Kim, H and Kang, HS}, title = {Synthetic Inducible Regulatory Systems Optimized for the Modulation of Secondary Metabolite Production in Streptomyces.}, journal = {ACS synthetic biology}, volume = {8}, number = {3}, pages = {577-586}, doi = {10.1021/acssynbio.9b00001}, pmid = {30807691}, issn = {2161-5063}, mesh = {Anthraquinones/metabolism ; CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; Gene Expression Regulation, Bacterial ; Genetic Engineering/*methods ; Multigene Family ; Piperidones/metabolism ; Promoter Regions, Genetic ; Saccharomyces cerevisiae/genetics ; Secondary Metabolism/*genetics ; Streptomyces/*genetics/*metabolism ; Synthetic Biology/*methods ; Transcription, Genetic ; }, abstract = {Biosynthesis of secondary metabolites is a highly complex process that often requires tight control of their production, as overproduction of metabolites could be toxic and also may cause metabolic burden to their hosts. Tight control of metabolite production could be achieved by expressing key biosynthetic genes under control of an inducible regulatory system. In this study, we employed the modular design approach to build a high performance synthetic inducible regulatory system that displays a large dynamic range and thus is well-suited for the modulation of secondary metabolite production in Streptomyces. To this end, an inducible regulatory system was divided into three separate functional modules: (1) the induction module, (2) the target expression module, and (3) the repressor expression module. Then, these three separate modules were individually optimized in a stepwise manner and assembled to a new system. First, the cumate (CMT) induction module was chosen as the best performing induction module based on the large dynamic range and moderate inducer sensitivity. Then the CMT induction module maintained its performance when combined with diverse constitutive target expression modules, in which overall dynamic ranges varied depending on maximum promoter strengths. Lastly, the repressor expression module was optimized to achieve complete elimination of leaky expression, further increasing the dynamic range of the system. We also demonstrate that any strong constitutive regulatory system could be converted into an inducible regulatory system by simple CRISPR/Cas9-aided markerless insertion of an operator sequence whenever tight control of gene expression is required. We believe that the synthetic inducible regulatory system we report here would become a useful tool in modulating secondary metabolite production in Streptomyces.}, }
@article {pmid30803822, year = {2019}, author = {Zhang, HX and Zhang, Y and Yin, H}, title = {Genome Editing with mRNA Encoding ZFN, TALEN, and Cas9.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {27}, number = {4}, pages = {735-746}, pmid = {30803822}, issn = {1525-0024}, mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/genetics ; DNA/genetics ; Drug Delivery Systems/methods ; Gene Editing/*methods ; Genome ; Humans ; RNA, Messenger/*genetics ; Transcription Activator-Like Effector Nucleases/*genetics ; Transcription, Genetic ; Zinc Finger Nucleases/*genetics ; }, abstract = {Genome-editing technologies based on programmable nucleases have significantly broadened our ability to make precise and direct changes in the genomic DNA of various species, including human cells. Delivery of programmable nucleases into the target tissue or cell is one of the pressing challenges in transforming the technology into medicine. In vitro-transcribed (IVT) mRNA-mediated delivery of nucleases has several advantages, such as transient expression with efficient in vivo and in vitro delivery, no genomic integration, a potentially low off-target rate, and high editing efficiency. This review focuses on key barriers related to IVT mRNA delivery, on developed modes of delivery, and on the application and future prospects of mRNA encoding nuclease-mediated genome editing in research and clinical trials.}, }
@article {pmid30773774, year = {2019}, author = {Miyamoto, T and Takada, R and Tobimatsu, Y and Takeda, Y and Suzuki, S and Yamamura, M and Osakabe, K and Osakabe, Y and Sakamoto, M and Umezawa, T}, title = {OsMYB108 loss-of-function enriches p-coumaroylated and tricin lignin units in rice cell walls.}, journal = {The Plant journal : for cell and molecular biology}, volume = {98}, number = {6}, pages = {975-987}, doi = {10.1111/tpj.14290}, pmid = {30773774}, issn = {1365-313X}, mesh = {Biomass ; CRISPR-Cas Systems ; Cell Wall/chemistry/metabolism ; Flavonoids/*metabolism ; Gene Editing ; *Gene Expression Regulation, Plant ; Gene Regulatory Networks ; Lignin/chemistry/*metabolism ; Loss of Function Mutation ; Oryza/chemistry/*genetics/growth &amp; development/metabolism ; Phylogeny ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Propionates/*metabolism ; Transcription Factors/genetics/*metabolism ; Up-Regulation ; }, abstract = {Breeding approaches to enrich lignins in biomass could be beneficial to improving the biorefinery process because lignins increase biomass heating value and represent a potent source of valuable aromatic chemicals. However, despite the fact that grasses are promising lignocellulose feedstocks, limited information is yet available for molecular-breeding approaches to upregulate lignin biosynthesis in grass species. In this study, we generated lignin-enriched transgenic rice (Oryza sativa), a model grass species, via targeted mutagenesis of the transcriptional repressor OsMYB108 using CRISPR/Cas9-mediated genome editing. The OsMYB108-knockout rice mutants displayed increased expressions of lignin biosynthetic genes and enhanced lignin deposition in culm cell walls. Chemical and two-dimensional nuclear magnetic resonance (NMR) analyses revealed that the mutant cell walls were preferentially enriched in γ-p-coumaroylated and tricin lignin units, both of which are typical and unique components in grass lignins. NMR analysis also showed that the relative abundances of major lignin linkage types were altered in the OsMYB108 mutants.}, }
@article {pmid30762338, year = {2019}, author = {Guo, Y and Bao, C and Ma, D and Cao, Y and Li, Y and Xie, Z and Li, S}, title = {Network-Based Combinatorial CRISPR-Cas9 Screens Identify Synergistic Modules in Human Cells.}, journal = {ACS synthetic biology}, volume = {8}, number = {3}, pages = {482-490}, doi = {10.1021/acssynbio.8b00237}, pmid = {30762338}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems ; Carcinogenesis/genetics/metabolism ; Cell Survival/drug effects ; Cell Transformation, Neoplastic/genetics ; Colorectal Neoplasms/etiology/genetics ; Computational Biology/*methods ; Drug Discovery/methods ; Drug Screening Assays, Antitumor/methods ; Gene Editing ; Gene Knockout Techniques ; *Gene Regulatory Networks ; HEK293 Cells ; Humans ; Inflammation/complications/genetics ; Inflammatory Bowel Diseases/complications/genetics ; RNA Interference ; Transcriptome ; Transfection ; Transforming Growth Factor beta1/genetics/metabolism ; }, abstract = {Tumorigenesis is a complex process that is driven by a combination of networks of genes and environmental factors; however, efficient approaches to identifying functional networks that are perturbed by the process of tumorigenesis are lacking. In this study, we provide a comprehensive network-based strategy for the systematic discovery of functional synergistic modules that are causal determinants of inflammation-induced tumorigenesis. Our approach prioritizes candidate genes selected by integrating clinical-based and network-based genome-wide gene prediction methods and identifies functional synergistic modules based on combinatorial CRISPR-Cas9 screening. On the basis of candidate genes inferred de novo from experimental and computational methods to be involved in inflammation and cancer, we used an existing TGFβ1-induced cellular transformation model in colonic epithelial cells and a new combinatorial CRISPR-Cas9 screening strategy to construct an inflammation-induced differential genetic interaction network. The inflammation-induced differential genetic interaction network that we generated yielded functional insights into the genes and functional module combinations, and showed varied responses to the inflammation agents as well as active traditional Chinese medicine compounds. We identified opposing differential genetic interactions of inflammation-induced tumorigenesis: synergistic promotion and suppression. The synergistic promotion state was primarily caused by deletions in the immune and metabolism modules; the synergistic suppression state was primarily induced by deletions in the proliferation and immune modules or in the proliferation and metabolism modules. These results provide insight into possible early combinational targets and biomarkers for inflammation-induced tumorigenesis and highlight the synergistic effects that occur among immune, proliferation, and metabolism modules. In conclusion, this approach deepens the understanding of the underlying mechanisms that cause inflammation to potentially increase the cancer risk of colonic epithelial cells and accelerate the translation into novel functional modules or synergistic module combinations that modulate complex disease phenotypes.}, }
@article {pmid30734978, year = {2019}, author = {Koizumi, Y and Fukushima, J and Kobayashi, Y and Kadowaki, A and Natsui, M and Yamaguchi, T and Imai, Y and Sugiyama, T and Kuba, K}, title = {Genome-Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin A1.}, journal = {Chembiochem : a European journal of chemical biology}, volume = {20}, number = {12}, pages = {1563-1568}, pmid = {30734978}, issn = {1439-7633}, mesh = {Aspergillus niger/metabolism ; CRISPR-Cas Systems ; Cell Survival/drug effects/genetics ; Humans ; Peptides, Cyclic/*pharmacology ; RNA, Guide/genetics ; Squalene Monooxygenase/*genetics/metabolism ; U937 Cells ; }, abstract = {Malformin A1 (MA1) is a fungus-produced cyclic pentapeptide. MA1 exhibits teratogenicity to plants, fibrinolysis-enhancing activity, and cytotoxicity to mammalian cells. To clarify the cytotoxic mechanism of MA1, we screened for the genes involved in the cytotoxicity of MA1 in monocytoid U937 cells by using a CRISPR/Cas9-based genome-wide knockout library. Screening was performed by positive selection for cells that were resistant to MA1 treatment, and single guide RNAs (sgRNAs) integrated into MA1-resistant cells were analyzed by high-throughput sequencing. As a result of the evaluation of sgRNAs that were enriched in MA1-resistant cells, SQLE, which encodes squalene epoxidase, was identified as a candidate gene. SQLE-depleted U937 cells were viable in the presence of MA1, and squalene epoxidase inhibitor conferred MA1 resistance to wild-type cells. These results indicate that squalene epoxidase is implicated in the cytotoxicity of MA1. This finding represents a new insight into applications of MA1 for treating ischemic diseases.}, }
@article {pmid30703321, year = {2019}, author = {Geng, P and Leonard, SP and Mishler, DM and Barrick, JE}, title = {Synthetic Genome Defenses against Selfish DNA Elements Stabilize Engineered Bacteria against Evolutionary Failure.}, journal = {ACS synthetic biology}, volume = {8}, number = {3}, pages = {521-531}, doi = {10.1021/acssynbio.8b00426}, pmid = {30703321}, issn = {2161-5063}, mesh = {Acinetobacter/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Transposable Elements/genetics ; Escherichia coli/*genetics ; *Evolution, Molecular ; Genetic Vectors ; Genome, Bacterial ; *Genomic Instability ; Metabolic Engineering/*methods ; Mutation ; Plasmids/genetics ; RNA Interference ; Repetitive Sequences, Nucleic Acid/*genetics ; Synthetic Biology/*methods ; }, abstract = {Mobile genetic elements drive evolution by disrupting genes and rearranging genomes. Eukaryotes have evolved epigenetic mechanisms, including DNA methylation and RNA interference, that silence mobile elements and thereby preserve the integrity of their genomes. We created an artificial reprogrammable epigenetic system based on CRISPR interference to give engineered bacteria a similar line of defense against transposons and other selfish elements in their genomes. We demonstrate that this CRISPR interference against mobile elements (CRISPRi-ME) approach can be used to simultaneously repress two different transposon families in Escherichia coli, thereby increasing the evolutionary stability of costly protein expression. We further show that silencing a transposon in Acinetobacter baylyi ADP1 reduces mutation rates by a factor of 5, nearly as much as deleting all copies of this element from its genome. By deploying CRISPRi-ME on a broad-host-range vector, we have created a generalizable platform for stabilizing the genomes of engineered bacterial cells for applications in metabolic engineering and synthetic biology.}, }
@article {pmid30618028, year = {2019}, author = {Mu, W and Tang, N and Cheng, C and Sun, W and Wei, X and Wang, H}, title = {In vitro transcribed sgRNA causes cell death by inducing interferon release.}, journal = {Protein &amp; cell}, volume = {10}, number = {6}, pages = {461-465}, pmid = {30618028}, issn = {1674-8018}, mesh = {*CRISPR-Cas Systems ; Cell Death ; Gene Editing ; HeLa Cells ; Hematopoietic Stem Cells/*cytology ; Humans ; Interferons ; RNA, Guide/*physiology ; T-Lymphocytes/*cytology ; }, }
@article {pmid30471466, year = {2019}, author = {White, CW and Johnstone, EKM and See, HB and Pfleger, KDG}, title = {NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing.}, journal = {Cellular signalling}, volume = {54}, number = {}, pages = {27-34}, doi = {10.1016/j.cellsig.2018.11.018}, pmid = {30471466}, issn = {1873-3913}, mesh = {Bioluminescence Resonance Energy Transfer Techniques/*methods ; CRISPR-Cas Systems ; HEK293 Cells ; Humans ; Luciferases/chemistry ; Receptor, Adenosine A2B/*analysis ; }, abstract = {Bioluminescence resonance energy transfer (BRET) is a versatile tool used to investigate membrane receptor signalling and function. We have recently developed a homogenous NanoBRET ligand binding assay to monitor interactions between G protein-coupled receptors and fluorescent ligands. However, this assay requires the exogenous expression of a receptor fused to the nanoluciferase (Nluc) and is thus not applicable to natively-expressed receptors. To overcome this limitation in HEK293 cells, we have utilised CRISPR/Cas9 genome engineering to insert Nluc in-frame with the endogenous ADORA2B locus this resulted in HEK293 cells expressing adenosine A2B receptors under endogenous promotion tagged on their N-terminus with Nluc. As expected, we found relatively low levels of endogenous (gene-edited) Nluc/A2B receptor expression compared to cells transiently transfected with expression vectors coding for Nluc/A2B. However, in cells expressing gene-edited Nluc/A2B receptors we observed clear saturable ligand binding of a non-specific fluorescent adenosine receptor antagonist XAC-X-BY630 (Kd = 21.4 nM). Additionally, at gene-edited Nluc/A2B receptors we derived pharmacological parameters of ligand binding; Kd as well as Kon and Koff for binding of XAC-X-BY630 by NanoBRET association kinetic binding assays. Lastly, cells expressing gene-edited Nluc/A2B were used to determine the pKi of unlabelled adenosine receptor ligands in competition ligand binding assays. Utilising CRISPR/Cas9 genome engineering here we show that NanoBRET ligand binding assays can be performed at gene-edited receptors under endogenous promotion in live cells, therefore overcoming a fundamental limitation of NanoBRET ligand assays.}, }
@article {pmid30378731, year = {2019}, author = {Adkar, SS and Wu, CL and Willard, VP and Dicks, A and Ettyreddy, A and Steward, N and Bhutani, N and Gersbach, CA and Guilak, F}, title = {Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing.}, journal = {Stem cells (Dayton, Ohio)}, volume = {37}, number = {1}, pages = {65-76}, pmid = {30378731}, issn = {1549-4918}, support = {R43 AR072166/AR/NIAMS NIH HHS/United States ; DP2 OD008586/OD/NIH HHS/United States ; R21 AR072870/AR/NIAMS NIH HHS/United States ; R01 DA036865/DA/NIDA NIH HHS/United States ; S10 OD010707/OD/NIH HHS/United States ; R01 AR048852/AR/NIAMS NIH HHS/United States ; R21 DA041878/DA/NIDA NIH HHS/United States ; R01 AG046927/AG/NIA NIH HHS/United States ; R01 AR048182/AR/NIAMS NIH HHS/United States ; T32 DK108742/DK/NIDDK NIH HHS/United States ; R01 AR070864/AR/NIAMS NIH HHS/United States ; R01 AR069085/AR/NIAMS NIH HHS/United States ; P01 AR050245/AR/NIAMS NIH HHS/United States ; R01 AG015768/AG/NIA NIH HHS/United States ; U01 HG007900/HG/NHGRI NIH HHS/United States ; R21 AR067467/AR/NIAMS NIH HHS/United States ; R41 GM119914/GM/NIGMS NIH HHS/United States ; R03 AR061042/AR/NIAMS NIH HHS/United States ; R21 NS103007/NS/NINDS NIH HHS/United States ; T32 EB018266/EB/NIBIB NIH HHS/United States ; R21 AR065956/AR/NIAMS NIH HHS/United States ; }, mesh = {Alleles ; CRISPR-Cas Systems/*genetics ; Cell Differentiation ; Chondrogenesis/*genetics ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; }, abstract = {The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. Here, we demonstrate cartilaginous matrix production in three unique hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors (CPs) produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified CPs demonstrated an improved chondrogenic capacity compared with unselected populations. The ability to enrich for CPs and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications. Stem Cells 2019;37:65-76.}, }
@article {pmid30269252, year = {2019}, author = {An, X and Li, L and Wu, S}, title = {In vivo tunable CRISPR mediates efficient somatic mutagenesis to generate tumor models.}, journal = {Protein &amp; cell}, volume = {10}, number = {6}, pages = {450-454}, pmid = {30269252}, issn = {1674-8018}, mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Gene Editing ; *Genetic Therapy ; HeLa Cells ; Humans ; Mice ; Mutagenesis ; *Neoplasms/genetics/therapy ; }, }
@article {pmid29449584, year = {2018}, author = {Callow, MG and Watanabe, C and Wickliffe, KE and Bainer, R and Kummerfield, S and Weng, J and Cuellar, T and Janakiraman, V and Chen, H and Chih, B and Liang, Y and Haley, B and Newton, K and Costa, MR}, title = {CRISPR whole-genome screening identifies new necroptosis regulators and RIPK1 alternative splicing.}, journal = {Cell death &amp; disease}, volume = {9}, number = {3}, pages = {261}, pmid = {29449584}, issn = {2041-4889}, mesh = {*Alternative Splicing ; Animals ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Fibroblasts/*enzymology/pathology ; Gene Expression Regulation, Enzymologic ; Gene Targeting/*methods ; HEK293 Cells ; HT29 Cells ; Heterogeneous-Nuclear Ribonucleoproteins/genetics/*metabolism ; Humans ; Mice ; *Necroptosis ; Polypyrimidine Tract-Binding Protein/genetics/*metabolism ; Receptor-Interacting Protein Serine-Threonine Kinases/genetics/*metabolism ; Signal Transduction ; }, abstract = {The necroptotic cell death pathway is a key component of human pathogen defense that can become aberrantly derepressed during tissue homeostasis to contribute to multiple types of tissue damage and disease. While formation of the necrosome kinase signaling complex containing RIPK1, RIPK3, and MLKL has been extensively characterized, additional mechanisms of its regulation and effector functions likely remain to be discovered. We screened 19,883 mouse protein-coding genes by CRISPR/Cas9-mediated gene knockout for resistance to cytokine-induced necroptosis and identified 112 regulators and mediators of necroptosis, including 59 new candidate pathway components with minimal or no effect on cell growth in the absence of necroptosis induction. Among these, we further characterized the function of PTBP1, an RNA binding protein whose activity is required to maintain RIPK1 protein abundance by regulating alternative splice-site selection.}, }
@article {pmid29449560, year = {2018}, author = {Cai, S and Chen, Y and Shang, Y and Cui, J and Li, Z and Li, Y}, title = {Knockout of zebrafish interleukin 7 receptor (IL7R) by the CRISPR/Cas9 system delays retinal neurodevelopment.}, journal = {Cell death &amp; disease}, volume = {9}, number = {3}, pages = {273}, pmid = {29449560}, issn = {2041-4889}, mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Developmental ; *Gene Knockdown Techniques ; Mutation ; *Neurogenesis ; Photic Stimulation ; Receptors, Interleukin-7/*deficiency/genetics ; Retinal Neurons/*metabolism ; Signal Transduction ; Vision, Ocular ; Zebrafish/embryology/genetics/*metabolism ; Zebrafish Proteins/*deficiency/genetics ; }, abstract = {Interleukin 7 receptor (il7r), a transmembrane receptor, belongs to the type I cytokine receptor family. Il7r is involved in the pathogenesis of neurodegenerative disorders, such as multiple sclerosis. Targeted knockdown of il7r leads to delayed myelination, highlighting the potential role of il7r in the development of the nervous system. Zebrafish is an ideal model for the study of neurogenesis; moreover, the il7r gene is highly conserved between zebrafish and human. The aim of the present study was to investigate the novel function of il7r in neurogenesis. First, an il7r -/- homozygous mutant line was generated by clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (CRISPR/Cas9) technology. Second, the gross development of il7r-/- mutants revealed remarkably smaller eyes and delayed retinal neurodifferentiation. Third, microarray analysis revealed that genes associated with the phototransduction signalling pathway were strongly down-regulated in il7r -/- mutants. Finally, the results from behavioural tests indicated that visual function was impaired in il7r -/- mutant larvae. Overall, our data demonstrate that a lack of il7r retards the development of the retina. Thus, il7r is an essential molecule for maintaining normal retinal development in zebrafish.}, }
@article {pmid32257106, year = {2020}, author = {Xu, M}, title = {CCR5-Δ32 biology, gene editing, and warnings for the future of CRISPR-Cas9 as a human and humane gene editing tool.}, journal = {Cell &amp; bioscience}, volume = {10}, number = {}, pages = {48}, doi = {10.1186/s13578-020-00410-6}, pmid = {32257106}, issn = {2045-3701}, abstract = {Background: Biomedical technologies have not just improved human health but also assisted in the creation of human life. Since the first birth of a healthy baby by in vitro fertilization (IVF) 40 years ago, IVF has been the mainstay treatment for couples struggling with infertility. This technology, in addition to increasingly accessible genetic testing, has made it possible for countless couples to have children. Since CRISPR-Cas9 gene editing was described in 2015, its potential for targeting genetic diseases has been much anticipated. However, the potential of using CRISPR-Cas9 for human germline modification has led to many fears of &quot;designer babies&quot; and widespread concerns for the impact of this technology on human evolution and its implications in Social Darwinism. In addition to these ethical/moral concerns, there remain many unknowns about CRISPR-Cas9 technology and endless unanticipated consequence to gene editing.<br><br>Methods: In this paper, we analyze the current progresses of CRISPR-Cas9 technology and discuss the theoretical advantages of certain allelic variances in the C-C chemokine receptor 5 gene (CCR5) in the setting of recent ethical/moral concerns regarding gene editing using the CRISPR-Cas9 system.<br><br>Results: These uncertainties have been highlighted recently by the birth of Chinese twins whose C-C chemokine receptor 5 (CCR5) gene had been inactivated via CRISPR-Cas9 to be theoretically protective against HIV infection. CCR5 signaling is critical for the successful infection of human immunodeficiency virus (HIV) and people with homozygous inactivating CCR5-Δ32 mutations have been shown to be protected against HIV infection. Those with the CCR5-Δ32/Δ32 mutation also have greater neuroplasticity, allowing for improved recovery from neurological trauma, and decreased Chagas cardiomyopathy. However, the CCR5-Δ32/Δ32 mutation has also been associated with earlier clinical manifestations for West Nile infection, ambiguous effects on osteoclast function, and a four-fold increased mortality from influenza infection. These detrimental health impacts, in addition to the confounding factor that these CRISPR babies do not carry this exact CCR5-Δ32/Δ32 mutation, lead to many questions regarding the children's future health and the moral conundrum of their birth. The creation and birth of these babies was not completed with any scientific, ethical, or governmental oversight, which has spurned the acceleration of talks regarding global regulations for human genetic editing.<br><br>Conclusions: Although we can try to regulate for ethical, health-related only use of this technology, moral and governmental oversights need to be supplemented by technical regulations. For instance, whole genome sequencing needs to be used to eliminate off-target mutations that could affect the health and safety of infants born to this process. Like Pandora's Box, we cannot pretend to forget CRISPR-Cas9 technology, all we can do is ensure a safe, moral, and equitable used of this technology.}, }
@article {pmid32256462, year = {2020}, author = {Álvarez, VE and Quiroga, MP and Galán, AV and Vilacoba, E and Quiroga, C and Ramírez, MS and Centrón, D}, title = {Crucial Role of the Accessory Genome in the Evolutionary Trajectory of Acinetobacter baumannii Global Clone 1.}, journal = {Frontiers in microbiology}, volume = {11}, number = {}, pages = {342}, doi = {10.3389/fmicb.2020.00342}, pmid = {32256462}, issn = {1664-302X}, abstract = {Acinetobacter baumannii is one of the most important nosocomial pathogens able to rapidly develop extensive drug resistance. Here, we study the role of accessory genome in the success of the globally disseminated clone 1 (GC1) with functional and genomic approaches. Comparative genomics was performed with available GC1 genomes (n = 106) against other A. baumannii high-risk and sporadic clones. Genetic traits related to accessory genome were found common and conserved along time as two novel regions of genome plasticity, and a CRISPR-Cas system acquired before clonal diversification located at the same loci as &quot;sedentary&quot; modules. Although identified within hotspot for recombination, other block of accessory genome was also &quot;sedentary&quot; in lineage 1 of GC1 with signs of microevolution as the AbaR0-type genomic island (GI) identified in A144 and in A155 strains which were maintained one month in independent experiments without antimicrobial pressure. The prophage YMC/09/02/B1251_ABA_BP was found to be &quot;mobile&quot; since, although it was shared by all GC1 genomes, it showed high intrinsic microevolution as well as mobility to different insertion sites. Interestingly, a wide variety of Insertion Sequences (IS), probably acquired by the flow of plasmids related to Rep_3 superfamily was found. These IS showed dissimilar genomic location amongst GC1 genomes presumably associated with promptly niche adaptation. On the other hand, a type VI secretion system and three efflux pumps were subjected to deep processes of genomic loss in A. baumannii but not in GC1. As a whole, these findings suggest that preservation of some genetic modules of accessory genome harbored by strains from different continents in combination with great plasticity of IS and varied flow of plasmids, may be central features of the genomic structure of GC1. Competition of A144 and A155 versus A118 (ST 404/ND) without antimicrobial pressure suggested a higher ability of GC1 to grow over a clone with sporadic behavior which explains, from an ecological perspective, the global achievement of this successful pandemic clone in the hospital habitat. Together, these data suggest an essential role of still unknown properties of &quot;mobile&quot; and &quot;sedentary&quot; accessory genome that is preserved over time under different antibiotic or stress conditions.}, }
@article {pmid32255179, year = {2020}, author = {Rabinowitz, R and Abadi, S and Almog, S and Offen, D}, title = {Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkaa215}, pmid = {32255179}, issn = {1362-4962}, abstract = {Base editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A deaminase enzyme is fused to a deactivated Cas and enables transition conversions. The diversified repertoire of base editors provides a wide range of base editing possibilities. However, existing base editors cannot induce transversion substitutions and activate only within a specified region relative to the binding site, thus, they cannot precisely correct every point mutation. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a point mutation. When a precise correction is impossible, BE-FF aims to mutate bystander nucleotides in order to induce synonymous corrections that will correct the coding sequence. To measure BE-FF practicality, we analysed a database of human pathogenic point mutations. Out of the transition mutations, 60.9% coding sequences could be corrected. Notably, 19.4% of the feasible corrections were not achieved by precise corrections but only by synonymous corrections. Moreover, 298 cases of transversion-derived pathogenic mutations were detected to be potentially repairable by base editing via synonymous corrections, although base editing is considered impractical for such mutations.}, }
@article {pmid32253539, year = {2020}, author = {Li, F and Liu, Q and Li, X and Zhang, C and Li, J and Sun, W and Liu, D and Xiao, D and Tian, C}, title = {Construction of a new thermophilic fungus Myceliophthora thermophila platform for enzyme production using a versatile 2A peptide strategy combined with efficient CRISPR-Cas9 system.}, journal = {Biotechnology letters}, volume = {}, number = {}, pages = {}, doi = {10.1007/s10529-020-02882-5}, pmid = {32253539}, issn = {1573-6776}, support = {2018YFA0900500//Ministry of Science and Technology of the People's Republic of China/ ; 31972878//National Natural Science Foundation of China/ ; 31771386//National Natural Science Foundation of China/ ; 31972879//National Natural Science Foundation of China/ ; 2019180//Youth Innovation Promotion Association of the Chinese Academy of Sciences/ ; 2020183//Youth Innovation Promotion Association of the Chinese Academy of Sciences/ ; }, abstract = {OBJECTIVE: To construct a new thermophilic platform for glucoamylase production through 2A peptide strategy combined with CRISPR-Cas9 system using Myceliophthora thermophila as host, thermophilic filamentous fungus with industrial attractiveness to produce enzymes and chemicals from biomass.<br><br>RESULTS: We adapted the viral 2A peptide approach for M. thermophila and constructed a bicistronic vector for co-expressing two heterologous genes MhglaA and egfp. We obtained positive transformants OE-MhglaA-gfp overexpressing MhGlaA-9 ×His-2A-eGFP through convenient fluorescence screening, western blotting and RT-qPCR. We purified and characterized the recombinant MhGlaA, which exhibited stability in a broader pH range of 3.0-9.0 and thermostable stability at 65 °C, suggesting its potential industrial application. Furthermore, to improve glucoamylase secretion, we genetically engineered the obtained strain OE-MhglaA-gfp through our efficient CRISPR/Cas9 system and generated the quintuple mutant OE-MhglaA-gfpOE-amyRΔalp-1Δres-1Δcre-1, in which protein productivity and amylase activity were increased by approximately 12.0- and 8.2-fold compared with WT.<br><br>CONCLUSIONS: The 2A peptide approach worked well in M. thermophila and can be used to heterologously co-express two different proteins, and thus in combination with efficient CRISPR-Cas system will accelerate establishing hyper-secretion platforms for biotechnological applications.}, }
@article {pmid32251383, year = {2020}, author = {Cheng, Q and Wei, T and Farbiak, L and Johnson, LT and Dilliard, SA and Siegwart, DJ}, title = {Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing.}, journal = {Nature nanotechnology}, volume = {}, number = {}, pages = {}, doi = {10.1038/s41565-020-0669-6}, pmid = {32251383}, issn = {1748-3395}, support = {R01 EB025192-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; R01 EB025192-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; R01 EB025192-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; R01 EB025192-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; R01 EB025192-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB)/ ; }, abstract = {CRISPR-Cas gene editing and messenger RNA-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed selective organ targeting (SORT) wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen- and liver-targeted SORT lipid nanoparticles were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells and hepatocytes. SORT is compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA/single guide RNA and Cas9 ribonucleoprotein complexes, and is envisioned to aid the development of protein replacement and gene correction therapeutics in targeted tissues.}, }
@article {pmid32245761, year = {2020}, author = {Meile, S and Sarbach, A and Du, J and Schuppler, M and Saez, C and Loessner, MJ and Kilcher, S}, title = {Engineered reporter phages for rapid bioluminescence-based detection and differentiation of viable Listeria cells.}, journal = {Applied and environmental microbiology}, volume = {}, number = {}, pages = {}, doi = {10.1128/AEM.00442-20}, pmid = {32245761}, issn = {1098-5336}, abstract = {The pathogen Listeria monocytogenes causes listeriosis, a severe foodborne disease associated with high mortality. Rapid and sensitive methods are required for specific detection of this pathogen during food production. Bioluminescence-based reporter bacteriophages are genetically engineered viruses that infect their host cells with high specificity and transduce a heterologous luciferase gene, whose activity can be detected with high sensitivity to indicate the presence of viable target cells. Here, we use synthetic biology for de novo genome assembly and activation as well as CRISPR-Cas-assisted phage engineering to construct a set of reporter phages for the detection and differentiation of viable Listeria cells. Based on a single phage backbone, we compare the performance of four reporter phages that encode different crustacean, cnidarian and bacterial luciferases. From this panel of reporter proteins, nanoluciferase (NLuc) was identified as a superior enzyme and was subsequently introduced into the genomes of a broad host-range phage (A511) and two serovar 1/2- and serovar 4b/6a-specific Listeria phages (A006 and A500, respectively). The broad-range NLuc-based phage A511::nlucCPS detects one CFU of Listeria monocytogenes in 25 g of artificially contaminated milk, cold cut, and lettuce within less than 24 hours. In addition, this reporter phage successfully detected Listeria spp. in potentially contaminated natural food samples without producing false-positive or false-negative results. Finally, A006::nluc and A500::nluc enable serovar-specific Listeria diagnostics. In conclusion, these NLuc-based reporter phages enable rapid, ultrasensitive detection and differentiation of viable Listeria cells using a simple protocol that is 72 hours faster than culture-dependent approaches.Importance Culture-dependent methods are the gold standard for sensitive and specific detection of pathogenic bacteria within the food production chain. In contrast to molecular approaches, these methods detect viable cells, which is a key advantage for foods generated from heat-inactivated source material. However, culture-based diagnostics are typically much slower than molecular or proteomic strategies. Reporter phage assays combine the best of both worlds and allow for near on-line assessment of microbial safety because phage replication is extremely fast, highly target specific, and restricted to metabolically active host cells. In addition, reporter phage assays are inexpensive and do not require highly trained personnel, facilitating their on-site implementation. The reporter phages presented in this study not only allow for rapid detection, but also enable an early estimation of the potential virulence of Listeria isolates from food production and processing sites.}, }
@article {pmid32244056, year = {2020}, author = {Araldi, RP and Khalil, C and Grignet, PH and Teixeira, MR and de Melo, TC and Módolo, DG and Fernandes, LGV and Ruiz, J and de Souza, EB}, title = {Medical Applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas) Tool: A Comprehensive Overview.}, journal = {Gene}, volume = {}, number = {}, pages = {144636}, doi = {10.1016/j.gene.2020.144636}, pmid = {32244056}, issn = {1879-0038}, abstract = {Since the discovery of the double helix and the introduction of genetic engineering, the possibility to develop new strategies to manipulate the genome has fascinated scientists around the world. Currently scientists have the knowledge andabilitytoedit the genomes. Several methodologies of gene editing have been established, all of them working like &quot;scissor&quot;, creating double strand breaks at specific spots. The introduction of a new technology, which was adapted from the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas bacterial immune system, has revolutionized the genetic therapy field, as it allows a much more precise editing of gene than the previously described tools and, therefore, to prevent and treat disease in humans. This review aims to revisit the genome editing history that led to the rediscovery of the CRISPR/Cas technology and to explore the technical aspects, applications and perspectives of this fascinating, powerful, precise, simpler and cheaper technology in different fields.}, }
@article {pmid32241913, year = {2020}, author = {Mekler, V and Kuznedelov, K and Severinov, K}, title = {Quantification of the affinities of CRISPR-Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1074/jbc.RA119.012239}, pmid = {32241913}, issn = {1083-351X}, abstract = {The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9/gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with gRNAs (SpCas9/gRNA, SaCas9/gRNA, and FnCas9/gRNA, respectively) and of three engineered SpCas9/gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a &quot;beacon&quot; assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9/gRNA binding to fluorescently labeled target DNA derivatives called &quot;Cas9 beacons.&quot; We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9/gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.}, }
@article {pmid32241274, year = {2020}, author = {Liu, E and Zhang, ZZ and Cheng, X and Liu, X and Cheng, L}, title = {SCNrank: spectral clustering for network-based ranking to reveal potential drug targets and its application in pancreatic ductal adenocarcinoma.}, journal = {BMC medical genomics}, volume = {13}, number = {Suppl 5}, pages = {50}, doi = {10.1186/s12920-020-0681-6}, pmid = {32241274}, issn = {1755-8794}, abstract = {BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic malignancy. Due to its wide heterogeneity, PDAC acts aggressively and responds poorly to most chemotherapies, causing an urgent need for the development of new therapeutic strategies. Cell lines have been used as the foundation for drug development and disease modeling. CRISPR-Cas9 plays a key role in every step-in drug discovery: from target identification and validation to preclinical cancer cell testing. Using cell-line models and CRISPR-Cas9 technology together make drug target prediction feasible. However, there is still a large gap between predicted results and actionable targets in real tumors. Biological network models provide great modus to mimic genetic interactions in real biological systems, which can benefit gene perturbation studies and potential target identification for treating PDAC. Nevertheless, building a network model that takes cell-line data and CRISPR-Cas9 data as input to accurately predict potential targets that will respond well on real tissue remains unsolved.<br><br>METHODS: We developed a novel algorithm 'Spectral Clustering for Network-based target Ranking' (SCNrank) that systematically integrates three types of data: expression profiles from tumor tissue, normal tissue and cell-line PDAC; protein-protein interaction network (PPI); and CRISPR-Cas9 data to prioritize potential drug targets for PDAC. The whole algorithm can be classified into three steps: 1. using STRING PPI network skeleton, SCNrank constructs tissue-specific networks with PDAC tumor and normal pancreas tissues from expression profiles; 2. With the same network skeleton, SCNrank constructs cell-line-specific networks using the cell-line PDAC expression profiles and CRISPR-Cas 9 data from pancreatic cancer cell-lines; 3. SCNrank applies a novel spectral clustering approach to reduce data dimension and generate gene clusters that carry common features from both networks. Finally, SCNrank applies a scoring scheme called 'Target Influence score' (TI), which estimates a given target's influence towards the cluster it belongs to, for scoring and ranking each drug target.<br><br>RESULTS: We applied SCNrank to analyze 263 expression profiles, CRPSPR-Cas9 data from 22 different pancreatic cancer cell-lines and the STRING protein-protein interaction (PPI) network. With SCNrank, we successfully constructed an integrated tissue PDAC network and an integrated cell-line PDAC network, both of which contain 4414 selected genes that are overexpressed in tumor tissue samples. After clustering, 4414 genes are distributed into 198 clusters, which include 367 targets of FDA approved drugs. These drug targets are all scored and ranked by their TI scores, which we defined to measure their influence towards the network. We validated top-ranked targets in three aspects: Firstly, mapping them onto the existing clinical drug targets of PDAC to measure the concordance. Secondly, we performed enrichment analysis to these drug targets and the clusters there are within, to reveal functional associations between clusters and PDAC; Thirdly, we performed survival analysis for the top-ranked targets to connect targets with clinical outcomes. Survival analysis reveals that overexpression of three top-ranked genes, PGK1, HMMR and POLE2, significantly increases the risk of death in PDAC patients.<br><br>CONCLUSION: SCNrank is an unbiased algorithm that systematically integrates multiple types of omics data to do potential drug target selection and ranking. SCNrank shows great capability in predicting drug targets for PDAC. Pancreatic cancer-associated gene candidates predicted by our SCNrank approach have the potential to guide genetics-based anti-pancreatic drug discovery.}, }
@article {pmid31852439, year = {2019}, author = {Cheng, Q and Dong, L and Su, T and Li, T and Gan, Z and Nan, H and Lu, S and Fang, C and Kong, L and Li, H and Hou, Z and Kou, K and Tang, Y and Lin, X and Zhao, X and Chen, L and Liu, B and Kong, F}, title = {CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean.}, journal = {BMC plant biology}, volume = {19}, number = {1}, pages = {562}, pmid = {31852439}, issn = {1471-2229}, support = {31901568//National Natural Science Foundation of China/ ; 31771815//National Natural Science Foundation of China/ ; 31701445//National Natural Science Foundation of China/ ; 31801384//National Natural Science Foundation of China/ ; 31725021//National Outstanding Youth Foundation of China/ ; 2017YFE0111000//Natural Key R&amp;D Program of China/ ; 2016YFD0100400//Natural Key R&amp;D Program of China/ ; }, mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genes, Plant ; *Mutagenesis ; Plants, Genetically Modified ; Soybeans/genetics/*growth &amp; development ; }, abstract = {BACKGROUND: Soybean (Glycine max) is an economically important oil and protein crop. Plant height is a key trait that significantly impacts the yield of soybean; however, research on the molecular mechanisms associated with soybean plant height is lacking. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated system 9) system is a recently developed technology for gene editing that has been utilized to edit the genomes of crop plants.<br><br>RESULTS: Here, we designed four gRNAs to mutate four LATE ELONGATED HYPOCOTYL (LHY) genes in soybean. In order to test whether the gRNAs could perform properly in transgenic soybean plants, we first tested the CRISPR construct in transgenic soybean hairy roots using Agrobacterium rhizogenes strain K599. Once confirmed, we performed stable soybean transformation and obtained 19 independent transgenic soybean plants. Subsequently, we obtained one T1 transgene-free homozygous quadruple mutant of GmLHY by self-crossing. The phenotypes of the T2-generation transgene-free quadruple mutant plants were observed, and the results showed that the quadruple mutant of GmLHY displayed reduced plant height and shortened internodes. The levels of endogenous gibberellic acid (GA3) in Gmlhy1a1b2a2b was lower than in the wild type (WT), and the shortened internode phenotype could be rescued by treatment with exogenous GA3. In addition, the relative expression levels of GA metabolic pathway genes in the quadruple mutant of GmLHY were significantly decreased in comparison to the WT. These results suggest that GmLHY encodes an MYB transcription factor that affects plant height through mediating the GA pathway in soybean. We also developed genetic markers for identifying mutants for application in breeding studies.<br><br>CONCLUSIONS: Our results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four GmLHY genes reduces soybean plant height and shortens internodes from 20 to 35 days after emergence (DAE). These findings provide insight into the mechanisms underlying plant height regulatory networks in soybean.}, }
@article {pmid31664995, year = {2019}, author = {Cook, NL and Pjanic, M and Emmerich, AG and Rao, AS and Hetty, S and Knowles, JW and Quertermous, T and Castillejo-López, C and Ingelsson, E}, title = {CRISPR-Cas9-mediated knockout of SPRY2 in human hepatocytes leads to increased glucose uptake and lipid droplet accumulation.}, journal = {BMC endocrine disorders}, volume = {19}, number = {1}, pages = {115}, pmid = {31664995}, issn = {1472-6823}, support = {20140422//Hjärt-Lungfonden/ ; R01 DK120565/DK/NIDDK NIH HHS/United States ; R01 DK106236-01A1/NH/NIH HHS/United States ; 2013.0126//Knut och Alice Wallenbergs Stiftelse/ ; R01DK107437/NH/NIH HHS/United States ; R01 DK106236/DK/NIDDK NIH HHS/United States ; P30 DK116074/DK/NIDDK NIH HHS/United States ; 2015-02907//Vetenskapsrådet/ ; }, mesh = {*CRISPR-Cas Systems ; Gene Expression Profiling ; Glucose/*metabolism ; Hep G2 Cells ; Hepatocytes/cytology/*metabolism ; Humans ; Intracellular Signaling Peptides and Proteins/*antagonists &amp; inhibitors/genetics/*metabolism ; Lipid Droplets/*metabolism ; *Lipogenesis ; Membrane Proteins/*antagonists &amp; inhibitors/genetics/*metabolism ; Phosphorylation ; Signal Transduction ; }, abstract = {BACKGROUND: The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic β-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance.<br><br>METHODS: CRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing.<br><br>RESULTS: The major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2.<br><br>CONCLUSION: These findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.}, }
@article {pmid31451668, year = {2019}, author = {Guo, P and Yang, J and Huang, J and Auguste, DT and Moses, MA}, title = {Therapeutic genome editing of triple-negative breast tumors using a noncationic and deformable nanolipogel.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {37}, pages = {18295-18303}, pmid = {31451668}, issn = {1091-6490}, support = {DP2 CA174495/CA/NCI NIH HHS/United States ; R01 CA185530/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Disease Models, Animal ; Drug Carriers/*chemistry ; Drug Delivery Systems/*methods ; Female ; *Gene Editing ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Genetic Therapy/methods ; Genetic Vectors ; Humans ; Intercellular Adhesion Molecule-1/genetics ; Lipids/chemistry ; Lipocalin-2/genetics ; Liposomes/chemistry ; Mice ; Mice, Nude ; Nanoparticles/*chemistry ; Triple Negative Breast Neoplasms/*drug therapy/*genetics ; }, abstract = {Triple-negative breast cancer (TNBC), which has the highest mortality rate of all breast cancer, is in urgent need of a therapeutic that hinders the spread and growth of cancer cells. CRISPR genome editing holds the promise of a potential cure for many genetic diseases, including TNBC; however, its clinical translation is being challenged by the lack of safe and effective nonviral delivery systems for in vivo therapeutic genome editing. Here we report the synthesis and application of a noncationic, deformable, and tumor-targeted nanolipogel system (tNLG) for CRISPR genome editing in TNBC tumors. We have demonstrated that tNLGs mediate a potent CRISPR knockout of Lipocalin 2 (Lcn2), a known breast cancer oncogene, in human TNBC cells in vitro and in vivo. The loss of Lcn2 significantly inhibits the migration and the mesenchymal phenotype of human TNBC cells and subsequently attenuates TNBC aggressiveness. In an orthotopic TNBC model, we have shown that systemically administered tNLGs mediated >81% CRISPR knockout of Lcn2 in TNBC tumor tissues, resulting in significant tumor growth suppression (>77%). Our proof-of-principle results provide experimental evidence that tNLGs can be used as a safe, precise, and effective delivery approach for in vivo CRISPR genome editing in TNBC.}, }
@article {pmid32239050, year = {2020}, author = {Lim, S and Kim, J and Kim, Y and Xu, D and Clark, DS}, title = {CRISPR/Cas-directed programmable assembly of multi-enzyme complexes.}, journal = {Chemical communications (Cambridge, England)}, volume = {}, number = {}, pages = {}, doi = {10.1039/d0cc01174f}, pmid = {32239050}, issn = {1364-548X}, abstract = {We describe a versatile CRISPR/Cas-based strategy to construct multi-enzyme complexes scaffolded on a DNA template in programmable patterns. Catalytically inactive dCas9 nuclease was used in combination with SpyCatcher-SpyTag chemistry to assemble enzymes in a highly modular fashion. Five enzymes comprising the violacein biosynthesis pathway were precisely organized in nanometer proximity; a notable increase in violacein production demonstrated the benefits of scaffolding.}, }
@article {pmid32238808, year = {2020}, author = {Fontana, J and Dong, C and Kiattisewee, C and Chavali, VP and Tickman, BI and Carothers, JM and Zalatan, JG}, title = {Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {1618}, doi = {10.1038/s41467-020-15454-y}, pmid = {32238808}, issn = {2041-1723}, abstract = {In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we lack predictive rules for designing effective gRNA target sites. Here, we identify multiple features of bacterial promoters that impose stringent requirements on CRISPRa target sites. Notably, we observe narrow, 2-4 base windows of effective sites with a periodicity corresponding to one helical turn of DNA, spanning ~40 bases and centered ~80 bases upstream of the TSS. However, we also identify two features suggesting the potential for broad scope: CRISPRa is effective at a broad range of σ70-family promoters, and an expanded PAM dCas9 allows the activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.}, }
@article {pmid32238445, year = {2020}, author = {Vigouroux, A and Bikard, D}, title = {CRISPR Tools To Control Gene Expression in Bacteria.}, journal = {Microbiology and molecular biology reviews : MMBR}, volume = {84}, number = {2}, pages = {}, doi = {10.1128/MMBR.00077-19}, pmid = {32238445}, issn = {1098-5557}, abstract = {SUMMARYCRISPR-Cas systems have been engineered as powerful tools to control gene expression in bacteria. The most common strategy relies on the use of Cas effectors modified to bind target DNA without introducing DNA breaks. These effectors can either block the RNA polymerase or recruit it through activation domains. Here, we discuss the mechanistic details of how Cas effectors can modulate gene expression by blocking transcription initiation or acting as transcription roadblocks. CRISPR-Cas tools can be further engineered to obtain fine-tuned control of gene expression or target multiple genes simultaneously. Several caveats in using these tools have also been revealed, including off-target effects and toxicity, making it important to understand the design rules of engineered CRISPR-Cas effectors in bacteria. Alternatively, some types of CRISPR-Cas systems target RNA and could be used to block gene expression at the posttranscriptional level. Finally, we review applications of these tools in high-throughput screens and the progress and challenges in introducing CRISPR knockdown to other species, including nonmodel bacteria with industrial or clinical relevance. A deep understanding of how CRISPR-Cas systems can be harnessed to control gene expression in bacteria and build powerful tools will certainly open novel research directions.}, }
@article {pmid32034392, year = {2020}, author = {Solomon, SM}, title = {Genome editing in animals: why FDA regulation matters.}, journal = {Nature biotechnology}, volume = {38}, number = {2}, pages = {142-143}, doi = {10.1038/s41587-020-0413-7}, pmid = {32034392}, issn = {1546-1696}, mesh = {Animals ; *CRISPR-Cas Systems ; Cattle ; *Gene Editing ; Genome ; Germ Cells ; Plasmids ; }, }
@article {pmid31844051, year = {2019}, author = {Valderrama, JA and Kulkarni, SS and Nizet, V and Bier, E}, title = {A bacterial gene-drive system efficiently edits and inactivates a high copy number antibiotic resistance locus.}, journal = {Nature communications}, volume = {10}, number = {1}, pages = {5726}, pmid = {31844051}, issn = {2041-1723}, support = {R01 GM117321/GM/NIGMS NIH HHS/United States ; }, mesh = {Anti-Bacterial Agents/pharmacology ; Biomedical Technology/methods ; Biotechnology/methods ; CRISPR-Cas Systems/genetics ; Colony Count, Microbial ; DNA Copy Number Variations/*genetics ; Drug Resistance, Bacterial/*genetics ; Escherichia coli/drug effects/genetics ; Gene Drive Technology/*methods ; Genes, Bacterial/*genetics ; Genetic Loci/*genetics ; Genetic Vectors/genetics ; Microbial Sensitivity Tests ; Plasmids/genetics ; RNA, Guide/genetics ; Transformation, Bacterial ; }, abstract = {Gene-drive systems in diploid organisms bias the inheritance of one allele over another. CRISPR-based gene-drive expresses a guide RNA (gRNA) into the genome at the site where the gRNA directs Cas9-mediated cleavage. In the presence of Cas9, the gRNA cassette and any linked cargo sequences are copied via homology-directed repair (HDR) onto the homologous chromosome. Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas9 cleavage site. This &quot;pro-active&quot; genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. Pro-AG can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine.}, }
@article {pmid31726032, year = {2019}, author = {Uribe, RV and van der Helm, E and Misiakou, MA and Lee, SW and Kol, S and Sommer, MOA}, title = {Discovery and Characterization of Cas9 Inhibitors Disseminated across Seven Bacterial Phyla.}, journal = {Cell host &amp; microbe}, volume = {26}, number = {5}, pages = {702}, doi = {10.1016/j.chom.2019.09.005}, pmid = {31726032}, issn = {1934-6069}, mesh = {*Bacteria ; *CRISPR-Cas Systems ; Metagenomics ; }, }
@article {pmid31722267, year = {2020}, author = {Kumbha, R and Hosny, N and Matson, A and Steinhoff, M and Hering, BJ and Burlak, C}, title = {Efficient production of GGTA1 knockout porcine embryos using a modified handmade cloning (HMC) method.}, journal = {Research in veterinary science}, volume = {128}, number = {}, pages = {59-68}, doi = {10.1016/j.rvsc.2019.10.021}, pmid = {31722267}, issn = {1532-2661}, mesh = {Animals ; *Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Galactosyltransferases/deficiency/*metabolism ; Gene Knockout Techniques ; Nuclear Transfer Techniques/*veterinary ; Oocytes/physiology ; *Sus scrofa ; Transplantation, Heterologous/*veterinary ; }, abstract = {Handmade cloning is a zona-free nuclear transfer approach and an economical, efficient, and simple micromanipulation-free alternative to dolly based traditional cloning (TC). In this study, based on handmade cloning with minor modifications, an optimized bi-oocyte fusion (BOF) cloning method was established to produce GGTA1 KO porcine embryos using the CRISPR/Cas9 gene editing system. The GGTA1 gene is responsible for the generation of Gal epitopes on the surface of porcine cells, triggering hyperacute immune rejection in preclinical porcine-to-human xenotransplantation. The purpose of the present study is to establish an efficient protocol for activation of porcine oocyte cytoplast-fibroblast fused constructs developed to GGTA1 KO blastocysts by the zona-free bi-oocyte fusion cloning method. High percentages of cleavage (90 ± 2.6%) and blastocyst rates (39 ± 4.0%) were achieved upon treatment with demecolcine-assisted oocyte enucleation followed by 6 V alternating current for proper alignment and single-step fusion technique using a single direct current pulse of 1.0 kV/cm for 9 μs duration, compared to the double-step fusion method with combined chemical activation using thimerosal and dithiothreitol. Overall blastocyst rate was higher for oocyte enucleation by demecolcine (0.4 μg/ml) and 45 min incubation (42 ± 1.5%) compared to without demecolcine incubation followed by complete chemical thimerosal/dithiothreitol activation (33 ± 1.1%). The blastocyst rate (39 ± 1.0%) was found to be significantly higher 1 h post-electrofusion, compared to at 0 and 4 h (28 ± 1.5 and 6 ± 1.5%, respectively). Blastocyst development rates for GGTA1 knockout embryos (38 ± 1.76%) were comparable to those obtained with wild-type embryos (41.1 ± 0.67%). In conclusion, we achieved high overall efficiency in production of GGTA1 KO blastocysts by modified HMC protocol.}, }
@article {pmid31557222, year = {2019}, author = {Lee, JE and Neumann, M and Duro, DI and Schmid, M}, title = {CRISPR-based tools for targeted transcriptional and epigenetic regulation in plants.}, journal = {PloS one}, volume = {14}, number = {9}, pages = {e0222778}, pmid = {31557222}, issn = {1932-6203}, mesh = {Arabidopsis/genetics ; Arabidopsis Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Epigenesis, Genetic/*genetics ; Feasibility Studies ; *Gene Expression Regulation, Plant ; Genetic Engineering/*methods ; Genetic Vectors/genetics ; Histone Code/genetics ; Plants, Genetically Modified/genetics ; Promoter Regions, Genetic/genetics ; *Transcriptional Activation ; }, abstract = {Programmable gene regulators that can modulate the activity of selected targets in trans are a useful tool for probing and manipulating gene function. CRISPR technology provides a convenient method for gene targeting that can also be adapted for multiplexing and other modifications to enable strong regulation by a range of different effectors. We generated a vector toolbox for CRISPR/dCas9-based targeted gene regulation in plants, modified with the previously described MS2 system to amplify the strength of regulation, and using Golden Gate-based cloning to enable rapid vector assembly with a high degree of flexibility in the choice of promoters, effectors and targets. We tested the system using the floral regulator FLOWERING LOCUS T (FT) as a target and a range of different effector domains including the transcriptional activator VP64, the H3K27 acetyltransferase p300 and the H3K9 methyltransferase KRYPTONITE. When transformed into Arabidopsis thaliana, several of the constructs caused altered flowering time phenotypes that were associated with changes in FT expression and/or epigenetic status, thus demonstrating the effectiveness of the system. The MS2-CRISPR/dCas9 system can be used to modulate transcriptional activity and epigenetic status of specific target genes in plants, and provides a versatile tool that can easily be used with different targets and types of regulation for a range of applications.}, }
@article {pmid31466822, year = {2019}, author = {Saito, H and Yokobayashi, Y}, title = {Editorial overview: Mammalian synthetic biology: from devices to multicellular systems.}, journal = {Current opinion in chemical biology}, volume = {52}, number = {}, pages = {A1-A2}, doi = {10.1016/j.cbpa.2019.07.010}, pmid = {31466822}, issn = {1879-0402}, mesh = {Animals ; CRISPR-Cas Systems ; Cell Differentiation ; Humans ; *Mammals ; *Synthetic Biology ; }, }
@article {pmid31283004, year = {2020}, author = {Dok, R and Bamps, M and Glorieux, M and Zhao, P and Sablina, A and Nuyts, S}, title = {Radiosensitization approaches for HPV-positive and HPV-negative head and neck squamous carcinomas.}, journal = {International journal of cancer}, volume = {146}, number = {4}, pages = {1075-1085}, pmid = {31283004}, issn = {1097-0215}, mesh = {Animals ; Benzimidazoles/administration &amp; dosage ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chemoradiotherapy/methods ; Chromones/administration &amp; dosage ; DNA Damage/drug effects/radiation effects ; DNA Repair/drug effects/radiation effects ; Female ; Head and Neck Neoplasms/pathology/*therapy/virology ; Humans ; Male ; Mice ; Middle Aged ; Morpholines/administration &amp; dosage ; Neoplasm Recurrence, Local/pathology/*therapy/virology ; Papillomaviridae/drug effects/genetics ; Papillomavirus Infections/pathology/*therapy/virology ; Radiation-Sensitizing Agents/*administration &amp; dosage ; Radiotherapy Dosage ; Squamous Cell Carcinoma of Head and Neck/pathology/*therapy/virology ; Treatment Outcome ; Xenograft Model Antitumor Assays ; }, abstract = {Radiotherapy is one of the most used treatment approaches for head and neck squamous cell carcinoma (HNSCC). Targeted inhibition of DNA repair machinery has the potential to improve treatment response by tailoring treatment to cancer cells lacking specific DNA repair pathways. Human papillomavirus (HPV)-negative and HPV-positive HNSCCs respond differently to radiotherapy treatment, suggesting that different approaches of DNA repair inhibition should be employed for these HNSCC groups. Here, we searched for optimal radiosensitization approaches for HPV-positive and HPV-negative HNSCCs by performing a targeted CRISPR-Cas9 screen. We found that inhibition of base excision repair resulted in a better radiotherapy response in HPV-positive HNSCC, which is correlated with upregulation of genes involved in base excision repair. In contrast, inhibition of nonhomologous end-joining and mismatch repair showed strong effects in both HNSCC groups. We validated the screen results by combining radiotherapy with targeted inhibition of DNA repair in several preclinical models including primary and recurrent patient-derived HNSCC xenografts. These findings underline the importance of stratifying HNSCC patients for combination treatments.}, }
@article {pmid31249142, year = {2019}, author = {Hou, L and Wang, Y and Liu, Y and Zhang, N and Shamovsky, I and Nudler, E and Tian, B and Dynlacht, BD}, title = {Paf1C regulates RNA polymerase II progression by modulating elongation rate.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {29}, pages = {14583-14592}, pmid = {31249142}, issn = {1091-6490}, support = {R01 GM126891/GM/NIGMS NIH HHS/United States ; R01 GM127267/GM/NIGMS NIH HHS/United States ; R01 GM084089/GM/NIGMS NIH HHS/United States ; R01 GM122395/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 GM129069/GM/NIGMS NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Carrier Proteins/genetics/*metabolism ; Cell Line ; Gene Knockout Techniques ; Histones/metabolism ; Mice ; Myoblasts ; Promoter Regions, Genetic/genetics ; RNA Polymerase II/*metabolism ; RNA, Small Interfering/metabolism ; *Transcription Elongation, Genetic ; *Transcription Termination, Genetic ; Ubiquitination/genetics ; }, abstract = {Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter-proximal regions and within the first 20 to 30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20 to 30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an &quot;accelerator&quot; over specific early gene body regions.}, }
@article {pmid31200335, year = {2019}, author = {Ishiguro, S and Mori, H and Yachie, N}, title = {DNA event recorders send past information of cells to the time of observation.}, journal = {Current opinion in chemical biology}, volume = {52}, number = {}, pages = {54-62}, doi = {10.1016/j.cbpa.2019.05.009}, pmid = {31200335}, issn = {1879-0402}, mesh = {Animals ; CRISPR-Cas Systems ; Cell Communication ; *Cells ; DNA/*genetics ; Humans ; Recombination, Genetic ; Transcriptome ; }, abstract = {While current omics and single cell technologies have enabled measurements of high-resolution molecular snapshots of cells at a large scale, these technologies all require destruction of samples and prevent us from analyzing dynamic changes in molecular profiles, phenotypes, and behaviors of individual cells in a complex system. One possible direction to overcome this issue is the development of a cell-embedded 'event recorder' system, whereby molecular and phenotypic information of a cell(s) can be obtained at the time of observation with their past event information stored in 'heritable polymers' of the same cell. This concept has been demonstrated by many synthetic cellular circuits that monitor and transmit a certain set of environmental and intracellular signals into DNA, and have now been further accelerated by recent CRISPR-related technologies. Notably, the discovery of the RT-Cas1-Cas2 system, which acquires sequences of cellular transcripts into a specific host genomic region, has enabled recording of a broader range of molecular profile histories in the DNA tapes of cells, to understand the dynamics of complex biological processes that cannot be addressed by current technologies.}, }
@article {pmid31171720, year = {2019}, author = {Jin, UH and Karki, K and Cheng, Y and Michelhaugh, SK and Mittal, S and Safe, S}, title = {The aryl hydrocarbon receptor is a tumor suppressor-like gene in glioblastoma.}, journal = {The Journal of biological chemistry}, volume = {294}, number = {29}, pages = {11342-11353}, pmid = {31171720}, issn = {1083-351X}, support = {P30 ES023512/ES/NIEHS NIH HHS/United States ; R01 CA202697/CA/NCI NIH HHS/United States ; R01 ES025713/ES/NIEHS NIH HHS/United States ; T32 ES026568/ES/NIEHS NIH HHS/United States ; }, mesh = {Animals ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism ; Brain Neoplasms/*genetics/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; Female ; Gene Knockdown Techniques ; *Genes, Tumor Suppressor ; Glioblastoma/*genetics/pathology ; Heterografts ; Humans ; Kynurenine/metabolism ; Mice ; Mice, Nude ; Protein Binding ; Receptors, Aryl Hydrocarbon/*genetics/metabolism ; }, abstract = {The aryl hydrocarbon receptor (AhR) plays an important role in maintaining cellular homeostasis and also in pathophysiology. For example, the interplay between the gut microbiome and microbially derived AhR ligands protects against inflammation along the gut-brain axis. The AhR and its ligands also inhibit colon carcinogenesis, but it has been reported that the AhR and its ligand kynurenine enhance glioblastoma (GBM). In this study, using both established and patient-derived GBM cells, we re-examined the role of kynurenine and the AhR in GBM, observing that kynurenine does not modulate AhR-mediated gene expression and does not affect invasion of GBM cells. Therefore, using an array of approaches, including ChIP, quantitative real-time PCR, and cell migration assays, we primarily focused on investigating the role of the AhR in GBM at the functional molecular and genomic levels. The results of transient and stable CRISPR/Cas9-mediated AhR knockdown in GBM cells indicated that loss of AhR enhances GBM tumor growth in a mouse xenograft model, increases GBM cell invasion, and up-regulates expression of pro-invasion/pro-migration genes, as determined by ingenuity pathway analysis of RNA-Seq data. We conclude that the AhR is a tumor suppressor-like gene in GBM; future studies are required to investigate whether the AhR could be a potential drug target for treating patients with GBM who express this receptor.}, }
@article {pmid31167792, year = {2019}, author = {Lessard, CB and Rodriguez, E and Ladd, TB and Minter, LM and Osborne, BA and Miele, L and Golde, TE and Ran, Y}, title = {Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells.}, journal = {The Journal of biological chemistry}, volume = {294}, number = {29}, pages = {11276-11285}, pmid = {31167792}, issn = {1083-351X}, support = {P01 CA166009/CA/NCI NIH HHS/United States ; U54 GM104940/GM/NIGMS NIH HHS/United States ; }, mesh = {Alzheimer Disease/genetics ; Amyloid Precursor Protein Secretases/metabolism ; Animals ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; HEK293 Cells ; Humans ; Hydrolysis ; Mice ; Presenilin-1/genetics/*physiology ; Presenilin-2/genetics/*physiology ; Substrate Specificity ; }, abstract = {Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.}, }
@article {pmid31167791, year = {2019}, author = {McGuire, CM and Collins, MP and Sun-Wada, G and Wada, Y and Forgac, M}, title = {Isoform-specific gene disruptions reveal a role for the V-ATPase subunit a4 isoform in the invasiveness of 4T1-12B breast cancer cells.}, journal = {The Journal of biological chemistry}, volume = {294}, number = {29}, pages = {11248-11258}, pmid = {31167791}, issn = {1083-351X}, support = {R01 GM034478/GM/NIGMS NIH HHS/United States ; }, mesh = {Breast Neoplasms/*enzymology/genetics/pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Membrane/enzymology ; Enzyme Inhibitors/pharmacology ; Female ; Humans ; Isoenzymes/antagonists &amp; inhibitors/genetics/*metabolism ; *Neoplasm Invasiveness ; Neoplasm Metastasis ; RNA, Messenger/genetics ; Vacuolar Proton-Translocating ATPases/antagonists &amp; inhibitors/genetics/*metabolism ; }, abstract = {The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump present in various intracellular membranes and at the plasma membrane of specialized cell types. Previous work has reported that plasma membrane V-ATPases are key players in breast cancer cell invasiveness. The two subunit a-isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, and expression of a3 has been shown to correlate with plasma membrane localization of the V-ATPase in various invasive human breast cancer cell lines. Here we analyzed the role of subunit a-isoforms in the invasive mouse breast cancer cell line, 4T1-12B. Quantitation of mRNA levels for each isoform by quantitative RT-PCR revealed that a4 is the dominant isoform expressed in these cells. Using a CRISPR/Cas9-based approach to disrupt the genes encoding each of the four V-ATPase subunit a-isoforms, we found that ablation of only the a4-encoding gene significantly inhibits invasion and migration of 4T1-12B cells. Additionally, cells with disrupted a4 exhibited reduced V-ATPase expression at the leading edge, suggesting that the a4 isoform is primarily responsible for targeting the V-ATPase to the plasma membrane in 4T1-12B cells. These findings suggest that different subunit a-isoforms may direct V-ATPases to the plasma membrane of different invasive breast cancer cell lines. They further suggest that expression of V-ATPases at the cell surface is the primary factor that promotes an invasive cancer cell phenotype.}, }
@article {pmid30642400, year = {2019}, author = {An, H and Skelt, L and Notaro, A and Highley, JR and Fox, AH and La Bella, V and Buchman, VL and Shelkovnikova, TA}, title = {ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional paraspeckles.}, journal = {Acta neuropathologica communications}, volume = {7}, number = {1}, pages = {7}, pmid = {30642400}, issn = {2051-5960}, support = {BUCHMAN/APR13/822-791/MNDA_/Motor Neurone Disease Association/United Kingdom ; Shelkovnikova/Oct17/968-799/MNDA_/Motor Neurone Disease Association/United Kingdom ; Buchman/Apr13/6096/MNDA_/Motor Neurone Disease Association/United Kingdom ; }, mesh = {Amyotrophic Lateral Sclerosis/genetics/*metabolism/*pathology ; CRISPR-Cas Systems ; Cell Line ; Cell Line, Tumor ; Cell Nucleus/*metabolism ; Humans ; Intranuclear Inclusion Bodies/*metabolism ; Loss of Function Mutation ; Protein Isoforms/metabolism ; RNA, Long Noncoding/*metabolism ; RNA-Binding Protein FUS/*genetics/*metabolism ; }, abstract = {Mutations in the FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). Mutant FUS is known to confer cytoplasmic gain of function but its effects in the nucleus are less understood. FUS is an essential component of paraspeckles, subnuclear bodies assembled on a lncRNA NEAT1. Paraspeckles may play a protective role specifically in degenerating spinal motor neurons. However it is still unknown how endogenous levels of mutant FUS would affect NEAT1/paraspeckles. Using novel cell lines with the FUS gene modified by CRISPR/Cas9 and human patient fibroblasts, we found that endogenous levels of mutant FUS cause accumulation of NEAT1 isoforms and paraspeckles. However, despite only mild cytoplasmic mislocalisation of FUS, paraspeckle integrity is compromised in these cells, as confirmed by reduced interaction of mutant FUS with core paraspeckle proteins NONO and SFPQ and increased NEAT1 extractability. This results in NEAT1 localisation outside paraspeckles, especially prominent under conditions of paraspeckle-inducing stress. Consistently, paraspeckle-dependent microRNA production, a readout for functionality of paraspeckles, is impaired in cells expressing mutant FUS. In line with the cellular data, we observed paraspeckle hyper-assembly in spinal neurons of ALS-FUS patients. Therefore, despite largely preserving its nuclear localisation, mutant FUS leads to loss (dysfunctional paraspeckles) and gain (excess of free NEAT1) of function in the nucleus. Perturbed fine structure and functionality of paraspeckles accompanied by accumulation of non-paraspeckle NEAT1 may contribute to the disease severity in ALS-FUS.}, }
@article {pmid32232367, year = {2020}, author = {Suárez, GA and Dugan, KR and Renda, BA and Leonard, SP and Gangavarapu, LS and Barrick, JE}, title = {Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkaa204}, pmid = {32232367}, issn = {1362-4962}, abstract = {One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1's native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.}, }
@article {pmid32232035, year = {2020}, author = {Calderón-Franco, D and Lin, Q and van Loosdrecht, MCM and Abbas, B and Weissbrodt, DG}, title = {Anticipating Xenogenic Pollution at the Source: Impact of Sterilizations on DNA Release From Microbial Cultures.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {8}, number = {}, pages = {171}, doi = {10.3389/fbioe.2020.00171}, pmid = {32232035}, issn = {2296-4185}, abstract = {The dissemination of DNA and xenogenic elements across waterways is under scientific and public spotlight due to new gene-editing tools, such as do-it-yourself (DIY) CRISPR-Cas kits deployable at kitchen table. Over decades, prevention of spread of genetically modified organisms (GMOs), antimicrobial resistances (AMR), and pathogens from transgenic systems has focused on microbial inactivation. However, sterilization methods have not been assessed for DNA release and integrity. Here, we investigated the fate of intracellular DNA from cultures of model prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae) cells that are traditionally used as microbial chassis for genetic modifications. DNA release was tracked during exposure of these cultures to conventional sterilization methods. Autoclaving, disinfection with glutaraldehyde, and microwaving are used to inactivate broths, healthcare equipment, and GMOs produced at kitchen table. DNA fragmentation and PCR-ability were measured on top of cell viability and morphology. Impact of these methods on DNA integrity was verified on a template of free λ DNA. Intense regular autoclaving (121°C, 20 min) resulted in the most severe DNA degradation and lowest household gene amplification capacity: 1.28 ± 0.11, 2.08 ± 0.03, and 4.96 ± 0.28 logs differences to the non-treated controls were measured from E. coli, S. cerevisiae, and λ DNA, respectively. Microwaving exerted strong DNA fragmentation after 100 s of exposure when free λ DNA was in solution (3.23 ± 0.06 logs difference) but a minor effect was observed when DNA was released from E. coli and S. cerevisiae (0.24 ± 0.14 and 1.32 ± 0.02 logs differences with the control, respectively). Glutaraldehyde prevented DNA leakage by preserving cell structures, while DNA integrity was not altered. The results show that current sterilization methods are effective on microorganism inactivation but do not safeguard an aqueous residue exempt of biologically reusable xenogenic material, being regular autoclaving the most severe DNA-affecting method. Reappraisal of sterilization methods is required along with risk assessment on the emission of DNA fragments in urban systems and nature.}, }
@article {pmid32230824, year = {2020}, author = {Zhang, Q and Zhang, L and Ross, P and Zhao, J and Zhang, H and Chen, W}, title = {Comparative Genomics of Lactobacillus crispatus from the Gut and Vagina Reveals Genetic Diversity and Lifestyle Adaptation.}, journal = {Genes}, volume = {11}, number = {4}, pages = {}, doi = {10.3390/genes11040360}, pmid = {32230824}, issn = {2073-4425}, support = {JUFSTR20180102//National First-class Discipline Program of Food Science and Technology/ ; No. 31820103010, 31530056//National Natural Science Foundation of China/ ; }, abstract = {Lactobacillus crispatus colonizes the human feces, human vagina, and the crops and ceca of chicken. To explore the genetic characteristics and evolutionary relationships of L. crispatus isolated from different niches, we selected 37 strains isolated from the human vagina (n = 17), human feces (n = 11), and chicken feces (n = 9), and used comparative genomics to explore the genetic information of L. crispatus from the feces and vagina. No significant difference was found in the three sources of genomic features such as genome size, GC content, and number of protein coding sequences (CDS). However, in a phylogenetic tree constructed based on core genes, vagina-derived L. crispatus and feces-derived strains were each clustered separately. Therefore, the niche exerted an important impact on the evolution of L. crispatus. According to gene annotation, the L. crispatus derived from the vagina possessed a high abundance of genes related to acid tolerance, redox reactions, pullulanase, and carbohydrate-binding modules (CBMs). These genes helped L. crispatus to better adapt to the acidic environment of the vagina and obtain more nutrients, maintaining its dominance in the vagina in competition with other strains. In feces-derived bacteria, more genes encoding CRISPR/Cas system, glycoside hydrolases (GHs) family, and tetracycline/lincomycin resistance genes were found to adapt to the complex intestinal environment. This study highlights the evolutionary relationship of L. crispatus strains isolated from the vagina and feces, and the adaptation of L. crispatus to the host environment.}, }
@article {pmid32078805, year = {2020}, author = {Komáromy, AM}, title = {CRISPR-Cas9 Disruption of Aquaporin 1: An Alternative to Glaucoma Eye Drop Therapy?.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {28}, number = {3}, pages = {706-708}, pmid = {32078805}, issn = {1525-0024}, mesh = {Aquaporin 1 ; *CRISPR-Cas Systems ; Ciliary Body ; Gene Editing ; Genetic Therapy ; *Glaucoma ; Humans ; Ophthalmic Solutions ; }, }
@article {pmid32029688, year = {2020}, author = {Ebright, RY and Lee, S and Wittner, BS and Niederhoffer, KL and Nicholson, BT and Bardia, A and Truesdell, S and Wiley, DF and Wesley, B and Li, S and Mai, A and Aceto, N and Vincent-Jordan, N and Szabolcs, A and Chirn, B and Kreuzer, J and Comaills, V and Kalinich, M and Haas, W and Ting, DT and Toner, M and Vasudevan, S and Haber, DA and Maheswaran, S and Micalizzi, DS}, title = {Deregulation of ribosomal protein expression and translation promotes breast cancer metastasis.}, journal = {Science (New York, N.Y.)}, volume = {367}, number = {6485}, pages = {1468-1473}, doi = {10.1126/science.aay0939}, pmid = {32029688}, issn = {1095-9203}, support = {R01 CA129933/CA/NCI NIH HHS/United States ; U01 EB012493/EB/NIBIB NIH HHS/United States ; U01 CA214297/CA/NCI NIH HHS/United States ; K12 CA087723/CA/NCI NIH HHS/United States ; T32 GM007753/GM/NIGMS NIH HHS/United States ; F30 CA232407/CA/NCI NIH HHS/United States ; F30 CA224588/CA/NCI NIH HHS/United States ; R01 GM100202/GM/NIGMS NIH HHS/United States ; T32 CA009361/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; }, mesh = {Animals ; Breast Neoplasms/genetics/*pathology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; *Neoplasm Metastasis ; Neoplasm Transplantation ; Neoplastic Cells, Circulating/*pathology ; Ribosomal Proteins/*genetics ; Sequence Analysis, RNA ; }, abstract = {Circulating tumor cells (CTCs) are shed into the bloodstream from primary tumors, but only a small subset of these cells generates metastases. We conducted an in vivo genome-wide CRISPR activation screen in CTCs from breast cancer patients to identify genes that promote distant metastasis in mice. Genes coding for ribosomal proteins and regulators of translation were enriched in this screen. Overexpression of RPL15, which encodes a component of the large ribosomal subunit, increased metastatic growth in multiple organs and selectively enhanced translation of other ribosomal proteins and cell cycle regulators. RNA sequencing of freshly isolated CTCs from breast cancer patients revealed a subset with strong ribosome and protein synthesis signatures; these CTCs expressed proliferation and epithelial markers and correlated with poor clinical outcome. Therapies targeting this aggressive subset of CTCs may merit exploration as potential suppressors of metastatic progression.}, }
@article {pmid31951830, year = {2020}, author = {Jang, HK and Bae, S}, title = {i-Silence, Please! An Alternative for Gene Disruption via Adenine Base Editors.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {28}, number = {2}, pages = {348-349}, pmid = {31951830}, issn = {1525-0024}, mesh = {*Adenine ; CRISPR-Cas Systems ; Codon, Initiator ; *Gene Editing ; Mutation ; }, }
@article {pmid32223887, year = {2020}, author = {Pyenson, NC and Marraffini, LA}, title = {Co-evolution within structured bacterial communities results in multiple expansion of CRISPR loci and enhanced immunity.}, journal = {eLife}, volume = {9}, number = {}, pages = {}, doi = {10.7554/eLife.53078}, pmid = {32223887}, issn = {2050-084X}, support = {DP1GM128184//NIH Office of the Director/ ; PATH Award//Burroughs Wellcome Fund/ ; Investigator/HHMI/Howard Hughes Medical Institute/United States ; }, abstract = {Type II CRISPR-Cas systems provide immunity against phages and plasmids that infect bacteria through the insertion of a short sequence from the invader's genome, known as the 'spacer', into the CRISPR locus. Spacers are transcribed into guide RNAs that direct the Cas9 nuclease to its target on the invader. In liquid cultures, most bacteria acquire a single spacer. Multiple spacer integration is a rare event which significance for immunity is poorly understood. Here, we found that when phage infections occur on solid media, a high proportion of the surviving colonies display complex morphologies that contain cells with multiple spacers. This is the result of the viral-host co-evolution, in which the immunity provided by the initial acquired spacer is easily overcome by escaper phages. Our results reveal the versatility of CRISPR-Cas immunity, which can respond with both single or multiple spacer acquisition schemes to solve challenges presented by different environments.}, }
@article {pmid31914134, year = {2020}, author = {Yamazaki, H and Shirakawa, K and Matsumoto, T and Kazuma, Y and Matsui, H and Horisawa, Y and Stanford, E and Sarca, AD and Shirakawa, R and Shindo, K and Takaori-Kondo, A}, title = {APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression.}, journal = {PloS one}, volume = {15}, number = {1}, pages = {e0223463}, pmid = {31914134}, issn = {1932-6203}, mesh = {Antibodies, Monoclonal, Humanized/pharmacology ; Bortezomib/pharmacology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Nucleus/genetics ; Cytidine Deaminase/*genetics ; DNA Damage/*genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Green Fluorescent Proteins/genetics ; Humans ; Lenalidomide/pharmacology ; Minor Histocompatibility Antigens/*genetics ; Multiple Myeloma/drug therapy/*genetics/pathology/radiotherapy ; Mutation/genetics ; Phosphorylcholine/analogs &amp; derivatives/pharmacology ; Polymethacrylic Acids/pharmacology ; RNA, Small Interfering/genetics ; Radiation ; Signal Transduction/drug effects ; }, abstract = {Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.}, }
@article {pmid31829959, year = {2019}, author = {Becú-Villalobos, D}, title = {[CRISPR-CAS9 in medicine, the saga continues].}, journal = {Medicina}, volume = {79}, number = {6}, pages = {522-523}, pmid = {31829959}, issn = {1669-9106}, mesh = {CRISPR-Cas Systems/*genetics ; Embryo, Mammalian ; Gene Editing ; Humans ; Mutation/genetics ; }, }
@article {pmid31124711, year = {2019}, author = {Straiton, J}, title = {Genetically modified humans: the X-Men of scientific research.}, journal = {BioTechniques}, volume = {66}, number = {6}, pages = {249-252}, doi = {10.2144/btn-2019-0056}, pmid = {31124711}, issn = {1940-9818}, mesh = {*CRISPR-Cas Systems ; DNA/*genetics ; Female ; Gene Editing/*methods ; Genetic Therapy/methods ; Genome, Human ; Human Genetics/methods ; Humans ; Male ; Organisms, Genetically Modified/genetics ; }, }
@article {pmid31039627, year = {2019}, author = {Campenhout, CV and Cabochette, P and Veillard, AC and Laczik, M and Zelisko-Schmidt, A and Sabatel, C and Dhainaut, M and Vanhollebeke, B and Gueydan, C and Kruys, V}, title = {Guidelines for optimized gene knockout using CRISPR/Cas9.}, journal = {BioTechniques}, volume = {66}, number = {6}, pages = {295-302}, doi = {10.2144/btn-2018-0187}, pmid = {31039627}, issn = {1940-9818}, mesh = {Animals ; *CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knockout Techniques/methods ; Genetic Loci ; HEK293 Cells ; Humans ; RNA, Guide/genetics ; Zebrafish/genetics ; }, abstract = {CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.}, }
@article {pmid30998092, year = {2019}, author = {Yang, B and Yang, L and Chen, J}, title = {Development and Application of Base Editors.}, journal = {The CRISPR journal}, volume = {2}, number = {2}, pages = {91-104}, doi = {10.1089/crispr.2019.0001}, pmid = {30998092}, issn = {2573-1602}, mesh = {APOBEC Deaminases ; Adenine ; Animals ; Bacteria/genetics ; CRISPR-Cas Systems ; Cytosine ; *Gene Editing ; Humans ; Plants/genetics ; }, abstract = {Base editing is emerging as a potent new strategy to achieve precise gene editing. By combining different nucleobase deaminases with Cas9 or Cpf1 proteins, several base editors have recently been developed to achieve targeted base conversions in different genomic contexts. Importantly, base editors have been successfully applied in animals, plants, and bacteria to induce precise substitutions at the single-base level with high efficiency. In this review, we summarize recent progress in the development and application of base editors and discuss some of the future directions of the technology.}, }
@article {pmid31889332, year = {2020}, author = {Borowicz, P and Chan, H and Medina, D and Gumpelmair, S and Kjelstrup, H and Spurkland, A}, title = {A simple and efficient workflow for generation of knock-in mutations in Jurkat T cells using CRISPR/Cas9.}, journal = {Scandinavian journal of immunology}, volume = {91}, number = {4}, pages = {e12862}, doi = {10.1111/sji.12862}, pmid = {31889332}, issn = {1365-3083}, support = {107561 - PR-2007-0193//Kreftforeningen/ ; //Anders Jahres Fond til Vitenskapens Fremme/ ; //Unifor/ ; //Universitetet i Oslo/ ; //Novo Nordisk Fonden/ ; }, mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Humans ; Jurkat Cells ; Mutation ; *T-Lymphocytes ; *Workflow ; }, abstract = {CRISPR/Cas9 is a powerful gene-editing tool allowing for specific gene manipulation at targeted sites in the genome. Here, we used CRISPR/Cas9-mediated gene editing to introduce single amino acid mutations into proteins involved in T cell receptor signalling pathways. Knock-in mutations were introduced in Jurkat T cells by homologous directed repair using single-stranded oligodeoxynucleotides. Specifically, we aimed to create targeted mutations at two loci within LCK, a constitutively expressed gene, and at three loci within SH2D2A, whose expression is induced upon T cell activation. Here, we present a simple workflow that can be applied by any laboratory equipped for cell culture work, utilizing basic flow cytometry, Western blotting and PCR techniques. Our data reveal that gene editing may be locus-dependent and can vary between target sites, also within a gene. In our two targeted genes, on average 2% of the clones harboured homozygous mutations as assessed by allele-specific PCR and subsequent sequencing. We highlight the importance of decreasing the clonal heterogeneity and developing robust screening methods to accurately select for correct knock-in mutations. Our workflow may be employed in other immune cell lines and acts as a useful approach for decoding functional mechanisms of proteins of interest.}, }
@article {pmid31831639, year = {2019}, author = {Guo, CJ and Allen, BM and Hiam, KJ and Dodd, D and Van Treuren, W and Higginbottom, S and Nagashima, K and Fischer, CR and Sonnenburg, JL and Spitzer, MH and Fischbach, MA}, title = {Depletion of microbiome-derived molecules in the host using Clostridium genetics.}, journal = {Science (New York, N.Y.)}, volume = {366}, number = {6471}, pages = {}, doi = {10.1126/science.aav1282}, pmid = {31831639}, issn = {1095-9203}, support = {R01 DK101674/DK/NIDDK NIH HHS/United States ; K08 DK110335/DK/NIDDK NIH HHS/United States ; DP1 DK113598/DK/NIDDK NIH HHS/United States ; DP5 OD023056/OD/NIH HHS/United States ; R01 DK085025/DK/NIDDK NIH HHS/United States ; S10 OD018040/OD/NIH HHS/United States ; R01 DK110174/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Clostridium/*genetics/*metabolism ; Gastrointestinal Microbiome/*genetics ; Gene Deletion ; Gene Editing/*methods ; *Host Microbial Interactions ; Metabolic Networks and Pathways/*genetics ; Mice ; Mice, Inbred Strains ; }, abstract = {The gut microbiota produce hundreds of molecules that are present at high concentrations in the host circulation. Unraveling the contribution of each molecule to host biology remains difficult. We developed a system for constructing clean deletions in Clostridium spp., the source of many molecules from the gut microbiome. By applying this method to the model commensal organism Clostridium sporogenes, we knocked out genes for 10 C. sporogenes-derived molecules that accumulate in host tissues. In mice colonized by a C. sporogenes for which the production of branched short-chain fatty acids was knocked out, we discovered that these microbial products have immunoglobulin A-modulatory activity.}, }
@article {pmid31661801, year = {2019}, author = {Yarra, R and Jin, L and Zhao, Z and Cao, H}, title = {Progress in Tissue Culture and Genetic Transformation of Oil Palm: An Overview.}, journal = {International journal of molecular sciences}, volume = {20}, number = {21}, pages = {}, pmid = {31661801}, issn = {1422-0067}, support = {(No.1630152019002),//Chinese Academy of Tropical Agricultural Sciences/ ; }, mesh = {Agrobacterium tumefaciens/genetics/metabolism ; Arecaceae/embryology/*genetics/*growth &amp; development ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Microinjections/methods ; Palm Oil/economics ; Plant Somatic Embryogenesis Techniques/methods ; Polyethylene Glycols/chemistry/pharmacology ; Protoplasts/cytology/drug effects ; Tissue Culture Techniques ; *Transformation, Genetic ; }, abstract = {Oil palm (Elaeis guineensis, Jacq.) is a prominent vegetable-oil-yielding crop. Cultivating high-yielding oil palm with improved traits is a pre-requisite to meet the increasing demands of palm oil consumption. However, tissue culture and biotechnological approaches can resolve these concerns. Over the past three decades, significant research has been carried out to develop tissue culture and genetic transformation protocols for oil palm. Somatic embryogenesis is an efficient platform for the micropropagation of oil palm on a large scale. In addition, various genetic transformation techniques, including microprojectile bombardment, Agrobacterium tumefaciens mediated, Polyethylene glycol mediated mediated, and DNA microinjection, have been developed by optimizing various parameters for the efficient genetic transformation of oil palm. This review mainly emphasizes the methods established for in vitro propagation and genetic transformation of oil palm. Finally, we propose the application of the genome editing tool CRISPR/Cas9 to improve the various traits in this oil yielding crop.}, }
@article {pmid31317418, year = {2019}, author = {Ermert, AL and Nogué, F and Stahl, F and Gans, T and Hughes, J}, title = {CRISPR/Cas9-Mediated Knockout of Physcomitrella patens Phytochromes.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2026}, number = {}, pages = {237-263}, doi = {10.1007/978-1-4939-9612-4_20}, pmid = {31317418}, issn = {1940-6029}, mesh = {Bryopsida/genetics/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair/genetics/physiology ; Phytochrome/*metabolism ; RNA, Guide/genetics ; Recombinational DNA Repair/genetics/physiology ; }, abstract = {Here we describe procedures for gene disruption and excision in Physcomitrella using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated 9) methods, exemplarily targeting phytochrome (PHY) gene loci. Thereby double-strand breaks (DSBs) are induced using a single guide RNA (sgRNA) with the Cas9 nuclease, leading to insertions or deletions (indels) due to incorrect repair by the nonhomologous-end joining (NHEJ) mechanism. We also include protocols for excision of smaller genomic fragments or whole genes either with or without homologous recombination-assisted repair. The protocol can be adapted to target several loci simultaneously, thereby allowing the physiological analysis of phenotypes that would be masked by functional redundancy. In our particular case, multiple PHY gene knockouts would likely be valuable in understanding phytochrome functions in mosses and, perhaps, higher plants too. Target sites for site-directed induction of DSBs are predicted with the CRISPOR online-tool and are inserted in silico into sequence matrices for the design of sgRNA expression cassettes. The resulting DNAs are cloned into Gateway DONOR vectors and the respective expression plasmids used for moss cotransformation with a Cas9 expression plasmid and a selectable marker (either on a separate plasmid or on one of the other plasmids). After the selection process, genomic DNA is extracted and transformants are analyzed by PCR fingerprinting.}, }
@article {pmid31267498, year = {2019}, author = {Ng, CL and Fidock, DA}, title = {Plasmodium falciparum In Vitro Drug Resistance Selections and Gene Editing.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2013}, number = {}, pages = {123-140}, pmid = {31267498}, issn = {1940-6029}, support = {R01 AI109023/AI/NIAID NIH HHS/United States ; R01 AI124678/AI/NIAID NIH HHS/United States ; R37 AI050234/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; Antimalarials/therapeutic use ; CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Malaria, Falciparum/*drug therapy/*prevention &amp; control ; Plasmodium falciparum/drug effects/*pathogenicity ; }, abstract = {Malaria continues to be a global health burden, threatening over 40% of the world's population. Drug resistance in Plasmodium falciparum, the etiological agent of the majority of human malaria cases, is compromising elimination efforts. New approaches to treating drug-resistant malaria benefit from defining resistance liabilities of known antimalarial agents and compounds in development and defining genetic changes that mediate loss of parasite susceptibility. Here, we present protocols for in vitro selection of drug-resistant parasites and for site-directed gene editing of candidate resistance mediators to test for causality.}, }
@article {pmid31260812, year = {2019}, author = {Hua, K and Zhang, J and Botella, JR and Ma, C and Kong, F and Liu, B and Zhu, JK}, title = {Perspectives on the Application of Genome-Editing Technologies in Crop Breeding.}, journal = {Molecular plant}, volume = {12}, number = {8}, pages = {1047-1059}, doi = {10.1016/j.molp.2019.06.009}, pmid = {31260812}, issn = {1752-9867}, mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics ; Gene Editing/*methods ; Genome, Plant/genetics/physiology ; Plant Breeding/*methods ; Plants, Genetically Modified/genetics/*metabolism/*physiology ; }, abstract = {Most conventional and modern crop-improvement methods exploit natural or artificially induced genetic variations and require laborious characterization of the progenies of multiple generations derived from time-consuming genetic crosses. Genome-editing systems, in contrast, provide the means to rapidly modify genomes in a precise and predictable way, making it possible to introduce improvements directly into elite varieties. Here, we describe the range of applications available to agricultural researchers using existing genome-editing tools. In addition to providing examples of genome-editing applications in crop breeding, we discuss the technical and social challenges faced by breeders using genome-editing tools for crop improvement.}, }
@article {pmid31220273, year = {2019}, author = {Wong, WR and Brugman, KI and Maher, S and Oh, JY and Howe, K and Kato, M and Sternberg, PW}, title = {Autism-associated missense genetic variants impact locomotion and neurodevelopment in Caenorhabditis elegans.}, journal = {Human molecular genetics}, volume = {28}, number = {13}, pages = {2271-2281}, pmid = {31220273}, issn = {1460-2083}, mesh = {Alleles ; Animals ; Autism Spectrum Disorder/*genetics ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Disease Models, Animal ; Fertility/genetics ; Genetic Association Studies ; Locomotion/genetics ; Mutation, Missense ; Neurodevelopmental Disorders/genetics ; Phenotype ; }, abstract = {Autism spectrum disorder (ASD) involves thousands of alleles in over 850 genes, but the current functional inference tools are not sufficient to predict phenotypic changes. As a result, the causal relationship of most of these genetic variants in the pathogenesis of ASD has not yet been demonstrated and an experimental method prioritizing missense alleles for further intensive analysis is crucial. For this purpose, we have designed a pipeline that uses Caenorhabditis elegans as a genetic model to screen for phenotype-changing missense alleles inferred from human ASD studies. We identified highly conserved human ASD-associated missense variants in their C. elegans orthologs, used a CRISPR/Cas9-mediated homology-directed knock-in strategy to generate missense mutants and analyzed their impact on behaviors and development via several broad-spectrum assays. All tested missense alleles were predicted to perturb protein function, but we found only 70% of them showed detectable phenotypic changes in morphology, locomotion or fecundity. Our findings indicate that certain missense variants in the C. elegans orthologs of human CACNA1D, CHD7, CHD8, CUL3, DLG4, GLRA2, NAA15, PTEN, SYNGAP1 and TPH2 impact neurodevelopment and movement functions, elevating these genes as candidates for future study into ASD. Our approach will help prioritize functionally important missense variants for detailed studies in vertebrate models and human cells.}, }
@article {pmid31216068, year = {2019}, author = {Sheng, Z and Wang, X and Ma, Y and Zhang, D and Yang, Y and Zhang, P and Zhu, H and Xu, N and Liang, S}, title = {MS-based strategies for identification of protein SUMOylation modification.}, journal = {Electrophoresis}, volume = {40}, number = {21}, pages = {2877-2887}, pmid = {31216068}, issn = {1522-2683}, support = {17ZD045//Health Commission of Sichuan Province/International ; 31470810//National Natural Science Foundation of China/International ; 2014AA020608//National 863 High Tech Foundation/International ; 31470810//National Natural Sciences Foundation of China/International ; 2017-GH02-00062-HZ//Chengdu Science &amp;Technology Program/International ; R01 AA020608/AA/NIAAA NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; Cells, Cultured ; Gene Editing ; Humans ; *Mass Spectrometry ; Models, Molecular ; *Peptides/analysis/chemistry/isolation &amp; purification ; Recombinant Fusion Proteins ; *Small Ubiquitin-Related Modifier Proteins ; *Sumoylation ; Ubiquitination ; }, abstract = {Protein SUMOylation modification conjugated with small ubiquitin-like modifiers (SUMOs) is one kind of PTMs, which exerts comprehensive roles in cellular functions, including gene expression regulation, DNA repair, intracellular transport, stress responses, and tumorigenesis. With the development of the peptide enrichment approaches and MS technology, more than 6000 SUMOylated proteins and about 40 000 SUMO acceptor sites have been identified. In this review, we summarize several popular approaches that have been developed for the identification of SUMOylated proteins in human cells, and further compare their technical advantages and disadvantages. And we also introduce identification approaches of target proteins which are co-modified by both SUMOylation and ubiquitylation. We highlight the emerging trends in the SUMOylation field as well. Especially, the advent of the clustered regularly interspaced short palindromic repeats/ Cas9 technique will facilitate the development of MS for SUMOylation identification.}, }
@article {pmid31209053, year = {2019}, author = {Darbellay, F and Bochaton, C and Lopez-Delisle, L and Mascrez, B and Tschopp, P and Delpretti, S and Zakany, J and Duboule, D}, title = {The constrained architecture of mammalian Hox gene clusters.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {27}, pages = {13424-13433}, pmid = {31209053}, issn = {1091-6490}, mesh = {Alleles ; Animals ; CCCTC-Binding Factor/metabolism ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation/genetics ; Genes, Homeobox/*genetics ; Genetic Loci/genetics ; Homeodomain Proteins/genetics ; Mammals/genetics ; Mice ; Multigene Family/*genetics ; Sequence Inversion ; Transcription Factors/genetics ; Transcription, Genetic/genetics ; }, abstract = {In many animal species with a bilateral symmetry, Hox genes are clustered either at one or at several genomic loci. This organization has a functional relevance, as the transcriptional control applied to each gene depends upon its relative position within the gene cluster. It was previously noted that vertebrate Hox clusters display a much higher level of genomic organization than their invertebrate counterparts. The former are always more compact than the latter, they are generally devoid of repeats and of interspersed genes, and all genes are transcribed by the same DNA strand, suggesting that particular factors constrained these clusters toward a tighter structure during the evolution of the vertebrate lineage. Here, we investigate the importance of uniform transcriptional orientation by engineering several alleles within the HoxD cluster, such as to invert one or several transcription units, with or without a neighboring CTCF site. We observe that the association between the tight structure of mammalian Hox clusters and their regulation makes inversions likely detrimental to the proper implementation of this complex genetic system. We propose that the consolidation of Hox clusters in vertebrates, including transcriptional polarity, evolved in conjunction with the emergence of global gene regulation via the flanking regulatory landscapes, to optimize a coordinated response of selected subsets of target genes in cis.}, }
@article {pmid31205318, year = {2019}, author = {Ciurkot, K and Vonk, B and Gorochowski, TE and Roubos, JA and Verwaal, R}, title = {CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {147}, pages = {}, doi = {10.3791/59350}, pmid = {31205318}, issn = {1940-087X}, support = {BB/L01386X/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; }, mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Saccharomyces cerevisiae/*pathogenicity ; }, abstract = {High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S. cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.}, }
@article {pmid31090950, year = {2019}, author = {Cheong, KH and Koh, JM and Jones, MC}, title = {Black Swans of CRISPR: Stochasticity and Complexity of Genetic Regulation.}, journal = {BioEssays : news and reviews in molecular, cellular and developmental biology}, volume = {41}, number = {7}, pages = {e1900032}, doi = {10.1002/bies.201900032}, pmid = {31090950}, issn = {1521-1878}, mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Gene Editing ; Gene Expression Regulation/*genetics ; Humans ; }, abstract = {Recent waves of controversies surrounding genetic engineering have spilled into popular science in Twitter battles between reputable scientists and their followers. Here, a cautionary perspective on the possible blind spots and risks of CRISPR and related biotechnologies is presented, focusing in particular on the stochastic nature of cellular control processes.}, }
@article {pmid31040209, year = {2019}, author = {Han, X and Wang, M and Duan, S and Franco, PJ and Kenty, JH and Hedrick, P and Xia, Y and Allen, A and Ferreira, LMR and Strominger, JL and Melton, DA and Meissner, TB and Cowan, CA}, title = {Generation of hypoimmunogenic human pluripotent stem cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {21}, pages = {10441-10446}, pmid = {31040209}, issn = {1091-6490}, support = {P30 DK040561/DK/NIDDK NIH HHS/United States ; }, mesh = {CRISPR-Cas Systems ; Cell Line ; Gene Knockout Techniques ; Genes, MHC Class I ; Genes, MHC Class II ; Genetic Engineering/*methods ; Humans ; Pluripotent Stem Cells/*immunology ; }, abstract = {Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage &quot;don't-eat me&quot; signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.}, }
@article {pmid30940750, year = {2019}, author = {Noble, C and Min, J and Olejarz, J and Buchthal, J and Chavez, A and Smidler, AL and DeBenedictis, EA and Church, GM and Nowak, MA and Esvelt, KM}, title = {Daisy-chain gene drives for the alteration of local populations.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {17}, pages = {8275-8282}, pmid = {30940750}, issn = {1091-6490}, support = {R00 DK102669/DK/NIDDK NIH HHS/United States ; }, mesh = {Animals ; Anopheles/genetics ; CRISPR-Cas Systems/*genetics ; Ecology ; Female ; Gene Drive Technology/*methods/*standards ; Genetic Engineering ; HEK293 Cells ; Humans ; Malaria/prevention &amp; control ; Male ; Organisms, Genetically Modified/*genetics ; RNA, Guide/genetics ; }, abstract = {If they are able to spread in wild populations, CRISPR-based gene-drive elements would provide new ways to address ecological problems by altering the traits of wild organisms, but the potential for uncontrolled spread tremendously complicates ethical development and use. Here, we detail a self-exhausting form of CRISPR-based drive system comprising genetic elements arranged in a daisy chain such that each drives the next. &quot;Daisy-drive&quot; systems can locally duplicate any effect achievable by using an equivalent self-propagating drive system, but their capacity to spread is limited by the successive loss of nondriving elements from one end of the chain. Releasing daisy-drive organisms constituting a small fraction of the local wild population can drive a useful genetic element nearly to local fixation for a wide range of fitness parameters without self-propagating spread. We additionally report numerous highly active guide RNA sequences sharing minimal homology that may enable evolutionarily stable daisy drive as well as self-propagating CRISPR-based gene drive. Especially when combined with threshold dependence, daisy drives could simplify decision-making and promote ethical use by enabling local communities to decide whether, when, and how to alter local ecosystems.}, }
@article {pmid30760561, year = {2019}, author = {Enderle, J and Dorn, A and Beying, N and Trapp, O and Puchta, H}, title = {The Protease WSS1A, the Endonuclease MUS81, and the Phosphodiesterase TDP1 Are Involved in Independent Pathways of DNA-protein Crosslink Repair in Plants.}, journal = {The Plant cell}, volume = {31}, number = {4}, pages = {775-790}, pmid = {30760561}, issn = {1532-298X}, mesh = {Arabidopsis/genetics/*metabolism ; Arabidopsis Proteins/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Endonucleases/genetics/metabolism ; Mutation/genetics ; }, abstract = {DNA-protein crosslinks (DPCs) represent a severe threat to the genome integrity; however, the main mechanisms of DPC repair were only recently elucidated in humans and yeast. Here we define the pathways for DPC repair in plants. Using CRISPR/Cas9, we could show that only one of two homologs of the universal repair proteases SPARTAN/ weak suppressor of smt3 (Wss1), WSS1A, is essential for DPC repair in Arabidopsis (Arabidopsis thaliana). WSS1A defective lines exhibit developmental defects and are hypersensitive to camptothecin (CPT) and cis-platin. Interestingly, the CRISPR/Cas9 mutants of TYROSYL-DNA PHOSPHODIESTERASE 1 (TDP1) are insensitive to CPT, and only the wss1A tdp1 double mutant reveals a higher sensitivity than the wss1A single mutant. This indicates that TDP1 defines a minor backup pathway in the repair of DPCs. Moreover, we found that knock out of the endonuclease METHYL METHANESULFONATE AND UV SENSITIVE PROTEIN 81 (MUS81) results in a strong sensitivity to DPC-inducing agents. The fact that wss1A mus81 and tdp1 mus81 double mutants exhibit growth defects and an increase in dead cells in root meristems after CPT treatment demonstrates that there are three independent pathways for DPC repair in Arabidopsis. These pathways are defined by their different biochemical specificities, as main actors, the DNA endonuclease MUS81 and the protease WSS1A, and the phosphodiesterase TDP1 as backup.}, }
@article {pmid30511203, year = {2019}, author = {Zhu, W and Saw, D and Weiss, M and Sun, Z and Wei, M and Shaligram, S and Wang, S and Su, H}, title = {Induction of Brain Arteriovenous Malformation Through CRISPR/Cas9-Mediated Somatic Alk1 Gene Mutations in Adult Mice.}, journal = {Translational stroke research}, volume = {10}, number = {5}, pages = {557-565}, pmid = {30511203}, issn = {1868-601X}, support = {R01 NS027713/NS/NINDS NIH HHS/United States ; R01 HL122774/HL/NHLBI NIH HHS/United States ; }, mesh = {Activin Receptors, Type II/*genetics ; Animals ; CRISPR-Cas Systems ; *Disease Models, Animal ; Endothelial Cells/metabolism ; Female ; Gene Expression ; Genetic Vectors ; Intracranial Arteriovenous Malformations/*genetics ; Male ; Mice, Inbred C57BL ; Mutation ; }, abstract = {Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. The pathogenesis of bAVM has not been fully understood. Animal models are important tools for dissecting bAVM pathogenesis and testing new therapies. We have developed several mouse bAVM models using genetically modified mice. However, due to the body size, mouse bAVM models have some limitations. Recent studies identified somatic mutations in sporadic human bAVM. To develop a feasible tool to create sporadic bAVM in rodent and animals larger than rodent, we made tests using the CRISPR/Cas9 technique to induce somatic gene mutations in mouse brain in situ. Two sequence-specific guide RNAs (sgRNAs) targeting mouse Alk1 exons 4 and 5 were cloned into pAd-Alk1e4sgRNA + e5sgRNA-Cas9 plasmid. These sgRNAs were capable to generate mutations in Alk1 gene in mouse cell lines. After packaged into adenovirus, Ad-Alk1e4sgRNA + e5sgRNA-Cas9 was co-injected with an adeno-associated viral vector expressing vascular endothelial growth factor (AAV-VEGF) into the brains of wild-type C57BL/6J mice. Eight weeks after viral injection, bAVMs were detected in 10 of 12 mice. Compared to the control (Ad-GFP/AAV-VEGF-injected) brain, 13% of Alk1 alleles were mutated and Alk1 expression was reduced by 26% in the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF-injected brains. Around the Ad-Alk1e4sgRNA + e5sgRNA-Cas9/AAV-VEGF injected site, Alk1-null endothelial cells were detected. Our data demonstrated that CRISPR/Cas9 is a feasible tool for generating bAVM model in animals.}, }
@article {pmid30362544, year = {2019}, author = {Khadempar, S and Familghadakchi, S and Motlagh, RA and Farahani, N and Dashtiahangar, M and Rezaei, H and Gheibi Hayat, SM}, title = {CRISPR-Cas9 in genome editing: Its function and medical applications.}, journal = {Journal of cellular physiology}, volume = {234}, number = {5}, pages = {5751-5761}, doi = {10.1002/jcp.27476}, pmid = {30362544}, issn = {1097-4652}, mesh = {Animals ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Gene Expression Regulation ; Gene Targeting/*methods ; Genetic Therapy/*methods ; Humans ; RNA, Guide/genetics/metabolism ; }, abstract = {The targeted genome modification using RNA-guided nucleases is associated with several advantages such as a rapid, easy, and efficient method that not only provides the manipulation and alteration of genes and functional studies for researchers, but also increases their awareness of the molecular basis of the disease and development of new and targeted therapeutic approaches. Different techniques have been emerged so far as the molecular scissors mediating targeted genome editing including zinc finger nuclease, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). CRISPR-Cas9 is a bacterial immune system against viruses in which the single-strand RNA-guided Cas9 nuclease is linked to the targeted complementary sequences to apply changes. The advances made in the transfer, modification, and emergence of specific solutions have led to the creation of different classes of CRISPR-Cas9. Since this robust tool is capable of direct correction of disease-causing mutations, its ability to treat genetic disorders has attracted the tremendous attention of researchers. Considering the reported cases of nonspecific targeting of Cas9 proteins, many studies focused on enhancing the Cas9 features. In this regard, significant advances have been made in choosing guide RNA, new enzymes and methods for identifying misplaced targeting. Here, we highlighted the history and various direct aspects of CRISPR-Cas9, such as precision in genomic targeting, system transfer and its control over correction events with its applications in future biological studies, and modern treatment of diseases.}, }
@article {pmid32221408, year = {2020}, author = {Wu, R and Chai, B and Cole, JR and Gunturu, SK and Guo, X and Tian, R and Gu, JD and Zhou, J and Tiedje, JM}, title = {Targeted assemblies of cas1 suggest CRISPR-Cas's response to soil warming.}, journal = {The ISME journal}, volume = {}, number = {}, pages = {}, doi = {10.1038/s41396-020-0635-1}, pmid = {32221408}, issn = {1751-7370}, support = {DE-SC0010715//U.S. Department of Energy (DOE)/ ; DE-FG02-99ER62848//U.S. Department of Energy (DOE)/ ; DBI-1356380//National Science Foundation (NSF)/ ; DBI-1759892//National Science Foundation (NSF)/ ; DBI-1356380//National Science Foundation (NSF)/ ; DBI-1759892//National Science Foundation (NSF)/ ; }, abstract = {There is an increasing interest in the clustered regularly interspaced short palindromic repeats CRISPR-associated protein (CRISPR-Cas) system to reveal potential virus-host dynamics. The universal and most conserved Cas protein, cas1 is an ideal marker to elucidate CRISPR-Cas ecology. We constructed eight Hidden Markov Models (HMMs) and assembled cas1 directly from metagenomes by a targeted-gene assembler, Xander, to improve detection capacity and resolve the diverse CRISPR-Cas systems. The eight HMMs were first validated by recovering all 17 cas1 subtypes from the simulated metagenome generated from 91 prokaryotic genomes across 11 phyla. We challenged the targeted method with 48 metagenomes from a tallgrass prairie in Central Oklahoma recovering 3394 cas1. Among those, 88 were near full length, 5 times more than in de-novo assemblies from the Oklahoma metagenomes. To validate the host assignment by cas1, the targeted-assembled cas1 was mapped to the de-novo assembled contigs. All the phylum assignments of those mapped contigs were assigned independent of CRISPR-Cas genes on the same contigs and consistent with the host taxonomies predicted by the mapped cas1. We then investigated whether 8 years of soil warming altered cas1 prevalence within the communities. A shift in microbial abundances was observed during the year with the biggest temperature differential (mean 4.16 °C above ambient). cas1 prevalence increased and even in the phyla with decreased microbial abundances over the next 3 years, suggesting increasing virus-host interactions in response to soil warming. This targeted method provides an alternative means to effectively mine cas1 from metagenomes and uncover the host communities.}, }
@article {pmid32221291, year = {2020}, author = {Garcia-Doval, C and Schwede, F and Berk, C and Rostøl, JT and Niewoehner, O and Tejero, O and Hall, J and Marraffini, LA and Jinek, M}, title = {Activation and self-inactivation mechanisms of the cyclic oligoadenylate-dependent CRISPR ribonuclease Csm6.}, journal = {Nature communications}, volume = {11}, number = {1}, pages = {1596}, doi = {10.1038/s41467-020-15334-5}, pmid = {32221291}, issn = {2041-1723}, support = {205321_169612//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; NCCR RNA and Disease//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; NCCR RNA &amp; Disease//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; DP1GM128184//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; ERC-CoG-820152//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; }, abstract = {Bacterial and archaeal CRISPR-Cas systems provide RNA-guided immunity against genetic invaders such as bacteriophages and plasmids. Upon target RNA recognition, type III CRISPR-Cas systems produce cyclic-oligoadenylate second messengers that activate downstream effectors, including Csm6 ribonucleases, via their CARF domains. Here, we show that Enteroccocus italicus Csm6 (EiCsm6) degrades its cognate cyclic hexa-AMP (cA6) activator, and report the crystal structure of EiCsm6 bound to a cA6 mimic. Our structural, biochemical, and in vivo functional assays reveal how cA6 recognition by the CARF domain activates the Csm6 HEPN domains for collateral RNA degradation, and how CARF domain-mediated cA6 cleavage provides an intrinsic off-switch to limit Csm6 activity in the absence of ring nucleases. These mechanisms facilitate rapid invader clearance and ensure termination of CRISPR interference to limit self-toxicity.}, }
@article {pmid32219408, year = {2020}, author = {George, AF and Jang, KS and Nyegaard, M and Neidleman, J and Spitzer, TL and Xie, G and Chen, JC and Herzig, E and Laustsen, A and Marques de Menezes, EG and Houshdaran, S and Pilcher, CD and Norris, PJ and Jakobsen, MR and Greene, WC and Giudice, LC and Roan, NR}, title = {Seminal plasma promotes decidualization of endometrial stromal fibroblasts in vitro from women with and without inflammatory disorders in a manner dependent on interleukin-11 signaling.}, journal = {Human reproduction (Oxford, England)}, volume = {}, number = {}, pages = {}, doi = {10.1093/humrep/deaa015}, pmid = {32219408}, issn = {1460-2350}, abstract = {STUDY QUESTION: Do seminal plasma (SP) and its constituents affect the decidualization capacity and transcriptome of human primary endometrial stromal fibroblasts (eSFs)?<br><br>SUMMARY ANSWER: SP promotes decidualization of eSFs from women with and without inflammatory disorders (polycystic ovary syndrome (PCOS), endometriosis) in a manner that is not mediated through semen amyloids and that is associated with a potent transcriptional response, including the induction of interleukin (IL)-11, a cytokine important for SP-induced decidualization.<br><br>WHAT IS KNOWN ALREADY: Clinical studies have suggested that SP can promote implantation, and studies in vitro have demonstrated that SP can promote decidualization, a steroid hormone-driven program of eSF differentiation that is essential for embryo implantation and that is compromised in women with the inflammatory disorders PCOS and endometriosis.<br><br>STUDY DESIGN, SIZE, DURATION: This is a cross-sectional study involving samples treated with vehicle alone versus treatment with SP or SP constituents. SP was tested for the ability to promote decidualization in vitro in eSFs from women with or without PCOS or endometriosis (n = 9). The role of semen amyloids and fractionated SP in mediating this effect and in eliciting transcriptional changes in eSFs was then studied. Finally, the role of IL-11, a cytokine with a key role in implantation and decidualization, was assessed as a mediator of the SP-facilitated decidualization.<br><br>eSFs and endometrial epithelial cells (eECs) were isolated from endometrial biopsies from women of reproductive age undergoing benign gynecologic procedures and maintained in vitro. Assays were conducted to assess whether the treatment of eSFs with SP or SP constituents affects the rate and extent of decidualization in women with and without inflammatory disorders. To characterize the response of the endometrium to SP and SP constituents, RNA was isolated from treated eSFs or eECs and analyzed by RNA sequencing (RNAseq). Secreted factors in conditioned media from treated cells were analyzed by Luminex and ELISA. The role of IL-11 in SP-induced decidualization was assessed through Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-9-mediated knockout experiments in primary eSFs.<br><br>SP promoted decidualization both in the absence and presence of steroid hormones (P < 0.05 versus vehicle) in a manner that required seminal proteins. Semen amyloids did not promote decidualization and induced weak transcriptomic and secretomic responses in eSFs. In contrast, fractionated SP enriched for seminal microvesicles (MVs) promoted decidualization. IL-11 was one of the most potently SP-induced genes in eSFs and was important for SP-facilitated decidualization.<br><br>LARGE SCALE DATA: RNAseq data were deposited in the Gene Expression Omnibus repository under series accession number GSE135640.<br><br>This study is limited to in vitro analyses.<br><br>Our results support the notion that SP promotes decidualization, including within eSFs from women with inflammatory disorders. Despite the general ability of amyloids to induce cytokines known to be important for implantation, semen amyloids poorly signaled to eSFs and did not promote their decidualization. In contrast, fractionated SP enriched for MVs promoted decidualization and induced a transcriptional response in eSFs that overlapped with that of SP. Our results suggest that SP constituents, possibly those associated with MVs, can promote decidualization of eSFs in an IL-11-dependent manner in preparation for implantation.<br><br>This project was supported by NIH (R21AI116252, R21AI122821 and R01AI127219) to N.R.R. and (P50HD055764) to L.C.G. The authors declare no conflict of interest.}, }
@article {pmid32218510, year = {2020}, author = {Mahendra, C and Christie, KA and Osuna, BA and Pinilla-Redondo, R and Kleinstiver, BP and Bondy-Denomy, J}, title = {Broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer.}, journal = {Nature microbiology}, volume = {5}, number = {4}, pages = {620-629}, doi = {10.1038/s41564-020-0692-2}, pmid = {32218510}, issn = {2058-5276}, support = {R01GM127489//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R00-CA218870//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01GM127489//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01GM127489//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R00-CA218870//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01GM127489//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, abstract = {CRISPR-Cas adaptive immune systems protect bacteria and archaea against their invading genetic parasites, including bacteriophages/viruses and plasmids. In response to this immunity, many phages have anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas targeting. To date, anti-CRISPR genes have primarily been discovered in phage or prophage genomes. Here, we uncovered acr loci on plasmids and other conjugative elements present in Firmicutes using the Listeria acrIIA1 gene as a marker. The four identified genes, found in Listeria, Enterococcus, Streptococcus and Staphylococcus genomes, can inhibit type II-A SpyCas9 or SauCas9, and are thus named acrIIA16-19. In Enterococcus faecalis, conjugation of a Cas9-targeted plasmid was enhanced by anti-CRISPRs derived from Enterococcus conjugative elements, highlighting a role for Acrs in the dissemination of plasmids. Reciprocal co-immunoprecipitation showed that each Acr protein interacts with Cas9, and Cas9-Acr complexes were unable to cleave DNA. Northern blotting suggests that these anti-CRISPRs manipulate single guide RNA length, loading or stability. Mirroring their activity in bacteria, AcrIIA16 and AcrIIA17 provide robust and highly potent broad-spectrum inhibition of distinct Cas9 proteins in human cells (for example, SpyCas9, SauCas9, SthCas9, NmeCas9 and CjeCas9). This work presents a focused analysis of non-phage Acr proteins, demonstrating a role in horizontal gene transfer bolstered by broad-spectrum CRISPR-Cas9 inhibition.}, }
@article {pmid32217751, year = {2020}, author = {Walton, RT and Christie, KA and Whittaker, MN and Kleinstiver, BP}, title = {Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.}, journal = {Science (New York, N.Y.)}, volume = {}, number = {}, pages = {}, doi = {10.1126/science.aba8853}, pmid = {32217751}, issn = {1095-9203}, abstract = {Manipulation of DNA by CRISPR-Cas enzymes requires the recognition of a protospacer adjacent motif (PAM), limiting target site recognition to a subset of sequences. To remove this constraint, we engineered variants of Streptococcus pyogenes Cas9 (SpCas9) to eliminate the NGG PAM requirement. We developed a variant named SpG capable of targeting an expanded set of NGN PAMs, and further optimized this enzyme to develop a near-PAMless SpCas9 variant named SpRY (NRN>NYN PAMs). SpRY nuclease and base-editor variants can target almost all PAMs, exhibiting robust activities on a wide range of sites with NRN PAMs in human cells and lower but substantial activity on those with NYN PAMs. Using SpG and SpRY, we generated previously inaccessible disease-relevant genetic variants, supporting the utility of high-resolution targeting across genome editing applications.}, }
@article {pmid32217510, year = {2020}, author = {Tang, LC and Gu, F}, title = {Next-generation CRISPR-Cas for genome editing: focusing on the Cas protein and PAM.}, journal = {Yi chuan = Hereditas}, volume = {42}, number = {3}, pages = {236-249}, doi = {10.16288/j.yczz.19-297}, pmid = {32217510}, issn = {0253-9772}, abstract = {The emergence of the gene editing technology, especial CRISPR-Cas (clustered regularly intersected short palindromic repeats and CRISPR associated proteins), has greatly promoted the ability of human beings to transform natural species. It has been widely harnessed for the engineering in the medical, industrial, agricultural and other fields. The key component of the CRISPR-Cas system, the Cas protein, possesses its specific features, including self-activity, recognition site, cutting end and guide RNA. PAM (protein assistant motif) is a number of nucleotides adjacent to the target site, which is very important for the Cas protein to recognize the target sequence and is also the key characteristic of CRISPR-Cas. There are several reported methods for identification of PAM. In this review, we summarize the searching for the Cas protein, the identification of Cas mutants with desired traits and the mapping of the PAM (including the extending of PAM spectrum), in order to provide a reference for the development and optimization of next-generation gene editing tools.}, }
@article {pmid32216029, year = {2020}, author = {Karunarathna, NL and Wang, H and Harloff, HJ and Jiang, L and Jung, C}, title = {Elevating seed oil content in a polypoid crop by induced mutations in SEED FATTY ACID REDUCER genes.}, journal = {Plant biotechnology journal}, volume = {}, number = {}, pages = {}, doi = {10.1111/pbi.13381}, pmid = {32216029}, issn = {1467-7652}, abstract = {Plant based oils are valuable agricultural products, and seed oil content (SOC) is the major yield component in oil crops. Increasing SOC has been successfully targeted through the selection and genetic modification of oil biosynthesis. The SOC in rapeseed declined during the seed maturation and eventually caused the final accumulated seed oil quantity. However, genes involved in oil degradation during seed maturity are not deeply studied so far. We performed a candidate gene association study using a world-wide collection of rapeseed germplasm. We identified SEED FATTY ACID REDUCER (SFAR) genes, which had a significant effect on SOC and fatty acid (FA) composition. SFAR genes belong to the GDSL lipases, and GDSL lipases have a broad range of functions in plants. After quantification of gene expression using RNA-seq and quantitative PCR, we used targeted (CRISPR-Cas mediated) and random (chemical) mutagenesis to modify turnover rates of seed oil in winter rapeseed. For the first time, we demonstrate significant increase of SOC in a crop after knocking out members of the BnSFAR4 and BnSFAR5 gene families without pleiotropic effects on seed germination, vigor, and oil mobilization. Our results offer new perspectives for improving oil yield by targeted mutagenesis.}, }
@article {pmid32209973, year = {2020}, author = {Meyer, M and Kuffner, K and Winter, J and Neumann, ID and Wetzel, CH and Jurek, B}, title = {Myocyte Enhancer Factor 2A (MEF2A) Defines Oxytocin-Induced Morphological Effects and Regulates Mitochondrial Function in Neurons.}, journal = {International journal of molecular sciences}, volume = {21}, number = {6}, pages = {}, doi = {10.3390/ijms21062200}, pmid = {32209973}, issn = {1422-0067}, support = {Ne465/27-1//Deutsche Forschungsgemeinschaft/ ; Ne465/31-1//Deutsche Forschungsgemeinschaft/ ; JU3039/1-1//Deutsche Forschungsgemeinschaft/ ; WE2289/10-1//Deutsche Forschungsgemeinschaft/ ; GRK2174//Deutsche Forschungsgemeinschaft/ ; 422182557//Deutsche Forschungsgemeinschaft/ ; }, abstract = {The neuropeptide oxytocin (OT) is a well-described modulator of socio-emotional traits, such as anxiety, stress, social behavior, and pair bonding. However, when dysregulated, it is associated with adverse psychiatric traits, such as various aspects of autism spectrum disorder (ASD). In this study, we identify the transcription factor myocyte enhancer factor 2A (MEF2A) as the common link between OT and cellular changes symptomatic for ASD, encompassing neuronal morphology, connectivity, and mitochondrial function. We provide evidence for MEF2A as the decisive factor defining the cellular response to OT: while OT induces neurite retraction in MEF2A expressing neurons, OT causes neurite outgrowth in absence of MEF2A. A CRISPR-Cas-mediated knockout of MEF2A and retransfection of an active version or permanently inactive mutant, respectively, validated our findings. We also identified the phosphatase calcineurin as the main upstream regulator of OT-induced MEF2A signaling. Further, MEF2A signaling dampens mitochondrial functioning in neurons, as MEF2A knockout cells show increased maximal cellular respiration, spare respiratory capacity, and total cellular ATP. In summary, we reveal a central role for OT-induced MEF2A activity as major regulator of cellular morphology as well as neuronal connectivity and mitochondrial functioning, with broad implications for a potential treatment of disorders based on morphological alterations or mitochondrial dysfunction.}, }
@article {pmid31821359, year = {2019}, author = {Matson, AW and Hosny, N and Swanson, ZA and Hering, BJ and Burlak, C}, title = {Optimizing sgRNA length to improve target specificity and efficiency for the GGTA1 gene using the CRISPR/Cas9 gene editing system.}, journal = {PloS one}, volume = {14}, number = {12}, pages = {e0226107}, pmid = {31821359}, issn = {1932-6203}, mesh = {Animals ; Binding Sites ; CRISPR-Cas Systems/*genetics ; DNA/genetics/*metabolism ; DNA Cleavage ; Galactosyltransferases/deficiency/*genetics ; Gene Editing/*methods ; RNA, Guide/chemistry/*metabolism ; Ribonucleoproteins/metabolism ; Swine ; }, abstract = {The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.}, }
@article {pmid31821343, year = {2019}, author = {Elso, CM and Scott, NA and Mariana, L and Masterman, EI and Sutherland, APR and Thomas, HE and Mannering, SI}, title = {Replacing murine insulin 1 with human insulin protects NOD mice from diabetes.}, journal = {PloS one}, volume = {14}, number = {12}, pages = {e0225021}, pmid = {31821343}, issn = {1932-6203}, mesh = {Animals ; CRISPR-Cas Systems ; Diabetes Mellitus, Type 1/*genetics/metabolism ; Disease Models, Animal ; Humans ; Insulin/*genetics/metabolism ; Insulin-Secreting Cells/*metabolism ; Islets of Langerhans/metabolism ; Mice ; Mice, Inbred NOD ; Pancreas/metabolism ; }, abstract = {Type 1, or autoimmune, diabetes is caused by the T-cell mediated destruction of the insulin-producing pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes akin to human type 1 diabetes. For this reason, the NOD mouse has been the preeminent murine model for human type 1 diabetes research for several decades. However, humanized mouse models are highly sought after because they offer both the experimental tractability of a mouse model and the clinical relevance of human-based research. Autoimmune T-cell responses against insulin, and its precursor proinsulin, play central roles in the autoimmune responses against pancreatic beta cells in both humans and NOD mice. As a first step towards developing a murine model of the human autoimmune response against pancreatic beta cells we set out to replace the murine insulin 1 gene (Ins1) with the human insulin gene (Ins) using CRISPR/Cas9. Here we describe a NOD mouse strain that expresses human insulin in place of murine insulin 1, referred to as HuPI. HuPI mice express human insulin, and C-peptide, in their serum and pancreata and have normal glucose tolerance. Compared with wild type NOD mice, the incidence of diabetes is much lower in HuPI mice. Only 15-20% of HuPI mice developed diabetes after 300 days, compared to more than 60% of unmodified NOD mice. Immune-cell infiltration into the pancreatic islets of HuPI mice was not detectable at 100 days but was clearly evident by 300 days. This work highlights the feasibility of using CRISPR/Cas9 to create mouse models of human diseases that express proteins pivotal to the human disease. Furthermore, it reveals that even subtle changes in proinsulin protect NOD mice from diabetes.}, }
@article {pmid31743770, year = {2020}, author = {Liu, X and Zhang, P and Zhang, X and Li, X and Bai, Y and Ao, Y and Hexig, B and Guo, X and Liu, D}, title = {Fgf21 knockout mice generated using CRISPR/Cas9 reveal genetic alterations that may affect hair growth.}, journal = {Gene}, volume = {733}, number = {}, pages = {144242}, doi = {10.1016/j.gene.2019.144242}, pmid = {31743770}, issn = {1879-0038}, mesh = {Animals ; CRISPR-Cas Systems/genetics ; Female ; Fibroblast Growth Factors/*genetics/metabolism ; Genetic Engineering/methods ; Hair/*metabolism ; Hair Follicle/*growth &amp; development/metabolism ; MAP Kinase Signaling System/physiology ; Male ; Mice ; Mice, Knockout ; Phosphatidylinositol 3-Kinases/metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Signal Transduction/physiology ; }, abstract = {OBJECTIVE: To investigate fibroblast growth factor 21 (Fgf21) alterations that may affect hair growth and the underlying molecular mechanisms by constructing Fgf21 global knockout (KO) mice using microinjection-mediated CRISPR/Cas9.<br><br>RESULTS: Following genomic DNA sequencing, we identified 18 mice carrying Ffg21 mutations among the total 63 offspring mice obtained by injecting 340 embryos, which yielded a mutation rate of 28.6 percent. Of these 18 mice, three had both alleles knocked out and 15 were monoallelic KO mice. Compared with the wild-type (WT) mice, the phenotypic analysis showed that the litter size of Fgf21 KO mice significantly reduced (p < 0.05), but physiological indexes of the birth weight, gender rate, body weight (0-8 week) and body weight of adult male and female were no significant difference (p > 0.05). Compared to WT mice, physiological anatomy indicated that the morphological characters of vital organs in Fgf21 KO mice were normal. Depilation experiments demonstrated that compared to the WT mice, the hair regrowth speed was reduced in the Fgf21 KO mice. The number of hair shafts in these mice considerably decreased, as indicated by the tissue sample analyses. Real-time quantitative PCR showed that Erk and Akt expression in the KO mice was significantly decreased (P < 0.05), whereas western blotting demonstrated that the expression of Erk and Akt proteins and their phosphorylation levels in KO mice decreased at different rates (P < 0.05).<br><br>CONCLUSIONS: Fgf21 was shown to affect hair follicle development and growth cycle, which may be associated with Pi3k/Akt and Mapk/Erk signaling pathways.}, }
@article {pmid31515362, year = {2019}, author = {Kaiser, J}, title = {CRISPR reveals some cancer drugs hit unexpected targets.}, journal = {Science (New York, N.Y.)}, volume = {365}, number = {6458}, pages = {1065}, doi = {10.1126/science.365.6458.1065}, pmid = {31515362}, issn = {1095-9203}, mesh = {Antineoplastic Agents/*pharmacology ; *CRISPR-Cas Systems ; Cyclin-Dependent Kinases/antagonists &amp; inhibitors ; *Drug Development ; Gene Silencing ; Humans ; Molecular Targeted Therapy ; Neoplasms/*drug therapy ; Quinolones/pharmacology ; }, }
@article {pmid31488703, year = {2019}, author = {Wang, H and Nakamura, M and Abbott, TR and Zhao, D and Luo, K and Yu, C and Nguyen, CM and Lo, A and Daley, TP and La Russa, M and Liu, Y and Qi, LS}, title = {CRISPR-mediated live imaging of genome editing and transcription.}, journal = {Science (New York, N.Y.)}, volume = {365}, number = {6459}, pages = {1301-1305}, doi = {10.1126/science.aax7852}, pmid = {31488703}, issn = {1095-9203}, support = {U01 EB021240/EB/NIBIB NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Cell Line, Tumor ; DNA/analysis ; DNA Breaks, Double-Stranded ; *Gene Editing ; Genetic Vectors ; HEK293 Cells ; Humans ; In Situ Hybridization, Fluorescence/*methods ; Microscopy, Fluorescence ; Molecular Imaging ; RNA/analysis ; RNA, Guide/genetics ; T-Lymphocytes ; }, abstract = {We report a robust, versatile approach called CRISPR live-cell fluorescent in situ hybridization (LiveFISH) using fluorescent oligonucleotides for genome tracking in a broad range of cell types, including primary cells. An intrinsic stability switch of CRISPR guide RNAs enables LiveFISH to accurately detect chromosomal disorders such as Patau syndrome in prenatal amniotic fluid cells and track multiple loci in human T lymphocytes. In addition, LiveFISH tracks the real-time movement of DNA double-strand breaks induced by CRISPR-Cas9-mediated editing and consequent chromosome translocations. Finally, by combining Cas9 and Cas13 systems, LiveFISH allows for simultaneous visualization of genomic DNA and RNA transcripts in living cells. The LiveFISH approach enables real-time live imaging of DNA and RNA during genome editing, transcription, and rearrangements in single cells.}, }
@article {pmid31416932, year = {2019}, author = {Wang, D and Stoveken, HM and Zucca, S and Dao, M and Orlandi, C and Song, C and Masuho, I and Johnston, C and Opperman, KJ and Giles, AC and Gill, MS and Lundquist, EA and Grill, B and Martemyanov, KA}, title = {Genetic behavioral screen identifies an orphan anti-opioid system.}, journal = {Science (New York, N.Y.)}, volume = {365}, number = {6459}, pages = {1267-1273}, pmid = {31416932}, issn = {1095-9203}, support = {U42 RR024244/RR/NCRR NIH HHS/United States ; F32 DA048579/DA/NIDA NIH HHS/United States ; R21 DA040406/DA/NIDA NIH HHS/United States ; P20 GM103638/GM/NIGMS NIH HHS/United States ; R01 DA036596/DA/NIDA NIH HHS/United States ; F32 DA047771/DA/NIDA NIH HHS/United States ; }, mesh = {Analgesia ; Animals ; Animals, Genetically Modified ; *Behavior, Animal ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Chromosome Mapping ; Female ; HEK293 Cells ; Humans ; Male ; Mice ; Mice, Knockout ; Morphine/pharmacology ; Nerve Tissue Proteins/*genetics ; Neurons/drug effects ; Orphan Nuclear Receptors/*genetics ; Receptors, G-Protein-Coupled/*genetics ; Receptors, Opioid, mu/*genetics ; Signal Transduction ; }, abstract = {Opioids target the μ-opioid receptor (MOR) to produce unrivaled pain management, but their addictive properties can lead to severe abuse. We developed a whole-animal behavioral platform for unbiased discovery of genes influencing opioid responsiveness. Using forward genetics in Caenorhabditis elegans, we identified a conserved orphan receptor, GPR139, with anti-opioid activity. GPR139 is coexpressed with MOR in opioid-sensitive brain circuits, binds to MOR, and inhibits signaling to heterotrimeric guanine nucleotide-binding proteins (G proteins). Deletion of GPR139 in mice enhanced opioid-induced inhibition of neuronal firing to modulate morphine-induced analgesia, reward, and withdrawal. Thus, GPR139 could be a useful target for increasing opioid safety. These results also demonstrate the potential of C. elegans as a scalable platform for genetic discovery of G protein-coupled receptor signaling principles.}, }
@article {pmid31358636, year = {2019}, author = {Davis, TH}, title = {Profile of Rodolphe Barrangou.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {116}, number = {32}, pages = {15754-15756}, pmid = {31358636}, issn = {1091-6490}, mesh = {CRISPR-Cas Systems/genetics ; Food Technology/*history ; History, 20th Century ; History, 21st Century ; }, }
@article {pmid31228115, year = {2019}, author = {Ali, Z and Zaidi, SS and Tashkandi, M and Mahfouz, MM}, title = {A Simplified Method to Engineer CRISPR/Cas9-Mediated Geminivirus Resistance in Plants.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2028}, number = {}, pages = {167-183}, doi = {10.1007/978-1-4939-9635-3_10}, pmid = {31228115}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; Cloning, Molecular ; DNA Mutational Analysis ; Disease Resistance/*genetics/immunology ; *Geminiviridae ; *Gene Editing ; *Genetic Engineering ; Genetic Vectors/genetics ; Plant Diseases/*genetics/immunology/*virology ; Plants, Genetically Modified ; RNA, Guide ; Sequence Analysis, DNA ; Viral Load ; }, abstract = {Throughout the world, geminiviruses cause devastating losses in economically important crops, including tomato, cotton, cassava, potato, chili, and cucumber; however, control mechanisms such as genetic resistance remain expensive and ineffective. CRISPR/Cas9 is an adaptive immunity mechanism used by prokaryotes to defend against invading nucleic acids of phages and plasmids. The CRISPR/Cas9 system has been harnessed for targeted genome editing in a variety of eukaryotic species, and in plants, CRISPR/Cas9 has been used to modify or introduce many traits, including virus resistance. Recently, we demonstrated that the CRISPR/Cas9 system could be used to engineer plant immunity against geminiviruses by directly targeting the viral genome for degradation. In this chapter, we describe a detailed method for engineering CRISPR/Cas9-mediated resistance against geminiviruses. This method may provide broad, durable viral resistance, as it can target conserved regions of the viral genome and can also be customized to emerging viral variants. Moreover, this method can be used in many crop species, as it requires little or no knowledge of the host plant's genome.}, }
@article {pmid31228114, year = {2019}, author = {Osmani, Z and Jin, S and Mikami, M and Endo, M and Atarashi, H and Fujino, K and Yamada, T and Nakahara, KS}, title = {CRISPR/Cas9-Mediated Editing of Genes Encoding rgs-CaM-like Proteins in Transgenic Potato Plants.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2028}, number = {}, pages = {153-165}, doi = {10.1007/978-1-4939-9635-3_9}, pmid = {31228114}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; DNA Mutational Analysis ; *Gene Editing ; Gene Targeting ; Genetic Vectors/genetics ; Mutagenesis ; Phenotype ; Plant Proteins/*genetics ; *Plants, Genetically Modified ; Solanum tuberosum/*genetics ; Transformation, Genetic ; }, abstract = {A tobacco calmodulin-like protein, rgs-CaM, has been shown to interact with viruses in a variety of ways; it contributes to geminivirus infections but is also involved in primed immunity to the cucumber mosaic virus. Sequence similarity searches revealed several calmodulin-like proteins similar to rgs-CaM (rCML) in Arabidopsis and other Solanaceae plants, including potato (Solanum tuberosum). To analyze the functions of each rCML, mutations were introduced into potato rCMLs using the CRISPR/Cas9 system. Here, we describe our protocol of the CRISPR/Cas9-mediated targeted mutagenesis in stably transformed potato plants.}, }
@article {pmid31107563, year = {2019}, author = {Baffoe-Bonnie, MS}, title = {A justice-based argument for including sickle cell disease in CRISPR/Cas9 clinical research.}, journal = {Bioethics}, volume = {33}, number = {6}, pages = {661-668}, doi = {10.1111/bioe.12589}, pmid = {31107563}, issn = {1467-8519}, mesh = {Anemia, Sickle Cell/*prevention &amp; control/therapy ; Biomedical Research ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Ethnic Groups ; *Gene Editing ; Genetic Therapy ; Humans ; *Social Justice ; }, abstract = {CRISPR/Cas9 is quickly becoming one of the most influential biotechnologies of the last five years. Clinical trials will soon be underway to test whether CRISPR/Cas9 can edit away the genetic mutations that cause sickle cell disease (SCD). This article will present the background of CRISPR/Cas9 gene editing and SCD, highlighting research that supports the application of CRISPR/Cas9 to SCD. While much has been written on why SCD is a good biological candidate for CRISPR/Cas9, less has been written on the ethical implications of including SCD in CRISPR/Cas9 research. This article will argue that there is a strong case in favor of including SCD. Three benefits are achieving distributive justice in research, continuing to repair the negative relationship between patients with SCD and the health-care system, and benefit-sharing for those who do not directly participate in CRISPR/Cas9 research. Opponents will argue that SCD is a risky candidate, that researchers will not find willing participants, and that the burden of SCD is low. Of this set of arguments, the first gives pause. However, on balance, the case in favor of including SCD in CRISPR/Cas9 research is stronger than the case against. Ultimately, this article will show that the historic and sociopolitical injustices that impede progress in treating and curing SCD can be alleviated through biotechnology.}, }
@article {pmid30928637, year = {2019}, author = {Zhong, Z and Sretenovic, S and Ren, Q and Yang, L and Bao, Y and Qi, C and Yuan, M and He, Y and Liu, S and Liu, X and Wang, J and Huang, L and Wang, Y and Baby, D and Wang, D and Zhang, T and Qi, Y and Zhang, Y}, title = {Improving Plant Genome Editing with High-Fidelity xCas9 and Non-canonical PAM-Targeting Cas9-NG.}, journal = {Molecular plant}, volume = {12}, number = {7}, pages = {1027-1036}, doi = {10.1016/j.molp.2019.03.011}, pmid = {30928637}, issn = {1752-9867}, mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRIS