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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 29 May 2020 at 01:37 Created: 

CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: "CRISPR.CAS" OR "crispr/cas" NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

RevDate: 2020-05-28

Geng X, Zhang S, He J, et al (2020)

The urea transporter UT-A1 plays a predominant role in a urea-dependent urine-concentrating mechanism.

The Journal of biological chemistry pii:RA120.013628 [Epub ahead of print].

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.

RevDate: 2020-05-28
CmpDate: 2020-05-28

Evanoff M, AC Komor (2019)

Base Editors: Modular Tools for the Introduction of Point Mutations in Living Cells.

Emerging topics in life sciences, 3(5):483-491.

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.

RevDate: 2020-05-28
CmpDate: 2020-05-28

Zoppo M, Luca MD, Villarreal SN, et al (2019)

A CRISPR/Cas9-based strategy to simultaneously inactivate the entire ALS gene family in Candida orthopsilosis.

Future microbiology, 14:1383-1396.

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.

RevDate: 2020-05-28
CmpDate: 2020-05-28

Feldman D, Singh A, Schmid-Burgk JL, et al (2019)

Optical Pooled Screens in Human Cells.

Cell, 179(3):787-799.e17.

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.

RevDate: 2020-05-28
CmpDate: 2020-05-28

Luteijn RD, van Diemen F, Blomen VA, et al (2019)

A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection.

Journal of virology, 93(13):.

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.

RevDate: 2020-05-28
CmpDate: 2020-05-28

Zhang W, Yang F, Zhu Z, et al (2019)

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 of virology, 93(13):.

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.

RevDate: 2020-05-27

Wang J, Dai W, Li J, et al (2020)

PaCRISPR: a server for predicting and visualizing anti-CRISPR proteins.

Nucleic acids research pii:5847774 [Epub ahead of print].

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/.

RevDate: 2020-05-27

Sledzinski P, Nowaczyk M, M Olejniczak (2020)

Computational Tools and Resources Supporting CRISPR-Cas Experiments.

Cells, 9(5): pii:cells9051288.

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.

RevDate: 2020-05-27
CmpDate: 2020-05-27

Chen Z, Song Z, Yang J, et al (2019)

Sp7/osterix positively regulates dlx2b and bglap to affect tooth development and bone mineralization in zebrafish larvae.

Journal of biosciences, 44(6):.

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.

RevDate: 2020-05-27
CmpDate: 2020-05-27

Morton AR, Dogan-Artun N, Faber ZJ, et al (2019)

Functional Enhancers Shape Extrachromosomal Oncogene Amplifications.

Cell, 179(6):1330-1341.e13.

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.

RevDate: 2020-05-27
CmpDate: 2020-05-27

Gentner B, L Naldini (2019)

In Vivo Selection for Gene-Corrected HSPCs Advances Gene Therapy for a Rare Stem Cell Disease.

Cell stem cell, 25(5):592-593.

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.

RevDate: 2020-05-27
CmpDate: 2020-05-27

Chen G, Park D, Magis AT, et al (2019)

Mcl-1 Interacts with Akt to Promote Lung Cancer Progression.

Cancer research, 79(24):6126-6138.

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.

RevDate: 2020-05-27
CmpDate: 2020-05-27

Memczak S, Shao Y, JC Izpisua Belmonte (2019)

Towards precise, safe genome editing.

Cell research, 29(9):687-689.

RevDate: 2020-05-27
CmpDate: 2020-05-27

Bryant JM, Baumgarten S, Glover L, et al (2019)

CRISPR in Parasitology: Not Exactly Cut and Dried!.

Trends in parasitology, 35(6):409-422.

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.

RevDate: 2020-05-27
CmpDate: 2020-05-27

Voss JE, Gonzalez-Martin A, Andrabi R, et al (2019)

Reprogramming the antigen specificity of B cells using genome-editing technologies.

eLife, 8:.

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.

RevDate: 2020-05-26

Reimann V, Ziemann M, Li H, et al (2020)

Specificities and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups.

RNA biology [Epub ahead of print].

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.

RevDate: 2020-05-26

Wada N, Ueta R, Osakabe Y, et al (2020)

Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering.

BMC plant biology, 20(1):234 pii:10.1186/s12870-020-02385-5.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Guo X, Aviles G, Liu Y, et al (2020)

Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway.

Nature, 579(7799):427-432.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

de Jong OG, Murphy DE, Mäger I, et al (2020)

A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA.

Nature communications, 11(1):1113 pii:10.1038/s41467-020-14977-8.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Harding HP, Ordonez A, Allen F, et al (2019)

The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells.

eLife, 8:.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Zeng H, Castillo-Cabrera J, Manser M, et al (2019)

Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC.

eLife, 8:.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Kang Y, Chu C, Wang F, et al (2019)

CRISPR/Cas9-mediated genome editing in nonhuman primates.

Disease models & mechanisms, 12(10):.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Mansourian S, Fandino RA, O Riabinina (2019)

Progress in the use of genetic methods to study insect behavior outside Drosophila.

Current opinion in insect science, 36:45-56.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Pacini V, Petit F, Querat B, et al (2019)

Identification of a pituitary ERα-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor.

Epigenetics & chromatin, 12(1):48.

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.

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.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Schneider D, Chua RL, Molitor N, et al (2019)

The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells.

Clinical epigenetics, 11(1):98.

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.

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.

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.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Sen M, Wang X, Hamdan FH, et al (2019)

ARID1A facilitates KRAS signaling-regulated enhancer activity in an AP1-dependent manner in colorectal cancer cells.

Clinical epigenetics, 11(1):92.

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.

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.

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.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Garcia-Leon JA, Vitorica J, A Gutierrez (2019)

Use of human pluripotent stem cell-derived cells for neurodegenerative disease modeling and drug screening platform.

Future medicinal chemistry, 11(11):1305-1322.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Ballard E, Weber J, Melchers WJG, et al (2019)

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.

Fungal genetics and biology : FG & B, 130:98-106.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Schuster M, R Kahmann (2019)

CRISPR-Cas9 genome editing approaches in filamentous fungi and oomycetes.

Fungal genetics and biology : FG & B, 130:43-53.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Darma R, Lutz A, Elliott CE, et al (2019)

Identification of a gene cluster for the synthesis of the plant hormone abscisic acid in the plant pathogen Leptosphaeria maculans.

Fungal genetics and biology : FG & B, 130:62-71.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Kunitake E, Tanaka T, Ueda H, et al (2019)

CRISPR/Cas9-mediated gene replacement in the basidiomycetous yeast Pseudozyma antarctica.

Fungal genetics and biology : FG & B, 130:82-90.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Shi Z, Xin H, Tian D, et al (2019)

Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 33(6):6962-6968.

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.

RevDate: 2020-05-26
CmpDate: 2020-05-26

Ko T, S Li (2019)

Genome-wide screening identifies novel genes and biological processes implicated in cisplatin resistance.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 33(6):7143-7154.

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.

RevDate: 2020-05-25
CmpDate: 2020-05-25

Bae T, Kim H, Kim JH, et al (2019)

Specificity Assessment of CRISPR Genome Editing of Oncogenic EGFR Point Mutation with Single-Base Differences.

Molecules (Basel, Switzerland), 25(1):.

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.

RevDate: 2020-05-22
CmpDate: 2020-05-22

Guo M, Ren K, Zhu Y, et al (2019)

Structural insights into a high fidelity variant of SpCas9.

Cell research, 29(3):183-192.

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.

RevDate: 2020-05-22

Kaneko T, S Tanaka (2020)

Improvement of genome editing by electroporation using embryos artificially removed cumulus cells in the oviducts.

Biochemical and biophysical research communications pii:S0006-291X(20)30942-6 [Epub ahead of print].

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).

RevDate: 2020-05-22

Hahn E, M Hiemenz (2020)

Therapeutic Gene Editing with CRISPR: A Laboratory Medicine Perspective.

Clinics in laboratory medicine, 40(2):205-219.

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.

RevDate: 2020-05-22
CmpDate: 2020-05-22

Palit SA, Vis D, Stelloo S, et al (2019)

TLE3 loss confers AR inhibitor resistance by facilitating GR-mediated human prostate cancer cell growth.

eLife, 8:.

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.

RevDate: 2020-05-21
CmpDate: 2020-05-21

Hayashida G, Shioi S, Hidaka K, et al (2019)

Differential genomic destabilisation in human cells with pathogenic MSH2 mutations introduced by genome editing.

Experimental cell research, 377(1-2):24-35.

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.

RevDate: 2020-05-21
CmpDate: 2020-05-21

Pettingill P, Weir GA, Wei T, et al (2019)

A causal role for TRESK loss of function in migraine mechanisms.

Brain : a journal of neurology, 142(12):3852-3867.

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.

RevDate: 2020-05-21
CmpDate: 2020-05-21

Jo DH, Song DW, Cho CS, et al (2019)

CRISPR-Cas9-mediated therapeutic editing of Rpe65 ameliorates the disease phenotypes in a mouse model of Leber congenital amaurosis.

Science advances, 5(10):eaax1210.

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.

RevDate: 2020-05-21
CmpDate: 2020-05-21

Nagy G, Vaz AG, Szebenyi C, et al (2019)

CRISPR-Cas9-mediated disruption of the HMG-CoA reductase genes of Mucor circinelloides and subcellular localization of the encoded enzymes.

Fungal genetics and biology : FG & B, 129:30-39.

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.

RevDate: 2020-05-21
CmpDate: 2020-05-21

Li X, Yu X, Chen X, et al (2019)

Localization of TMC1 and LHFPL5 in auditory hair cells in neonatal and adult mice.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 33(6):6838-6851.

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.

RevDate: 2020-05-20

Roberts A, R Barrangou (2020)

Applications of CRISPR-Cas systems in Lactic Acid Bacteria.

FEMS microbiology reviews pii:5841184 [Epub ahead of print].

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.

RevDate: 2020-05-19

Singhal J, Chikara S, Horne D, et al (2020)

Targeting RLIP with CRISPR/Cas9 controls tumor growth.

Carcinogenesis pii:5840500 [Epub ahead of print].

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 "guardian of the genome" 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.

RevDate: 2020-05-19
CmpDate: 2020-05-19

Abbott TR, Dhamdhere G, Liu Y, et al (2020)

Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza.

Cell, 181(4):865-876.e12.

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.

RevDate: 2020-05-20
CmpDate: 2020-05-20

Szentes S, Zsibrita N, Koncz M, et al (2020)

I-Block: a simple Escherichia coli-based assay for studying sequence-specific DNA binding of proteins.

Nucleic acids research, 48(5):e28.

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.

RevDate: 2020-05-20
CmpDate: 2020-05-20

Tian B, Minero GAS, Fock J, et al (2020)

CRISPR-Cas12a based internal negative control for nonspecific products of exponential rolling circle amplification.

Nucleic acids research, 48(5):e30.

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.

RevDate: 2020-05-20
CmpDate: 2020-05-20

Yu J, Xiang X, Huang J, et al (2020)

Haplotyping by CRISPR-mediated DNA circularization (CRISPR-hapC) broadens allele-specific gene editing.

Nucleic acids research, 48(5):e25.

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.

RevDate: 2020-05-20
CmpDate: 2020-05-20

Li J, Li HY, Gu SY, et al (2020)

One-step generation of zebrafish carrying a conditional knockout-knockin visible switch via CRISPR/Cas9-mediated intron targeting.

Science China. Life sciences, 63(1):59-67.

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.

RevDate: 2020-05-19
CmpDate: 2020-05-19

Wang X, Ma Y, Wang F, et al (2020)

Disruption of nicotinic acetylcholine receptor α6 mediated by CRISPR/Cas9 confers resistance to spinosyns in Plutella xylostella.

Pest management science, 76(5):1618-1625.

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.

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.

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.

RevDate: 2020-05-20
CmpDate: 2020-05-20

Hong W, Huang M, Wei Y, et al (2019)

A new and promising application of gene editing: CRISPR-controlled smart materials for tissue engineering, bioelectronics, and diagnostics.

Science China. Life sciences, 62(11):1547-1549.

RevDate: 2020-05-20
CmpDate: 2020-05-20

Li G, Liu YG, Y Chen (2019)

Genome-editing technologies: the gap between application and policy.

Science China. Life sciences, 62(11):1534-1538.

RevDate: 2020-05-19
CmpDate: 2020-05-19

Zhang Y, Zhang Z, Zhang H, et al (2020)

PADS Arsenal: a database of prokaryotic defense systems related genes.

Nucleic acids research, 48(D1):D590-D598.

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.

RevDate: 2020-05-20
CmpDate: 2020-05-20

Dandage R, CR Landry (2019)

Paralog dependency indirectly affects the robustness of human cells.

Molecular systems biology, 15(9):e8871.

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.

RevDate: 2020-05-20
CmpDate: 2020-05-20

Lou WP, Wang L, Long C, et al (2019)

Direct Gene Knock-out of Axolotl Spinal Cord Neural Stem Cells via Electroporation of CAS9 Protein-gRNA Complexes.

Journal of visualized experiments : JoVE.

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.

RevDate: 2020-05-20
CmpDate: 2020-05-20

Liu X, Zheng G, Wang G, et al (2019)

Overexpression of the diguanylate cyclase CdgD blocks developmental transitions and antibiotic biosynthesis in Streptomyces coelicolor.

Science China. Life sciences, 62(11):1492-1505.

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.

RevDate: 2020-05-20
CmpDate: 2020-05-20

Lee HJ, Yoon JW, Jung KM, et al (2019)

Targeted gene insertion into Z chromosome of chicken primordial germ cells for avian sexing model development.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 33(7):8519-8529.

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.

RevDate: 2020-05-20
CmpDate: 2020-05-20

González-Romero E, Martínez-Valiente C, García-Ruiz C, et al (2019)

CRISPR to fix bad blood: a new tool in basic and clinical hematology.

Haematologica, 104(5):881-893.

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.

RevDate: 2020-05-19
CmpDate: 2020-05-19

Foss DV, Hochstrasser ML, RC Wilson (2019)

Clinical applications of CRISPR-based genome editing and diagnostics.

Transfusion, 59(4):1389-1399.

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.

RevDate: 2020-05-18

Hashmat R, Yousaf MZ, Rahman Z, et al (2020)

CRISPR-CAS Replacing Antiviral Drugs against HIV: An Update.

Critical reviews in eukaryotic gene expression, 30(1):77-83.

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.

RevDate: 2020-05-18

Ashfaq UA, H Khalid (2020)

CRISPR/CAS9-Mediated Antiviral Activity: A Tool to Combat Viral Infection.

Critical reviews in eukaryotic gene expression, 30(1):45-56.

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.

RevDate: 2020-05-18

Garrett S, Shiimori M, Watts EA, et al (2020)

Primed CRISPR DNA uptake in Pyrococcus furiosus.

Nucleic acids research pii:5838857 [Epub ahead of print].

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.

RevDate: 2020-05-18

Bouchaut B, L Asveld (2020)

Safe-by-Design: Stakeholders' Perceptions and Expectations of How to Deal with Uncertain Risks of Emerging Biotechnologies in the Netherlands.

Risk analysis : an official publication of the Society for Risk Analysis [Epub ahead of print].

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.

RevDate: 2020-05-18
CmpDate: 2020-05-18

Ding N, Qin Q, Wu X, et al (2020)

Antagonistic regulation of axillary bud outgrowth by the BRANCHED genes in tobacco.

Plant molecular biology, 103(1-2):185-196.

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.

RevDate: 2020-05-18
CmpDate: 2020-05-18

Shockey J (2020)

Gene editing in plants: assessing the variables through a simplified case study.

Plant molecular biology, 103(1-2):75-89.

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.

RevDate: 2020-05-18
CmpDate: 2020-05-18

Echave MC, Domingues RMA, Gómez-Florit M, et al (2019)

Biphasic Hydrogels Integrating Mineralized and Anisotropic Features for Interfacial Tissue Engineering.

ACS applied materials & interfaces, 11(51):47771-47784.

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.

RevDate: 2020-05-18
CmpDate: 2020-05-18

Li S, Song Z, Liu C, et al (2019)

Biomimetic Mineralization-Based CRISPR/Cas9 Ribonucleoprotein Nanoparticles for Gene Editing.

ACS applied materials & interfaces, 11(51):47762-47770.

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.

RevDate: 2020-05-18
CmpDate: 2020-05-18

Brandt ZJ, North PN, BA Link (2019)

Somatic Mutations of lats2 Cause Peripheral Nerve Sheath Tumors in Zebrafish.

Cells, 8(9):.

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.

RevDate: 2020-05-19
CmpDate: 2020-05-18

Towers CG, Fitzwalter BE, Regan D, et al (2019)

Cancer Cells Upregulate NRF2 Signaling to Adapt to Autophagy Inhibition.

Developmental cell, 50(6):690-703.e6.

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.

RevDate: 2020-05-18
CmpDate: 2020-05-18

Ouyang Y (2019)

Understanding and breaking down the reproductive barrier between Asian and African cultivated rice: a new start for hybrid rice breeding.

Science China. Life sciences, 62(8):1114-1116.

RevDate: 2020-05-18
CmpDate: 2020-05-18

Fan J, Xia Y, GL Wang (2019)

An improved heteroduplex analysis for rapid genotyping of SNPs and single base pair indels.

BioTechniques, 67(1):6-10.

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.

RevDate: 2020-05-18
CmpDate: 2020-05-12

Wang Z, Yang L, Qu S, et al (2019)

CRISPR-mediated gene editing to rescue haploinsufficient obesity syndrome.

Protein & cell, 10(10):705-708.

RevDate: 2020-05-18
CmpDate: 2020-05-18

Sun Q, Wang Y, Dong N, et al (2019)

Application of CRISPR/Cas9-Based Genome Editing in Studying the Mechanism of Pandrug Resistance in Klebsiella pneumoniae.

Antimicrobial agents and chemotherapy, 63(7):.

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.

RevDate: 2020-05-18
CmpDate: 2020-05-12

Chin JS, Chooi WH, Wang H, et al (2019)

Scaffold-mediated non-viral delivery platform for CRISPR/Cas9-based genome editing.

Acta biomaterialia, 90:60-70.

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.

RevDate: 2020-05-18
CmpDate: 2020-05-18

Dunbar GL, Koneru S, Kolli N, et al (2019)

Silencing of the Mutant Huntingtin Gene through CRISPR-Cas9 Improves the Mitochondrial Biomarkers in an In Vitro Model of Huntington's Disease.

Cell transplantation, 28(4):460-463.

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 "clustered regularly interspaced short palindromic repeats" (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.

RevDate: 2020-05-18
CmpDate: 2020-05-18

Tapscott T, Guarnieri MT, CA Henard (2019)

Development of a CRISPR/Cas9 System for Methylococcus capsulatus In Vivo Gene Editing.

Applied and environmental microbiology, 85(11):.

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.

RevDate: 2020-05-18
CmpDate: 2020-05-18

Xu H, Wang B, Ono M, et al (2019)

Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility.

Cell stem cell, 24(4):566-578.e7.

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.

RevDate: 2020-05-18
CmpDate: 2020-05-12

Chang J, Chen X, Glass Z, et al (2019)

Integrating Combinatorial Lipid Nanoparticle and Chemically Modified Protein for Intracellular Delivery and Genome Editing.

Accounts of chemical research, 52(3):665-675.

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.

RevDate: 2020-05-17

Levesque S, Agudelo D, Y Doyon (2020)

Rewired Cas9s with Minimal Sequence Constraints.

Trends in pharmacological sciences pii:S0165-6147(20)30095-X [Epub ahead of print].

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.

RevDate: 2020-05-16

Wan T, Y Ping (2020)

Delivery of genome-editing biomacromolecules for treatment of lung genetic disorders.

Advanced drug delivery reviews pii:S0169-409X(20)30035-1 [Epub ahead of print].

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.

RevDate: 2020-05-15

Liu D, Zhao X, Tang A, et al (2020)

Potential of CRISPR screen in target discovery for cancer immunotherapy.

Biochimica et biophysica acta. Reviews on cancer pii:S0304-419X(20)30097-4 [Epub ahead of print].

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.

RevDate: 2020-05-15

Boonekamp FJ, Dashko S, Duiker D, et al (2020)

Design and experimental evaluation of a minimal, innocuous watermarking strategy to distinguish near-identical DNA and RNA sequences.

ACS synthetic biology [Epub ahead of print].

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.

RevDate: 2020-05-15

Lin J, Feng M, Zhang H, et al (2020)

Characterization of a novel type III CRISPR-Cas effector provides new insights into the allosteric activation and suppression of the Cas10 DNase.

Cell discovery, 6:29 pii:160.

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.

RevDate: 2020-05-15

Collins MP, Stransky LA, M Forgac (2020)

AKT Ser/Thr kinase increases V-ATPase-dependent lysosomal acidification in response to amino acid starvation in mammalian cells.

The Journal of biological chemistry pii:RA120.013223 [Epub ahead of print].

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.

RevDate: 2020-05-15

Morimoto K, Suzuki N, Tanida I, et al (2020)

Blood group P1 antigen-bearing glycoproteins are functional but less efficient receptors of Shiga toxin than conventional glycolipid-based receptors.

The Journal of biological chemistry pii:RA120.013926 [Epub ahead of print].

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.

RevDate: 2020-05-15

Pan M, Nethery MA, Hidalgo-Cantabrana C, et al (2020)

Comprehensive Mining and Characterization of CRISPR-Cas Systems in Bifidobacterium.

Microorganisms, 8(5): pii:microorganisms8050720.

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.

RevDate: 2020-05-15
CmpDate: 2020-05-15

Zhang S, Zhang F, Chen Q, et al (2019)

CRISPR/Cas9-mediated knockout of NSD1 suppresses the hepatocellular carcinoma development via the NSD1/H3/Wnt10b signaling pathway.

Journal of experimental & clinical cancer research : CR, 38(1):467.

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.

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.

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.

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.

RevDate: 2020-05-15
CmpDate: 2020-05-15

Ji X, Wang D, C Gao (2019)

CRISPR editing-mediated antiviral immunity: a versatile source of resistance to combat plant virus infections.

Science China. Life sciences, 62(9):1246-1249.

RevDate: 2020-05-15
CmpDate: 2020-05-15

Xu RG, Wang X, Shen D, et al (2019)

Perspectives on gene expression regulation techniques in Drosophila.

Journal of genetics and genomics = Yi chuan xue bao, 46(4):213-220.

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.

RevDate: 2020-05-15
CmpDate: 2020-05-15

Liu Q, Qi Y, Liang Q, et al (2019)

Targeted disruption of tyrosinase causes melanin reduction in Carassius auratus cuvieri and its hybrid progeny.

Science China. Life sciences, 62(9):1194-1202.

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.

RevDate: 2020-05-14
CmpDate: 2020-05-14

Zhang L, Xu W, Gao X, et al (2020)

lncRNA Sensing of a Viral Suppressor of RNAi Activates Non-canonical Innate Immune Signaling in Drosophila.

Cell host & microbe, 27(1):115-128.e8.

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.

RevDate: 2020-05-14
CmpDate: 2020-05-14

Pizon V, Gaudin N, Poteau M, et al (2020)

hVFL3/CCDC61 is a component of mother centriole subdistal appendages required for centrosome cohesion and positioning.

Biology of the cell, 112(1):22-37.

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.

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.

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.

RevDate: 2020-05-14
CmpDate: 2020-05-14

Dong L, Lin X, Yu D, et al (2020)

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 of industrial microbiology & biotechnology, 47(1):133-144.

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.

RevDate: 2020-05-14
CmpDate: 2020-05-14

Li ZH, Meng H, Ma B, et al (2020)

Immediate, multiplexed and sequential genome engineering facilitated by CRISPR/Cas9 in Saccharomyces cerevisiae.

Journal of industrial microbiology & biotechnology, 47(1):83-96.

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.

RevDate: 2020-05-14
CmpDate: 2020-05-14

Cali CP, Park DS, EB Lee (2019)

Targeted DNA methylation of neurodegenerative disease genes via homology directed repair.

Nucleic acids research, 47(22):11609-11622.

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.

RevDate: 2020-05-14
CmpDate: 2020-05-14

Cohen J (2019)

Parents weigh promise and risks of germline editing.

Science (New York, N.Y.), 366(6465):564-565.

RevDate: 2020-05-14
CmpDate: 2020-05-14

Bilousova G (2019)

Gene Therapy for Skin Fragility Diseases: The New Generation.

The Journal of investigative dermatology, 139(8):1634-1637.

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.

RevDate: 2020-05-14
CmpDate: 2020-05-14

Nimura T, Itoh T, Hagio H, et al (2019)

Role of Reelin in cell positioning in the cerebellum and the cerebellum-like structure in zebrafish.

Developmental biology, 455(2):393-408.

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.

RevDate: 2020-05-14
CmpDate: 2020-05-14

Wang F, Chang C, Li R, et al (2019)

Retinol binding protein 4 mediates MEHP-induced glucometabolic abnormalities in HepG2 cells.

Toxicology, 424:152236.

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.

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ESP Quick Facts

ESP Origins

In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.

ESP Support

In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.

ESP Rationale

Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.

ESP Goal

In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.

ESP Usage

Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.

ESP Content

When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.

ESP Help

Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.

ESP Plans

With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.

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CRISPR-Cas

By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

Electronic Scholarly Publishing
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Bellingham, WA 98226

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Papers in Classical Genetics

The ESP began as an effort to share a handful of key papers from the early days of classical genetics. Now the collection has grown to include hundreds of papers, in full-text format.

Digital Books

Along with papers on classical genetics, ESP offers a collection of full-text digital books, including many works by Darwin (and even a collection of poetry — Chicago Poems by Carl Sandburg).

Timelines

ESP now offers a much improved and expanded collection of timelines, designed to give the user choice over subject matter and dates.

Biographies

Biographical information about many key scientists.

Selected Bibliographies

Bibliographies on several topics of potential interest to the ESP community are now being automatically maintained and generated on the ESP site.

ESP Picks from Around the Web (updated 07 JUL 2018 )