@article {pmid32497618,
year = {2020},
author = {Galani, V and Papagiannitsis, CC and Petinaki, E},
title = {First description of ST409 OXA-23-producing Acinetobacter baumannii, carrying a CST8 CRISPR/Cas system, in Central Greece.},
journal = {Journal of global antimicrobial resistance},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgar.2020.05.008},
pmid = {32497618},
issn = {2213-7173},
}

@article {pmid32496535,
year = {2020},
author = {Mullally, G and van Aelst, K and Naqvi, MM and Diffin, FM and Karvelis, T and Gasiunas, G and Siksnys, V and Szczelkun, MD},
title = {5' modifications to CRISPR-Cas9 gRNA can change the dynamics and size of R-loops and inhibit DNA cleavage.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaa477},
pmid = {32496535},
issn = {1362-4962},
abstract = {A key aim in exploiting CRISPR-Cas is gRNA engineering to introduce additional functionalities, ranging from individual nucleotide changes that increase efficiency of on-target binding to the inclusion of larger functional RNA aptamers or ribonucleoproteins (RNPs). Cas9-gRNA interactions are crucial for complex assembly, but several distinct regions of the gRNA are amenable to modification. We used in vitro ensemble and single-molecule assays to assess the impact of gRNA structural alterations on RNP complex formation, R-loop dynamics, and endonuclease activity. Our results indicate that RNP formation was unaffected by any of our modifications. R-loop formation and DNA cleavage activity were also essentially unaffected by modification of the Upper Stem, first Hairpin and 3' end. In contrast, we found that 5' additions of only two or three nucleotides could reduce R-loop formation and cleavage activity of the RuvC domain relative to a single nucleotide addition. Such modifications are a common by-product of in vitro transcribed gRNA. We also observed that addition of a 20 nt RNA hairpin to the 5' end of a gRNA still supported RNP formation but produced a stable ∼9 bp R-loop that could not activate DNA cleavage. Consideration of these observations will assist in successful gRNA design.},
}

@article {pmid32493587,
year = {2020},
author = {Uygun, ZO and Yeniay, L and Gi Rgi N Sağın, F},
title = {CRISPR-dCas9 powered impedimetric biosensor for label-free detection of circulating tumor DNAs.},
journal = {Analytica chimica acta},
volume = {1121},
number = {},
pages = {35-41},
doi = {10.1016/j.aca.2020.04.009},
pmid = {32493587},
issn = {1873-4324},
abstract = {Label-free biosensors which can be integrated into lab-on-a-chip platforms have the advantage of using small volumes for rapid and inexpensive measurements contrary to label-based technologies which are often more costly and time-consuming. In this study, graphene oxide screen printed electrodes (GPHOXE) were modified by deactivated Cas9 (dCas9) proteins and synthetic guide RNA (sgRNA) as the biorecognition receptor for label-free detection of circulating tumor DNAs (ctDNA). This was achieved by detection of a tumor related mutation (PIK3CA exon 9 mutation) via sequence-specific recognition followed by electrochemical impedance spectroscopy (EIS) analysis. The biosensor showed high specificity as there was no impedance signal for other ctDNA sequences, even the single nucleotide mismatch. dCas9-sgRNA modified biosensor demonstrated linear detection limits between 2 and 20 nM for 120 bp ctDNA's in 40 s. The calibration curve showed good linearity, LOD was calculated as 0.65 nM and LOQ was calculated as 1.92 nM. Selectivity and repeatability studies were carried out in real blood samples and the recovery was higher than 96%. In conclusion, dCas9-sgRNA was effectively immobilized and optimized on GPHOXE as the selective biorecognition receptor of this ultrafast impedimetric biosensor. The CRISPR-dCas9 powered impedimetric system showed good selectivity, high repeatability and good recovery properties. This is the first literature to report the use of CRISPR/Cas technology as a label-free tool that can be used in an impedimetric system for detection of ctDNA's.},
}

@article {pmid32493511,
year = {2020},
author = {Song, L and Xie, K},
title = {Engineering CRISPR/Cas9 to mitigate abundant host contamination for 16S rRNA gene-based amplicon sequencing.},
journal = {Microbiome},
volume = {8},
number = {1},
pages = {80},
doi = {10.1186/s40168-020-00859-0},
pmid = {32493511},
issn = {2049-2618},
support = {31622047//National Natural Science Foundation of China/ ; 2018ZX08010-05B//National Transgenic Science and Technology Program/ ; },
abstract = {BACKGROUND: High-throughput sequencing of bacterial 16S rRNA gene (16S-seq) is a useful and common method for studying bacterial community structures. However, contamination of the 16S rRNA genes from the mitochondrion and plastid hinders the sensitive bacterial 16S-seq in plant microbiota profiling, especially for some plant species such as rice. To date, efficiently mitigating such host contamination without a bias is challenging in 16S rRNA gene-based amplicon sequencing.

RESULTS: We developed Cas-16S-seq method to reduce abundant host contamination for plant microbiota profiling. This method utilizes the Cas9 nuclease and specific guide RNA (gRNA) to cut 16S rRNA targets during library construction, thereby removing host contamination in 16S-seq. We used rice as an example to validate the feasibility and effectiveness of Cas-16S-seq. We established a bioinformatics pipeline to design gRNAs that specifically target rice 16S rRNA genes without bacterial 16S rRNA off-targets. We compared the effectiveness of Cas-16S-seq with that of the commonly used 16S-seq method for artificially mixed 16S rRNA gene communities, paddy soil, rice root, and phyllosphere samples. The results showed that Cas-16S-seq substantially reduces the fraction of rice 16S rRNA gene sequences from 63.2 to 2.9% in root samples and from 99.4 to 11.6% in phyllosphere samples on average. Consequently, Cas-16S-seq detected more bacterial species than the 16S-seq in plant samples. Importantly, when analyzing soil samples, Cas-16S-seq and 16S-seq showed almost identical bacterial communities, suggesting that Cas-16S-seq with host-specific gRNAs that we designed has no off-target in rice microbiota profiling.

CONCLUSION: Our Cas-16S-seq can efficiently remove abundant host contamination without a bias for 16S rRNA gene-based amplicon sequencing, thereby enabling deeper bacterial community profiling with a low cost and high flexibility. Thus, we anticipate that this method would be a useful tool for plant microbiomics. Video Abstract.},
}

@article {pmid31941712,
year = {2020},
author = {Chen, X and Chen, Y and Xin, H and Wan, T and Ping, Y},
title = {Near-infrared optogenetic engineering of photothermal nanoCRISPR for programmable genome editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {5},
pages = {2395-2405},
pmid = {31941712},
issn = {1091-6490},
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems/genetics/radiation effects ; Gene Editing/*methods ; Gold/chemistry ; HEK293 Cells ; Humans ; Infrared Rays ; Nanotubes/*chemistry ; *Optogenetics ; Plasmids/genetics ; Promoter Regions, Genetic ; },
abstract = {We herein report an optogenetically activatable CRISPR-Cas9 nanosystem for programmable genome editing in the second near-infrared (NIR-II) optical window. The nanosystem, termed nanoCRISPR, is composed of a cationic polymer-coated Au nanorod (APC) and Cas9 plasmid driven by a heat-inducible promoter. The APC not only serves as a carrier for intracellular plasmid delivery but also can harvest external NIR-II photonic energy and convert it into local heat to induce the gene expression of the Cas9 endonuclease. Due to high transfection activity, the APC shows strong ability to induce a significant level of disruption in different genomic loci upon optogenetic activation. Moreover, the precise control of genome-editing activity can be simply programmed by finely tuning exposure time and irradiation time in vitro and in vivo and also enables editing at multiple time points, thus proving the sensitivity and inducibility of such an editing modality. The NIR-II optical feature of nanoCRISPR enables therapeutic genome editing at deep tissue, by which treatment of deep tumor and rescue of fulminant hepatic failure are demonstrated as proof-of-concept therapeutic examples. Importantly, this modality of optogenetic genome editing can significantly minimize the off-target effect of CRISPR-Cas9 in most potential off-target sites. The optogenetically activatable CRISPR-Cas9 nanosystem we have developed offers a useful tool to expand the current applications of CRISPR-Cas9, and also defines a programmable genome-editing strategy toward high precision and spatial specificity.},
}

CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems/genetics/radiation effects
Gene Editing/*methods
Gold/chemistry
HEK293 Cells
Humans
Infrared Rays
Nanotubes/*chemistry
*Optogenetics
Plasmids/genetics
Promoter Regions, Genetic

@article {pmid31840076,
year = {2019},
author = {Rui, Y and Wilson, DR and Choi, J and Varanasi, M and Sanders, K and Karlsson, J and Lim, M and Green, JJ},
title = {Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing.},
journal = {Science advances},
volume = {5},
number = {12},
pages = {eaay3255},
pmid = {31840076},
issn = {2375-2548},
support = {S10 OD016374/OD/NIH HHS/United States ; R01 CA228133/CA/NCI NIH HHS/United States ; R01 EB022148/EB/NIBIB NIH HHS/United States ; P30 EY001765/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cytosol/chemistry ; Gene Editing ; *Gene Transfer Techniques ; Glioma/genetics/pathology/*therapy ; Humans ; Mice ; Nanoparticles/chemistry ; Polymers/chemistry/*pharmacology ; Ribonucleoproteins/genetics ; },
abstract = {Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.},
}

Animals
CRISPR-Cas Systems/*genetics
Cytosol/chemistry
Gene Editing
*Gene Transfer Techniques
Glioma/genetics/pathology/*therapy
Humans
Mice
Nanoparticles/chemistry
Polymers/chemistry/*pharmacology
Ribonucleoproteins/genetics

@article {pmid31597782,
year = {2019},
author = {Passos, V and Zillinger, T and Casartelli, N and Wachs, AS and Xu, S and Malassa, A and Steppich, K and Schilling, H and Franz, S and Todt, D and Steinmann, E and Sutter, K and Dittmer, U and Bohne, J and Schwartz, O and Barchet, W and Goffinet, C},
title = {Characterization of Endogenous SERINC5 Protein as Anti-HIV-1 Factor.},
journal = {Journal of virology},
volume = {93},
number = {24},
pages = {},
pmid = {31597782},
issn = {1098-5514},
mesh = {Anti-HIV Agents/*pharmacology ; CRISPR-Cas Systems ; Cell Line ; Cell Membrane/drug effects/metabolism ; Gene Editing ; Gene Expression Regulation ; Gene Knockout Techniques ; Genotype ; HEK293 Cells ; HIV Infections/virology ; HIV-1/*drug effects ; Host-Pathogen Interactions/physiology ; Humans ; Interferon-alpha ; Membrane Proteins/genetics/*metabolism/*pharmacology ; Pyrazoles/pharmacology ; T-Lymphocytes/virology ; Virion/metabolism ; nef Gene Products, Human Immunodeficiency Virus/genetics/metabolism ; },
abstract = {When expressed in virus-producing cells, the cellular multipass transmembrane protein SERINC5 reduces the infectivity of HIV-1 particles and is counteracted by HIV-1 Nef. Due to the unavailability of an antibody of sufficient specificity and sensitivity, investigation of SERINC5 protein expression and subcellular localization has been limited to heterologously expressed SERINC5. We generated, via CRISPR/Cas9-assisted gene editing, Jurkat T-cell clones expressing endogenous SERINC5 bearing an extracellularly exposed hemagglutinin (HA) epitope [Jurkat SERINC5(iHA knock-in) T cells]. This modification enabled quantification of endogenous SERINC5 protein levels and demonstrated a predominant localization in lipid rafts. Interferon alpha (IFN-α) treatment enhanced cell surface levels of SERINC5 in a ruxolitinib-sensitive manner in the absence of modulation of mRNA and protein quantities. Parental and SERINC5(iHA knock-in) T cells shared the ability to produce infectious wild-type HIV-1 but not an HIV-1 Δnef mutant. SERINC5-imposed reduction of infectivity involved a modest reduction of virus fusogenicity. An association of endogenous SERINC5 protein with HIV-1 Δnef virions was consistently detectable as a 35-kDa species, as opposed to heterologous SERINC5, which presented as a 51-kDa species. Nef-mediated functional counteraction did not correlate with virion exclusion of SERINC5, arguing for the existence of additional counteractive mechanisms of Nef that act on virus-associated SERINC5. In HIV-1-infected cells, Nef triggered the internalization of SERINC5 in the absence of detectable changes of steady-state protein levels. These findings establish new properties of endogenous SERINC5 expression and subcellular localization, challenge existing concepts of HIV-1 Nef-mediated antagonism of SERINC5, and uncover an unprecedented role of IFN-α in modulating SERINC5 through accumulation at the cell surface.IMPORTANCE SERINC5 is the long-searched-for antiviral factor that is counteracted by the HIV-1 accessory gene product Nef. Here, we engineered, via CRISPR/Cas9 technology, T-cell lines that express endogenous SERINC5 alleles tagged with a knocked-in HA epitope. This genetic modification enabled us to study basic properties of endogenous SERINC5 and to verify proposed mechanisms of HIV-1 Nef-mediated counteraction of SERINC5. Using this unique resource, we identified the susceptibility of endogenous SERINC5 protein to posttranslational modulation by type I IFNs and suggest uncoupling of Nef-mediated functional antagonism from SERINC5 exclusion from virions.},
}

Anti-HIV Agents/*pharmacology
CRISPR-Cas Systems
Cell Line
Cell Membrane/drug effects/metabolism
Gene Editing
Gene Expression Regulation
Gene Knockout Techniques
Genotype
HEK293 Cells
HIV Infections/virology
HIV-1/*drug effects
Host-Pathogen Interactions/physiology
Humans
Interferon-alpha
Membrane Proteins/genetics/*metabolism/*pharmacology
Pyrazoles/pharmacology
T-Lymphocytes/virology
Virion/metabolism
nef Gene Products, Human Immunodeficiency Virus/genetics/metabolism

@article {pmid30880083,
year = {2019},
author = {Palermo, G and Casalino, L and Magistrato, A and Andrew McCammon, J},
title = {Understanding the mechanistic basis of non-coding RNA through molecular dynamics simulations.},
journal = {Journal of structural biology},
volume = {206},
number = {3},
pages = {267-279},
pmid = {30880083},
issn = {1095-8657},
support = {R01 GM031749/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*trends ; Humans ; Molecular Dynamics Simulation ; *Nucleic Acid Conformation ; Proteins/chemistry/genetics ; RNA Splicing/genetics ; RNA, Untranslated/*genetics/ultrastructure ; },
abstract = {Noncoding RNA (ncRNA) has a key role in regulating gene expression, mediating fundamental processes and diseases via a variety of yet unknown mechanisms. Here, we review recent applications of conventional and enhanced Molecular Dynamics (MD) simulations methods to address the mechanistic function of large biomolecular systems that are tightly involved in the ncRNA function and that are of key importance in life sciences. This compendium focuses of three biomolecular systems, namely the CRISPR-Cas9 genome editing machinery, group II intron ribozyme and the ribonucleoprotein complex of the spliceosome, which edit and process ncRNA. We show how the application of a novel accelerated MD simulations method has been key in disclosing the conformational transitions underlying RNA binding in the CRISPR-Cas9 complex, suggesting a mechanism for RNA recruitment and clarifying the conformational changes required for attaining genome editing. As well, we discuss the use of mixed quantum-classical MD simulations in deciphering the catalytic mechanism of RNA splicing as operated by group II intron ribozyme, one of the largest ncRNA structures crystallized so far. Finally, we debate the future challenges and opportunities in the field, discussing the recent application of MD simulations for unraveling the functional biophysics of the spliceosome, a multi-mega Dalton complex of proteins and small nuclear RNAs that performs RNA splicing in humans. This showcase of applications highlights the current talent of MD simulations to dissect atomic-level details of complex biomolecular systems instrumental for the design of finely engineered genome editing machines. As well, this review aims at inspiring future investigations of several other ncRNA regulatory systems, such as micro and small interfering RNAs, which achieve their function and specificity using RNA-based recognition and targeting strategies.},
}

CRISPR-Cas Systems/*genetics
Gene Editing/*trends
Humans
Molecular Dynamics Simulation
*Nucleic Acid Conformation
Proteins/chemistry/genetics
RNA Splicing/genetics
RNA, Untranslated/*genetics/ultrastructure

@article {pmid32490186,
year = {2020},
author = {Moroz-Omori, EV and Satyapertiwi, D and Ramel, MC and Høgset, H and Sunyovszki, IK and Liu, Z and Wojciechowski, JP and Zhang, Y and Grigsby, CL and Brito, L and Bugeon, L and Dallman, MJ and Stevens, MM},
title = {Photoswitchable gRNAs for Spatiotemporally Controlled CRISPR-Cas-Based Genomic Regulation.},
journal = {ACS central science},
volume = {6},
number = {5},
pages = {695-703},
doi = {10.1021/acscentsci.9b01093},
pmid = {32490186},
issn = {2374-7943},
abstract = {The recently discovered CRISPR-Cas gene editing system and its derivatives have found numerous applications in fundamental biology research and pharmaceutical sciences. The need for precise external control over the gene editing and regulatory events has driven the development of inducible CRISPR-Cas systems. While most of the light-controllable CRISPR-Cas systems are based on protein engineering, we developed an alternative synthetic approach based on modification of crRNA/tracrRNA duplex (guide RNA or gRNA) with photocaging groups, preventing the gRNA from recognizing its genome target sequence until its deprotection is induced within seconds of illumination. This approach relies on a straightforward solid-phase synthesis of the photocaged gRNAs, with simpler purification and characterization processes in comparison to engineering a light-responsive protein. We have demonstrated the feasibility of photocaging of gRNAs and light-mediated DNA cleavage upon brief exposure to light in vitro. We have achieved light-mediated spatiotemporally resolved gene editing as well as gene activation in cells, whereas photocaged gRNAs showed virtually no detectable gene editing or activation in the absence of light irradiation. Finally, we have applied this system to spatiotemporally control gene editing in zebrafish embryos in vivo, enabling the use of this strategy for developmental biology and tissue engineering applications.},
}

@article {pmid32486913,
year = {2020},
author = {Curti, LA and Pereyra-Bonnet, F and Repizo, GD and Fay, JV and Salvatierra, K and Blariza, MJ and Ibañez-Alegre, D and Rinflerch, AR and Miretti, M and Gimenez, CA},
title = {CRISPR-based platform for carbapenemases and emerging viruses detection using Cas12a (Cpf1) effector nuclease.},
journal = {Emerging microbes & infections},
volume = {9},
number = {1},
pages = {1140-1148},
doi = {10.1080/22221751.2020.1763857},
pmid = {32486913},
issn = {2222-1751},
abstract = {CRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precise double-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activity upon target recognition. This feature allows CRISPR-Cas to be coupled with a ssDNA reporter and generate a fast, accurate and ultrasensitive molecular detection method. Here, we demonstrate that Cas12a was able to detect DNA target sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with the addition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTV genomes. In all cases, assay run time was less than two hours. Additionally, we report attomolar levels of detection. This methodology was validated using clinical samples from patients infected with Dengue virus. Reactions were visualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicate that Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect all types of pathogens.},
}

@article {pmid32486152,
year = {2020},
author = {Maule, G and Arosio, D and Cereseto, A},
title = {Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing.},
journal = {International journal of molecular sciences},
volume = {21},
number = {11},
pages = {},
doi = {10.3390/ijms21113903},
pmid = {32486152},
issn = {1422-0067},
support = {2019 #3//Fondazione per la Ricerca sulla Fibrosi Cistica/ ; UPGRADE//Horizon 2020 Framework Programme/ ; 2018.256//Fondazione Cassa Di Risparmio Di Trento E Rovereto/ ; },
abstract = {Since the early days of its conceptualization and application, human gene transfer held the promise of a permanent solution to genetic diseases including cystic fibrosis (CF). This field went through alternated periods of enthusiasm and distrust. The development of refined technologies allowing site specific modification with programmable nucleases highly revived the gene therapy field. CRISPR nucleases and derived technologies tremendously facilitate genome manipulation offering diversified strategies to reverse mutations. Here we discuss the advancement of gene therapy, from therapeutic nucleic acids to genome editing techniques, designed to reverse genetic defects in CF. We provide a roadmap through technologies and strategies tailored to correct different types of mutations in the cystic fibrosis transmembrane regulator (CFTR) gene, and their applications for the development of experimental models valuable for the advancement of CF therapies.},
}

@article {pmid32486059,
year = {2020},
author = {Laanto, E and Mäkelä, K and Hoikkala, V and Ravantti, JJ and Sundberg, LR},
title = {Adapting a Phage to Combat Phage Resistance.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {9},
number = {6},
pages = {},
doi = {10.3390/antibiotics9060291},
pmid = {32486059},
issn = {2079-6382},
support = {321985//Academy of Finland/ ; 314939//Academy of Finland/ ; },
abstract = {Phage therapy is becoming a widely recognized alternative for fighting pathogenic bacteria due to increasing antibiotic resistance problems. However, one of the common concerns related to the use of phages is the evolution of bacterial resistance against the phages, putatively disabling the treatment. Experimental adaptation of the phage (phage training) to infect a resistant host has been used to combat this problem. Yet, there is very little information on the trade-offs of phage infectivity and host range. Here we co-cultured a myophage FCV-1 with its host, the fish pathogen Flavobacteriumcolumnare, in lake water and monitored the interaction for a one-month period. Phage resistance was detected within one day of co-culture in the majority of the bacterial isolates (16 out of the 18 co-evolved clones). The primary phage resistance mechanism suggests defense via surface modifications, as the phage numbers rose in the first two days of the experiment and remained stable thereafter. However, one bacterial isolate had acquired a spacer in its CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas locus, indicating that also CRISPR-Cas defense was employed in the phage-host interactions. After a week of co-culture, a phage isolate was obtained that was able to infect 18 out of the 32 otherwise resistant clones isolated during the experiment. Phage genome sequencing revealed several mutations in two open reading frames (ORFs) likely to be involved in the regained infectivity of the evolved phage. Their location in the genome suggests that they encode tail genes. Characterization of this evolved phage, however, showed a direct cost for the ability to infect several otherwise resistant clones-adsorption was significantly lower than in the ancestral phage. This work describes a method for adapting the phage to overcome phage resistance in a fish pathogenic system.},
}

@article {pmid32053639,
year = {2020},
author = {Jin, J and Xu, Y and Huo, L and Ma, L and Scott, AW and Pizzi, MP and Li, Y and Wang, Y and Yao, X and Song, S and Ajani, JA},
title = {An improved strategy for CRISPR/Cas9 gene knockout and subsequent wildtype and mutant gene rescue.},
journal = {PloS one},
volume = {15},
number = {2},
pages = {e0228910},
pmid = {32053639},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Fluorescent Dyes ; Gene Editing ; Gene Knockout Techniques/*methods ; Genetic Engineering/*methods ; Genetic Vectors ; Humans ; Lentivirus/genetics ; Plasmids ; RNA, Guide/genetics ; },
abstract = {A fluorescence marker mOrange was inserted to the popular pLentiCrispr-V2 to create pLentiCrispr-V2-mOrange (V2mO) that contained both a puromycin selection and a fluorescent marker, making viral production and target transduction visible. Lentiviruses packaged with this plasmid and appropriate guide RNAs (gRNAs) successfully knocked out the genes RhoA, Gli1, and Gal3 in human gastric cancer cell lines. Cas9-gRNA editing efficiency could be estimated directly from Sanger electropherograms of short polymerase chain reaction products around the gRNA regions in Cas9-gRNA transduced cells. Single cloning of transduced target cell pools must be performed to establish stable knockout clones. Rescue of wildtype (RhoA and Gal3) and mutant (RhoA.Y42C) genes into knockout cells was successful only when cDNAs, where gRNAs bind, were modified by three nucleotides while the amino acid sequences remained unchanged. Stringent on-target CRISPR/Cas9 editing was observed in Gal3 gene, but not in RhoA gene since RhoA.Y42C already presented a nucleotide change in gRNA5 binding site. In summary, our improved strategy added these advantages: adding visual marker to the popular lentiviral system, monitoring lentiviral production and transduction efficiencies, cell-sorting Cas9+ cells in target cells by fluorescence-activated cell sorting, direct estimation of gene editing efficiency of target cell pools by short PCR electropherograms around gRNA binding sites, and successful rescue of wildtype and mutant genes in knockout cells, overcoming Cas9 editing by modifying cDNAs.},
}

CRISPR-Cas Systems/genetics
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Fluorescent Dyes
Gene Editing
Gene Knockout Techniques/*methods
Genetic Engineering/*methods
Genetic Vectors
Humans
Lentivirus/genetics
Plasmids
RNA, Guide/genetics

@article {pmid31883579,
year = {2020},
author = {Wu, H and He, JS and Zhang, F and Ping, J and Wu, J},
title = {Contamination-free visual detection of CaMV35S promoter amplicon using CRISPR/Cas12a coupled with a designed reaction vessel: Rapid, specific and sensitive.},
journal = {Analytica chimica acta},
volume = {1096},
number = {},
pages = {130-137},
doi = {10.1016/j.aca.2019.10.042},
pmid = {31883579},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; Colorimetry ; DNA Primers/genetics ; Nucleic Acid Amplification Techniques ; Plants, Genetically Modified/*genetics ; *Promoter Regions, Genetic ; RNA, Guide/genetics ; Soybeans/*genetics ; Zea mays/*genetics ; },
abstract = {An ultrafast and convenient method for visually detecting CaMV35S promoter amplicon (amplified products) was established by using CRISPR/Cas12a system coupled with a designed reaction vessel. Genetically modified (GM) soybean (Roundup Ready®) powders containing CaMV35S promoter were employed as detection targets, which were amplified by loop-mediated isothermal amplification (LAMP). The CRISPR/Cas12a system directly mixed with amplified products at 37 °C for 5 min and detection results could be clearly identified by the naked eye under UV light (254 nm). A designed reaction vessel was employed to make operation easier and could effectively prevent contamination at the source. The CRISPR/Cas12a detection system was optimized in our study and the concentration of magnesium ions was proved to be important for the work of CRISPR/Cas12a system. The optimized concentration range of magnesium ions was between 10 mM and 12 mM. Besides, the activated Bst DNA polymerase also had little effects on CRISPR/Cas12a system. The developed method could significantly distinguish the specific and non-specific amplification. And as low as 0.05% transgenic contents in soybean powders could be detected. It would have the potential to be complementary to instrument-based ultrahigh sensitive method and provide a new solution for on-site rapid detection.},
}

*CRISPR-Cas Systems
Colorimetry
DNA Primers/genetics
Nucleic Acid Amplification Techniques
Plants, Genetically Modified/*genetics
*Promoter Regions, Genetic
RNA, Guide/genetics
Soybeans/*genetics
Zea mays/*genetics

@article {pmid31724704,
year = {2019},
author = {Pflueger, C and Swain, T and Lister, R},
title = {Harnessing targeted DNA methylation and demethylation using dCas9.},
journal = {Essays in biochemistry},
volume = {63},
number = {6},
pages = {813-825},
doi = {10.1042/EBC20190029},
pmid = {31724704},
issn = {1744-1358},
mesh = {Animals ; Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; DNA/*metabolism ; *DNA Demethylation ; *DNA Methylation ; Epigenomics/methods ; Gene Editing/methods ; Humans ; Streptococcus pyogenes/enzymology ; },
abstract = {DNA methylation is an essential DNA modification that plays a crucial role in genome regulation during differentiation and development, and is disrupted in a range of disease states. The recent development of CRISPR/catalytically dead CRISPR/Cas9 (dCas9)-based targeted DNA methylation editing tools has enabled new insights into the roles and functional relevance of this modification, including its importance at regulatory regions and the role of aberrant methylation in various diseases. However, while these tools are advancing our ability to understand and manipulate this regulatory layer of the genome, they still possess a variety of limitations in efficacy, implementation, and targeting specificity. Effective targeted DNA methylation editing will continue to advance our fundamental understanding of the role of this modification in different genomic and cellular contexts, and further improvements may enable more accurate disease modeling and possible future treatments. In this review, we discuss strategies, considerations, and future directions for targeted DNA methylation editing.},
}

Animals
Bacterial Proteins/metabolism
CRISPR-Associated Protein 9/*metabolism
*CRISPR-Cas Systems
DNA/*metabolism
*DNA Demethylation
*DNA Methylation
Epigenomics/methods
Gene Editing/methods
Humans
Streptococcus pyogenes/enzymology

@article {pmid31167905,
year = {2019},
author = {Wang, C and Jiang, S and Zhang, L and Li, D and Liang, J and Narita, Y and Hou, I and Zhong, Q and Gewurz, BE and Teng, M and Zhao, B},
title = {TAF Family Proteins and MEF2C Are Essential for Epstein-Barr Virus Super-Enhancer Activity.},
journal = {Journal of virology},
volume = {93},
number = {16},
pages = {},
pmid = {31167905},
issn = {1098-5514},
support = {R35 CA047006/CA/NCI NIH HHS/United States ; R01 CA047006/CA/NCI NIH HHS/United States ; R01 AI123420/AI/NIAID NIH HHS/United States ; R01 AI137337/AI/NIAID NIH HHS/United States ; P30 CA076292/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/genetics ; Enhancer Elements, Genetic ; Epstein-Barr Virus Infections/*virology ; Gene Editing ; Gene Expression ; *Gene Expression Regulation, Viral ; Gene Knockout Techniques ; Genes, myc ; Herpesvirus 4, Human/*physiology ; Histones/metabolism ; Host-Pathogen Interactions ; Humans ; MEF2 Transcription Factors/genetics/metabolism ; TATA-Binding Protein Associated Factors/*metabolism ; },
abstract = {Super-enhancers (SEs) are clusters of enhancers marked by extraordinarily high and broad chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) signals for H3K27ac or other transcription factors (TFs). SEs play pivotal roles in development and oncogenesis. Epstein-Barr virus (EBV) super-enhancers (ESEs) are co-occupied by all essential EBV oncogenes and EBV-activated NF-κB subunits. Perturbation of ESEs stops lymphoblastoid cell line (LCL) growth. To further characterize ESEs and identify proteins critical for ESE function, MYC ESEs were cloned upstream of a green fluorescent protein (GFP) reporter. Reporters driven by MYC ESEs 525 kb and 428 kb upstream of MYC (525ESE and 428ESE) had very high activities in LCLs but not in EBV-negative BJAB cells. EBNA2 activated MYC ESE-driven luciferase reporters. CRISPRi targeting 525ESE significantly decreased MYC expression. Genome-wide CRISPR screens identified factors essential for ESE activity. TBP-associated factor (TAF) family proteins, including TAF8, TAF11, and TAF3, were essential for the activity of the integrated 525ESE-driven reporter in LCLs. TAF8 and TAF11 knockout significantly decreased 525ESE activity and MYC transcription. MEF2C was also identified to be essential for 525ESE activity. Depletion of MEF2C decreased 525ESE reporter activity, MYC expression, and LCL growth. MEF2C cDNA resistant to CRIPSR cutting rescued MEF2C knockout and restored 525ESE reporter activity and MYC expression. MEF2C depletion decreased IRF4, EBNA2, and SPI1 binding to 525ESE in LCLs. MEF2C depletion also affected the expression of other ESE target genes, including the ETS1 and BCL2 genes. These data indicated that in addition to EBNA2, TAF family members and MEF2C are essential for ESE activity, MYC expression, and LCL growth.IMPORTANCE SEs play critical roles in cancer development. Since SEs assemble much bigger protein complexes on enhancers than typical enhancers (TEs), they are more sensitive than TEs to perturbations. Understanding the protein composition of SEs that are linked to key oncogenes may identify novel therapeutic targets. A genome-wide CRISPR screen specifically identified proteins essential for MYC ESE activity but not simian virus 40 (SV40) enhancer. These proteins not only were essential for the reporter activity but also were also important for MYC expression and LCL growth. Targeting these proteins may lead to new therapies for EBV-associated cancers.},
}

CRISPR-Cas Systems
Cell Line, Tumor
Cell Survival/genetics
Enhancer Elements, Genetic
Epstein-Barr Virus Infections/*virology
Gene Editing
Gene Expression
*Gene Expression Regulation, Viral
Gene Knockout Techniques
Genes, myc
Herpesvirus 4, Human/*physiology
Histones/metabolism
Host-Pathogen Interactions
Humans
MEF2 Transcription Factors/genetics/metabolism
TATA-Binding Protein Associated Factors/*metabolism

@article {pmid31166175,
year = {2019},
author = {Liao, M and Tong, T and Zong, Y and Zhou, X and Cheng, L and Huang, R and Ren, B and Alterovitz, G},
title = {Application of Omics and Bioinformatics Tools in Streptococcus Research.},
journal = {Current issues in molecular biology},
volume = {32},
number = {},
pages = {327-376},
doi = {10.21775/cimb.032.327},
pmid = {31166175},
issn = {1467-3045},
mesh = {Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems ; Chromosome Mapping ; Computational Biology/*methods ; DNA, Bacterial/genetics/metabolism ; Drug Resistance, Multiple, Bacterial/*genetics ; *Gene Expression Regulation, Bacterial ; Gene Transfer, Horizontal ; *Genome, Bacterial ; High-Throughput Nucleotide Sequencing ; Host-Pathogen Interactions/genetics ; Humans ; Phylogeny ; Streptococcal Infections/drug therapy/microbiology/pathology ; Streptococcus/classification/drug effects/*genetics/pathogenicity ; Virulence ; },
abstract = {Researchers used to focus on analyzing single gene or protein expression of the microbes. But recently, genome, transcriptome, proteome and metabolome have gained more and more attention. Based on technologies of omics, including genomics, transcriptomics and metabolomics, a large quantity of information about cells, microbes and human, such as the information about phylogeny, virulence, antibiotic resistance and other aspects, has been revealed. Genus Streptococcus is one of the most invasive groups of bacteria that cause both human and animal diseases, threatening public health. In this review, we summarize the application of omics to analyze this genus-Streptococcus.},
}

Anti-Bacterial Agents/pharmacology
Bacterial Proteins/*genetics/metabolism
CRISPR-Cas Systems
Chromosome Mapping
Computational Biology/*methods
DNA, Bacterial/genetics/metabolism
Drug Resistance, Multiple, Bacterial/*genetics
*Gene Expression Regulation, Bacterial
Gene Transfer, Horizontal
*Genome, Bacterial
High-Throughput Nucleotide Sequencing
Host-Pathogen Interactions/genetics
Humans
Phylogeny
Streptococcal Infections/drug therapy/microbiology/pathology
Streptococcus/classification/drug effects/*genetics/pathogenicity
Virulence

@article {pmid30968393,
year = {2019},
author = {Iyer, N and Tcheuyap, VT and Schneider, S and Marshall, V and Jagadeeswaran, P},
title = {Knockout of von Willebrand factor in Zebrafish by CRISPR/Cas9 mutagenesis.},
journal = {British journal of haematology},
volume = {186},
number = {4},
pages = {e76-e80},
pmid = {30968393},
issn = {1365-2141},
support = {R15 DK117384/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Exons ; *Gene Editing ; Gene Knockout Techniques ; Gene Targeting ; *Mutagenesis ; RNA, Guide ; Sequence Analysis, DNA ; Zebrafish/*genetics ; von Willebrand Factor/*genetics ; },
}

Animals
*CRISPR-Cas Systems
Exons
*Gene Editing
Gene Knockout Techniques
Gene Targeting
*Mutagenesis
RNA, Guide
Sequence Analysis, DNA
Zebrafish/*genetics
von Willebrand Factor/*genetics

@article {pmid30680376,
year = {2019},
author = {Alberti, F and Corre, C},
title = {Editing streptomycete genomes in the CRISPR/Cas9 age.},
journal = {Natural product reports},
volume = {36},
number = {9},
pages = {1237-1248},
doi = {10.1039/c8np00081f},
pmid = {30680376},
issn = {1460-4752},
support = {BB/M017982/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Genome, Bacterial/genetics ; Streptomyces/*genetics ; },
abstract = {Covering: up to December 2018 This article aims to highlight advantages, drawbacks and issues that users should consider when implementing the use of CRISPR/Cas9-tools for genome editing in streptomycetes, the most prolific source of antimicrobial natural products to date. Here, we examine four toolkits that have so far been made available for streptomycete in vivo-engineering and one for in vitro-editing, and review how they have been applied over the last three years. Our critical evaluation of these toolkits intends to support potential users in determining what they could achieve, what they should consider and what system they should select/optimise for their application.},
}

CRISPR-Associated Protein 9/*genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Genome, Bacterial/genetics
Streptomyces/*genetics

@article {pmid32485207,
year = {2020},
author = {Ely, A and Singh, P and Smith, TS and Arbuthnot, P},
title = {In vitro transcribed MRNA for expression of designer nucleases: advantages as a novel therapeutic for the management of chronic HBV infection.},
journal = {Advanced drug delivery reviews},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.addr.2020.05.010},
pmid = {32485207},
issn = {1872-8294},
abstract = {Chronic infection with the hepatitis B virus (HBV) remains a significant worldwide medical problem. While diseases caused by HIV infection, tuberculosis and malaria are on the decline, new cases of chronic hepatitis B are on the rise. Because often fatal complications of cirrhosis and hepatocellular carcinoma are associated with chronic hepatitis B, the need for a cure is as urgent as ever. Currently licensed therapeutics fail to eradicate the virus and this is attributable to persistence of the viral replication intermediate comprising covalently closed circular DNA (cccDNA). Elimination or inactivation of the viral cccDNA is thus a goal of research aimed at hepatitis B cure. Ability to engineer nucleases that are capable of specific cleavage of a DNA sequence now provides the means to disable cccDNA permanently. The scientific literature is replete with many examples of using designer zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs) to inactivate HBV. However, important concerns about safety, dose control and efficient delivery need to be addressed before the technology is employed in a clinical setting. Use of in vitro transcribed mRNA to express therapeutic gene editors goes some way to overcoming these concerns. The labile nature of RNA limits off-target effects and enables dose control. Compatibility with hepatotropic non-viral vectors is convenient for the large scale preparation that will be required for advancing gene editing as a mode of curing chronic hepatitis B.},
}

@article {pmid32485068,
year = {2020},
author = {Zeng, D and Liu, T and Ma, X and Wang, B and Zheng, Z and Zhang, Y and Xie, X and Yang, B and Zhao, Z and Zhu, Q and Liu, YG},
title = {Quantitative regulation of Waxy expression by CRISPR/Cas9-based promoter and 5'UTR-intron editing improves grain quality in rice.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13427},
pmid = {32485068},
issn = {1467-7652},
abstract = {In cereal crops, grain starches are composed of different proportions of amylose and amylopectin, which determine the cooking and eating qualities. The amylose synthesis is controlled by the Waxy (Wx) gene encoding a granulebound NDP-glucose-starch glucosyltransferase (Shure et al., 1983). In rice (Oryza sativa L.), the varied activities of natural Wx alleles regulate different amylose contents (AC), gel consistency (GC), and pasting viscosity of grain starches; these factors together influence the grain appearance, cooking/eating quality, and starch physical characters (Zhang et al., 2019).},
}

@article {pmid32483454,
year = {2020},
author = {Liu, H and Li, H and Liang, Y and Du, X and Yang, C and Yang, L and Xie, J and Zhao, R and Tong, Y and Qiu, S and Song, H},
title = {Phage-delivered sensitisation with subsequent antibiotic treatment reveals sustained effect against antimicrobial resistant bacteria.},
journal = {Theranostics},
volume = {10},
number = {14},
pages = {6310-6321},
doi = {10.7150/thno.42573},
pmid = {32483454},
issn = {1838-7640},
abstract = {Temperate phages integrated with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas systems have been gaining attention as potential strategies for combating bacteria resistant to antimicrobials. To further advance this technology, phage recombination procedure should be improved, and the bactericidal effect should be examined in detail and compared with conventional lytic phage strategy. The possibility of the emergence of mutational resistance, a phenomenon commonly observed with lytic phage therapy, should be illustrated. Methods: Here, we developed a novel one-step cloning method to fulfil the recombination of CRISPR/Cas9 system within the genome of a new isolated lysogenic Escherichia coli phage. Then, we proposed and developed a phage-delivered resistance eradication with subsequent antibiotic treatment (PRESA) strategy. The removal efficiency and antimicrobial effect of the plasmids were analysed. Long-term antimicrobial effect was evaluated by continued OD600 monitoring for 240 hours to illustrate the potential mutational resistance, compared with the lytic phage strategy. The treatment effect of PRESA was evaluated in vivo by determining bacterial loads in the skin and intestine of infected mice, in contrast with lytic phage therapy. Genome sequencing was performed to identify mutations in bacterial cells treated with phage strategies. Results: Phage-delivered CRISPR targeting efficiently eradicated and blocked the transfer of the antibiotic resistance plasmid. PRESA decreased the bacterial load by over 6- and 5-logs in vitro and in vivo, respectively. Importantly, while lytic phages induced mutational phage resistance at 24 h in vitro and 48 hours in vivo, PRESA demonstrated a constant effect and revealed no resistant mutants. Genes involved in DNA mismatch repair were upregulated in cells undergoing Cas9-based plasmid cleavage, which may reduce the development of mutations. Conclusion: The PRESA strategy for eradicating resistant bacteria showed high bactericidal efficacy and a sustained inhibition effect against resistant bacteria. By restoring the efficacy of low-cost antibiotics, PRESA could be developed as an efficient and economical therapy for infections of antibiotic resistant bacteria.},
}

@article {pmid32483187,
year = {2020},
author = {Hirschi, M and Lu, WT and Santiago-Frangos, A and Wilkinson, R and Golden, SM and Davidson, AR and Lander, GC and Wiedenheft, B},
title = {AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided surveillance complex.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {2730},
doi = {10.1038/s41467-020-16512-1},
pmid = {32483187},
issn = {2041-1723},
support = {R35GM134867//U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; S10OD021634//U.S. Department of Health & Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; },
abstract = {Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.},
}

@article {pmid31733438,
year = {2019},
author = {Überbacher, C and Obergasteiger, J and Volta, M and Venezia, S and Müller, S and Pesce, I and Pizzi, S and Lamonaca, G and Picard, A and Cattelan, G and Malpeli, G and Zoli, M and Beccano-Kelly, D and Flynn, R and Wade-Martins, R and Pramstaller, PP and Hicks, AA and Cowley, SA and Corti, C},
title = {Application of CRISPR/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons.},
journal = {Stem cell research},
volume = {41},
number = {},
pages = {101656},
doi = {10.1016/j.scr.2019.101656},
pmid = {31733438},
issn = {1876-7753},
support = {MR/L023784/1/MRC_/Medical Research Council/United Kingdom ; MC_EX_MR/N50192X/1/MRC_/Medical Research Council/United Kingdom ; MR/L023784/2/MRC_/Medical Research Council/United Kingdom ; MR/M024962/1/MRC_/Medical Research Council/United Kingdom ; WTISSF121302/WT_/Wellcome Trust/United Kingdom ; J-0901/PUK_/Parkinson's UK/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Dopaminergic Neurons/cytology/*metabolism ; *Gene Editing ; *Gene Knock-In Techniques ; Green Fluorescent Proteins/*biosynthesis/genetics ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; Microscopy, Fluorescence ; *Polymerase Chain Reaction ; *Transgenes ; },
abstract = {Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase - enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.},
}

*CRISPR-Cas Systems
Cell Line
Dopaminergic Neurons/cytology/*metabolism
*Gene Editing
*Gene Knock-In Techniques
Green Fluorescent Proteins/*biosynthesis/genetics
Humans
Induced Pluripotent Stem Cells/cytology/*metabolism
Microscopy, Fluorescence
*Polymerase Chain Reaction
*Transgenes

@article {pmid31217244,
year = {2019},
author = {Staller, E and Sheppard, CM and Neasham, PJ and Mistry, B and Peacock, TP and Goldhill, DH and Long, JS and Barclay, WS},
title = {ANP32 Proteins Are Essential for Influenza Virus Replication in Human Cells.},
journal = {Journal of virology},
volume = {93},
number = {17},
pages = {},
pmid = {31217244},
issn = {1098-5514},
support = {/WT_/Wellcome Trust/United Kingdom ; BB/K002465/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 205100/WT_/Wellcome Trust/United Kingdom ; BB/R013071/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 104931/Z/14/Z/WT_/Wellcome Trust/United Kingdom ; BB/L015129/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Cycle Proteins/chemistry/*genetics/metabolism ; Cell Line ; Disease Models, Animal ; Humans ; Influenza A virus/*physiology ; Influenzavirus B/*physiology ; Mice ; Nerve Tissue Proteins/chemistry/*genetics/metabolism ; Nuclear Proteins/chemistry/*genetics/metabolism ; Point Mutation ; Protein Domains ; RNA Replicase/*metabolism ; RNA-Binding Proteins/chemistry/*genetics/metabolism ; Viral Proteins/metabolism ; Virus Replication ; },
abstract = {ANP32 proteins have been implicated in supporting influenza virus replication, but most of the work to date has focused on the ability of avian Anp32 proteins to overcome restriction of avian influenza polymerases in human cells. Using a CRISPR approach, we show that the human acidic nuclear phosphoproteins (ANPs) ANP32A and ANP32B are functionally redundant but essential host factors for mammalian-adapted influenza A virus (IAV) and influenza B virus (IBV) replication in human cells. When both proteins are absent from human cells, influenza polymerases are unable to replicate the viral genome, and infectious virus cannot propagate. Provision of exogenous ANP32A or ANP32B recovers polymerase activity and virus growth. We demonstrate that this redundancy is absent in the murine Anp32 orthologues; murine Anp32A is incapable of recovering IAV polymerase activity, while murine Anp32B can do so. Intriguingly, IBV polymerase is able to use murine Anp32A. We show, using a domain swap and point mutations, that the leucine-rich repeat (LRR) 5 region comprises an important functional domain for mammalian ANP32 proteins. Our approach has identified a pair of essential host factors for influenza virus replication and can be harnessed to inform future interventions.IMPORTANCE Influenza virus is the etiological agent behind some of the most devastating infectious disease pandemics to date, and influenza outbreaks still pose a major threat to public health. Influenza virus polymerase, the molecule that copies the viral RNA genome, hijacks cellular proteins to support its replication. Current anti-influenza drugs are aimed against viral proteins, including the polymerase, but RNA viruses like influenza tend to become resistant to such drugs very rapidly. An alternative strategy is to design therapeutics that target the host proteins that are necessary for virus propagation. Here, we show that the human proteins ANP32A and ANP32B are essential for influenza A and B virus replication, such that in their absence cells become impervious to the virus. We map the proviral activity of ANP32 proteins to one region in particular, which could inform future intervention.},
}

Animals
CRISPR-Cas Systems
Cell Cycle Proteins/chemistry/*genetics/metabolism
Cell Line
Disease Models, Animal
Humans
Influenza A virus/*physiology
Influenzavirus B/*physiology
Mice
Nerve Tissue Proteins/chemistry/*genetics/metabolism
Nuclear Proteins/chemistry/*genetics/metabolism
Point Mutation
Protein Domains
RNA Replicase/*metabolism
RNA-Binding Proteins/chemistry/*genetics/metabolism
Viral Proteins/metabolism
Virus Replication

@article {pmid31189706,
year = {2019},
author = {Zhang, Y and Tang, N and Luo, J and Teng, M and Moffat, K and Shen, Z and Watson, M and Nair, V and Yao, Y},
title = {Marek's Disease Virus-Encoded MicroRNA 155 Ortholog Critical for the Induction of Lymphomas Is Not Essential for the Proliferation of Transformed Cell Lines.},
journal = {Journal of virology},
volume = {93},
number = {17},
pages = {},
pmid = {31189706},
issn = {1098-5514},
support = {BBS/E/I/00007032/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R007632/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/R007896/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Transformed ; Cell Line, Tumor ; Cell Proliferation ; Cell Transformation, Viral/*genetics ; Humans ; Lymphoma/*virology ; Mardivirus/genetics/*pathogenicity ; MicroRNAs/*genetics ; RNA, Viral/genetics ; },
abstract = {MicroRNAs (miRNAs) are small noncoding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) and Marek's disease virus (MDV), respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and in lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, those studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative reverse transcription-PCR (qRT-PCR), and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is not essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumor cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gaining further insights into the biology of oncogenic herpesviruses.IMPORTANCE Marek's disease virus (MDV) is an alphaherpesvirus associated with Marek's disease (MD), a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumors, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this was the first study in which precise editing of an oncogenic miRNA was carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype.},
}

Animals
CRISPR-Cas Systems
Cell Line, Transformed
Cell Line, Tumor
Cell Proliferation
Cell Transformation, Viral/*genetics
Humans
Lymphoma/*virology
Mardivirus/genetics/*pathogenicity
MicroRNAs/*genetics
RNA, Viral/genetics

@article {pmid32479612,
year = {2020},
author = {Wu, F and Shim, J and Tan, C},
title = {Orthogonal tuning of gene expression noise using CRISPR-Cas.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaa451},
pmid = {32479612},
issn = {1362-4962},
abstract = {The control of gene expression noise is important for improving drug treatment and the performance of synthetic biological systems. Previous work has tuned gene expression noise by changing the rate of transcription initiation, mRNA degradation, and mRNA translation. However, these methods are invasive: they require changes to the target genetic components. Here, we create an orthogonal system based on CRISPR-dCas9 to tune gene expression noise. Specifically, we modulate the gene expression noise of a reporter gene in Escherichia coli by incorporating CRISPR activation and repression (CRISPRar) simultaneously in a single cell. The CRISPRar uses a single dCas9 that recognizes two different single guide RNAs (sgRNA). We build a library of sgRNA variants with different expression activation and repression strengths. We find that expression noise and mean of a reporter gene can be tuned independently by CRISPRar. Our results suggest that the expression noise is tuned by the competition between two sgRNAs that modulate the binding of RNA polymerase to promoters. The CRISPRar may change how we tune expression noise at the genomic level. Our work has broad impacts on the study of gene functions, phenotypical heterogeneity, and genetic circuit control.},
}

@article {pmid32478351,
year = {2020},
author = {Hsieh, K and Zhao, G and Wang, TH},
title = {Applying biosensor development concepts to improve preamplification-free CRISPR/Cas12a-Dx.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d0an00664e},
pmid = {32478351},
issn = {1364-5528},
abstract = {Development of CRISPR/Cas-based in vitro diagnostic devices, or CRISPR/Cas-Dx, has become an intensely researched area. Among the different classes of CRISPR/Cas-Dx, the class based on the Cas12a enzyme (i.e., CRISPR/Cas12a-Dx or simply Cas12a-Dx), is predominantly employed for detecting DNA targets. Current research in Cas12a-Dx has focused on appending Cas12a-Dx to preamplification techniques or coupling Cas12a-Dx to different detection modalities, which has inevitably overshadowed the detection performance of Cas12a-Dx and overlooked its intrinsic detection capability without preamplification. We recognize that Cas12a-Dx, which relies on DNA-activated Cas12a to cleave single-stranded DNA, shares significant similarity with other nuclease-based DNA biosensors, whose performances can be influenced by parameters ranging from the reaction buffer to the reaction temperature. We are thus inspired to probe the limits of preamplification-free Cas12a-Dx by exploring and systematically evaluating several potential parameters that may impact its detection sensitivity and time. Using a previously reported fluorescence-based Cas12a-Dx as the test bed, we have identified that the Cas12a enzyme, the reaction buffer, the substrate label, the substrate concentration, and the reaction temperature can be optimized to significantly improve the signal-to-background ratio and the reaction rate of Cas12a-Dx. Based on these findings, we have improved the limit of detection (LOD) of the Cas12a-Dx to 100 fM, while reduced the time-to-positive to <46 min, representing the most sensitive LOD without preamplification and the fastest time-to-positive for this LOD to date. More broadly, our work provides a roadmap for further advancing Cas12a-Dx and perhaps other classes of CRISPR/Cas-Dx.},
}

@article {pmid32476557,
year = {2020},
author = {O'Shea, P and Wildenhain, J and Leveridge, M and Revankar, C and Yang, JP and Bradley, J and Firth, M and Pilling, J and Piper, D and Chesnut, J and Isherwood, B},
title = {A Novel Screening Approach for the Dissection of Cellular Regulatory Networks of NF-κB Using Arrayed CRISPR gRNA Libraries.},
journal = {SLAS discovery : advancing life sciences R & D},
volume = {},
number = {},
pages = {2472555220926160},
doi = {10.1177/2472555220926160},
pmid = {32476557},
issn = {2472-5560},
abstract = {CRISPR/Cas9 is increasingly being used as a tool to prosecute functional genomic screens. However, it is not yet possible to apply the approach at scale across a full breadth of cell types and endpoints. In order to address this, we developed a novel and robust workflow for array-based lentiviral CRISPR/Cas9 screening. We utilized a β-lactamase reporter gene assay to investigate mediators of TNF-α-mediated NF-κB signaling. The system was adapted for CRISPR/Cas9 through the development of a cell line stably expressing Cas9 and application of a lentiviral gRNA library comprising mixtures of four gRNAs per gene. We screened a 743-gene kinome library whereupon hits were independently ranked by percent inhibition, Z' score, strictly standardized mean difference, and T statistic. A consolidated and optimized ranking was generated using Borda-based methods. Screening data quality was above acceptable limits (Z' ≥ 0.5). In order to determine the contribution of individual gRNAs and to better understand false positives and negatives, a subset of gRNAs, against 152 genes, were profiled in singlicate format. We highlight the use of known reference genes and high-throughput, next-generation amplicon and RNA sequencing to assess screen data quality. Screening with singlicate gRNAs was more successful than screening with mixtures at identifying genes with known regulatory roles in TNF-α-mediated NF-κB signaling and was found to be superior to previous RNAi-based methods. These results add to the available data on TNF-α-mediated NF-κB signaling and establish a high-throughput functional genomic screening approach, utilizing a vector-based arrayed gRNA library, applicable across a wide variety of endpoints and cell types at a genome-wide scale.},
}

@article {pmid32165308,
year = {2020},
author = {Rothschild, SC and Ingram, SR and Lu, FI and Thisse, B and Thisse, C and Parkerson, JA and Tombes, RM},
title = {Genetic compensation of γ CaMKII, an evolutionarily conserved gene.},
journal = {Gene},
volume = {742},
number = {},
pages = {144567},
doi = {10.1016/j.gene.2020.144567},
pmid = {32165308},
issn = {1879-0038},
mesh = {Animals ; Animals, Genetically Modified ; Biological Evolution ; CRISPR-Cas Systems/genetics ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/*genetics ; Embryo, Nonmammalian ; *Gene Expression Regulation, Developmental ; Loss of Function Mutation ; Mutagenesis ; Zebrafish/*genetics/growth & development ; Zebrafish Proteins/*genetics ; },
abstract = {CaMKII is a Ca2+/CaM-dependent protein kinase encoded by a family of conserved genes found throughout all metazoan species and expressed from fertilization into adulthood. One of these genes, camk2g1, is particularly important during early development as determined by pharmacologic, dominant negative and antisense morpholino approaches in zebrafish. Four other teleost fish species (cavefish, medaka, stickleback, and tilapia), exhibit sequence conservation of camk2g1 and duplication of the same CaMKII genes. A homozygous mutant of camk2g1 was generated in zebrafish using TALEN technology but yielded none of the phenotypic alterations seen using all other approaches and was reproductively viable. However, these camk2g1 mutant embryos showed a 4-fold over-expression of its paralog camk2g2. None of the other camk2 genes showed such transcriptional elevation, in fact, some of these genes were suppressed to 10% of wild type levels. In contrast, G0 camk2g1 CRISPR/Cas9 embryos recapitulated nearly all of the altered phenotypes observed in camk2g1 morphants, including renal, aural and ciliary defects. These findings validate the importance of this gene family during early zebrafish development and provide evidence for gene-specific transcriptional cross-talk consistent with genetic compensation.},
}

Animals
Animals, Genetically Modified
Biological Evolution
CRISPR-Cas Systems/genetics
Calcium-Calmodulin-Dependent Protein Kinase Type 2/*genetics
Embryo, Nonmammalian
*Gene Expression Regulation, Developmental
Loss of Function Mutation
Mutagenesis
Zebrafish/*genetics/growth & development
Zebrafish Proteins/*genetics

@article {pmid31964810,
year = {2020},
author = {Koslová, A and Trefil, P and Mucksová, J and Reinišová, M and Plachý, J and Kalina, J and Kučerová, D and Geryk, J and Krchlíková, V and Lejčková, B and Hejnar, J},
title = {Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {117},
number = {4},
pages = {2108-2112},
pmid = {31964810},
issn = {1091-6490},
mesh = {Animals ; Animals, Genetically Modified/genetics/immunology/virology ; Avian Leukosis/genetics/*immunology/virology ; Avian Leukosis Virus/classification/*genetics/physiology ; Avian Proteins/*genetics/immunology ; CRISPR-Cas Systems ; Chickens ; Disease Resistance ; Female ; Gene Editing ; Male ; Poultry Diseases/genetics/*immunology/virology ; Sodium-Hydrogen Exchanger 1/*genetics/immunology ; },
abstract = {Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na+/H+ exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J-resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J-susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.},
}

Animals
Animals, Genetically Modified/genetics/immunology/virology
Avian Leukosis/genetics/*immunology/virology
Avian Leukosis Virus/classification/*genetics/physiology
Avian Proteins/*genetics/immunology
CRISPR-Cas Systems
Chickens
Disease Resistance
Female
Gene Editing
Male
Poultry Diseases/genetics/*immunology/virology
Sodium-Hydrogen Exchanger 1/*genetics/immunology

@article {pmid31860398,
year = {2019},
author = {Flotte, TR and Gao, G},
title = {Prime Editing: A Novel Cas9-Reverse Transcriptase Fusion May Revolutionize Genome Editing.},
journal = {Human gene therapy},
volume = {30},
number = {12},
pages = {1445-1446},
doi = {10.1089/hum.2019.29098.trf},
pmid = {31860398},
issn = {1557-7422},
support = {P01 HL131471/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; DNA/biosynthesis/*genetics ; Gene Editing/*methods ; Genetic Therapy/*trends ; Humans ; RNA-Directed DNA Polymerase/genetics ; Recombinational DNA Repair/*genetics ; },
}

CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems/genetics
DNA/biosynthesis/*genetics
Gene Editing/*methods
Genetic Therapy/*trends
Humans
RNA-Directed DNA Polymerase/genetics
Recombinational DNA Repair/*genetics

@article {pmid31842424,
year = {2019},
author = {Volkova, EI and Andreyenkova, NG and Andreyenkov, OV and Sidorenko, DS and Zhimulev, IF and Demakov, SA},
title = {Structural and Functional Dissection of the 5' Region of the Notch Gene in Drosophila melanogaster.},
journal = {Genes},
volume = {10},
number = {12},
pages = {},
pmid = {31842424},
issn = {2073-4425},
mesh = {5' Untranslated Regions/genetics ; Animals ; CRISPR-Cas Systems ; Chromosome Structures/genetics ; Drosophila Proteins/*genetics/*metabolism ; Drosophila melanogaster/genetics ; Gene Expression Regulation/genetics ; Homologous Recombination/genetics ; Polytene Chromosomes/genetics ; Receptors, Notch/*genetics/*metabolism ; Structure-Activity Relationship ; },
abstract = {Notch is a key factor of a signaling cascade which regulates cell differentiation in all multicellular organisms. Numerous investigations have been directed mainly at studying the mechanism of Notch protein action; however, very little is known about the regulation of activity of the gene itself. Here, we provide the results of targeted 5'-end editing of the Drosophila Notch gene in its native environment and genetic and cytological effects of these changes. Using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) system in combination with homologous recombination, we obtained a founder fly stock in which a 4-kb fragment, including the 5' nontranscribed region, the first exon, and a part of the first intron of Notch, was replaced by an attachment Phage (attP) site. Then, fly lines carrying a set of six deletions within the 5'untranscribed region of the gene were obtained by ΦC31-mediated integration of transgenic constructs. Part of these deletions does not affect gene activity, but their combinations with transgenic construct in the first intron of the gene cause defects in the Notch target tissues. At the polytene chromosome level we defined a DNA segment (~250 bp) in the Notch5'-nontranscribed region which when deleted leads to disappearance of the 3C6/C7 interband and elimination of CTC-Factor (CTCF) and Chromator (CHRIZ) insulator proteins in this region.},
}

5' Untranslated Regions/genetics
Animals
CRISPR-Cas Systems
Chromosome Structures/genetics
Drosophila Proteins/*genetics/*metabolism
Drosophila melanogaster/genetics
Gene Expression Regulation/genetics
Homologous Recombination/genetics
Polytene Chromosomes/genetics
Receptors, Notch/*genetics/*metabolism
Structure-Activity Relationship

@article {pmid31829168,
year = {2019},
author = {Liu, W and Meridew, JA and Aravamudhan, A and Ligresti, G and Tschumperlin, DJ and Tan, Q},
title = {Targeted regulation of fibroblast state by CRISPR-mediated CEBPA expression.},
journal = {Respiratory research},
volume = {20},
number = {1},
pages = {281},
pmid = {31829168},
issn = {1465-993X},
support = {R01 HL092961/HL/NHLBI NIH HHS/United States ; R01 HL142596/HL/NHLBI NIH HHS/United States ; },
mesh = {Adipogenesis ; CCAAT-Enhancer-Binding Proteins/genetics/*metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Case-Control Studies ; Cells, Cultured ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Fibroblasts/drug effects/*metabolism/pathology ; Fibrosis ; *Gene Editing ; Gene Expression Regulation ; Humans ; Idiopathic Pulmonary Fibrosis/genetics/*metabolism/pathology ; Lung/drug effects/*metabolism/pathology ; Phenotype ; RNA Interference ; Signal Transduction ; Transforming Growth Factor beta1/pharmacology ; },
abstract = {BACKGROUND: Fibroblasts regulate tissue homeostasis and the balance between tissue repair and fibrosis. CCAAT/enhancer-binding protein alpha (CEBPA) is a key transcription factor that regulates adipogenesis. CEBPA has been shown to be essential for lung maturation, and deficiency of CEBPA expression leads to abnormal lung architecture. However, its specific role in lung fibroblast regulation and fibrosis has not yet been elucidated.

METHODS: Lung fibroblast CEBPA expression, pro-fibrotic and lipofibroblast gene expression were assessed by qRT-PCR. CEBPA gain and loss of function experiments were carried out to evaluate the role of CEBPA in human lung fibroblast activation with and without TGF-β1 treatment. Adipogenesis assay was used to measure the adiopogenic potential of lung fibroblasts. Finally, CRISPR activation system was used to enhance endogenous CEBPA expression.

RESULTS: We found that CEBPA gene expression is significantly decreased in IPF-derived fibroblasts compared to normal lung fibroblasts. CEBPA knockdown in normal human lung fibroblasts enhanced fibroblast pro-fibrotic activation and ECM production. CEBPA over-expression by transient transfection in IPF-derived fibroblasts significantly reduced pro-fibrotic gene expression, ECM deposition and αSMA expression and promoted the formation of lipid droplets measured by Oil Red O staining and increased lipofibroblast gene expression. Inhibition of the histone methyl transferase G9a enhanced CEBPA expression, and the anti-fibrotic effects of G9a inhibition were partially mediated by CEBPA expression. Finally, targeted CRISPR-mediated activation of CEBPA resulted in fibroblasts switching from fibrogenic to lipofibroblast states.

CONCLUSIONS: CEBPA expression is reduced in human IPF fibroblasts and its deficiency reduces adipogenic potential and promotes fibrogenic activation. CEBPA expression can be rescued via an inhibitor of epigenetic repression or by targeted CRISPR activation, leading to reduced fibrogenic activation.},
}

Adipogenesis
CCAAT-Enhancer-Binding Proteins/genetics/*metabolism
CRISPR-Associated Protein 9/genetics/metabolism
*CRISPR-Cas Systems
Case-Control Studies
Cells, Cultured
*Clustered Regularly Interspaced Short Palindromic Repeats
Fibroblasts/drug effects/*metabolism/pathology
Fibrosis
*Gene Editing
Gene Expression Regulation
Humans
Idiopathic Pulmonary Fibrosis/genetics/*metabolism/pathology
Lung/drug effects/*metabolism/pathology
Phenotype
RNA Interference
Signal Transduction
Transforming Growth Factor beta1/pharmacology

@article {pmid31722988,
year = {2019},
author = {Anderson, W and Thorpe, J and Long, SA and Rawlings, DJ},
title = {Efficient CRISPR/Cas9 Disruption of Autoimmune-Associated Genes Reveals Key Signaling Programs in Primary Human T Cells.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {203},
number = {12},
pages = {3166-3178},
pmid = {31722988},
issn = {1550-6606},
support = {DP3 DK111802/DK/NIDDK NIH HHS/United States ; TL1 TR002318/TR/NCATS NIH HHS/United States ; },
mesh = {Alleles ; Autoimmunity/*genetics ; Blood Donors ; CD4-Positive T-Lymphocytes/*metabolism ; CRISPR-Cas Systems/*immunology ; Cells, Cultured ; Gene Editing ; Genome-Wide Association Study ; Humans ; Interleukin-2/metabolism ; Phenotype ; Polymorphism, Single Nucleotide ; Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics/metabolism ; Protein Tyrosine Phosphatase, Non-Receptor Type 22/genetics ; Receptors, Antigen, T-Cell/metabolism ; Signal Transduction/genetics ; Suppressor of Cytokine Signaling 3 Protein/metabolism ; ZAP-70 Protein-Tyrosine Kinase/genetics ; },
abstract = {Risk of autoimmunity is associated with multiple genetic variants. Genome-wide association studies have linked single-nucleotide polymorphisms in the phosphatases PTPN22 (rs2476601) and PTPN2 (rs1893217) to increased risk for multiple autoimmune diseases. Previous mouse studies of loss of function or risk variants in these genes revealed hyperactive T cell responses, whereas studies of human lymphocytes revealed contrasting phenotypes. To better understand this dichotomy, we established a robust gene editing platform to rapidly address the consequences of loss of function of candidate genes in primary human CD4+ T cells. Using CRISPR/Cas9, we obtained efficient gene disruption (>80%) of target genes encoding proteins involved in Ag and cytokine receptor signaling pathways including PTPN22 and PTPN2 Loss-of-function data in all genes studied correlated with previous data from mouse models. Further analyses of PTPN2 gene-disrupted T cells demonstrated dynamic effects, by which hyperactive IL-2R signaling promoted compensatory transcriptional events, eventually resulting in T cells that were hyporesponsive to IL-2. These results imply that altered phosphatase activity promotes evolving phenotypes based on Ag experience and/or other programming signals. This approach enables the discovery of molecular mechanisms modulating risk of autoimmunity that have been difficult to parse in traditional mouse models or cross-sectional human studies.},
}

Alleles
Autoimmunity/*genetics
Blood Donors
CD4-Positive T-Lymphocytes/*metabolism
CRISPR-Cas Systems/*immunology
Cells, Cultured
Gene Editing
Genome-Wide Association Study
Humans
Interleukin-2/metabolism
Phenotype
Polymorphism, Single Nucleotide
Protein Tyrosine Phosphatase, Non-Receptor Type 2/genetics/metabolism
Protein Tyrosine Phosphatase, Non-Receptor Type 22/genetics
Receptors, Antigen, T-Cell/metabolism
Signal Transduction/genetics
Suppressor of Cytokine Signaling 3 Protein/metabolism
ZAP-70 Protein-Tyrosine Kinase/genetics

@article {pmid31615875,
year = {2019},
author = {Achuthankutty, D and Thakur, RS and Haahr, P and Hoffmann, S and Drainas, AP and Bizard, AH and Weischenfeldt, J and Hickson, ID and Mailand, N},
title = {Regulation of ETAA1-mediated ATR activation couples DNA replication fidelity and genome stability.},
journal = {The Journal of cell biology},
volume = {218},
number = {12},
pages = {3943-3953},
pmid = {31615875},
issn = {1540-8140},
mesh = {Antigens, Surface/*metabolism ; Ataxia Telangiectasia Mutated Proteins/*metabolism ; CRISPR-Cas Systems ; Cell Cycle ; Cell Line, Tumor ; Chromosome Aberrations ; DNA Damage ; *DNA Replication ; *Gene Expression Regulation, Neoplastic ; Genome, Human ; *Genomic Instability ; HCT116 Cells ; HEK293 Cells ; HeLa Cells ; Humans ; Mitosis ; Nuclear Proteins/metabolism ; Phosphorylation ; Protein Binding ; Signal Transduction ; },
abstract = {The ATR kinase is a master regulator of the cellular response to DNA replication stress. Activation of ATR relies on dual pathways involving the TopBP1 and ETAA1 proteins, both of which harbor ATR-activating domains (AADs). However, the exact contribution of the recently discovered ETAA1 pathway to ATR signaling in different contexts remains poorly understood. Here, using an unbiased CRISPR-Cas9-based genome-scale screen, we show that the ATR-stimulating function of ETAA1 becomes indispensable for cell fitness and chromosome stability when the fidelity of DNA replication is compromised. We demonstrate that the ATR-activating potential of ETAA1 is controlled by cell cycle- and replication stress-dependent phosphorylation of highly conserved residues within its AAD, and that the stimulatory impact of these modifications is required for the ability of ETAA1 to prevent mitotic chromosome abnormalities following replicative stress. Our findings suggest an important role of ETAA1 in protecting against genome instability arising from incompletely duplicated DNA via regulatory control of its ATR-stimulating potential.},
}

Antigens, Surface/*metabolism
Ataxia Telangiectasia Mutated Proteins/*metabolism
CRISPR-Cas Systems
Cell Cycle
Cell Line, Tumor
Chromosome Aberrations
DNA Damage
*DNA Replication
*Gene Expression Regulation, Neoplastic
Genome, Human
*Genomic Instability
HCT116 Cells
HEK293 Cells
HeLa Cells
Humans
Mitosis
Nuclear Proteins/metabolism
Phosphorylation
Protein Binding
Signal Transduction

@article {pmid31462429,
year = {2019},
author = {Song, X and Wan, X and Huang, T and Zeng, C and Sastry, N and Wu, B and James, CD and Horbinski, C and Nakano, I and Zhang, W and Hu, B and Cheng, SY},
title = {SRSF3-Regulated RNA Alternative Splicing Promotes Glioblastoma Tumorigenicity by Affecting Multiple Cellular Processes.},
journal = {Cancer research},
volume = {79},
number = {20},
pages = {5288-5301},
pmid = {31462429},
issn = {1538-7445},
support = {R21 CA209345/CA/NCI NIH HHS/United States ; R01 NS095642/NS/NINDS NIH HHS/United States ; R01 NS107071/NS/NINDS NIH HHS/United States ; R01 NS102669/NS/NINDS NIH HHS/United States ; UL1 TR001422/TR/NCATS NIH HHS/United States ; R01 NS093843/NS/NINDS NIH HHS/United States ; P50 CA221747/CA/NCI NIH HHS/United States ; F31 CA232630/CA/NCI NIH HHS/United States ; },
mesh = {*Alternative Splicing ; Animals ; Brain Neoplasms/*genetics/metabolism/pathology ; CRISPR-Cas Systems ; Cell Division ; Cell Line, Tumor ; Cell Self Renewal ; DNA-Binding Proteins/genetics ; Disease Progression ; *Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Glioblastoma/*genetics/metabolism/pathology ; HEK293 Cells ; Heterografts ; Humans ; Mice ; Mice, Nude ; Microtubule-Associated Proteins/genetics ; Neoplasm Proteins/biosynthesis/genetics/*physiology ; Neoplasm Transplantation ; Phosphorylation ; Prognosis ; Protein Isoforms/physiology ; Protein Processing, Post-Translational ; RNA, Messenger/*biosynthesis/genetics ; Serine-Arginine Splicing Factors/antagonists & inhibitors/genetics/*physiology ; Spindle Apparatus/metabolism ; Transcription Factors/genetics ; },
abstract = {Misregulated alternative RNA splicing (AS) contributes to the tumorigenesis and progression of human cancers, including glioblastoma (GBM). Here, we showed that a major splicing factor, serine and arginine rich splicing factor 3 (SRSF3), was frequently upregulated in clinical glioma specimens and that elevated SRSF3 was associated with tumor progression and a poor prognosis for patients with glioma. In patient-derived glioma stem-like cells (GSC), SRSF3 expression promoted cell proliferation, self-renewal, and tumorigenesis. Transcriptomic profiling identified more than 1,000 SRSF3-affected AS events, with a preference for exon skipping in genes involved with cell mitosis. Motif analysis identified the sequence of CA(G/C/A)CC(C/A) as a potential exonic splicing enhancer for these SRSF3-regulated exons. To evaluate the biological impact of SRSF3-affected AS events, four candidates were selected whose AS correlated with SRSF3 expression in glioma tissues, and their splicing pattern was modified using a CRISPR/Cas9 approach. Two functionally validated AS candidates were further investigated for the mechanisms underlying their isoform-specific functions. Specifically, following knockout of SRSF3, transcription factor ETS variant 1 (ETV1) gene showed exon skipping at exon 7, while nudE neurodevelopment protein 1 (NDE1) gene showed replacement of terminal exon 9 with a mutually exclusive exon 9'. SRSF3-regulated AS of these two genes markedly increased their oncogenic activity in GSCs. Taken together, our data demonstrate that SRSF3 is a key regulator of AS in GBM and that understanding mechanisms of misregulated AS could provide critical insights for developing effective therapeutic strategies against GBMs. SIGNIFICANCE: SRSF3 is a significant regulator of glioma-associated alternative splicing, implicating SRSF3 as an oncogenic factor that contributes to the tumor biology of GBM.},
}

*Alternative Splicing
Animals
Brain Neoplasms/*genetics/metabolism/pathology
CRISPR-Cas Systems
Cell Division
Cell Line, Tumor
Cell Self Renewal
DNA-Binding Proteins/genetics
Disease Progression
*Gene Expression Regulation, Neoplastic
Gene Knockdown Techniques
Gene Knockout Techniques
Glioblastoma/*genetics/metabolism/pathology
HEK293 Cells
Heterografts
Humans
Mice
Mice, Nude
Microtubule-Associated Proteins/genetics
Neoplasm Proteins/biosynthesis/genetics/*physiology
Neoplasm Transplantation
Phosphorylation
Prognosis
Protein Isoforms/physiology
Protein Processing, Post-Translational
RNA, Messenger/*biosynthesis/genetics
Serine-Arginine Splicing Factors/antagonists & inhibitors/genetics/*physiology
Spindle Apparatus/metabolism
Transcription Factors/genetics

@article {pmid31444400,
year = {2019},
author = {Cazzola, A and Schlegel, C and Jansen, I and Bochtler, T and Jauch, A and Krämer, A},
title = {TP53 deficiency permits chromosome abnormalities and karyotype heterogeneity in acute myeloid leukemia.},
journal = {Leukemia},
volume = {33},
number = {11},
pages = {2619-2627},
doi = {10.1038/s41375-019-0550-5},
pmid = {31444400},
issn = {1476-5551},
mesh = {Aneuploidy ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; *Chromosome Aberrations ; DNA Damage ; Gene Deletion ; Humans ; In Situ Hybridization, Fluorescence ; Karyotyping ; Leukemia, Myeloid, Acute/diagnosis/*genetics ; Mutation ; Prognosis ; Tumor Suppressor Protein p53/*genetics ; },
abstract = {Abnormal karyotypes are common in cancer cells and frequently observed in acute myeloid leukemia (AML), in which complex karyotype aberrations are associated with poor prognosis. How exactly abnormal karyotypes arise and are propagated in AML is unclear. TP53 mutations and deletions are frequent in complex karyotype AML, suggesting a role of TP53 alterations in the development of chromosome abnormalities. Here, we generated isogenic TP53-knockout versions of the euploid AML cell line EEB to investigate the impact of TP53 on karyotype stability. We show that chromosome abnormalities spontaneously arise in TP53-deficient cells. Numerical aneuploidy could, to some extent, be propagated in a TP53-proficient setting, indicating that it does not necessarily trigger TP53 activation. In contrast, tolerance to structural chromosome aberrations was almost entirely restricted to TP53-knockout clones, all of which were able to continue proliferation in the presence of damaged DNA. Mechanistically, as a source of chromosome aberrations, limited numerical but not structural chromosomal instability was tolerated by TP53-wildtype cells. In contrast, structural instability was found only in TP53-knockout cells. Together, in myeloid cells TP53 loss allows for the development of complex karyotype aberrations and karyotype heterogeneity by perpetuation of chromosome segregation errors.},
}

Aneuploidy
CRISPR-Cas Systems
Cell Line, Tumor
Cell Proliferation
*Chromosome Aberrations
DNA Damage
Gene Deletion
Humans
In Situ Hybridization, Fluorescence
Karyotyping
Leukemia, Myeloid, Acute/diagnosis/*genetics
Mutation
Prognosis
Tumor Suppressor Protein p53/*genetics

@article {pmid31199673,
year = {2019},
author = {Lu, FI and Wang, YT and Wang, YS and Wu, CY and Li, CC},
title = {Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {9},
pages = {9959-9973},
doi = {10.1096/fj.201900342RR},
pmid = {31199673},
issn = {1530-6860},
mesh = {ADP-Ribosylation Factor 1/antagonists & inhibitors/physiology ; Animals ; CRISPR-Cas Systems ; Cell Movement ; Embryo, Nonmammalian/blood supply ; Embryonic Development ; Endothelial Cells/cytology/metabolism ; Gene Knockdown Techniques ; Genes, Reporter ; Guanine Nucleotide Exchange Factors/antagonists & inhibitors/genetics/*physiology ; Human Umbilical Vein Endothelial Cells ; Humans ; Neovascularization, Physiologic/genetics/*physiology ; RNA Interference ; RNA, Small Interfering/genetics/pharmacology ; Vascular Endothelial Growth Factor A/*biosynthesis/genetics ; Zebrafish/embryology/genetics ; Zebrafish Proteins/genetics/physiology ; },
abstract = {VEGF stimulates the formation of new blood vessels by inducing endothelial cell (EC) proliferation and migration. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (BIG)1 and 2 accelerate the replacement of bound GDP with GTP to activate ADP-ribosylation factor (Arf)1, which regulates vesicular transport between the Golgi and plasma membrane. Although it has been reported that treating cells with BFA interferes with Arf1 activation to inhibit VEGF secretion, the role of BIG1 and BIG2 in VEGF trafficking and expression, EC migration and proliferation, and vascular development remains unknown. Here, we found that inactivation of Arf1 reduced VEGF secretion but did not affect the levels of VEGF protein. Interestingly, however, BIG1 and BIG2 knockdown significantly decreased the levels of VEGF mRNA and protein in glioblastoma U251 cells and HUVECs. Furthermore, depletion of BIG1 and BIG2 inhibited HUVEC angiogenesis by diminishing cell migration. Angioblast migration and intersegmental vessel sprouting were also impaired when the BIG2 homolog, Arf guanine nucleotide exchange factor (arfgef)2, was knocked down in zebrafish with endothelial expression of green fluorescent protein (GFP). Depletion of arfgef2 by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) also caused defects in vascular development of zebrafish embryos. Taken together, these data reveal that BIG1 and BIG2 participate in endothelial cell angiogenesis.-Lu, F.-I., Wang, Y.-T., Wang, Y.-S., Wu, C.-Y., Li, C.-C. Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.},
}

ADP-Ribosylation Factor 1/antagonists & inhibitors/physiology
Animals
CRISPR-Cas Systems
Cell Movement
Embryo, Nonmammalian/blood supply
Embryonic Development
Endothelial Cells/cytology/metabolism
Gene Knockdown Techniques
Genes, Reporter
Guanine Nucleotide Exchange Factors/antagonists & inhibitors/genetics/*physiology
Human Umbilical Vein Endothelial Cells
Humans
Neovascularization, Physiologic/genetics/*physiology
RNA Interference
RNA, Small Interfering/genetics/pharmacology
Vascular Endothelial Growth Factor A/*biosynthesis/genetics
Zebrafish/embryology/genetics
Zebrafish Proteins/genetics/physiology

@article {pmid31071267,
year = {2019},
author = {Liu, Z and Shan, H and Chen, S and Chen, M and Zhang, Q and Lai, L and Li, Z},
title = {Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {8},
pages = {9210-9219},
doi = {10.1096/fj.201900476RR},
pmid = {31071267},
issn = {1530-6860},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Mutation/genetics ; Polymerase Chain Reaction ; Rabbits ; Rats ; Zygote ; },
abstract = {Cytidine base editors, which are composed of a cytidine deaminase fused to clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) nickase, enable the efficient conversion of the C·G base pair to T·A in various organisms. However, the currently used rat apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1(rA1)-based BE3 is often inefficient in target Cs that are immediately downstream of a G (GC context). Here, we observed that, with an 11-nt editing window, an optimized activation-induced cytidine deaminase (AID)-Cas9 fusion can efficiently convert C to T in a variety of sequence contexts in rabbits. Strikingly, the enhanced AID-Cas9 fusion (eAID-BE4max) has significant effectiveness of inducing Tyr p.R299H mutation in GC contexts (from 16.67 to 83.33%) in comparison with BE3 in founder rabbits. Furthermore, the engineered AID-Cas9 variants were produced with reduced bystander activity [eAID (N51G)-BE4max] and increased genome-targeting scope (eAID-NG-BE4max). Overall, this work provides a series of improved tools that were generated using optimized AID-Cas9 fusions and associated engineered variants that can be used for efficient and versatile C-to-T base editing, especially in GC contexts.-Liu, Z., Shan, H., Chen, S., Chen, M., Zhang, Q., Lai, L., Li, Z. Improved base editor for efficient editing in GC contexts in rabbits with an optimized AID-Cas9 fusion.},
}

Animals
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/methods
Mutation/genetics
Polymerase Chain Reaction
Rabbits
Rats
Zygote

@article {pmid30962579,
year = {2019},
author = {Song, Y and Park, PMC and Wu, L and Ray, A and Picaud, S and Li, D and Wimalasena, VK and Du, T and Filippakopoulos, P and Anderson, KC and Qi, J and Chauhan, D},
title = {Development and preclinical validation of a novel covalent ubiquitin receptor Rpn13 degrader in multiple myeloma.},
journal = {Leukemia},
volume = {33},
number = {11},
pages = {2685-2694},
doi = {10.1038/s41375-019-0467-z},
pmid = {30962579},
issn = {1476-5551},
support = {P01 CA155258/CA/NCI NIH HHS/United States ; R01 CA207237/CA/NCI NIH HHS/United States ; P50 CA100707/CA/NCI NIH HHS/United States ; MR/N010051/1/MRC_/Medical Research Council/United Kingdom ; R01 CA222218/CA/NCI NIH HHS/United States ; R01 CA050947/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/pharmacology ; Apoptosis ; Bortezomib/pharmacology ; CRISPR-Cas Systems ; Caspases/metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cell Survival ; Dendritic Cells/cytology ; Drug Evaluation, Preclinical ; HCT116 Cells ; Humans ; Intracellular Signaling Peptides and Proteins/*antagonists & inhibitors/*metabolism ; Lenalidomide/pharmacology ; Mice ; Mice, SCID ; Multiple Myeloma/*metabolism/therapy ; Neoplasm Transplantation ; Proteasome Endopeptidase Complex/metabolism ; Proteasome Inhibitors/*pharmacology ; RNA Interference ; Ubiquitin/chemistry ; },
abstract = {Proteasome inhibition is an effective treatment for multiple myeloma (MM); however, targeting different components of the ubiquitin-proteasome system (UPS) remains elusive. Our RNA-interference studies identified proteasome-associated ubiquitin-receptor Rpn13 as a mediator of MM cell growth and survival. Here, we developed the first degrader of Rpn13, WL40, using a small-molecule-induced targeted protein degradation strategy to selectively degrade this component of the UPS. WL40 was synthesized by linking the Rpn13 covalent inhibitor RA190 with the cereblon (CRBN) binding ligand thalidomide. We show that WL40 binds to both Rpn13 and CRBN and triggers degradation of cellular Rpn13, and is therefore first-in-class in exploiting a covalent inhibitor for the development of degraders. Biochemical and cellular studies show that WL40-induced Rpn13 degradation is both CRBN E3 ligase- and Rpn13-dependent. Importantly, WL40 decreases viability in MM cell lines and patient MM cells, even those resistant to bortezomib. Mechanistically, WL40 interrupts Rpn13 function and activates caspase apoptotic cascade, ER stress response and p53/p21 signaling. In animal model studies, WL40 inhibits xenografted human MM cell growth and prolongs survival. Overall, our data show the development of the first UbR Rpn13 degrader with potent anti-MM activity, and provide proof of principle for the development of degraders targeting components of the UPS for therapeutic application.},
}

Animals
Antineoplastic Agents/pharmacology
Apoptosis
Bortezomib/pharmacology
CRISPR-Cas Systems
Caspases/metabolism
Cell Line, Tumor
Cell Proliferation
Cell Survival
Dendritic Cells/cytology
Drug Evaluation, Preclinical
HCT116 Cells
Humans
Intracellular Signaling Peptides and Proteins/*antagonists & inhibitors/*metabolism
Lenalidomide/pharmacology
Mice
Mice, SCID
Multiple Myeloma/*metabolism/therapy
Neoplasm Transplantation
Proteasome Endopeptidase Complex/metabolism
Proteasome Inhibitors/*pharmacology
RNA Interference
Ubiquitin/chemistry

@article {pmid30831133,
year = {2019},
author = {Takashima, S and Shinkuma, S and Fujita, Y and Nomura, T and Ujiie, H and Natsuga, K and Iwata, H and Nakamura, H and Vorobyev, A and Abe, R and Shimizu, H},
title = {Efficient Gene Reframing Therapy for Recessive Dystrophic Epidermolysis Bullosa with CRISPR/Cas9.},
journal = {The Journal of investigative dermatology},
volume = {139},
number = {8},
pages = {1711-1721.e4},
doi = {10.1016/j.jid.2019.02.015},
pmid = {30831133},
issn = {1523-1747},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Collagen Type VII/chemistry/*genetics ; *DNA End-Joining Repair ; Epidermolysis Bullosa Dystrophica/genetics/*therapy ; Exons/genetics ; Fibroblasts/transplantation ; Frameshift Mutation ; Gene Editing/methods ; Genes, Recessive/genetics ; Genetic Therapy/*methods ; HEK293 Cells ; Humans ; Infant ; Mice ; Mutagenesis, Site-Directed ; Mutation ; Primary Cell Culture ; Protein Conformation, alpha-Helical/genetics ; Recombinant Proteins/chemistry/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system induces site-specific double-strand breaks, which stimulate cellular DNA repair through either the homologous recombination or non-homologous end-joining pathways. The non-homologous end-joining pathway, which is activated more frequently than homologous recombination, is prone to introducing small insertions and/or deletions at the double-strand break site, leading to changes in the reading frame. We hypothesized that the non-homologous end-joining pathway is applicable to genetic diseases caused by a frameshift mutation through restoration of the reading frame. Recessive dystrophic epidermolysis bullosa is a hereditary skin disorder caused by mutations in COL7A1. In this study, we applied gene reframing therapy to a recurrent frameshift mutation, c.5819delC, in COL7A1, which results in a premature termination codon. CRISPR/Cas9 targeting this specific mutation site was delivered to recessive dystrophic epidermolysis bullosa patient fibroblasts. After genotyping a large collection of gene-edited fibroblast clones, we identified a significant number (17/50) of clones in which the frameshift in COL7A1 was restored. The reframed COL7 was functional, as shown by triple-helix formation assay in vitro, and was correctly distributed in the basement membrane zone in mice. Our data suggest that mutation site-specific non-homologous end-joining might be a highly efficient gene therapy for inherited disorders caused by frameshift mutations.},
}

Animals
CRISPR-Cas Systems/*genetics
Collagen Type VII/chemistry/*genetics
*DNA End-Joining Repair
Epidermolysis Bullosa Dystrophica/genetics/*therapy
Exons/genetics
Fibroblasts/transplantation
Frameshift Mutation
Gene Editing/methods
Genes, Recessive/genetics
Genetic Therapy/*methods
HEK293 Cells
Humans
Infant
Mice
Mutagenesis, Site-Directed
Mutation
Primary Cell Culture
Protein Conformation, alpha-Helical/genetics
Recombinant Proteins/chemistry/genetics

@article {pmid30830215,
year = {2019},
author = {Franca, MM and Han, X and Funari, MFA and Lerario, AM and Nishi, MY and Fontenele, EGP and Domenice, S and Jorge, AAL and Garcia-Galiano, D and Elias, CF and Mendonca, BB},
title = {Exome Sequencing Reveals the POLR3H Gene as a Novel Cause of Primary Ovarian Insufficiency.},
journal = {The Journal of clinical endocrinology and metabolism},
volume = {104},
number = {7},
pages = {2827-2841},
pmid = {30830215},
issn = {1945-7197},
support = {P30 CA046592/CA/NCI NIH HHS/United States ; P30 DK034933/DK/NIDDK NIH HHS/United States ; R01 HD069702/HD/NICHD NIH HHS/United States ; R21 HD090567/HD/NICHD NIH HHS/United States ; },
mesh = {Adolescent ; Animals ; CRISPR-Cas Systems ; Child ; Female ; Forkhead Box Protein O3/metabolism ; Gene Knockout Techniques ; Heterozygote ; Homozygote ; Humans ; Infertility/genetics ; Litter Size ; Loss of Function Mutation ; Male ; Mice ; Mutation, Missense ; Ovary/metabolism ; Primary Ovarian Insufficiency/*genetics ; RNA Polymerase III/*genetics ; Sexual Maturation/genetics ; Time-to-Pregnancy ; Whole Exome Sequencing ; },
abstract = {CONTEXT: Primary ovarian insufficiency (POI) is a cause of female infertility. However, the genetic etiology of this disorder remains unknown in most patients with POI.

OBJECTIVE: To investigate the genetic etiology of idiopathic POI.

PATIENTS AND METHODS: We performed whole-exome sequencing of 11 families with idiopathic POI. To gain insights into the potential mechanisms associated with this mutation, we generated two mouse lines via clustered regularly interspaced short palindromic repeats/Cas9 technology.

RESULTS: A pathogenic homozygous missense mutation (c.149A>G; p.Asp50Gly) in the POLR3H gene in two unrelated families was identified. Pathogenic mutations in this subunit have not been associated with human disorders. Loss-of-function Polr3h mutation in mice caused early embryonic lethality. Mice with homozygous point mutation (Polr3hD50G) were viable but showed delayed pubertal development, characterized by late first estrus or preputial separation. The Polr3hD50G female and male mice showed decreased fertility later in life, associated with small litter size and increased time to pregnancy or to impregnate a female. Polr3hD50G mice displayed decreased expression of ovarian Foxo3a and lower numbers of primary follicles.

CONCLUSION: Our manuscript provides a case of POI caused by missense mutation in POLR3H, expanding the knowledge of molecular pathways of the ovarian function and human infertility. Screening of the POLR3H gene may elucidate POI cases without previously identified genetic causes, supporting approaches of genetic counseling.},
}

Adolescent
Animals
CRISPR-Cas Systems
Child
Female
Forkhead Box Protein O3/metabolism
Gene Knockout Techniques
Heterozygote
Homozygote
Humans
Infertility/genetics
Litter Size
Loss of Function Mutation
Male
Mice
Mutation, Missense
Ovary/metabolism
Primary Ovarian Insufficiency/*genetics
RNA Polymerase III/*genetics
Sexual Maturation/genetics
Time-to-Pregnancy
Whole Exome Sequencing

@article {pmid32474853,
year = {2020},
author = {Xie, P and Mo, JL and Liu, JH and Li, X and Tan, LM and Zhang, W and Zhou, HH and Liu, ZQ},
title = {Pharmacogenomics of 5-fluorouracil in colorectal cancer: review and update.},
journal = {Cellular oncology (Dordrecht)},
volume = {},
number = {},
pages = {},
doi = {10.1007/s13402-020-00529-1},
pmid = {32474853},
issn = {2211-3436},
support = {2016YFC1306900//the National Key Research and Development Program of China/ ; 81573508//National Natural Science Foundation of China/ ; 81874327//National Natural Science Foundation of China/ ; ZLXD2017003//The Strategy-Oriented Special Project of Central South University in China/ ; SZSM201811057//Sanming Project of Medicine in Shenzhen/ ; },
abstract = {BACKGROUND: Colorectal cancer (CRC) is a disease with high morbidity and mortality rates. 5-fluorouracil (5-FU) is the first-line recommended drug for chemotherapy in patients with CRC, and it has a good effect on a variety of other solid tumors as well. Unfortunately, however, due to the emergence of drug resistance the effectiveness of treatment may be greatly reduced. In the past decade, major progress has been made in the field of 5-FU drug resistance in terms of molecular mechanisms, pre-clinical (animal) models and clinical trials.

CONCLUSIONS: In this article we systematically review and update current knowledge on 5-FU pharmacogenomics related to drug uptake and activation, the expression and activity of target enzymes (DPD, TS and MTHFR) and key signaling pathways in CRC. Furthermore, a summary of drug combination strategies aimed at targeting specific genes and/or pathways to reverse 5-FU resistance is provided. Based on this, we suggest that causal relationships between genes, pathways and drug sensitivity should be systematically considered from a multidimensional perspective. In the design of research methods, emerging technologies such as CRISPR-Cas, TALENS and patient-derived xenograft models should be applied as far as possible to improve the accuracy of clinically relevant results.},
}

@article {pmid32473055,
year = {2020},
author = {Merker, L and Schindele, P and Huang, TK and Wolter, F and Puchta, H},
title = {Enhancing in planta gene targeting efficiencies in Arabidopsis using temperature-tolerant CRISPR/LbCas12a.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13426},
pmid = {32473055},
issn = {1467-7652},
abstract = {The application of the CRISPR/Cas-system paved the way for the era of genome engineering and quickly became the standardly applied tool for targeted non-homologous end joining (NHEJ) based mutagenesis. Despite various efforts, homologous recombination (HR) based gene targeting (GT) still requires further improvement regarding efficiency for general applicability in plants (Huang and Puchta, 2019). We developed the in planta GT (ipGT) method aimed to establish a technology for crops with meagre transformation efficiencies (Fauser et al., 2012).},
}

@article {pmid32472422,
year = {2020},
author = {Safari, I and Baradaran-Rafii, A and Issazadeh-Navikas, S and Elahi, E},
title = {CHST6 mutations identified in Iranian MCD patients and CHST6 mutations reported worldwide identify targets for gene editing approaches including the CRISPR/Cas system.},
journal = {International ophthalmology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s10792-020-01401-9},
pmid = {32472422},
issn = {1573-2630},
abstract = {PURPOSE: To identify CHST6 mutations in Iranians macular corneal dystrophy (MCD) patients and also to assess distribution of amino acids in the encoded protein that are affected by CHST6 mutations reported hitherto in various populations in order to predict gene regions that may be appropriate targets for gene editing approaches including the CRISPR/Cas system. The analysis will also reveal biologically and functionally important regions of the protein.

METHODS: Mutation screening of CHST6 by sequencing was performed on 21 Iranian MCD-affected probands. Previously reported MCD causing CHST6 mutations were identified by searches in NCBI.

RESULTS: Nineteen CHST6 mutations were found among the 21 Iranian patients, most of which were missense mutations and six of which were novel. Totally, 189 mutations among 375 MCD patients have been found worldwide, and 134 of these are missense mutations. The distribution of 88 amino acids affected by missense mutations along the length of the encoded protein was not random, and four regions of possible mutation clustering were noted. 25% of patients harbored mutations in a DNA region consisting of only 36 nucleotides.

CONCLUSION: Similar to most populations, CHST6 mutations among Iranians are very heterogeneous as indicated by finding 19 different mutations among 21 MCD patients. Nevertheless, identification of four potential mutation clusters identifies regions that are most suitable for gene therapy targeting by the CRISPR/Cas approach. Additionally, the mutation clusters identify regions with potential structural and/or functional importance. Consistent with this, the amino acids in these regions are well conserved among various membrane-bound sulfotransferases.},
}

@article {pmid32467331,
year = {2020},
author = {Meeske, AJ and Jia, N and Cassel, AK and Kozlova, A and Liao, J and Wiedmann, M and Patel, DJ and Marraffini, LA},
title = {A phage-encoded anti-CRISPR enables complete evasion of type VI-A CRISPR-Cas immunity.},
journal = {Science (New York, N.Y.)},
volume = {},
number = {},
pages = {},
doi = {10.1126/science.abb6151},
pmid = {32467331},
issn = {1095-9203},
abstract = {The crRNA-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. How viruses can counteract this immunity is not known. We describe a listeriophage (ϕLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivated the type VI-A CRISPR system of Listeria seeligeri Using genetics, biochemistry and structural biology we found that AcrVIA1 interacted with the guide-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion could completely dismantle type VI-A CRISPR-mediated immunity.},
}

@article {pmid32467232,
year = {2020},
author = {Long, J and Galvan, DL and Mise, K and Kanwar, YS and Li, L and Poungvarin, N and Overbeek, PA and Chang, BH and Danesh, FR},
title = {Role for carbohydrate response element-binding protein (ChREBP) in high glucose-mediated repression of long noncoding RNA Tug1.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA120.013228},
pmid = {32467232},
issn = {1083-351X},
abstract = {Long-noncoding RNAs (lncRNAs) have been shown to play key roles in a variety of biological activities of the cell. However, less is known about how lncRNAs respond to environmental cues, and what transcriptional mechanisms regulate their expression. Studies from our laboratory have shown that the lncRNA Tug1 (taurine upregulated gene 1) is crucial for progression of diabetic kidney disease, a major microvascular complication of diabetes. Using a combination of proximity labeling with the engineered soybean ascorbate peroxidase (APEX2), ChIP-qPCR, biotin-labeled oligo-nucleotides pulldown, and classical promoter luciferase assays in kidney podocytes, we extend our initial observations in the current study, and now provide a detailed analysis on how high glucose milieu down-regulates Tug1 expression in podocytes. Our results revealed an essential role for the transcription factor carbohydrate response element binding protein (ChREBP) in controlling Tug1 transcription in the podocytes in response to increased glucose levels. Along with ChREBP, other co-regulators, including MAX dimerization protein (MLX), MAX dimerization protein 1 (MXD1), and histone deacetylase 1 (HDAC1) were enriched at the Tug1 promoter under the high-glucose conditions. These observations provide the first characterization of the mouse Tug1 promoter's response to the high glucose milieu. Our findings illustrate a molecular mechanism by which ChREBP can coordinate glucose homeostasis with the expression of the lncRNA Tug1, and further our understanding of dynamic transcriptional regulation of lncRNAs in a disease state.},
}

@article {pmid31657874,
year = {2020},
author = {Bae, SJ and Park, BG and Kim, BG and Hahn, JS},
title = {Multiplex Gene Disruption by Targeted Base Editing of Yarrowia lipolytica Genome Using Cytidine Deaminase Combined with the CRISPR/Cas9 System.},
journal = {Biotechnology journal},
volume = {15},
number = {1},
pages = {e1900238},
doi = {10.1002/biot.201900238},
pmid = {31657874},
issn = {1860-7314},
support = {//National Research Foundation of Korea/ ; NRF-2017R1E1A1A01073523//Ministry of Science, ICT & Future Planning/ ; },
mesh = {CRISPR-Cas Systems/*genetics ; *Cytidine Deaminase/genetics/metabolism ; Gene Editing/*methods ; Genome, Bacterial/*genetics ; Recombinant Fusion Proteins/genetics/metabolism ; Uracil-DNA Glycosidase/genetics ; Yarrowia/*genetics ; },
abstract = {The oleaginous yeast Yarrowia lipolytica has a tendency to use the non-homologous end joining repair (NHEJ) over the homology directed recombination as double-strand breaks (DSB) repair system, making it difficult to edit the genome using homologous recombination. A recently developed Target-AID (activation-induced cytidine deaminase) base editor, designed to recruit cytidine deaminase (CDA) to the target DNA locus via the CRISPR/Cas9 system, can directly induce C to T mutation without DSB and donor DNA. In this study, this system is adopted in Y. lipolytica for multiplex gene disruption. Target-specific gRNA(s) and a fusion protein consisting of a nickase Cas9, pmCDA1, and uracil DNA glycosylase inhibitor are expressed from a single plasmid to disrupt target genes by introducing a stop codon via C to T mutation within the mutational window. Deletion of the KU70 gene involved in the NHEJ prevents the generation of indels by base excision repair following cytidine deamination, increasing the accuracy of genome editing. Using this Target-AID system with optimized expression levels of the base editor, single gene disruption and simultaneous double gene disruption are achieved with the efficiencies up to 94% and 31%, respectively, demonstrating this base editing system as a convenient genome editing tool in Y. lipolytica.},
}

CRISPR-Cas Systems/*genetics
*Cytidine Deaminase/genetics/metabolism
Gene Editing/*methods
Genome, Bacterial/*genetics
Recombinant Fusion Proteins/genetics/metabolism
Uracil-DNA Glycosidase/genetics
Yarrowia/*genetics

@article {pmid31436510,
year = {2019},
author = {Sukhdev, M},
title = {CRISPRcon: What Does Rome Look Like?.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {193-195},
doi = {10.1089/crispr.2019.29068.msu},
pmid = {31436510},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems ; Gene Editing/ethics/*legislation & jurisprudence ; Policy Making ; },
}

*CRISPR-Cas Systems
Gene Editing/ethics/*legislation & jurisprudence
Policy Making

@article {pmid31436509,
year = {2019},
author = {Davies, K and Bolt, A},
title = {The Director's Cut: An Interview with Adam Bolt.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {188-192},
doi = {10.1089/crispr.2019.29067.abo},
pmid = {31436509},
issn = {2573-1602},
mesh = {CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*history/methods ; History, 21st Century ; *Motion Pictures ; },
}

CRISPR-Associated Proteins
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing/*history/methods
History, 21st Century
*Motion Pictures

@article {pmid31436507,
year = {2019},
author = {Barrangou, R},
title = {Bringing CRISPR to the Cinema.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {187},
doi = {10.1089/crispr.2019.29070.rba},
pmid = {31436507},
issn = {2573-1602},
mesh = {CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; *Motion Pictures ; },
}

CRISPR-Associated Proteins
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing
*Motion Pictures

@article {pmid31436506,
year = {2019},
author = {Standage-Beier, K and Brookhouser, N and Balachandran, P and Zhang, Q and Brafman, DA and Wang, X},
title = {RNA-Guided Recombinase-Cas9 Fusion Targets Genomic DNA Deletion and Integration.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {209-222},
pmid = {31436506},
issn = {2573-1602},
support = {R01 GM106081/GM/NIGMS NIH HHS/United States ; R01 GM131405/GM/NIGMS NIH HHS/United States ; R21 AG056706/AG/NIA NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/*metabolism ; *CRISPR-Cas Systems ; Gene Editing/*methods ; HEK293 Cells ; Humans ; Recombinant Fusion Proteins ; Saccharomyces cerevisiae/genetics ; Transposon Resolvases ; },
abstract = {CRISPR-based technologies have become central to genome engineering. However, CRISPR-based editing strategies are dependent on the repair of DNA breaks via endogenous DNA repair mechanisms, which increases susceptibility to unwanted mutations. Here we complement Cas9 with a recombinase's functionality by fusing a hyperactive mutant resolvase from transposon Tn3, a member of serine recombinases, to a catalytically inactive Cas9, which we term integrase Cas9 (iCas9). We demonstrate iCas9 targets DNA deletion and integration. First, we validate iCas9's function in Saccharomyces cerevisiae using a genome-integrated reporter. Cooperative targeting by CRISPR RNAs at spacings of 22 or 40 bp enables iCas9-mediated recombination. Next, iCas9's ability to target DNA deletion and integration in human HEK293 cells is demonstrated using dual GFP-mCherry fluorescent reporter plasmid systems. Finally, we show that iCas9 is capable of targeting integration into a genomic reporter locus. We envision targeting and design concepts of iCas9 will contribute to genome engineering and synthetic biology.},
}

CRISPR-Associated Protein 9/*metabolism
*CRISPR-Cas Systems
Gene Editing/*methods
HEK293 Cells
Humans
Recombinant Fusion Proteins
Saccharomyces cerevisiae/genetics
Transposon Resolvases

@article {pmid31436505,
year = {2019},
author = {Gilmore, MS},
title = {The CRISPR-Antibiotic Resistance Connection.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {199-200},
doi = {10.1089/crispr.2019.29065.msg},
pmid = {31436505},
issn = {2573-1602},
mesh = {Bacteria/genetics ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Microbial/*genetics ; Gene Editing/*history ; History, 21st Century ; },
}

Bacteria/genetics
CRISPR-Associated Proteins
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Drug Resistance, Microbial/*genetics
Gene Editing/*history
History, 21st Century

@article {pmid31436504,
year = {2019},
author = {Sun, N and Petiwala, S and Lu, C and Hutti, JE and Hu, M and Hu, M and Domanus, MH and Mitra, D and Addo, SN and Miller, CP and Chung, N},
title = {VHL Synthetic Lethality Signatures Uncovered by Genotype-Specific CRISPR-Cas9 Screens.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {230-245},
doi = {10.1089/crispr.2019.0018},
pmid = {31436504},
issn = {2573-1602},
mesh = {CRISPR-Cas Systems ; Carcinoma, Renal Cell/genetics/metabolism ; Cell Line, Tumor ; DNA Damage ; *DNA Repair ; Gene Editing ; Humans ; Kidney Neoplasms/genetics/metabolism ; Selenocysteine/*biosynthesis ; Sequence Analysis, RNA ; *Signal Transduction ; Von Hippel-Lindau Tumor Suppressor Protein/genetics/*metabolism ; von Hippel-Lindau Disease/genetics/metabolism ; },
abstract = {Genome-wide CRISPR-Cas9 essentiality screening represents a powerful approach to identify genetic vulnerabilities in cancer cells. Here, we applied this technology and designed a strategy to identify target genes that are synthetic lethal (SL) with von Hippel-Lindau (VHL) tumor suppressor gene. Inactivation of VHL has been frequently found in clear cell renal cell carcinoma. Its SL partners serve as potential drug targets for the development of targeted cancer therapies. We performed parallel genome-wide CRISPR screens in two pairs of isogenic clear cell renal cell carcinoma cell lines that differ only in the VHL status. Comparative analyses of screening results not only confirmed a well-known role for mTOR signaling in renal carcinoma, but also identified DNA damage response and selenocysteine biosynthesis pathways as novel SL targets in VHL-inactivated cancer cells. Follow-up studies provided cellular and mechanistic insights into SL interactions of these pathway genes with the VHL gene. Our CRISPR and RNA-seq datasets provide a rich resource for future investigation of the function of the VHL tumor suppressor protein. Our work demonstrates the efficiency of CRISPR-based synthetic lethality screening in human isogenic cell pairs. Similar strategies could be employed to unveil SL partners with other oncogenic drivers.},
}

CRISPR-Cas Systems
Carcinoma, Renal Cell/genetics/metabolism
Cell Line, Tumor
DNA Damage
*DNA Repair
Gene Editing
Humans
Kidney Neoplasms/genetics/metabolism
Selenocysteine/*biosynthesis
Sequence Analysis, RNA
*Signal Transduction
Von Hippel-Lindau Tumor Suppressor Protein/genetics/*metabolism
von Hippel-Lindau Disease/genetics/metabolism

@article {pmid31436503,
year = {2019},
author = {LeMieux, J},
title = {CRISPR-Accelerated Gene Drives Pump the Brakes.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {196-198},
doi = {10.1089/crispr.2019.29069.jlm},
pmid = {31436503},
issn = {2573-1602},
mesh = {Animals ; *Animals, Genetically Modified ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Culicidae/*genetics ; *Gene Drive Technology ; Gene Editing ; Mosquito Control/*methods ; },
}

Animals
*Animals, Genetically Modified
CRISPR-Associated Proteins
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Culicidae/*genetics
*Gene Drive Technology
Gene Editing
Mosquito Control/*methods

@article {pmid31328964,
year = {2019},
author = {Xu, L and Liu, Y and Han, R},
title = {BEAT: A Python Program to Quantify Base Editing from Sanger Sequencing.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {223-229},
pmid = {31328964},
issn = {2573-1602},
support = {R01 AR064241/AR/NIAMS NIH HHS/United States ; R01 HL116546/HL/NHLBI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; HEK293 Cells ; Humans ; Sequence Analysis, DNA/*methods ; *Software ; },
abstract = {Through fusing CRISPR-Cas9 nickases with cytidine or adenine deaminases, a new paradigm-shifting class of genome-editing technology, termed "base editors," has recently been developed. Base editors mediate highly efficient, targeted single-base conversion without introducing double-stranded breaks. Analysis of base editing outcomes typically relies on imprecise enzymatic mismatch cleavage assays, time-consuming single-colony sequencing, or expensive next-generation deep sequencing. To overcome these limitations, several groups have recently developed computer programs to measure base-editing efficiency from fluorescence-based Sanger sequencing data such as Edit deconvolution by inference of traces in R (EditR), TIDER, and ICE. These approaches have greatly simplified the quantitation of base-editing experiments. However, the current Sanger sequencing tools lack the capability of batch analysis and producing high-quality images for publication. Here, we provide a base editing analysis tool (BEAT) written in Python to analyze and quantify the base-editing events from Sanger sequencing data in a batch manner, which can also produce intuitive, publication-ready base-editing images.},
}

*CRISPR-Cas Systems
*Gene Editing
HEK293 Cells
Humans
Sequence Analysis, DNA/*methods
*Software

@article {pmid31253581,
year = {2019},
author = {Lo Scrudato, M and Poulard, K and Sourd, C and Tomé, S and Klein, AF and Corre, G and Huguet, A and Furling, D and Gourdon, G and Buj-Bello, A},
title = {Genome Editing of Expanded CTG Repeats within the Human DMPK Gene Reduces Nuclear RNA Foci in the Muscle of DM1 Mice.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {8},
pages = {1372-1388},
pmid = {31253581},
issn = {1525-0024},
mesh = {Alternative Splicing ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Cell Nucleus ; Disease Models, Animal ; Fluorescent Antibody Technique ; *Gene Editing ; Gene Expression ; Gene Targeting ; Genetic Vectors/genetics ; Humans ; Mice ; Mice, Knockout ; Muscle, Skeletal/metabolism/pathology ; Myotonic Dystrophy/genetics/therapy ; Myotonin-Protein Kinase/*genetics ; RNA, Guide ; *RNA, Nuclear ; Transduction, Genetic ; *Trinucleotide Repeat Expansion ; },
abstract = {Myotonic dystrophy type 1 (DM1) is caused by a CTG repeat expansion located in the 3' UTR of the DMPK gene. Expanded DMPK transcripts aggregate into nuclear foci and alter the function of RNA-binding proteins, leading to defects in the alternative splicing of numerous pre-mRNAs. To date, there is no curative treatment for DM1. Here we investigated a gene-editing strategy using the CRISPR-Cas9 system from Staphylococcus aureus (Sa) to delete the CTG repeats in the human DMPK locus. Co-expression of SaCas9 and selected pairs of single-guide RNAs (sgRNAs) in cultured DM1 patient-derived muscle line cells carrying 2,600 CTG repeats resulted in targeted DNA deletion, ribonucleoprotein foci disappearance, and correction of splicing abnormalities in various transcripts. Furthermore, a single intramuscular injection of recombinant AAV vectors expressing CRISPR-SaCas9 components in the tibialis anterior muscle of DMSXL (myotonic dystrophy mouse line carrying the human DMPK gene with >1,000 CTG repeats) mice decreased the number of pathological RNA foci in myonuclei. These results establish the proof of concept that genome editing of a large trinucleotide expansion is feasible in muscle and may represent a useful strategy to be further developed for the treatment of myotonic dystrophy.},
}

Alternative Splicing
Animals
Base Sequence
CRISPR-Cas Systems
Cell Nucleus
Disease Models, Animal
Fluorescent Antibody Technique
*Gene Editing
Gene Expression
Gene Targeting
Genetic Vectors/genetics
Humans
Mice
Mice, Knockout
Muscle, Skeletal/metabolism/pathology
Myotonic Dystrophy/genetics/therapy
Myotonin-Protein Kinase/*genetics
RNA, Guide
*RNA, Nuclear
Transduction, Genetic
*Trinucleotide Repeat Expansion

@article {pmid31225756,
year = {2019},
author = {Boggio, A and Knoppers, BM and Almqvist, J and Romano, CPR},
title = {The Human Right to Science and the Regulation of Human Germline Engineering.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {134-142},
doi = {10.1089/crispr.2018.0053},
pmid = {31225756},
issn = {2573-1602},
mesh = {CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*legislation & jurisprudence ; Germ Cells ; *Human Rights ; Humans ; },
abstract = {There is currently no international consensus on how human germline engineering should be regulated. Existing national legislation fails to provide the governance framework necessary to regulate germline engineering in the CRISPR era. This is an obstacle to scientific and clinical advancements and inconsistent with human rights requirements. To move forward, we suggest that the human right to science is an ideal starting point for building consensus, at the national and international levels, on governing principles that promote responsible scientific and technological advancements. Regulatory frameworks must recognize the international nature of modern germline genome engineering research, the need for shared governance rather than tech-locked prohibitions, and the fact that humans are not their germline.},
}

CRISPR-Associated Proteins
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing/*legislation & jurisprudence
Germ Cells
*Human Rights
Humans

@article {pmid31225754,
year = {2019},
author = {Abudayyeh, OO and Gootenberg, JS and Kellner, MJ and Zhang, F},
title = {Nucleic Acid Detection of Plant Genes Using CRISPR-Cas13.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {165-171},
pmid = {31225754},
issn = {2573-1602},
support = {R01 MH110049/MH/NIMH NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; F30 CA210382/CA/NCI NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA, Plant/*analysis ; Endodeoxyribonucleases ; *Genes, Plant ; Genetic Testing/*methods ; Soybeans/*genetics ; },
abstract = {Nucleic acid detection is vital for agricultural applications including trait detection during breeding, pest surveillance, and pathogen identification. Here, we use a modified version of the CRISPR-based nucleic acid detection platform SHERLOCK to quantify levels of a glyphosate resistance gene in a mixture of soybeans and to detect multiple plant genes in a single reaction. SHERLOCK is rapid (∼15 min), quantitative, and portable, and can process crude soybean extracts as input material for minimal nucleic acid sample preparation. This field-ready SHERLOCK platform with color-based lateral flow readout can be applied for detection and quantitation of genes in a range of agricultural applications.},
}

*CRISPR-Cas Systems
DNA, Plant/*analysis
Endodeoxyribonucleases
*Genes, Plant
Genetic Testing/*methods
Soybeans/*genetics

@article {pmid31225751,
year = {2019},
author = {Fox, R},
title = {Too Much Compromise in Today's CRISPR Pipelines.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {143-145},
pmid = {31225751},
issn = {2573-1602},
mesh = {Animals ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Eukaryota ; Gene Editing/*methods ; Humans ; },
abstract = {CRISPR-based editing is a revolutionary tool for genome engineering and discovery. The pace of innovation in this young field is truly astonishing, and yet significant gaps remain in the set of capabilities offered. In particular, scalable (massively parallel and multiplex) editing is only available for a small variety of edit types, placing fundamental limits on the kinds of studies researchers can perform. Additional advancements are needed to realize the full potential of the technology.},
}

Animals
CRISPR-Associated Proteins
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Eukaryota
Gene Editing/*methods
Humans

@article {pmid31225750,
year = {2019},
author = {Barrangou, R},
title = {Taking CRISPR to New Heights.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {133},
doi = {10.1089/crispr.2019.29064.rba},
pmid = {31225750},
issn = {2573-1602},
mesh = {CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing ; },
}

CRISPR-Associated Proteins
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Editing

@article {pmid31225748,
year = {2019},
author = {Newsham, W},
title = {Now on Course, CRISPR J.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {155-156},
doi = {10.1089/crispr.2019.29057.wne},
pmid = {31225748},
issn = {2573-1602},
mesh = {Animals ; *Animals, Genetically Modified ; CRISPR-Associated Proteins ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Horses/genetics ; },
}

Animals
*Animals, Genetically Modified
CRISPR-Associated Proteins
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing/*methods
Horses/genetics

@article {pmid31225747,
year = {2019},
author = {Huston, NC and Tycko, J and Tillotson, EL and Wilson, CJ and Myer, VE and Jayaram, H and Steinberg, BE},
title = {Identification of Guide-Intrinsic Determinants of Cas9 Specificity.},
journal = {The CRISPR journal},
volume = {2},
number = {},
pages = {172-185},
pmid = {31225747},
issn = {2573-1602},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; Staphylococcus aureus/*enzymology ; Substrate Specificity ; },
abstract = {Considerable effort has been devoted to developing a comprehensive understanding of CRISPR nuclease specificity. In silico predictions and multiple genome-wide cellular and biochemical approaches have revealed a basic understanding of the Cas9 specificity profile. However, none of these approaches has delivered a model that allows accurate prediction of a CRISPR nuclease's ability to cleave a site based entirely on the sequence of the guide RNA (gRNA) and the target. We describe a library-based biochemical assay that directly reports the cleavage efficiency of a particular Cas9-guide complex by measuring both uncleaved and cleaved target molecules over a wide range of mismatched library members. We applied our assay using libraries of targets to evaluate the specificity of Staphylococcus aureus Cas9 under a variety of experimental conditions. Surprisingly, our data show an unexpectedly high variation in the random gRNA:target DNA mismatch tolerance when cleaving with different gRNAs, indicating guide-intrinsic mismatch permissiveness and challenging the assumption of universal specificity models. We use data generated by our assay to create the first off-target, guide-specific cleavage models. The barcoded libraries of targets approach is rapid, highly modular, and capable of generating protein- and guide-specific models, as well as illuminating the biophysics of Cas9 binding versus cutting. These models may be useful in identifying potential off-targets, and the gRNA-intrinsic nature of mismatch tolerance argues for coupling these specificity models with orthogonal methods for a more complete assessment of gRNA specificity.},
}

CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems
DNA/metabolism
Staphylococcus aureus/*enzymology
Substrate Specificity

@article {pmid31178391,
year = {2019},
author = {Romero, Z and Lomova, A and Said, S and Miggelbrink, A and Kuo, CY and Campo-Fernandez, B and Hoban, MD and Masiuk, KE and Clark, DN and Long, J and Sanchez, JM and Velez, M and Miyahira, E and Zhang, R and Brown, D and Wang, X and Kurmangaliyev, YZ and Hollis, RP and Kohn, DB},
title = {Editing the Sickle Cell Disease Mutation in Human Hematopoietic Stem Cells: Comparison of Endonucleases and Homologous Donor Templates.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {8},
pages = {1389-1406},
pmid = {31178391},
issn = {1525-0024},
support = {R25 GM055052/GM/NIGMS NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/*genetics/metabolism/therapy ; CRISPR-Cas Systems ; Endonucleases/genetics ; *Gene Editing ; Gene Expression ; Gene Targeting ; Genetic Therapy ; Genetic Vectors/genetics ; Hematopoietic Stem Cells/*metabolism ; Humans ; *Mutation ; Parvovirinae/genetics ; Tissue Donors ; Transduction, Genetic ; Zinc Finger Nucleases/genetics ; beta-Globins/*genetics ; },
abstract = {Site-specific correction of a point mutation causing a monogenic disease in autologous hematopoietic stem and progenitor cells (HSPCs) can be used as a treatment of inherited disorders of the blood cells. Sickle cell disease (SCD) is an ideal model to investigate the potential use of gene editing to transvert a single point mutation at the β-globin locus (HBB). We compared the activity of zinc-finger nucleases (ZFNs) and CRISPR/Cas9 for editing, and homologous donor templates delivered as single-stranded oligodeoxynucleotides (ssODNs), adeno-associated virus serotype 6 (AAV6), integrase-deficient lentiviral vectors (IDLVs), and adenovirus 5/35 serotype (Ad5/35) to transvert the base pair responsible for SCD in HBB in primary human CD34+ HSPCs. We found that the ZFNs and Cas9 directed similar frequencies of nuclease activity. In vitro, AAV6 led to the highest frequencies of homology-directed repair (HDR), but levels of base pair transversions were significantly reduced when analyzing cells in vivo in immunodeficient mouse xenografts, with similar frequencies achieved with either AAV6 or ssODNs. AAV6 also caused significant impairment of colony-forming progenitors and human cell engraftment. Gene correction in engrafting hematopoietic stem cells may be limited by the capacity of the cells to mediate HDR, suggesting additional manipulations may be needed for high-efficiency gene correction in HSPCs.},
}

Anemia, Sickle Cell/*genetics/metabolism/therapy
CRISPR-Cas Systems
Endonucleases/genetics
*Gene Editing
Gene Expression
Gene Targeting
Genetic Therapy
Genetic Vectors/genetics
Hematopoietic Stem Cells/*metabolism
Humans
*Mutation
Parvovirinae/genetics
Tissue Donors
Transduction, Genetic
Zinc Finger Nucleases/genetics
beta-Globins/*genetics

@article {pmid31129119,
year = {2019},
author = {Xu, L and Lau, YS and Gao, Y and Li, H and Han, R},
title = {Life-Long AAV-Mediated CRISPR Genome Editing in Dystrophic Heart Improves Cardiomyopathy without Causing Serious Lesions in mdx Mice.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {27},
number = {8},
pages = {1407-1414},
pmid = {31129119},
issn = {1525-0024},
support = {R01 AR064241/AR/NIAMS NIH HHS/United States ; R01 HL116546/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cardiomyopathies/*etiology/metabolism/pathology/therapy ; DNA Repair ; Dependovirus/*genetics ; Disease Models, Animal ; Dystrophin/*genetics/metabolism ; Fluorescent Antibody Technique ; *Gene Editing ; Gene Expression ; *Genetic Therapy/methods ; Genetic Vectors/administration & dosage/*genetics ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal/metabolism/pathology ; Muscular Dystrophy, Duchenne/complications/genetics/therapy ; RNA, Guide/genetics ; Transduction, Genetic ; },
abstract = {Previous studies from others and us have demonstrated that CRISPR genome editing could offer a promising therapeutic strategy to restore dystrophin expression and function in the skeletal muscle and heart of Duchenne muscular dystrophy (DMD) mouse models. However, the long-term efficacy and safety of CRISPR genome-editing therapy for DMD has not been well established. We packaged both SaCas9 and guide RNA (gRNA) together into one AAVrh.74 vector, injected two such vectors (targeting intron 20 and intron 23, respectively) into mdx pups at day 3 and evaluated the mice at 19 months. We found that AAVrh.74-mediated life-long CRISPR genome editing in mdx mice restored dystrophin expression and improved cardiac function without inducing serious adverse effects. PCR analysis and targeted deep sequencing showed that the DSBs were mainly repaired by the precise ligation of the two cut sites. Serological and histological examination of major vital organs did not reveal any signs of tumor development or other deleterious defects arising from CRISPR genome editing. These results support that in vivo CRISPR genome editing could be developed as a safe therapeutic treatment for DMD and potentially other diseases.},
}

Animals
*CRISPR-Cas Systems
Cardiomyopathies/*etiology/metabolism/pathology/therapy
DNA Repair
Dependovirus/*genetics
Disease Models, Animal
Dystrophin/*genetics/metabolism
Fluorescent Antibody Technique
*Gene Editing
Gene Expression
*Genetic Therapy/methods
Genetic Vectors/administration & dosage/*genetics
Mice
Mice, Inbred mdx
Muscle, Skeletal/metabolism/pathology
Muscular Dystrophy, Duchenne/complications/genetics/therapy
RNA, Guide/genetics
Transduction, Genetic

@article {pmid30796544,
year = {2020},
author = {Ordon, J and Bressan, M and Kretschmer, C and Dall'Osto, L and Marillonnet, S and Bassi, R and Stuttmann, J},
title = {Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation.},
journal = {Functional & integrative genomics},
volume = {20},
number = {1},
pages = {151-162},
pmid = {30796544},
issn = {1438-7948},
support = {CRC 648//Deutsche Forschungsgemeinschaft/ ; STU 642-1/1//Deutsche Forschungsgemeinschaft/ ; Program CooperInt2017 and HuntingLight Ricerca di Base 2015//Universit? degli Studi di Verona/ ; Program CooperInt2017 and HuntingLight Ricerca di Base 2015//Universit? degli Studi di Verona/ ; },
mesh = {Alleles ; Arabidopsis/*genetics ; CRISPR-Associated Protein 9/*genetics/metabolism ; CRISPR-Cas Systems ; Gene Deletion ; Gene Editing/*methods ; Genome, Plant ; Mutation ; Promoter Regions, Genetic ; Ubiquitin/genetics ; },
abstract = {Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~ 1.6% in the T2 generation, through PCR-based screening of numerous individuals. However, we failed to isolate a line lacking Lhcb1 genes, which are present in five copies organized at two loci in the Arabidopsis genome. To improve efficiency of our Cas9-based nuclease system, regulatory sequences controlling Cas9 expression levels and timing were systematically compared. Indeed, use of DD45 and RPS5a promoters improved efficiency of our genome editing system by approximately 25-30-fold in comparison to the previous ubiquitin promoter. Using an optimized genome editing system with RPS5a promoter-driven Cas9, putatively quintuple mutant lines lacking detectable amounts of Lhcb1 protein represented approximately 30% of T1 transformants. These results show how improved genome editing systems facilitate the isolation of complex mutant alleles, previously considered impossible to generate, at high frequency even in a single (T1) generation.},
}

Alleles
Arabidopsis/*genetics
CRISPR-Associated Protein 9/*genetics/metabolism
CRISPR-Cas Systems
Gene Deletion
Gene Editing/*methods
Genome, Plant
Mutation
Promoter Regions, Genetic
Ubiquitin/genetics

@article {pmid32461256,
year = {2020},
author = {Geng, X and Zhang, S and He, J and Ma, A and Li, Y and Li, M and Zhou, H and Chen, G and Yang, B},
title = {The urea transporter UT-A1 plays a predominant role in a urea-dependent urine-concentrating mechanism.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA120.013628},
pmid = {32461256},
issn = {1083-351X},
abstract = {Urea transporters are a family of urea-selective channel proteins expressed in multiple tissues and play an important role in the urine-concentrating mechanism of the mammalian kidney. Previous studies have shown that knockout of urea transporter-B (UT-B), UT-A1/A3, or all-UT lead to urea-selective diuresis, indicating that urea transporters have important roles in urine concentration. Here, we sought to determine the role of UT-A1 in the urine-concentrating mechanism in a newly developed UT-A1-knockout mouse model. Phenotypically, daily urine output in UT-A1-knockout mice was nearly 3-fold that of wild-type mice and 82% of all-UT-knockout mice, and the UT-A1-knockout mice had significantly lower urine osmolality than wild-type mice. After 24 h water restriction, acute urea loading, or high-protein (40%) intake, UT-A1 knockout mice were unable to increase urine-concentrating ability. Compared with all-UT-knockout mice, the UT-A1-null mice exhibited similarly elevated daily urine output and decreased urine osmolality, indicating impaired urea-selective urine concentration. Our experimental findings reveal that UT-A1 has a predominant role in urea-dependent urine-concentrating mechanisms, suggesting that UT-A1 represents a promising diuretic target.},
}

@article {pmid32270050,
year = {2019},
author = {Evanoff, M and Komor, AC},
title = {Base Editors: Modular Tools for the Introduction of Point Mutations in Living Cells.},
journal = {Emerging topics in life sciences},
volume = {3},
number = {5},
pages = {483-491},
pmid = {32270050},
issn = {2397-8554},
support = {T32 GM007240/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Line ; Cytidine Deaminase/chemistry/genetics ; DNA, Single-Stranded/chemistry/metabolism ; Endonucleases/chemistry/*genetics ; Gene Editing/*methods ; Genetic Enhancement ; *Point Mutation ; RNA, Guide/chemistry/metabolism ; Recombination, Genetic/genetics ; Synthetic Biology ; Transcription, Genetic ; },
abstract = {Base editors are a new family of programmable genome editing tools that fuse ssDNA (single stranded DNA) modifying enzymes to catalytically inactive CRISPR-associated (Cas) endonucleases to induce highly efficient single base changes. With dozens of base editors now reported, it is apparent that these tools are highly modular; many combinations of ssDNA modifying enzymes and Cas proteins have resulted in a variety of base editors, each with its own unique properties and potential uses. In this perspective, we describe currently available base editors, highlighting their modular nature and describing the various options available for each component. Furthermore, we briefly discuss applications in synthetic biology and genome engineering where base editors have presented unique advantages over alternative techniques.},
}

CRISPR-Cas Systems
Cell Line
Cytidine Deaminase/chemistry/genetics
DNA, Single-Stranded/chemistry/metabolism
Endonucleases/chemistry/*genetics
Gene Editing/*methods
Genetic Enhancement
*Point Mutation
RNA, Guide/chemistry/metabolism
Recombination, Genetic/genetics
Synthetic Biology
Transcription, Genetic

@article {pmid31659913,
year = {2019},
author = {Zoppo, M and Luca, MD and Villarreal, SN and Poma, N and Barrasa, MI and Bottai, D and Vyas, VK and Tavanti, A},
title = {A CRISPR/Cas9-based strategy to simultaneously inactivate the entire ALS gene family in Candida orthopsilosis.},
journal = {Future microbiology},
volume = {14},
number = {},
pages = {1383-1396},
doi = {10.2217/fmb-2019-0168},
pmid = {31659913},
issn = {1746-0921},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Candida parapsilosis/*genetics/growth & development ; Candidiasis/microbiology ; Cell Adhesion ; Cells, Cultured ; Epithelial Cells/microbiology ; Gene Editing/*methods ; *Genes, Fungal ; Humans ; Hyphae/growth & development ; Mouth/cytology ; Multigene Family ; RNA, Guide/genetics ; },
abstract = {Aim: In this study, the CRISPR gene-editing approach was used to simultaneously inactivate all three members of the ALS gene family in the opportunistic pathogen Candida orthopsilosis. Materials & methods: Using a single gRNA and repair template, CRISPR-edited clones were successfully generated in a one-step process in both C. orthopsilosis reference and clinical strains. Results: The phenotypic characterization of the ALS triple-edited strains revealed no impact on growth in liquid or solid media. However, pseudohyphal formation and the ability to adhere to human buccal epithelial cells were significantly decreased in triple-edited clones. Conclusion: Our CRISPR/Cas9 system is a powerful tool for simultaneous editing of fungal gene families, which greatly accelerates the generation of multiple gene-edited Candida strains. Data deposition: Nucleotide sequence data are available in the GenBank databases under the accession numbers MK875971, MK875972, MK875973, MK875974, MK875975, MK875976, MK875977.},
}

Base Sequence
CRISPR-Associated Protein 9/*genetics
*CRISPR-Cas Systems
Candida parapsilosis/*genetics/growth & development
Candidiasis/microbiology
Cell Adhesion
Cells, Cultured
Epithelial Cells/microbiology
Gene Editing/*methods
*Genes, Fungal
Humans
Hyphae/growth & development
Mouth/cytology
Multigene Family
RNA, Guide/genetics

@article {pmid31626775,
year = {2019},
author = {Feldman, D and Singh, A and Schmid-Burgk, JL and Carlson, RJ and Mezger, A and Garrity, AJ and Zhang, F and Blainey, PC},
title = {Optical Pooled Screens in Human Cells.},
journal = {Cell},
volume = {179},
number = {3},
pages = {787-799.e17},
doi = {10.1016/j.cell.2019.09.016},
pmid = {31626775},
issn = {1097-4172},
support = {R01 HG009283/HG/NHGRI NIH HHS/United States ; R01 MH110049/MH/NIMH NIH HHS/United States ; DP1 HL141201/HL/NHLBI NIH HHS/United States ; P50 HG006193/HG/NHGRI NIH HHS/United States ; R01 HG009761/HG/NHGRI NIH HHS/United States ; RM1 HG006193/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Cell Nucleus/genetics/metabolism ; *Genetic Testing ; *Genomics ; Humans ; Mediator Complex/genetics ; NF-kappa B/*genetics ; RNA, Guide/genetics ; Transcription Factor RelA/*genetics ; },
abstract = {Genetic screens are critical for the systematic identification of genes underlying cellular phenotypes. Pooling gene perturbations greatly improves scalability but is not compatible with imaging of complex and dynamic cellular phenotypes. Here, we introduce a pooled approach for optical genetic screens in mammalian cells. We use targeted in situ sequencing to demultiplex a library of genetic perturbations following image-based phenotyping. We screened a set of 952 genes across millions of cells for involvement in nuclear factor κB (NF-κB) signaling by imaging the translocation of RelA (p65) to the nucleus. Screening at a single time point across 3 cell lines recovered 15 known pathway components, while repeating the screen with live-cell imaging revealed a role for Mediator complex subunits in regulating the duration of p65 nuclear retention. These results establish a highly multiplexed approach to image-based screens of spatially and temporally defined phenotypes with pooled libraries.},
}

Animals
CRISPR-Cas Systems
Cell Line
Cell Nucleus/genetics/metabolism
*Genetic Testing
*Genomics
Humans
Mediator Complex/genetics
NF-kappa B/*genetics
RNA, Guide/genetics
Transcription Factor RelA/*genetics

@article {pmid30996093,
year = {2019},
author = {Luteijn, RD and van Diemen, F and Blomen, VA and Boer, IGJ and Manikam Sadasivam, S and van Kuppevelt, TH and Drexler, I and Brummelkamp, TR and Lebbink, RJ and Wiertz, EJ},
title = {A Genome-Wide Haploid Genetic Screen Identifies Heparan Sulfate-Associated Genes and the Macropinocytosis Modulator TMED10 as Factors Supporting Vaccinia Virus Infection.},
journal = {Journal of virology},
volume = {93},
number = {13},
pages = {},
pmid = {30996093},
issn = {1098-5514},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; Cowpox virus/genetics ; DNA Viruses ; Gene Knockout Techniques ; Genetic Testing ; Golgi Apparatus ; HEK293 Cells ; *Haploidy ; HeLa Cells ; Heparitin Sulfate/*genetics/*isolation & purification/metabolism ; Host Specificity ; Host-Pathogen Interactions ; Humans ; Membrane Proteins ; Monkeypox virus/genetics ; N-Acetylglucosaminyltransferases ; Phosphatidylserines/metabolism ; Pinocytosis/*physiology ; Poxviridae/genetics ; Vaccinia/*virology ; Vaccinia virus/*genetics/*metabolism ; Vesicular Transport Proteins/*metabolism ; Virus Attachment ; },
abstract = {Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.},
}

CRISPR-Cas Systems
Cell Line, Tumor
Cowpox virus/genetics
DNA Viruses
Gene Knockout Techniques
Genetic Testing
Golgi Apparatus
HEK293 Cells
*Haploidy
HeLa Cells
Heparitin Sulfate/*genetics/*isolation & purification/metabolism
Host Specificity
Host-Pathogen Interactions
Humans
Membrane Proteins
Monkeypox virus/genetics
N-Acetylglucosaminyltransferases
Phosphatidylserines/metabolism
Pinocytosis/*physiology
Poxviridae/genetics
Vaccinia/*virology
Vaccinia virus/*genetics/*metabolism
Vesicular Transport Proteins/*metabolism
Virus Attachment

@article {pmid30996089,
year = {2019},
author = {Zhang, W and Yang, F and Zhu, Z and Yang, Y and Wang, Z and Cao, W and Dang, W and Li, L and Mao, R and Liu, Y and Tian, H and Zhang, K and Liu, X and Ma, J and Zheng, H},
title = {Cellular DNAJA3, a Novel VP1-Interacting Protein, Inhibits Foot-and-Mouth Disease Virus Replication by Inducing Lysosomal Degradation of VP1 and Attenuating Its Antagonistic Role in the Beta Interferon Signaling Pathway.},
journal = {Journal of virology},
volume = {93},
number = {13},
pages = {},
pmid = {30996089},
issn = {1098-5514},
mesh = {Animals ; Antiviral Agents/metabolism/pharmacology ; CRISPR-Cas Systems ; Capsid Proteins/*metabolism ; Cell Line ; Foot-and-Mouth Disease Virus/*drug effects ; Gene Knockout Techniques ; HEK293 Cells ; HSP40 Heat-Shock Proteins/*antagonists & inhibitors/chemistry/genetics/*metabolism ; Host-Pathogen Interactions ; Humans ; Interferon Regulatory Factor-3 ; Interferon-beta/*metabolism ; Lysosomes/*metabolism ; Phosphorylation ; Proteasome Endopeptidase Complex ; Protein Interaction Domains and Motifs ; Signal Transduction/*drug effects ; Viral Proteins/metabolism ; Virus Replication/*drug effects ; },
abstract = {DnaJ heat shock protein family (Hsp40) member A3 (DNAJA3) plays an important role in viral infections. However, the role of DNAJA3 in replication of foot-and-mouth-disease virus (FMDV) remains unknown. In this study, DNAJA3, a novel binding partner of VP1, was identified using yeast two-hybrid screening. The DNAJA3-VP1 interaction was further confirmed by coimmunoprecipitation and colocalization in FMDV-infected cells. The J domain of DNAJA3 (amino acids 1 to 168) and the lysine at position 208 (K208) of VP1 were shown to be critical for the DNAJA3-VP1 interaction. Overexpression of DNAJA3 dramatically dampened FMDV replication, whereas loss of function of DNAJA3 elicited opposing effects against FMDV replication. Mechanistical study demonstrated that K208 of VP1 was critical for reducing virus titer caused by DNAJA3 using K208A mutant virus. DNAJA3 induced lysosomal degradation of VP1 by interacting with LC3 to enhance the activation of lysosomal pathway. Meanwhile, we discovered that VP1 suppressed the beta interferon (IFN-β) signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. This inhibitory effect was considerably boosted in DNAJA3-knockout cells. In contrast, overexpression of DNAJA3 markedly attenuated VP1-mediated suppression on the IFN-β signaling pathway. Poly(I⋅C)-induced phosphorylation of IRF3 was also decreased in DNAJA3-knockout cells compared to that in the DNAJA3-WT cells. In conclusion, our study described a novel role for DNAJA3 in the host's antiviral response by inducing the lysosomal degradation of VP1 and attenuating the VP1-induced suppressive effect on the IFN-β signaling pathway.IMPORTANCE This study pioneeringly determined the antiviral role of DNAJA3 in FMDV. DNAJA3 was found to interact with FMDV VP1 and trigger its degradation via the lysosomal pathway. In addition, this study is also the first to clarify the mechanism by which VP1 suppressed IFN-β signaling pathway by inhibiting the phosphorylation, dimerization, and nuclear translocation of IRF3. Moreover, DNAJA3 significantly abrogated VP1-induced inhibitive effect on the IFN-β signaling pathway. These data suggested that DNAJA3 plays an important antiviral role against FMDV by both degrading VP1 and restoring of IFN-β signaling pathway.},
}

Animals
Antiviral Agents/metabolism/pharmacology
CRISPR-Cas Systems
Capsid Proteins/*metabolism
Cell Line
Foot-and-Mouth Disease Virus/*drug effects
Gene Knockout Techniques
HEK293 Cells
HSP40 Heat-Shock Proteins/*antagonists & inhibitors/chemistry/genetics/*metabolism
Host-Pathogen Interactions
Humans
Interferon Regulatory Factor-3
Interferon-beta/*metabolism
Lysosomes/*metabolism
Phosphorylation
Proteasome Endopeptidase Complex
Protein Interaction Domains and Motifs
Signal Transduction/*drug effects
Viral Proteins/metabolism
Virus Replication/*drug effects

@article {pmid32459325,
year = {2020},
author = {Wang, J and Dai, W and Li, J and Xie, R and Dunstan, RA and Stubenrauch, C and Zhang, Y and Lithgow, T},
title = {PaCRISPR: a server for predicting and visualizing anti-CRISPR proteins.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaa432},
pmid = {32459325},
issn = {1362-4962},
abstract = {Anti-CRISPRs are widespread amongst bacteriophage and promote bacteriophage infection by inactivating the bacterial host's CRISPR-Cas defence system. Identifying and characterizing anti-CRISPR proteins opens an avenue to explore and control CRISPR-Cas machineries for the development of new CRISPR-Cas based biotechnological and therapeutic tools. Past studies have identified anti-CRISPRs in several model phage genomes, but a challenge exists to comprehensively screen for anti-CRISPRs accurately and efficiently from genome and metagenome sequence data. Here, we have developed an ensemble learning based predictor, PaCRISPR, to accurately identify anti-CRISPRs from protein datasets derived from genome and metagenome sequencing projects. PaCRISPR employs different types of feature recognition united within an ensemble framework. Extensive cross-validation and independent tests show that PaCRISPR achieves a significantly more accurate performance compared with homology-based baseline predictors and an existing toolkit. The performance of PaCRISPR was further validated in discovering anti-CRISPRs that were not part of the training for PaCRISPR, but which were recently demonstrated to function as anti-CRISPRs for phage infections. Data visualization on anti-CRISPR relationships, highlighting sequence similarity and phylogenetic considerations, is part of the output from the PaCRISPR toolkit, which is freely available at http://pacrispr.erc.monash.edu/.},
}

@article {pmid32455882,
year = {2020},
author = {Sledzinski, P and Nowaczyk, M and Olejniczak, M},
title = {Computational Tools and Resources Supporting CRISPR-Cas Experiments.},
journal = {Cells},
volume = {9},
number = {5},
pages = {},
doi = {10.3390/cells9051288},
pmid = {32455882},
issn = {2073-4409},
support = {2018/29/B/NZ1/00293//National Science Center, Poland/ ; },
abstract = {The CRISPR-Cas system has become a cutting-edge technology that revolutionized genome engineering. The use of Cas9 nuclease is currently the method of choice in most tasks requiring a specific DNA modification. The rapid development in the field of CRISPR-Cas is reflected by the constantly expanding ecosystem of computational tools aimed at facilitating experimental design and result analysis. The first group of CRISPR-Cas-related tools that we review is dedicated to aid in guide RNA design by prediction of their efficiency and specificity. The second, relatively new group of tools exploits the observed biases in repair outcomes to predict the results of CRISPR-Cas edits. The third class of tools is developed to assist in the evaluation of the editing outcomes by analysis of the sequencing data. These utilities are accompanied by relevant repositories and databases. Here we present a comprehensive and updated overview of the currently available CRISPR-Cas-related tools, from the perspective of a user who needs a convenient and reliable means to facilitate genome editing experiments at every step, from the guide RNA design to analysis of editing outcomes. Moreover, we discuss the current limitations and challenges that the field must overcome for further improvement in the CRISPR-Cas endeavor.},
}

@article {pmid31894108,
year = {2019},
author = {Chen, Z and Song, Z and Yang, J and Huang, J and Jiang, H},
title = {Sp7/osterix positively regulates dlx2b and bglap to affect tooth development and bone mineralization in zebrafish larvae.},
journal = {Journal of biosciences},
volume = {44},
number = {6},
pages = {},
pmid = {31894108},
issn = {0973-7138},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Calcification, Physiologic/genetics ; Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; Homeodomain Proteins/genetics ; Humans ; Larva/genetics/growth & development ; Osteocalcin/genetics ; Osteogenesis/*genetics ; Sp7 Transcription Factor/*genetics ; Tooth/growth & development ; Transcription Factors/genetics ; Zebrafish/*genetics/growth & development ; Zebrafish Proteins/*genetics ; },
abstract = {Osterix (or Sp7) is an important transcription factor that promotes osteoblast differentiation by modulating the expression of a range of target genes. Although many studies have focused on Osterix/Sp7 regulatory mechanisms, the detailed functions have not been fully elucidated. Toward this end, in this study, we used CRISPR/Cas9 technology to knock out the zebrafish sp7 gene, and then analyzed its phenotype and biological function. Two knockout sp7 mutant lines were successfully obtained. The bone mineralization level was significantly reduced in the zebrafish sp7-/- homozygote, resulting in abnormal tooth development in the larvae. Quantitative real-time polymerase chain reaction showed that loss of sp7 led to down-regulated expression of the dlx2b and bglap genes related to tooth development and bone mineralization, respectively. Moreover, cell transfection experiments demonstrated that Sp7 directly regulates the expression of dlx2b and bglap through Sp7-binding sites on the promoter regions of these two genes. Overall, this study provides new insight into the role of Sp7 in bone mineralization and tooth development.},
}

Animals
CRISPR-Cas Systems/genetics
Calcification, Physiologic/genetics
Cell Differentiation/genetics
Gene Expression Regulation, Developmental
Homeodomain Proteins/genetics
Humans
Larva/genetics/growth & development
Osteocalcin/genetics
Osteogenesis/*genetics
Sp7 Transcription Factor/*genetics
Tooth/growth & development
Transcription Factors/genetics
Zebrafish/*genetics/growth & development
Zebrafish Proteins/*genetics

@article {pmid31761532,
year = {2019},
author = {Morton, AR and Dogan-Artun, N and Faber, ZJ and MacLeod, G and Bartels, CF and Piazza, MS and Allan, KC and Mack, SC and Wang, X and Gimple, RC and Wu, Q and Rubin, BP and Shetty, S and Angers, S and Dirks, PB and Sallari, RC and Lupien, M and Rich, JN and Scacheri, PC},
title = {Functional Enhancers Shape Extrachromosomal Oncogene Amplifications.},
journal = {Cell},
volume = {179},
number = {6},
pages = {1330-1341.e13},
pmid = {31761532},
issn = {1097-4172},
support = {R01 CA169117/CA/NCI NIH HHS/United States ; R01 CA204279/CA/NCI NIH HHS/United States ; R01 CA160356/CA/NCI NIH HHS/United States ; TL1 TR000441/TR/NCATS NIH HHS/United States ; R01 DA043980/DA/NIDA NIH HHS/United States ; R01 CA143237/CA/NCI NIH HHS/United States ; R01 NS087913/NS/NINDS NIH HHS/United States ; R01 NS103434/NS/NINDS NIH HHS/United States ; R01 CA193677/CA/NCI NIH HHS/United States ; R01 CA171652/CA/NCI NIH HHS/United States ; R01 NS089272/NS/NINDS NIH HHS/United States ; F30 CA236313/CA/NCI NIH HHS/United States ; R35 CA197718/CA/NCI NIH HHS/United States ; T32 GM007250/GM/NIGMS NIH HHS/United States ; R01 CA154130/CA/NCI NIH HHS/United States ; },
mesh = {Acetylation ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/genetics ; Chromatin/metabolism ; Chromosomes, Human/*genetics ; DNA, Neoplasm/genetics ; *Enhancer Elements, Genetic ; ErbB Receptors/genetics/metabolism ; *Gene Amplification ; Genes, Neoplasm ; Genetic Loci ; Glioblastoma/genetics/pathology ; Histones/metabolism ; Humans ; Neuroglia/metabolism ; *Oncogenes ; },
abstract = {Non-coding regions amplified beyond oncogene borders have largely been ignored. Using a computational approach, we find signatures of significant co-amplification of non-coding DNA beyond the boundaries of amplified oncogenes across five cancer types. In glioblastoma, EGFR is preferentially co-amplified with its two endogenous enhancer elements active in the cell type of origin. These regulatory elements, their contacts, and their contribution to cell fitness are preserved on high-level circular extrachromosomal DNA amplifications. Interrogating the locus with a CRISPR interference screening approach reveals a diversity of additional elements that impact cell fitness. The pattern of fitness dependencies mirrors the rearrangement of regulatory elements and accompanying rewiring of the chromatin topology on the extrachromosomal amplicon. Our studies indicate that oncogene amplifications are shaped by regulatory dependencies in the non-coding genome.},
}

Acetylation
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Cell Survival/genetics
Chromatin/metabolism
Chromosomes, Human/*genetics
DNA, Neoplasm/genetics
*Enhancer Elements, Genetic
ErbB Receptors/genetics/metabolism
*Gene Amplification
Genes, Neoplasm
Genetic Loci
Glioblastoma/genetics/pathology
Histones/metabolism
Humans
Neuroglia/metabolism
*Oncogenes

@article {pmid31703769,
year = {2019},
author = {Gentner, B and Naldini, L},
title = {In Vivo Selection for Gene-Corrected HSPCs Advances Gene Therapy for a Rare Stem Cell Disease.},
journal = {Cell stem cell},
volume = {25},
number = {5},
pages = {592-593},
doi = {10.1016/j.stem.2019.10.004},
pmid = {31703769},
issn = {1875-9777},
mesh = {CRISPR-Cas Systems ; DNA Breaks ; *Fanconi Anemia ; Genetic Therapy ; Genetic Vectors ; *Hematopoietic Stem Cell Transplantation ; Humans ; Lentivirus/genetics ; Mutation ; },
abstract = {Two recent papers (one by Román-Rodríguez et al., 2019 in this issue of Cell Stem Cell) highlight how the power of biological selection on hematopoietic stem cell fitness can facilitate gene therapies for Fanconi Anemia. A clinical trial using lentiviral gene replacement and a proof-of-concept targeted genome editing study show robust engraftment and expansion of gene-corrected cells at levels reaching therapeutic relevance.},
}

CRISPR-Cas Systems
DNA Breaks
*Fanconi Anemia
Genetic Therapy
Genetic Vectors
*Hematopoietic Stem Cell Transplantation
Humans
Lentivirus/genetics
Mutation

@article {pmid31662324,
year = {2019},
author = {Chen, G and Park, D and Magis, AT and Behera, M and Ramalingam, SS and Owonikoko, TK and Sica, GL and Ye, K and Zhang, C and Chen, Z and Curran, WJ and Deng, X},
title = {Mcl-1 Interacts with Akt to Promote Lung Cancer Progression.},
journal = {Cancer research},
volume = {79},
number = {24},
pages = {6126-6138},
pmid = {31662324},
issn = {1538-7445},
support = {R01 CA136534/CA/NCI NIH HHS/United States ; R01 CA193828/CA/NCI NIH HHS/United States ; R01 CA200905/CA/NCI NIH HHS/United States ; P50 CA217691/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Carcinoma, Non-Small-Cell Lung/drug therapy/mortality/*pathology ; Cell Line, Tumor ; Disease Progression ; Follow-Up Studies ; Gene Knockout Techniques ; Humans ; Kaplan-Meier Estimate ; Lung/pathology ; Lung Neoplasms/drug therapy/mortality/*pathology ; Male ; Mice ; Molecular Docking Simulation ; Myeloid Cell Leukemia Sequence 1 Protein/genetics/*metabolism ; Prognosis ; Protein Binding/drug effects ; Protein Domains/drug effects ; Proto-Oncogene Proteins c-akt/antagonists & inhibitors/*metabolism ; Signal Transduction/drug effects ; Tissue Array Analysis ; Xenograft Model Antitumor Assays ; },
abstract = {Mcl-1 is a unique antiapoptotic Bcl2 family protein that functions as a gatekeeper in manipulating apoptosis and survival in cancer cells. Akt is an oncogenic kinase that regulates multiple cellular functions and its activity is significantly elevated in human cancers. Here we discovered a cross-talk between Mcl-1 and Akt in promoting lung cancer cell growth. Depletion of endogenous Mcl-1 from human lung cancer cells using CRISPR/Cas9 or Mcl-1 shRNA significantly decreased Akt activity, leading to suppression of lung cancer cell growth in vitro and in xenografts. Mechanistically, Mcl-1 directly interacted via its PEST domain with Akt at the pleckstrin homology (PH) domain. It is known that the interactions between the PH domain and kinase domain (KD) are important for maintaining Akt in an inactive state. The binding of Mcl-1/PH domain disrupted intramolecular PH/KD interactions to activate Akt. Intriguingly, Mcl-1 expression correlated with Akt activity in tumor tissues from patients with non-small cell lung cancer. Using the Mcl-1-binding PH domain of Akt as a docking site, we identified a novel small molecule, PH-687, that directly targets the PH domain and disrupts Mcl-1/Akt binding, leading to suppression of Akt activity and growth inhibition of lung cancer in vitro and in vivo. By targeting the Mcl-1/Akt interaction, this mechanism-driven agent provides a highly attractive strategy for the treatment of lung cancer. SIGNIFICANCE: These findings indicate that targeting Mcl-1/Akt interaction by employing small molecules such as PH-687 represents a potentially new and effective strategy for cancer treatment.},
}

Animals
Antineoplastic Agents/*pharmacology/therapeutic use
CRISPR-Cas Systems/genetics
Carcinoma, Non-Small-Cell Lung/drug therapy/mortality/*pathology
Cell Line, Tumor
Disease Progression
Follow-Up Studies
Gene Knockout Techniques
Humans
Kaplan-Meier Estimate
Lung/pathology
Lung Neoplasms/drug therapy/mortality/*pathology
Male
Mice
Molecular Docking Simulation
Myeloid Cell Leukemia Sequence 1 Protein/genetics/*metabolism
Prognosis
Protein Binding/drug effects
Protein Domains/drug effects
Proto-Oncogene Proteins c-akt/antagonists & inhibitors/*metabolism
Signal Transduction/drug effects
Tissue Array Analysis
Xenograft Model Antitumor Assays

@article {pmid31201381,
year = {2019},
author = {Memczak, S and Shao, Y and Izpisua Belmonte, JC},
title = {Towards precise, safe genome editing.},
journal = {Cell research},
volume = {29},
number = {9},
pages = {687-689},
pmid = {31201381},
issn = {1748-7838},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; *Gene Editing ; RNA Editing ; Transcriptome ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA
*Gene Editing
RNA Editing
Transcriptome

@article {pmid31006600,
year = {2019},
author = {Bryant, JM and Baumgarten, S and Glover, L and Hutchinson, S and Rachidi, N},
title = {CRISPR in Parasitology: Not Exactly Cut and Dried!.},
journal = {Trends in parasitology},
volume = {35},
number = {6},
pages = {409-422},
doi = {10.1016/j.pt.2019.03.004},
pmid = {31006600},
issn = {1471-5007},
mesh = {Animals ; *CRISPR-Cas Systems ; Congresses as Topic ; Humans ; Parasites/genetics ; Parasitology/*trends ; Research/trends ; },
abstract = {CRISPR/Cas9 technology has been developing rapidly in the field of parasitology, allowing for the dissection of molecular processes with unprecedented efficiency. Optimization and implementation of a new technology like CRISPR, especially in nonmodel organisms, requires communication and collaboration throughout the field. Recently, a 'CRISPR in Parasitology' symposium was held at the Institut Pasteur Paris, bringing together scientists studying Leishmania, Plasmodium, Trypanosoma, and Anopheles. Here we share technological advances and challenges in using CRISPR/Cas9 in the parasite and vector systems that were discussed. As CRISPR/Cas9 continues to be applied to diverse parasite systems, the community should now focus on improvement and standardization of the technique as well as expanding the CRISPR toolkit to include Cas9 alternatives/derivatives for more advanced applications like genome-wide functional screens.},
}

Animals
*CRISPR-Cas Systems
Congresses as Topic
Humans
Parasites/genetics
Parasitology/*trends
Research/trends

@article {pmid30648968,
year = {2019},
author = {Voss, JE and Gonzalez-Martin, A and Andrabi, R and Fuller, RP and Murrell, B and McCoy, LE and Porter, K and Huang, D and Li, W and Sok, D and Le, K and Briney, B and Chateau, M and Rogers, G and Hangartner, L and Feeney, AJ and Nemazee, D and Cannon, P and Burton, DR},
title = {Reprogramming the antigen specificity of B cells using genome-editing technologies.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {30648968},
issn = {2050-084X},
support = {5R01DE025167-05/NH/NIH HHS/United States ; UM1 AI100663/AI/NIAID NIH HHS/United States ; MR/R008698/1/MRC_/Medical Research Council/United Kingdom ; UL1 TR001114/TR/NCATS NIH HHS/United States ; R01 AI073148/AI/NIAID NIH HHS/United States ; R01 DE025167/DE/NIDCR NIH HHS/United States ; FP7-PEOPLE-2013-IOF//FP7 People: Marie-Curie Actions/International ; Ramón y Cajal Merit Award RYC-2016-21155//Ministerio de Ciencia, Innovacion y Universidades/International ; R37 AI059714/AI/NIAID NIH HHS/United States ; RYC-2016-21155//Ramón y Cajal Merit Award, Ministerio de Ciencia, Innovacion y Universidades/International ; FP7-PEOPLE-2013-IOF//Marie-Curie Fellowship/International ; OPP1183956//Bill and Melinda Gates Foundation/International ; },
mesh = {Antibodies, Neutralizing/immunology ; Antibody Specificity ; Antigen-Antibody Reactions/*genetics ; B-Lymphocytes/*immunology ; CRISPR-Cas Systems ; Cell Line ; Cytidine Deaminase/metabolism ; Gene Editing/*methods ; HIV Antibodies/immunology ; Humans ; Immunoglobulin Heavy Chains/genetics ; Receptors, Antigen, B-Cell/genetics/immunology ; },
abstract = {We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.},
}

Antibodies, Neutralizing/immunology
Antibody Specificity
Antigen-Antibody Reactions/*genetics
B-Lymphocytes/*immunology
CRISPR-Cas Systems
Cell Line
Cytidine Deaminase/metabolism
Gene Editing/*methods
HIV Antibodies/immunology
Humans
Immunoglobulin Heavy Chains/genetics
Receptors, Antigen, B-Cell/genetics/immunology

@article {pmid32453626,
year = {2020},
author = {Reimann, V and Ziemann, M and Li, H and Zhu, T and Behler, J and Lu, X and Hess, WR},
title = {Specificities and functional coordination between the two Cas6 maturation endonucleases in Anabaena sp. PCC 7120 assign orphan CRISPR arrays to three groups.},
journal = {RNA biology},
volume = {},
number = {},
pages = {},
doi = {10.1080/15476286.2020.1774197},
pmid = {32453626},
issn = {1555-8584},
abstract = {Many bacteria and archaea possess an RNA-guided adaptive and inheritable immune system that consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. In most CRISPR-Cas systems, the maturation of CRISPR-derived small RNAs (crRNAs) is essential for functionality. Cas6 endonucleases function as the most frequent CRISPR RNA maturation enzymes. In the cyanobacterium Anabaena sp. PCC 7120, ten CRISPR loci are present, but only two cas gene cassettes plus a Tn7-associated eleventh array. In this study, we deleted the two cas6 genes alr1482 (Type III-D) or alr1566 (Type I-D) and tested the specificities of the two corresponding enzymes in the resulting mutant strains, as recombinant proteins and in a cell-free transcription-translation system. The results assign the direct repeats (DRs) to three different groups. While Alr1566 is specific for one group, Alr1482 has a higher preference for the DRs of the second group but can also cleave those of the first group. We found that this cross-recognition limits crRNA accumulation for the Type I-D system in vivo. We also show that the DR of the cas gene-free CRISPR array of cyanophage N-1 is processed by these enzymes, suggesting that it is fully competent in association with host-encoded Cas proteins. The data support the functionality of CRISPR arrays that frequently appear fragmented to multiple genomic loci in multicellular cyanobacteria and disfavor other possibilities, such as the nonfunctionality of these orphan repeat-spacer arrays. Our results show the functional coordination of Cas6 endonucleases with both neighboring and remote repeat-spacer arrays in the CRISPR-Cas system of cyanobacteria.},
}

@article {pmid32450802,
year = {2020},
author = {Wada, N and Ueta, R and Osakabe, Y and Osakabe, K},
title = {Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering.},
journal = {BMC plant biology},
volume = {20},
number = {1},
pages = {234},
doi = {10.1186/s12870-020-02385-5},
pmid = {32450802},
issn = {1471-2229},
support = {JP19H02932//Japan Society for the Promotion of Science/ ; },
abstract = {Traditionally, generation of new plants with improved or desirable features has relied on laborious and time-consuming breeding techniques. Genome-editing technologies have led to a new era of genome engineering, enabling an effective, precise, and rapid engineering of the plant genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a new genome-editing tool, extensively applied in various organisms, including plants. The use of CRISPR/Cas9 allows generating transgene-free genome-edited plants ("null segregants") in a short period of time. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9 derived technologies for inducing mutations at target sites in the genome and controlling the expression of target genes. We highlight the major breakthroughs in applying CRISPR/Cas9 to plant engineering, and challenges toward the production of null segregants. We also provide an update on the efforts of engineering Cas9 proteins, newly discovered Cas9 variants, and novel CRISPR/Cas systems for use in plants. The application of CRISPR/Cas9 and related technologies in plant engineering will not only facilitate molecular breeding of crop plants but also accelerate progress in basic research.},
}

@article {pmid32132707,
year = {2020},
author = {Guo, X and Aviles, G and Liu, Y and Tian, R and Unger, BA and Lin, YT and Wiita, AP and Xu, K and Correia, MA and Kampmann, M},
title = {Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway.},
journal = {Nature},
volume = {579},
number = {7799},
pages = {427-432},
doi = {10.1038/s41586-020-2078-2},
pmid = {32132707},
issn = {1476-4687},
support = {1S10OD010786-01/NH/NIH HHS/United States ; R01 GM044037/GM/NIGMS NIH HHS/United States ; R01 DK026506/DK/NIDDK NIH HHS/United States ; DP2 GM123500/GM/NIGMS NIH HHS/United States ; DK26506/NH/NIH HHS/United States ; OD022552/NH/NIH HHS/United States ; GM44037/NH/NIH HHS/United States ; DP2 GM119139/GM/NIGMS NIH HHS/United States ; GM119139/NH/NIH HHS/United States ; },
mesh = {Activating Transcription Factor 4/biosynthesis/metabolism ; CRISPR-Cas Systems ; Cell Line ; Cytosol/enzymology/*metabolism ; Enzyme Activation ; Eukaryotic Initiation Factor-2/metabolism ; Humans ; Male ; Metalloendopeptidases/*metabolism ; Mitochondria/*metabolism/*pathology ; Mitochondrial Proteins/chemistry/*metabolism ; Molecular Chaperones/metabolism ; Phosphorylation ; Protein Binding ; *Stress, Physiological ; eIF-2 Kinase/*metabolism ; },
abstract = {In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) results in the induction of the transcription factor ATF41-3. However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2α kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2α kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2α phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.},
}

Activating Transcription Factor 4/biosynthesis/metabolism
CRISPR-Cas Systems
Cell Line
Cytosol/enzymology/*metabolism
Enzyme Activation
Eukaryotic Initiation Factor-2/metabolism
Humans
Male
Metalloendopeptidases/*metabolism
Mitochondria/*metabolism/*pathology
Mitochondrial Proteins/chemistry/*metabolism
Molecular Chaperones/metabolism
Phosphorylation
Protein Binding
*Stress, Physiological
eIF-2 Kinase/*metabolism

@article {pmid32111843,
year = {2020},
author = {de Jong, OG and Murphy, DE and Mäger, I and Willms, E and Garcia-Guerra, A and Gitz-Francois, JJ and Lefferts, J and Gupta, D and Steenbeek, SC and van Rheenen, J and El Andaloussi, S and Schiffelers, RM and Wood, MJA and Vader, P},
title = {A CRISPR-Cas9-based reporter system for single-cell detection of extracellular vesicle-mediated functional transfer of RNA.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1113},
doi = {10.1038/s41467-020-14977-8},
pmid = {32111843},
issn = {2041-1723},
support = {BB/M024393/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Biological Transport ; *CRISPR-Cas Systems ; Cell Communication ; Cell Line ; Endocytosis/genetics ; Extracellular Vesicles/genetics/*metabolism ; Fluorescence ; Genes, Reporter/genetics ; HEK293 Cells ; Humans ; RNA, Guide/genetics/metabolism ; RNA, Small Untranslated/genetics/*metabolism ; },
abstract = {Extracellular vesicles (EVs) form an endogenous transport system for intercellular transfer of biological cargo, including RNA, that plays a pivotal role in physiological and pathological processes. Unfortunately, whereas biological effects of EV-mediated RNA transfer are abundantly studied, regulatory pathways and mechanisms remain poorly defined due to a lack of suitable readout systems. Here, we describe a highly-sensitive CRISPR-Cas9-based reporter system that allows direct functional study of EV-mediated transfer of small non-coding RNA molecules at single-cell resolution. Using this CRISPR operated stoplight system for functional intercellular RNA exchange (CROSS-FIRE) we uncover various genes involved in EV subtype biogenesis that play a regulatory role in RNA transfer. Moreover we identify multiple genes involved in endocytosis and intracellular membrane trafficking that strongly regulate EV-mediated functional RNA delivery. Altogether, this approach allows the elucidation of regulatory mechanisms in EV-mediated RNA transfer at the level of EV biogenesis, endocytosis, intracellular trafficking, and RNA delivery.},
}

Biological Transport
*CRISPR-Cas Systems
Cell Communication
Cell Line
Endocytosis/genetics
Extracellular Vesicles/genetics/*metabolism
Fluorescence
Genes, Reporter/genetics
HEK293 Cells
Humans
RNA, Guide/genetics/metabolism
RNA, Small Untranslated/genetics/*metabolism

@article {pmid31749445,
year = {2019},
author = {Harding, HP and Ordonez, A and Allen, F and Parts, L and Inglis, AJ and Williams, RL and Ron, D},
title = {The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31749445},
issn = {2050-084X},
support = {C14801/A21211/CRUK_/Cancer Research UK/United Kingdom ; Wellcome 100140//Wellcome/International ; Wellcome 200848/Z/16/Z//Wellcome/International ; },
mesh = {Amino Acids/*metabolism ; Animals ; CHO Cells ; CRISPR-Cas Systems ; Cricetulus ; Endoplasmic Reticulum/metabolism ; Gene Expression Regulation, Enzymologic ; HeLa Cells ; Humans ; Kinetics ; Ligands ; Mice ; Models, Molecular ; Mutagenesis ; Phosphorylation ; Protein Binding ; Protein Conformation ; Protein Unfolding ; Protein-Serine-Threonine Kinases/chemistry/genetics/*metabolism ; RNA, Transfer/metabolism ; Ribosomes/chemistry/*metabolism ; Signal Transduction ; Starvation/*metabolism ; Transcriptome ; eIF-2 Kinase/genetics/metabolism ; },
abstract = {The eukaryotic translation initiation factor 2α (eIF2α) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs. We report on a mammalian CHO cell-based CRISPR-Cas9 mutagenesis screen for genes that contribute to ISR activation by amino acid starvation. Disruption of genes encoding components of the ribosome P-stalk, uL10 and P1, selectively attenuated GCN2-mediated ISR activation by amino acid starvation or interference with tRNA charging without affecting the endoplasmic reticulum unfolded protein stress-induced ISR, mediated by the related eIF2α kinase PERK. Wildtype ribosomes isolated from CHO cells, but not those with P-stalk lesions, stimulated GCN2-dependent eIF2α phosphorylation in vitro. These observations support a model whereby lack of a cognate charged tRNA exposes a latent capacity of the ribosome P-stalk to activate GCN2 in cells and help explain the emerging link between ribosome stalling and ISR activation.},
}

Amino Acids/*metabolism
Animals
CHO Cells
CRISPR-Cas Systems
Cricetulus
Endoplasmic Reticulum/metabolism
Gene Expression Regulation, Enzymologic
HeLa Cells
Humans
Kinetics
Ligands
Mice
Models, Molecular
Mutagenesis
Phosphorylation
Protein Binding
Protein Conformation
Protein Unfolding
Protein-Serine-Threonine Kinases/chemistry/genetics/*metabolism
RNA, Transfer/metabolism
Ribosomes/chemistry/*metabolism
Signal Transduction
Starvation/*metabolism
Transcriptome
eIF-2 Kinase/genetics/metabolism

@article {pmid31741433,
year = {2019},
author = {Zeng, H and Castillo-Cabrera, J and Manser, M and Lu, B and Yang, Z and Strande, V and Begue, D and Zamponi, R and Qiu, S and Sigoillot, F and Wang, Q and Lindeman, A and Reece-Hoyes, JS and Russ, C and Bonenfant, D and Jiang, X and Wang, Y and Cong, F},
title = {Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
pmid = {31741433},
issn = {2050-084X},
mesh = {A549 Cells ; Adaptor Proteins, Signal Transducing/metabolism ; Animals ; *CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/*genetics ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cullin Proteins ; ErbB Receptors/genetics ; Female ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Guanine Nucleotide Exchange Factors/genetics ; HEK293 Cells ; Humans ; Methionyl Aminopeptidases/metabolism ; Mice ; Mice, Nude ; Receptors, Lysophosphatidic Acid/metabolism ; Signal Transduction ; Transcription Factors/metabolism ; Transcriptome ; Ubiquitin-Protein Ligases/genetics ; rhoA GTP-Binding Protein/metabolism ; },
abstract = {EGFR-mutant NSCLCs frequently respond to EGFR tyrosine kinase inhibitors (TKIs). However, the responses are not durable, and the magnitude of tumor regression is variable, suggesting the existence of genetic modifiers of EGFR dependency. Here, we applied a genome-wide CRISPR-Cas9 screening to identify genetic determinants of EGFR TKI sensitivity and uncovered putative candidates. We show that knockout of RIC8A, essential for G-alpha protein activation, enhanced EGFR TKI-induced cell death. Mechanistically, we demonstrate that RIC8A is a positive regulator of YAP signaling, activation of which rescued the EGFR TKI sensitizing phenotype resulting from RIC8A knockout. We also show that knockout of ARIH2, or other components in the Cullin-5 E3 complex, conferred resistance to EGFR inhibition, in part by promoting nascent protein synthesis through METAP2. Together, these data uncover a spectrum of previously unidentified regulators of EGFR TKI sensitivity in EGFR-mutant human NSCLC, providing insights into the heterogeneity of EGFR TKI treatment responses.},
}

A549 Cells
Adaptor Proteins, Signal Transducing/metabolism
Animals
*CRISPR-Cas Systems
Carcinoma, Non-Small-Cell Lung/*genetics
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats
Cullin Proteins
ErbB Receptors/genetics
Female
Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
Guanine Nucleotide Exchange Factors/genetics
HEK293 Cells
Humans
Methionyl Aminopeptidases/metabolism
Mice
Mice, Nude
Receptors, Lysophosphatidic Acid/metabolism
Signal Transduction
Transcription Factors/metabolism
Transcriptome
Ubiquitin-Protein Ligases/genetics
rhoA GTP-Binding Protein/metabolism

@article {pmid31636095,
year = {2019},
author = {Kang, Y and Chu, C and Wang, F and Niu, Y},
title = {CRISPR/Cas9-mediated genome editing in nonhuman primates.},
journal = {Disease models & mechanisms},
volume = {12},
number = {10},
pages = {},
pmid = {31636095},
issn = {1754-8411},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; Humans ; Mosaicism ; Mutation/genetics ; Primates/*genetics ; },
abstract = {Owing to their high similarity to humans, non-human primates (NHPs) provide an exceedingly suitable model for the study of human disease. In this Review, we summarize the history of transgenic NHP models and the progress of CRISPR/Cas9-mediated genome editing in NHPs, from the first proof-of-principle green fluorescent protein-expressing monkeys to sophisticated NHP models of human neurodegenerative disease that accurately phenocopy several complex disease features. We discuss not only the breakthroughs and advantages, but also the potential shortcomings of the application of the CRISPR/Cas9 system to NHPs that have emerged from the expanded understanding of this technology in recent years. Although off-target and mosaic mutations are the main concerns in CRISPR/Cas9-mediated NHP modeling, recent progress in genome editing techniques make it likely that these technical limitations will be overcome soon, bringing excellent prospects to human disease studies.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems/*genetics
*Gene Editing
Humans
Mosaicism
Mutation/genetics
Primates/*genetics

@article {pmid31494407,
year = {2019},
author = {Mansourian, S and Fandino, RA and Riabinina, O},
title = {Progress in the use of genetic methods to study insect behavior outside Drosophila.},
journal = {Current opinion in insect science},
volume = {36},
number = {},
pages = {45-56},
doi = {10.1016/j.cois.2019.08.001},
pmid = {31494407},
issn = {2214-5753},
mesh = {Animals ; Appetitive Behavior ; *Behavior, Animal ; CRISPR-Cas Systems ; Insecta/*genetics/*physiology ; Mutagenesis, Site-Directed ; Oviposition ; RNA Interference ; Social Behavior ; },
abstract = {In the span of a decade we have seen a rapid progress in the application of genetic tools and genome editing approaches in 'non-model' insects. It is now possible to target sensory receptor genes and neurons, explore their functional roles and manipulate behavioral responses in these insects. In this review, we focus on the latest examples from Diptera, Lepidoptera and Hymenoptera of how applications of genetic tools advanced our understanding of diverse behavioral phenomena. We further discuss genetic methods that could be applied to study insect behavior in the future.},
}

Animals
Appetitive Behavior
*Behavior, Animal
CRISPR-Cas Systems
Insecta/*genetics/*physiology
Mutagenesis, Site-Directed
Oviposition
RNA Interference
Social Behavior

@article {pmid31391075,
year = {2019},
author = {Pacini, V and Petit, F and Querat, B and Laverriere, JN and Cohen-Tannoudji, J and L'hôte, D},
title = {Identification of a pituitary ERα-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor.},
journal = {Epigenetics & chromatin},
volume = {12},
number = {1},
pages = {48},
pmid = {31391075},
issn = {1756-8935},
support = {Allocation de recherche 2018//Société Française d'Endocrinologie/International ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; Chromatin/metabolism ; Chromatin Assembly and Disassembly ; DNA-Directed RNA Polymerases/metabolism ; Enhancer Elements, Genetic ; Estrogen Receptor alpha/*metabolism ; Gonadotrophs/cytology/metabolism ; Histones/metabolism ; Humans ; Mice ; Pituitary Gland/growth & development/metabolism ; Promoter Regions, Genetic ; Sequence Alignment ; Steroidogenic Factor 1/genetics/*metabolism ; Transcription, Genetic ; },
abstract = {BACKGROUND: Gonadotrope lineage differentiation is a stepwise process taking place during pituitary development. The early step of gonadotrope lineage specification is characterized by the expression of the Nr5a1 transcription factor, a crucial factor for gonadotrope cell fate determination. Abnormalities affecting Nr5a1 expression lead to hypogonadotropic hypogonadism and infertility. Although significant knowledge has been gained on the signaling and transcriptional events controlling gonadotrope differentiation, epigenetic mechanisms regulating Nr5a1 expression during early gonadotrope lineage specification are still poorly understood.

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.},
}

Animals
Base Sequence
CRISPR-Cas Systems/genetics
Cell Differentiation
Cell Line
Chromatin/metabolism
Chromatin Assembly and Disassembly
DNA-Directed RNA Polymerases/metabolism
Enhancer Elements, Genetic
Estrogen Receptor alpha/*metabolism
Gonadotrophs/cytology/metabolism
Histones/metabolism
Humans
Mice
Pituitary Gland/growth & development/metabolism
Promoter Regions, Genetic
Sequence Alignment
Steroidogenic Factor 1/genetics/*metabolism
Transcription, Genetic

@article {pmid31266541,
year = {2019},
author = {Schneider, D and Chua, RL and Molitor, N and Hamdan, FH and Rettenmeier, EM and Prokakis, E and Mishra, VK and Kari, V and Wegwitz, F and Johnsen, SA and Kosinsky, RL},
title = {The E3 ubiquitin ligase RNF40 suppresses apoptosis in colorectal cancer cells.},
journal = {Clinical epigenetics},
volume = {11},
number = {1},
pages = {98},
pmid = {31266541},
issn = {1868-7083},
mesh = {Apoptosis ; CRISPR-Cas Systems ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms/*genetics ; Epigenesis, Genetic ; Gene Expression Profiling/*methods ; Gene Expression Regulation, Neoplastic ; *Gene Silencing ; HCT116 Cells ; HT29 Cells ; Histones/metabolism ; Humans ; Ubiquitin-Protein Ligases/*genetics ; Ubiquitination ; },
abstract = {BACKGROUND: Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The loss of monoubiquitinated histone H2B (H2Bub1) was correlated with poor prognosis of CRC patients and, accordingly, H2Bub1 was suggested as a tumor-suppressive mark. Surprisingly, our previous work revealed that the H2B ubiquitin ligase RING finger protein 40 (RNF40) might exert tumor-promoting functions. Here, we investigated the effect of RNF40 loss on tumorigenic features of CRC cells and their survival in vitro.

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.},
}

Apoptosis
CRISPR-Cas Systems
Cell Cycle
Cell Line, Tumor
Cell Proliferation
Colorectal Neoplasms/*genetics
Epigenesis, Genetic
Gene Expression Profiling/*methods
Gene Expression Regulation, Neoplastic
*Gene Silencing
HCT116 Cells
HT29 Cells
Histones/metabolism
Humans
Ubiquitin-Protein Ligases/*genetics
Ubiquitination

@article {pmid31217031,
year = {2019},
author = {Sen, M and Wang, X and Hamdan, FH and Rapp, J and Eggert, J and Kosinsky, RL and Wegwitz, F and Kutschat, AP and Younesi, FS and Gaedcke, J and Grade, M and Hessmann, E and Papantonis, A and Strӧbel, P and Johnsen, SA},
title = {ARID1A facilitates KRAS signaling-regulated enhancer activity in an AP1-dependent manner in colorectal cancer cells.},
journal = {Clinical epigenetics},
volume = {11},
number = {1},
pages = {92},
pmid = {31217031},
issn = {1868-7083},
mesh = {CRISPR-Cas Systems ; Cell Proliferation ; Colorectal Neoplasms/*genetics/metabolism ; *DNA Methylation ; DNA-Binding Proteins/*genetics/*metabolism ; Enhancer Elements, Genetic ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Genome-Wide Association Study ; HCT116 Cells ; HT29 Cells ; Humans ; MAP Kinase Signaling System ; Mutation ; Proto-Oncogene Proteins p21(ras)/*genetics ; Signal Transduction ; Transcription Factor AP-1/*genetics ; Transcription Factors/*genetics/*metabolism ; },
abstract = {BACKGROUND: ARID1A (AT-rich interactive domain-containing protein 1A) is a subunit of the BAF chromatin remodeling complex and plays roles in transcriptional regulation and DNA damage response. Mutations in ARID1A that lead to inactivation or loss of expression are frequent and widespread across many cancer types including colorectal cancer (CRC). A tumor suppressor role of ARID1A has been established in a number of tumor types including CRC where the genetic inactivation of Arid1a alone led to the formation of invasive colorectal adenocarcinomas in mice. Mechanistically, ARID1A has been described to largely function through the regulation of enhancer activity.

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.},
}

CRISPR-Cas Systems
Cell Proliferation
Colorectal Neoplasms/*genetics/metabolism
*DNA Methylation
DNA-Binding Proteins/*genetics/*metabolism
Enhancer Elements, Genetic
Gene Editing
Gene Expression Regulation, Neoplastic
Genome-Wide Association Study
HCT116 Cells
HT29 Cells
Humans
MAP Kinase Signaling System
Mutation
Proto-Oncogene Proteins p21(ras)/*genetics
Signal Transduction
Transcription Factor AP-1/*genetics
Transcription Factors/*genetics/*metabolism

@article {pmid31161803,
year = {2019},
author = {Garcia-Leon, JA and Vitorica, J and Gutierrez, A},
title = {Use of human pluripotent stem cell-derived cells for neurodegenerative disease modeling and drug screening platform.},
journal = {Future medicinal chemistry},
volume = {11},
number = {11},
pages = {1305-1322},
doi = {10.4155/fmc-2018-0520},
pmid = {31161803},
issn = {1756-8927},
mesh = {Animals ; CRISPR-Cas Systems ; Drug Evaluation, Preclinical/*methods ; Gene Editing/methods ; Humans ; Neurodegenerative Diseases/*drug therapy/genetics/pathology ; Neurogenesis/drug effects ; Neurons/*cytology/drug effects/metabolism/pathology ; Pluripotent Stem Cells/*cytology/metabolism/pathology ; },
abstract = {Most neurodegenerative diseases are characterized by a complex and mostly still unresolved pathology. This fact, together with the lack of reliable disease models, has precluded the development of effective therapies counteracting the disease progression. The advent of human pluripotent stem cells has revolutionized the field allowing the generation of disease-relevant neural cell types that can be used for disease modeling, drug screening and, possibly, cell transplantation purposes. In this Review, we discuss the applications of human pluripotent stem cells, the development of efficient protocols for the derivation of the different neural cells and their applicability for robust in vitro disease modeling and drug screening platforms for most common neurodegenerative conditions.},
}

Animals
CRISPR-Cas Systems
Drug Evaluation, Preclinical/*methods
Gene Editing/methods
Humans
Neurodegenerative Diseases/*drug therapy/genetics/pathology
Neurogenesis/drug effects
Neurons/*cytology/drug effects/metabolism/pathology
Pluripotent Stem Cells/*cytology/metabolism/pathology

@article {pmid31128273,
year = {2019},
author = {Ballard, E and Weber, J and Melchers, WJG and Tammireddy, S and Whitfield, PD and Brakhage, AA and Brown, AJP and Verweij, PE and Warris, A},
title = {Recreation of in-host acquired single nucleotide polymorphisms by CRISPR-Cas9 reveals an uncharacterised gene playing a role in Aspergillus fumigatus azole resistance via a non-cyp51A mediated resistance mechanism.},
journal = {Fungal genetics and biology : FG & B},
volume = {130},
number = {},
pages = {98-106},
pmid = {31128273},
issn = {1096-0937},
support = {MR/M026663/1/MRC_/Medical Research Council/United Kingdom ; MR/N006364/1/MRC_/Medical Research Council/United Kingdom ; BB/M010996/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Antifungal Agents/pharmacology ; Aspergillosis/microbiology ; Aspergillus fumigatus/*drug effects/*genetics/growth & development/isolation & purification ; Azoles/*pharmacology ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Drug Resistance, Multiple, Fungal/*genetics ; Ergosterol ; Fungal Proteins/genetics ; Genotype ; Host-Pathogen Interactions ; Humans ; Itraconazole/pharmacology ; Microbial Sensitivity Tests ; Mycelium/drug effects/growth & development ; Phenotype ; *Polymorphism, Single Nucleotide ; },
abstract = {The human host comprises a range of specific niche environments. In order to successfully persist, pathogens such as Aspergillus fumigatus must adapt to these environments. One key example of in-host adaptation is the development of resistance to azole antifungals. Azole resistance in A. fumigatus is increasingly reported worldwide and the most commonly reported mechanisms are cyp51A mediated. Using a unique series of A. fumigatus isolates, obtained from a patient suffering from persistent and recurrent invasive aspergillosis over 2 years, this study aimed to gain insight into the genetic basis of in-host adaptation. Single nucleotide polymorphisms (SNPs) unique to a single isolate in this series, which had developed multi-azole resistance in-host, were identified. Two nonsense SNPs were recreated using CRISPR-Cas9; these were 213* in svf1 and 167* in uncharacterised gene AFUA_7G01960. Phenotypic analyses including antifungal susceptibility testing, mycelial growth rate assessment, lipidomics analysis and statin susceptibility testing were performed to associate genotypes to phenotypes. This revealed a role for svf1 in A. fumigatus oxidative stress sensitivity. In contrast, recapitulation of 167* in AFUA_7G01960 resulted in increased itraconazole resistance. Comprehensive lipidomics analysis revealed decreased ergosterol levels in strains containing this SNP, providing insight to the observed itraconazole resistance. Decreases in ergosterol levels were reflected in increased resistance to lovastatin and nystatin. Importantly, this study has identified a SNP in an uncharacterised gene playing a role in azole resistance via a non-cyp51A mediated resistance mechanism. This mechanism is of clinical importance, as this SNP was identified in a clinical isolate, which acquired azole resistance in-host.},
}

Antifungal Agents/pharmacology
Aspergillosis/microbiology
Aspergillus fumigatus/*drug effects/*genetics/growth & development/isolation & purification
Azoles/*pharmacology
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Drug Resistance, Multiple, Fungal/*genetics
Ergosterol
Fungal Proteins/genetics
Genotype
Host-Pathogen Interactions
Humans
Itraconazole/pharmacology
Microbial Sensitivity Tests
Mycelium/drug effects/growth & development
Phenotype
*Polymorphism, Single Nucleotide

@article {pmid31048007,
year = {2019},
author = {Schuster, M and Kahmann, R},
title = {CRISPR-Cas9 genome editing approaches in filamentous fungi and oomycetes.},
journal = {Fungal genetics and biology : FG & B},
volume = {130},
number = {},
pages = {43-53},
doi = {10.1016/j.fgb.2019.04.016},
pmid = {31048007},
issn = {1096-0937},
mesh = {*CRISPR-Cas Systems ; Fungi/*genetics ; *Gene Editing ; Genome, Fungal ; Mutation ; Oomycetes/*genetics ; },
abstract = {Due to their biotechnological relevance as well as their importance as disease agents, filamentous fungi and oomycetes have been prime candidates for genetic selection and in vitro manipulation for decades. With the advent of new genome editing technologies such manipulations have reached a new level of speed and sophistication. The CRISPR-Cas9 genome editing technology in particular has revolutionized the ways how desired mutations can be introduced. To date, the CRISPR-Cas9 genome editing system has been established in more than 40 different species of filamentous fungi and oomycetes. In this review we describe the various approaches taken to assure expression of the components necessary for editing and describe the varying strategies used to achieve gene disruptions, gene replacements and precise editing. We discuss potential problems faced when establishing the system, propose ways to circumvent them and suggest future approaches not yet realized in filamentous fungi or oomycetes.},
}

*CRISPR-Cas Systems
Fungi/*genetics
*Gene Editing
Genome, Fungal
Mutation
Oomycetes/*genetics

@article {pmid31034868,
year = {2019},
author = {Darma, R and Lutz, A and Elliott, CE and Idnurm, A},
title = {Identification of a gene cluster for the synthesis of the plant hormone abscisic acid in the plant pathogen Leptosphaeria maculans.},
journal = {Fungal genetics and biology : FG & B},
volume = {130},
number = {},
pages = {62-71},
doi = {10.1016/j.fgb.2019.04.015},
pmid = {31034868},
issn = {1096-0937},
mesh = {Abscisic Acid/*metabolism ; Ascomycota/*genetics/*metabolism/pathogenicity ; Brassica napus/microbiology ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; Gene Expression Regulation, Fungal ; Genes, Fungal/*genetics ; Multigene Family/*genetics ; Plant Diseases/microbiology ; Plant Growth Regulators/*biosynthesis/*genetics ; Polyketide Synthases/genetics ; Secondary Metabolism ; Transcription Factors/genetics ; Up-Regulation ; Virulence/genetics ; },
abstract = {Leptosphaeria maculans is an ascomycetous fungus that causes the disease blackleg on Brassica napus (canola). In spite of the importance of the disease worldwide, the mechanisms of disease development are poorly understood. Secondary metabolites, which are one of the common virulence factors of pathogenic fungi, have not been extensively explored from this fungus. An RNA-seq dataset was examined to find genes responsible for secondary metabolite synthesis by this fungus during infection. One polyketide synthase gene, pks5, was found to be upregulated during the early biotrophic stage of development. In addition to pks5, six other genes adjacent to the pks5 gene, including one encoding a Zn(II)2Cys6 transcription factor abscisic acid-like 7 gene (abl7), were also upregulated during that time. A striking feature of the L. maculans genome is that it contains large AT-rich regions that are gene-poor and large GC-rich regions that are gene rich. This set of seven co-regulated genes is embedded within and separated by two such AT-rich regions. Three of the genes in the cluster have similarities to those known to be involved in the synthesis of abscisic acid (ABA) in other fungi. When L. maculans is grown in axenic culture the genes in this cluster are not expressed and ABA is not produced. Overexpressing abl7, encoding the putative transcription factor, resulted in the transcription of the six adjacent genes in axenic culture and in the production of ABA, as detected by liquid chromatography quadrupole-time-of-flight mass spectrometry analysis. Mutation of two genes of the cluster using CRISPR/Cas9 did not affect pathogenicity on canola cotyledons. The characterization of the ABA gene cluster has led to the discovery of the co-regulation of genes within an AT-rich region by a transcription factor, and the first report of the plant hormone abscisic acid being produced by L. maculans.},
}

Abscisic Acid/*metabolism
Ascomycota/*genetics/*metabolism/pathogenicity
Brassica napus/microbiology
CRISPR-Cas Systems
Fungal Proteins/genetics/metabolism
Gene Expression Regulation, Fungal
Genes, Fungal/*genetics
Multigene Family/*genetics
Plant Diseases/microbiology
Plant Growth Regulators/*biosynthesis/*genetics
Polyketide Synthases/genetics
Secondary Metabolism
Transcription Factors/genetics
Up-Regulation
Virulence/genetics

@article {pmid31026589,
year = {2019},
author = {Kunitake, E and Tanaka, T and Ueda, H and Endo, A and Yarimizu, T and Katoh, E and Kitamoto, H},
title = {CRISPR/Cas9-mediated gene replacement in the basidiomycetous yeast Pseudozyma antarctica.},
journal = {Fungal genetics and biology : FG & B},
volume = {130},
number = {},
pages = {82-90},
doi = {10.1016/j.fgb.2019.04.012},
pmid = {31026589},
issn = {1096-0937},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Fungal/genetics ; Fungal Proteins/genetics ; Gene Editing/*methods ; Gene Targeting/*methods ; Genes, Fungal/genetics ; Genetic Loci ; Homologous Recombination ; Ribonucleoproteins/genetics ; Transformation, Genetic ; Ustilaginales/*genetics ; },
abstract = {The basidiomycetous yeast, Pseudozyma antarctica, has the ability to express industrially beneficial biodegradable plastic-degrading enzyme (PaE) and glycolipids. In this study, we developed a highly efficient gene-targeting method in P. antarctica using a CRISPR/Cas9 gene-editing approach. Transformation of protoplast cells was achieved by incubation with a ribonucleoprotein (RNP) complex prepared by mixing the Cas9 protein with a single-guide RNA together with donor DNA (dDNA) containing a selectable marker in vitro. The PaE gene was selected as the targeted locus for gene disruption and gene-disrupted colonies were readily detected by their ability to degrade polybutylene succinate-co-adipate on solid media. The accuracy of the gene conversion event was confirmed by colony PCR. An increase in the RNP mix increased both transformation and gene disruption efficiencies. Examining the effect of the homology arm length of the dDNA revealed that dDNA with homology arms longer than 0.1 kb induced efficient homologous recombination in our system. Furthermore, this system was successful in another targeted locus, PaADE2. Following the creation of RNP-induced double-strand break of the chromosomal DNA, dDNA could be inserted into the target locus even in the absence of homology arms.},
}

Base Sequence
CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA, Fungal/genetics
Fungal Proteins/genetics
Gene Editing/*methods
Gene Targeting/*methods
Genes, Fungal/genetics
Genetic Loci
Homologous Recombination
Ribonucleoproteins/genetics
Transformation, Genetic
Ustilaginales/*genetics

@article {pmid30844313,
year = {2019},
author = {Shi, Z and Xin, H and Tian, D and Lian, J and Wang, J and Liu, G and Ran, R and Shi, S and Zhang, Z and Shi, Y and Deng, Y and Hou, C and Chen, Y},
title = {Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {33},
number = {6},
pages = {6962-6968},
doi = {10.1096/fj.201802661R},
pmid = {30844313},
issn = {1530-6860},
mesh = {Abnormalities, Multiple/*genetics ; Albinism, Oculocutaneous/*genetics ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Female ; Genotype ; Heart Defects, Congenital/*genetics ; Heart Septal Defects, Atrial/*genetics ; Humans ; Lower Extremity Deformities, Congenital/*genetics ; Male ; *Point Mutation ; Upper Extremity Deformities, Congenital/*genetics ; Xenopus/*embryology ; },
abstract = {Precise single-base editing in Xenopus tropicalis would greatly expand the utility of this true diploid frog for modeling human genetic diseases caused by point mutations. Here, we report the efficient conversion of C-to-T or G-to-A in X. tropicalis using the rat apolipoprotein B mRNA editing enzyme catalytic subunit 1-XTEN-clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9) nickase-uracil DNA glycosylase inhibitor-nuclear localization sequence base editor [base editor 3 (BE3)]. Coinjection of guide RNA and the Cas9 mutant complex mRNA into 1-cell stage X. tropicalis embryos caused precise C-to-T or G-to-A substitution in 14 out of 19 tested sites with efficiencies of 5-75%, which allowed for easy establishment of stable lines. Targeting the conserved T-box 5 R237 and Tyr C28 residues in X. tropicalis with the BE3 system mimicked human Holt-Oram syndrome and oculocutaneous albinism type 1A, respectively. Our data indicate that BE3 is an easy and efficient tool for precise base editing in X. tropicalis.-Shi, Z., Xin, H., Tian, D., Lian, J., Wang, J., Liu, G., Ran, R., Shi, S., Zhang, Z., Shi, Y., Deng, Y., Hou, C., Chen, Y. Modeling human point mutation diseases in Xenopus tropicalis with a modified CRISPR/Cas9 system.},
}