@article {pmid32755000,
year = {2020},
author = {Fantoni, N and McGorman, B and Molphy, Z and Singleton, D and Walsh, S and El-Sagheer, AH and McKee, V and Brown, T and Kellett, A},
title = {Development of Gene-Targeted Polypyridyl Triplex Forming Oligonucleotide Hybrids.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {},
number = {},
pages = {},
doi = {10.1002/cbic.202000408},
pmid = {32755000},
issn = {1439-7633},
abstract = {In the field of nucleic acid therapy there is major interest in the development of libraries of DNA-reactive small molecules which are tethered to vectors that recognize and bind specific genes. This approach mimics enzymatic gene-editors, such as ZFNs, TALENs and CRISPR-Cas, but overcomes limitations imposed by the delivery of a large protein endonuclease which is required for DNA cleavage. Here, we introduce a chemistry-based DNA cleavage system comprising an artificial metallo-nuclease (AMN) that oxidatively cuts DNA, and a triplex forming oligonucleotide (TFO) that sequence-specifically recognises duplex DNA. The AMN-TFO hybrids coordinate Cu(II) ions to form chimeric catalytic complexes that are programmable-based on the TFO sequence employed-to bind and cut specific DNA sequences. Use of the alkyne-azide cycloaddition 'click' reaction allows scalable and high-throughput generation of hybrid libraries that can be tuned for specific reactivity and gene-of-interest knockout. As a first approach, we demonstrate targeted cleavage of purine-rich sequences, optimisation of the hybrid system to enhance stability, and discrimination between target and off-target sequences. Our results highlight the potential of this approach where the cutting unit, which mimics the endonuclease cleavage machinery, is directly bound to a TFO guide by click chemistry.},
}

@article {pmid32749110,
year = {2020},
author = {Ahmed, B and Arif, M and Qadir, MI},
title = {CRISPR/Cas: A Successful Tool for Genome Editing in Animal Models.},
journal = {Critical reviews in eukaryotic gene expression},
volume = {30},
number = {3},
pages = {239-243},
doi = {10.1615/CritRevEukaryotGeneExpr.2020028791},
pmid = {32749110},
issn = {1045-4403},
abstract = {CRISPR/Cas9 is an innovative molecular tool that is utilized in advanced biological applications. This review focuses on modifying the genomes of a wide range of animals by CRISPR/Cas9 for greater usability and higher efficiency, providing an overview of the function and mechanism of this system and the utilization of this system for medicinal research. The type II CRISPR-Cas system is found in the Enterobacteriaceae bacteria family, which uses this system as a defense against invading phages and plasmids. This system can be engineered to direct its action toward a targeted site for the modification of a specific genome.},
}

@article {pmid32748081,
year = {2020},
author = {Huang, X and Wang, Y and Xu, J and Wang, N},
title = {Development of multiplex genome editing toolkits for citrus with high efficacy in biallelic and homozygous mutations.},
journal = {Plant molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s11103-020-01043-6},
pmid = {32748081},
issn = {1573-5028},
support = {2017-67013-26527//National Institute of Food and Agriculture/ ; },
abstract = {KEY MESSAGE: We have developed multiplex genome editing toolkits for citrus that significantly improve citrus genome editing efficacy. CRISPR/Cas systems have been engineered for genome editing in many organisms, including plants. However, the gene editing efficiency in citrus via CRISPR technology remains too low to be implemented for genetic improvement in practice. Moreover, it is very difficult to obtain homozygous or biallelic knockout mutants in citrus. Here, we have developed multiplex genome editing toolkits for citrus including PEG-mediated protoplast transformation, a GFP reporter system that allows the rapid assessment of CRISPR constructs, citrus U6 promoters with improved efficacy, and tRNA-mediated or Csy4-mediated multiplex genome editing. Using the toolkits, we successfully conducted genome modification of embryogenic protoplast cells and epicotyl tissues. We have achieved a biallelic mutation rate of 44.4% and a homozygous mutation rate of 11.1%, representing a significant improvement in citrus genome editing efficacy. In addition, our study lays the foundation for nontransgenic genome editing of citrus.},
}

@article {pmid32747749,
year = {2020},
author = {Sun, W and Wang, H},
title = {Recent advances of genome editing and related technologies in China.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41434-020-0181-5},
pmid = {32747749},
issn = {1476-5462},
support = {31722036//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {Genome editing is a powerful tool, enabling scientists to alter DNA sequence at virtually any genome locus in any species. Different technologies have been developed employing programmable nucleases including meganuclease, zinc-finger nucleases, transcription activator-like effector nucleases, and most recently CRISPR-Cas systems. Chinese research groups are making important contributions at an increasing speed in genome editing field in recent years. In this review, we summarize recent progress made by Chinese scientists on the technological development of genome editing and beyond, focusing on the optimization and expanded application of existing genome editing tools, as well as the exploration of novel proteins as potential genome editing tools.},
}

@article {pmid32415249,
year = {2020},
author = {Mullard, A},
title = {Gene-editing pipeline takes off.},
journal = {Nature reviews. Drug discovery},
volume = {19},
number = {6},
pages = {367-372},
doi = {10.1038/d41573-020-00096-y},
pmid = {32415249},
issn = {1474-1784},
mesh = {CRISPR-Cas Systems/*genetics ; Computational Biology/*methods/trends ; *Gene Editing ; Humans ; Medical Oncology/*methods/trends ; },
}

CRISPR-Cas Systems/*genetics
Computational Biology/*methods/trends
*Gene Editing
Humans
Medical Oncology/*methods/trends

@article {pmid32415248,
year = {2020},
author = {Mullard, A},
title = {Jennifer Doudna.},
journal = {Nature reviews. Drug discovery},
volume = {19},
number = {6},
pages = {380-381},
doi = {10.1038/d41573-020-00095-z},
pmid = {32415248},
issn = {1474-1784},
mesh = {CRISPR-Cas Systems/genetics ; Drug Discovery/*methods/*trends ; Gene Editing ; *Genetic Research ; Genome-Wide Association Study ; },
}

CRISPR-Cas Systems/genetics
Drug Discovery/*methods/*trends
Gene Editing
*Genetic Research
Genome-Wide Association Study

@article {pmid32313011,
year = {2020},
author = {Després, PC and Dubé, AK and Seki, M and Yachie, N and Landry, CR},
title = {Perturbing proteomes at single residue resolution using base editing.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1871},
pmid = {32313011},
issn = {2041-1723},
support = {364920//CIHR/Canada ; 384483//CIHR/Canada ; 387697//CIHR/Canada ; },
mesh = {Base Sequence ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing/*methods ; Genes, Essential ; Genome ; Genome, Fungal ; High-Throughput Nucleotide Sequencing ; *High-Throughput Screening Assays ; Molecular Sequence Data ; Mutagenesis ; Mutation ; Proteome/*genetics ; RNA, Guide/genetics ; Yeasts/*genetics ; },
abstract = {Base editors derived from CRISPR-Cas9 systems and DNA editing enzymes offer an unprecedented opportunity for the precise modification of genes, but have yet to be used at a genome-scale throughput. Here, we test the ability of the Target-AID base editor to systematically modify genes genome-wide by targeting yeast essential genes. We mutate around 17,000 individual sites in parallel across more than 1500 genes. We identify over 700 sites at which mutations have a significant impact on fitness. Using previously determined and preferred Target-AID mutational outcomes, we find that gRNAs with significant effects on fitness are enriched in variants predicted to be deleterious based on residue conservation and predicted protein destabilization. We identify key features influencing effective gRNAs in the context of base editing. Our results show that base editing is a powerful tool to identify key amino acid residues at the scale of proteomes.},
}

Base Sequence
CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing/*methods
Genes, Essential
Genome
Genome, Fungal
High-Throughput Nucleotide Sequencing
*High-Throughput Screening Assays
Molecular Sequence Data
Mutagenesis
Mutation
Proteome/*genetics
RNA, Guide/genetics
Yeasts/*genetics

@article {pmid31576005,
year = {2020},
author = {Meyer, T and Jahn, N and Lindner, S and Röhner, L and Dolnik, A and Weber, D and Scheffold, A and Köpff, S and Paschka, P and Gaidzik, VI and Heckl, D and Wiese, S and Ebert, BL and Döhner, H and Bullinger, L and Döhner, K and Krönke, J},
title = {Functional characterization of BRCC3 mutations in acute myeloid leukemia with t(8;21)(q22;q22.1).},
journal = {Leukemia},
volume = {34},
number = {2},
pages = {404-415},
pmid = {31576005},
issn = {1476-5551},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line ; Cell Proliferation/drug effects/genetics ; Cytokines/genetics ; DNA Damage/drug effects/genetics ; Deubiquitinating Enzymes/*genetics ; Doxorubicin/pharmacology ; Granulocyte Colony-Stimulating Factor/genetics ; HEK293 Cells ; Humans ; Inflammasomes/genetics ; Leukemia, Myeloid, Acute/drug therapy/*genetics ; Mice ; Mutation/*genetics ; },
abstract = {BRCA1/BRCA2-containing complex 3 (BRCC3) is a Lysine 63-specific deubiquitinating enzyme (DUB) involved in inflammasome activity, interferon signaling, and DNA damage repair. Recurrent mutations in BRCC3 have been reported in myelodysplastic syndromes (MDS) but not in de novo AML. In one of our recent studies, we found BRCC3 mutations selectively in 9/191 (4.7%) cases with t(8;21)(q22;q22.1) AML but not in 160 cases of inv(16)(p13.1q22) AML. Clinically, AML patients with BRCC3 mutations had an excellent outcome with an event-free survival of 100%. Inactivation of BRCC3 by CRISPR/Cas9 resulted in improved proliferation in t(8;21)(q22;q22.1) positive AML cell lines and together with expression of AML1-ETO induced unlimited self-renewal in mouse hematopoietic progenitor cells in vitro. Mutations in BRCC3 abrogated its deubiquitinating activity on IFNAR1 resulting in an impaired interferon response and led to diminished inflammasome activity. In addition, BRCC3 inactivation increased release of several cytokines including G-CSF which enhanced proliferation of AML cell lines with t(8;21)(q22;q22.1). Cell lines and primary mouse cells with inactivation of BRCC3 had a higher sensitivity to doxorubicin due to an impaired DNA damage response providing a possible explanation for the favorable outcome of BRCC3 mutated AML patients.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Line
Cell Proliferation/drug effects/genetics
Cytokines/genetics
DNA Damage/drug effects/genetics
Deubiquitinating Enzymes/*genetics
Doxorubicin/pharmacology
Granulocyte Colony-Stimulating Factor/genetics
HEK293 Cells
Humans
Inflammasomes/genetics
Leukemia, Myeloid, Acute/drug therapy/*genetics
Mice
Mutation/*genetics

@article {pmid31471561,
year = {2020},
author = {Balligand, T and Achouri, Y and Pecquet, C and Gaudray, G and Colau, D and Hug, E and Rahmani, Y and Stroobant, V and Plo, I and Vainchenker, W and Kralovics, R and Van den Eynde, BJ and Defour, JP and Constantinescu, SN},
title = {Knock-in of murine Calr del52 induces essential thrombocythemia with slow-rising dominance in mice and reveals key role of Calr exon 9 in cardiac development.},
journal = {Leukemia},
volume = {34},
number = {2},
pages = {510-521},
doi = {10.1038/s41375-019-0538-1},
pmid = {31471561},
issn = {1476-5551},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Calreticulin/*genetics ; Exons/*genetics ; Female ; Frameshift Mutation/genetics ; Heart/*physiology ; Hematopoiesis/genetics ; Homozygote ; Male ; Mice ; Primary Myelofibrosis/genetics ; Receptors, Thrombopoietin/genetics ; Thrombocythemia, Essential/*genetics ; Thrombocytosis/genetics ; },
abstract = {Frameshifting mutations (-1/+2) of the calreticulin (CALR) gene are responsible for the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF). The mutant CALR proteins activate the thrombopoietin receptor (TpoR) inducing cytokine-independent megakaryocyte progenitor proliferation. Here, we generated via CRISPR/Cas9 technology two knock-in mouse models that are heterozygous for a type-I murine Calr mutation. These mice exhibit an ET phenotype with elevated circulating platelets compared with wild-type controls, consistent with our previous results showing that murine CALR mutants activate TpoR. We also show that the mutant CALR proteins can be detected in plasma. The phenotype of Calr del52 is transplantable, and the Calr mutated hematopoietic cells have a slow-rising advantage over wild-type hematopoiesis. Importantly, a homozygous state of a type-1 Calr mutation is lethal at a late embryonic development stage, showing narrowed ventricular myocardium walls, similar to the murine Calr knockout phenotype, pointing to the C terminus of CALR as crucial for heart development.},
}

Animals
CRISPR-Cas Systems/genetics
Calreticulin/*genetics
Exons/*genetics
Female
Frameshift Mutation/genetics
Heart/*physiology
Hematopoiesis/genetics
Homozygote
Male
Mice
Primary Myelofibrosis/genetics
Receptors, Thrombopoietin/genetics
Thrombocythemia, Essential/*genetics
Thrombocytosis/genetics

@article {pmid31444197,
year = {2019},
author = {Zhou, D and Jiang, Z and Pang, Q and Zhu, Y and Wang, Q and Qi, Q},
title = {CRISPR/Cas9-Assisted Seamless Genome Editing in Lactobacillus plantarum and Its Application in N-Acetylglucosamine Production.},
journal = {Applied and environmental microbiology},
volume = {85},
number = {21},
pages = {},
pmid = {31444197},
issn = {1098-5336},
mesh = {Acetylglucosamine/*biosynthesis ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; DNA, Single-Stranded ; Gene Editing/*methods ; Gene Knockout Techniques ; Genes, Bacterial/genetics ; Genetic Engineering ; Genome, Bacterial ; Lactobacillus plantarum/*genetics ; Metabolic Engineering/*methods ; Metabolic Networks and Pathways/genetics ; Recombination, Genetic ; },
abstract = {Lactobacillus plantarum is a potential starter and health-promoting probiotic bacterium. Effective, precise, and diverse genome editing of Lactobacillus plantarum without introducing exogenous genes or plasmids is of great importance. In this study, CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering was established in L. plantarum WCFS1 to seamlessly edit the genome, including gene knockouts, insertions, and point mutations. To optimize our editing method, phosphorothioate modification was used to improve the dsDNA insertion, and adenine-specific methyltransferase was used to improve the ssDNA recombination efficiency. These strategies were applied to engineer L. plantarum WCFS1 toward producing N-acetylglucosamine (GlcNAc). nagB was truncated to eliminate the reverse reaction of fructose-6-phosphate (F6P) to glucosamine 6-phosphate (GlcN-6P). Riboswitch replacement and point mutation in glmS1 were introduced to relieve feedback repression. The resulting strain produced 797.3 mg/liter GlcNAc without introducing exogenous genes or plasmids. This strategy may contribute to the available methods for precise and diverse genetic engineering in lactic acid bacteria and boost strain engineering for more applications.IMPORTANCE CRISPR/Cas9-assisted recombineering is restricted in lactic acid bacteria because of the lack of available antibiotics and vectors. In this study, a seamless genome editing method was carried out in Lactobacillus plantarum using CRISPR/Cas9-assisted double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) recombineering, and recombination efficiency was effectively improved by endogenous adenine-specific methyltransferase overexpression. L. plantarum WCFS1 produced 797.3 mg/liter N-acetylglucosamine (GlcNAc) through reinforcement of the GlcNAc pathway, without introducing exogenous genes or plasmids. This seamless editing strategy, combined with the potential exogenous GlcNAc-producing pathway, makes this strain an attractive candidate for industrial use in the future.},
}

Acetylglucosamine/*biosynthesis
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA
DNA, Single-Stranded
Gene Editing/*methods
Gene Knockout Techniques
Genes, Bacterial/genetics
Genetic Engineering
Genome, Bacterial
Lactobacillus plantarum/*genetics
Metabolic Engineering/*methods
Metabolic Networks and Pathways/genetics
Recombination, Genetic

@article {pmid31038202,
year = {2019},
author = {Schultz, JC and Cao, M and Zhao, H},
title = {Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {8},
pages = {2103-2109},
doi = {10.1002/bit.27001},
pmid = {31038202},
issn = {1097-0290},
mesh = {Basidiomycota/*genetics ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Deletion ; Gene Editing/methods ; Genes, Fungal ; Metabolic Engineering/methods ; RNA, Guide/genetics ; },
abstract = {The oleaginous yeast Rhodosporidium toruloides is considered a promising candidate for production of chemicals and biofuels thanks to its ability to grow on lignocellulosic biomass, and its high production of lipids and carotenoids. However, efforts to engineer this organism are hindered by a lack of suitable genetic tools. Here we report the development of a CRISPR/Cas9 system for genome editing in R. toruloides based on a fusion 5S rRNA-tRNA promoter for guide RNA (gRNA) expression, capable of greater than 95% gene knockout for various genetic targets. Additionally, multiplexed double-gene knockout mutants were obtained using this method with an efficiency of 78%. This tool can be used to accelerate future metabolic engineering work in this yeast.},
}

Basidiomycota/*genetics
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Gene Deletion
Gene Editing/methods
Genes, Fungal
Metabolic Engineering/methods
RNA, Guide/genetics

@article {pmid32332735,
year = {2020},
author = {Jiang, T and Henderson, JM and Coote, K and Cheng, Y and Valley, HC and Zhang, XO and Wang, Q and Rhym, LH and Cao, Y and Newby, GA and Bihler, H and Mense, M and Weng, Z and Anderson, DG and McCaffrey, AP and Liu, DR and Xue, W},
title = {Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1979},
pmid = {32332735},
issn = {2041-1723},
support = {P01 HL131471/HL/NHLBI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; P30 CA014051/CA/NCI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB022376/EB/NIBIB NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; DP2 HL137167/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Adenine/*chemistry ; Alleles ; Animals ; *CRISPR-Cas Systems ; Cell Line ; Codon ; Codon, Nonsense ; Cystic Fibrosis/pathology ; Gene Editing ; HEK293 Cells ; Humans ; Mice ; Mutation ; Nucleotides ; Phenotype ; Plasmids ; RNA, Guide/*chemistry ; RNA, Messenger/*chemistry ; Transfection ; Uridine/analogs & derivatives/chemistry ; },
abstract = {CRISPR-Cas9-associated base editing is a promising tool to correct pathogenic single nucleotide mutations in research or therapeutic settings. Efficient base editing requires cellular exposure to levels of base editors that can be difficult to attain in hard-to-transfect cells or in vivo. Here we engineer a chemically modified mRNA-encoded adenine base editor that mediates robust editing at various cellular genomic sites together with moderately modified guide RNA, and show its therapeutic potential in correcting pathogenic single nucleotide mutations in cell and animal models of diseases. The optimized chemical modifications of adenine base editor mRNA and guide RNA expand the applicability of CRISPR-associated gene editing tools in vitro and in vivo.},
}

Adenine/*chemistry
Alleles
Animals
*CRISPR-Cas Systems
Cell Line
Codon
Codon, Nonsense
Cystic Fibrosis/pathology
Gene Editing
HEK293 Cells
Humans
Mice
Mutation
Nucleotides
Phenotype
Plasmids
RNA, Guide/*chemistry
RNA, Messenger/*chemistry
Transfection
Uridine/analogs & derivatives/chemistry

@article {pmid32317636,
year = {2020},
author = {Ulgherait, M and Chen, A and McAllister, SF and Kim, HX and Delventhal, R and Wayne, CR and Garcia, CJ and Recinos, Y and Oliva, M and Canman, JC and Picard, M and Owusu-Ansah, E and Shirasu-Hiza, M},
title = {Circadian regulation of mitochondrial uncoupling and lifespan.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1927},
pmid = {32317636},
issn = {2041-1723},
support = {T32 HL120826/HL/NHLBI NIH HHS/United States ; R01 GM117407/GM/NIGMS NIH HHS/United States ; P40 OD018537/OD/NIH HHS/United States ; R35 GM119793/GM/NIGMS NIH HHS/United States ; R21 DK112074/DK/NIDDK NIH HHS/United States ; F31 GM125363/GM/NIGMS NIH HHS/United States ; R21 AR077312/AR/NIAMS NIH HHS/United States ; T32 DK007328/DK/NIDDK NIH HHS/United States ; R35 GM127049/GM/NIGMS NIH HHS/United States ; R35 GM124717/GM/NIGMS NIH HHS/United States ; R01 AG045842/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Carcinogenesis ; Cell Proliferation ; Circadian Clocks ; *Circadian Rhythm ; Drosophila Proteins/*metabolism ; Drosophila melanogaster/*metabolism ; Homeostasis ; Intestines/pathology ; Longevity ; Male ; Membrane Potential, Mitochondrial ; Membrane Transport Proteins/*metabolism ; Mitochondria/*metabolism ; Mutation ; Oxidative Stress/physiology ; Oxygen Consumption ; Period Circadian Proteins/*metabolism ; Uncoupling Protein 1/metabolism ; },
abstract = {Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, upregulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents overproliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadian-regulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target.},
}

Animals
CRISPR-Cas Systems
Carcinogenesis
Cell Proliferation
Circadian Clocks
*Circadian Rhythm
Drosophila Proteins/*metabolism
Drosophila melanogaster/*metabolism
Homeostasis
Intestines/pathology
Longevity
Male
Membrane Potential, Mitochondrial
Membrane Transport Proteins/*metabolism
Mitochondria/*metabolism
Mutation
Oxidative Stress/physiology
Oxygen Consumption
Period Circadian Proteins/*metabolism
Uncoupling Protein 1/metabolism

@article {pmid32276963,
year = {2020},
author = {Bian, T and Tagmount, A and Vulpe, C and Vijendra, KC and Xing, C},
title = {CXL146, a Novel 4H-Chromene Derivative, Targets GRP78 to Selectively Eliminate Multidrug-Resistant Cancer Cells.},
journal = {Molecular pharmacology},
volume = {97},
number = {6},
pages = {402-408},
pmid = {32276963},
issn = {1521-0111},
support = {R01 CA163864/CA/NCI NIH HHS/United States ; },
mesh = {Antineoplastic Agents/*pharmacology ; Benzopyrans/*pharmacology ; CRISPR-Cas Systems ; Cell Line, Tumor ; Drug Resistance, Multiple/drug effects ; Drug Resistance, Neoplasm/*drug effects ; Drug Screening Assays, Antitumor ; HL-60 Cells ; Heat-Shock Proteins/drug effects/*metabolism ; Humans ; Mitoxantrone/pharmacology ; },
abstract = {The 78-kDa glucose-regulated protein (GRP78), an endoplasmic reticulum (ER) chaperone, is a master regulator of the ER stress. A number of studies revealed that high levels of GRP78 protein in cancer cells confer multidrug resistance (MDR) to therapeutic treatment. Therefore, drug candidate that reduces GRP78 may represent a novel approach to eliminate MDR cancer cells. Our earlier studies showed that a set of 4H-chromene derivatives induced selective cytotoxicity in MDR cancer cells. In the present study, we elucidated its selective mechanism in four MDR cancer cell lines with one lead candidate (CXL146). Cytotoxicity results confirmed the selective cytotoxicity of CXL146 toward the MDR cancer cell lines. We noted significant overexpression of GRP78 in all four MDR cell lines compared with the parental cell lines. Unexpectedly, CXL146 treatment rapidly and dose-dependently reduced GRP78 protein in MDR cancer cell lines. Using human leukemia (HL) 60/mitoxantrone (MX) 2 cell line as the model, we demonstrated that CXL146 treatment activated the unfolded protein response (UPR); as evidenced by the activation of inositol-requiring enzyme 1α, protein kinase R-like ER kinase, and activating transcription factor 6. CXL146-induced UPR activation led to a series of downstream events, including extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase activation, which contributed to CXL146-induced apoptosis. Targeted reduction in GRP78 resulted in reduced sensitivity of HL60/MX2 toward CXL146. Long-term sublethal CXL146 exposure also led to reduction in GRP78 in HL60/MX2. These data collectively support GRP78 as the target of CXL146 in MDR treatment. Interestingly, HL60/MX2 upon long-term sublethal CXL146 exposure regained sensitivity to mitoxantrone treatment. Therefore, further exploration of CXL146 as a novel therapy in treating MDR cancer cells is warranted. SIGNIFICANCE STATEMENT: Multidrug resistance is one major challenge to cancer treatment. This study provides evidence that cancer cells overexpress 78-kDa glucose-regulated protein (GRP78) as a mechanism to acquire resistance to standard cancer therapies. A chromene-based small molecule, CXL146, selectively eliminates cancer cells with GRP78 overexpression via activating unfolded protein response-mediated apoptosis. Further characterization indicates that CXL146 and standard therapies complementarily target different populations of cancer cells, supporting the potential of CXL146 to overcome multidrug resistance in cancer treatment.},
}

Antineoplastic Agents/*pharmacology
Benzopyrans/*pharmacology
CRISPR-Cas Systems
Cell Line, Tumor
Drug Resistance, Multiple/drug effects
Drug Resistance, Neoplasm/*drug effects
Drug Screening Assays, Antitumor
HL-60 Cells
Heat-Shock Proteins/drug effects/*metabolism
Humans
Mitoxantrone/pharmacology

@article {pmid32234808,
year = {2020},
author = {Vanderboor, CMG and Thibeault, PE and Nixon, KCJ and Gros, R and Kramer, J and Ramachandran, R},
title = {Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA and β-Arrestin.},
journal = {Molecular pharmacology},
volume = {97},
number = {6},
pages = {365-376},
doi = {10.1124/mol.119.118232},
pmid = {32234808},
issn = {1521-0111},
support = {376560//CIHR/Canada ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Membrane/*metabolism/*pathology ; Cell Shape ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Muscle, Smooth, Vascular/metabolism ; Rats ; Rats, Inbred WKY ; Receptors, G-Protein-Coupled/metabolism ; Receptors, Thrombin/agonists/*metabolism ; Signal Transduction ; beta-Arrestins/*metabolism ; rhoA GTP-Binding Protein/*metabolism ; },
abstract = {Proteinase-activated receptors (PARs) are a four-member family of G-protein-coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin, and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types, including platelets, vascular smooth muscle cells, and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane, with spherical membrane protrusions that resemble plasma membrane blebbing. Since nonapoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release, we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gαq/11- and Gαi-signaling pathways and is dependent on signaling via the β-arrestin-1/2 and Ras homolog family member A (RhoA) signaling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. SIGNIFICANCE STATEMENT: We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA-and β-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4.},
}

Animals
CRISPR-Cas Systems
Cell Membrane/*metabolism/*pathology
Cell Shape
Gene Knockout Techniques
HEK293 Cells
Humans
Muscle, Smooth, Vascular/metabolism
Rats
Rats, Inbred WKY
Receptors, G-Protein-Coupled/metabolism
Receptors, Thrombin/agonists/*metabolism
Signal Transduction
beta-Arrestins/*metabolism
rhoA GTP-Binding Protein/*metabolism

@article {pmid32015155,
year = {2020},
author = {Cole, KA},
title = {Targeting ATRX Loss through Inhibition of the Cell-Cycle Checkpoint Mediator WEE1.},
journal = {Cancer research},
volume = {80},
number = {3},
pages = {375-376},
doi = {10.1158/0008-5472.CAN-19-3587},
pmid = {32015155},
issn = {1538-7445},
mesh = {Adult ; CRISPR-Cas Systems ; Cell Cycle Proteins/genetics ; Child ; Humans ; *Neoplasms ; Nuclear Proteins ; *Protein-Tyrosine Kinases ; Pyrimidinones ; X-linked Nuclear Protein ; },
abstract = {In this issue of Cancer Research, Liang and colleagues perform a genome-wide CRISPR-Cas9-negative loss-of-function screen and identify WEE1 kinase as a therapeutic vulnerability in cells depleted of the ATRX chromatin remodeler gene. Because ATRX mutations are frequently mutated across a variety of pediatric and adult malignancies, this work may contribute to the preclinical rationale for a precision medicine trial of the WEE1 inhibitor AZD1775 (adavosertib) for patients whose tumors demonstrate ATRX loss.See related article by Liang et al., p. 510.},
}

Adult
CRISPR-Cas Systems
Cell Cycle Proteins/genetics
Child
Humans
*Neoplasms
Nuclear Proteins
*Protein-Tyrosine Kinases
Pyrimidinones
X-linked Nuclear Protein

@article {pmid31935373,
year = {2020},
author = {Chen, CC and Li, B and Millman, SE and Chen, C and Li, X and Morris, JP and Mayle, A and Ho, YJ and Loizou, E and Liu, H and Qin, W and Shah, H and Violante, S and Cross, JR and Lowe, SW and Zhang, L},
title = {Vitamin B6 Addiction in Acute Myeloid Leukemia.},
journal = {Cancer cell},
volume = {37},
number = {1},
pages = {71-84.e7},
pmid = {31935373},
issn = {1878-3686},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; P30 CA045508/CA/NCI NIH HHS/United States ; R01 CA190261/CA/NCI NIH HHS/United States ; U01 HL127522/HL/NHLBI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Proliferation ; GTP Phosphohydrolases/metabolism ; Gene Expression Regulation, Leukemic ; Humans ; Leukemia, Myeloid, Acute/*enzymology ; Membrane Proteins/metabolism ; Mice ; Monomeric GTP-Binding Proteins/metabolism ; Phosphotransferases/genetics/*metabolism ; Polyamines/metabolism ; Pyridoxal Phosphate/*metabolism ; RNA, Small Interfering/metabolism ; Vitamin B 6/*metabolism ; },
abstract = {Cancer cells rely on altered metabolism to support abnormal proliferation. We performed a CRISPR/Cas9 functional genomic screen targeting metabolic enzymes and identified PDXK-an enzyme that produces pyridoxal phosphate (PLP) from vitamin B6-as an acute myeloid leukemia (AML)-selective dependency. PDXK kinase activity is required for PLP production and AML cell proliferation, and pharmacological blockade of the vitamin B6 pathway at both PDXK and PLP levels recapitulated PDXK disruption effects. PDXK disruption reduced intracellular concentrations of key metabolites needed for cell division. Furthermore, disruption of PLP-dependent enzymes ODC1 or GOT2 selectively inhibited AML cell proliferation and their downstream products partially rescued PDXK disruption induced proliferation blockage. Our work identifies the vitamin B6 pathway as a pharmacologically actionable dependency in AML.},
}

Animals
CRISPR-Cas Systems
Cell Line, Tumor
Cell Proliferation
GTP Phosphohydrolases/metabolism
Gene Expression Regulation, Leukemic
Humans
Leukemia, Myeloid, Acute/*enzymology
Membrane Proteins/metabolism
Mice
Monomeric GTP-Binding Proteins/metabolism
Phosphotransferases/genetics/*metabolism
Polyamines/metabolism
Pyridoxal Phosphate/*metabolism
RNA, Small Interfering/metabolism
Vitamin B 6/*metabolism

@article {pmid31663334,
year = {2019},
author = {Ruiz, E and Talenton, V and Dubrana, MP and Guesdon, G and Lluch-Senar, M and Salin, F and Sirand-Pugnet, P and Arfi, Y and Lartigue, C},
title = {CReasPy-Cloning: A Method for Simultaneous Cloning and Engineering of Megabase-Sized Genomes in Yeast Using the CRISPR-Cas9 System.},
journal = {ACS synthetic biology},
volume = {8},
number = {11},
pages = {2547-2557},
doi = {10.1021/acssynbio.9b00224},
pmid = {31663334},
issn = {2161-5063},
mesh = {CRISPR-Associated Protein 9/metabolism ; *CRISPR-Cas Systems ; Chromosomes, Bacterial/genetics ; Cloning, Molecular/*methods ; DNA Cleavage ; DNA, Bacterial/genetics ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genetic Loci ; Genome, Bacterial ; Mycoplasma pneumoniae/genetics ; Plasmids/genetics ; RNA, Guide/metabolism ; Saccharomyces cerevisiae/*genetics ; },
abstract = {Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (<50) number of clones, allowing for significantly shortened genome engineering cycle times.},
}

CRISPR-Associated Protein 9/metabolism
*CRISPR-Cas Systems
Chromosomes, Bacterial/genetics
Cloning, Molecular/*methods
DNA Cleavage
DNA, Bacterial/genetics
Gene Editing/*methods
Genetic Engineering/*methods
Genetic Loci
Genome, Bacterial
Mycoplasma pneumoniae/genetics
Plasmids/genetics
RNA, Guide/metabolism
Saccharomyces cerevisiae/*genetics

@article {pmid31551363,
year = {2020},
author = {Liang, J and Zhao, H and Diplas, BH and Liu, S and Liu, J and Wang, D and Lu, Y and Zhu, Q and Wu, J and Wang, W and Yan, H and Zeng, YX and Wang, X and Jiao, Y},
title = {Genome-Wide CRISPR-Cas9 Screen Reveals Selective Vulnerability of ATRX-Mutant Cancers to WEE1 Inhibition.},
journal = {Cancer research},
volume = {80},
number = {3},
pages = {510-523},
doi = {10.1158/0008-5472.CAN-18-3374},
pmid = {31551363},
issn = {1538-7445},
mesh = {CRISPR-Cas Systems ; Carcinoma, Hepatocellular/*genetics ; Cell Cycle Proteins/genetics ; Humans ; Liver Neoplasms/*genetics ; Nuclear Proteins/genetics ; Protein-Tyrosine Kinases ; Pyrimidinones ; X-linked Nuclear Protein ; },
abstract = {The tumor suppressor gene ATRX is frequently mutated in a variety of tumors including gliomas and liver cancers, which are highly unresponsive to current therapies. Here, we performed a genome-wide synthetic lethal screen, using CRISPR-Cas9 genome editing, to identify potential therapeutic targets specific for ATRX-mutated cancers. In isogenic hepatocellular carcinoma (HCC) cell lines engineered for ATRX loss, we identified 58 genes, including the checkpoint kinase WEE1, uniquely required for the cell growth of ATRX null cells. Treatment with the WEE1 inhibitor AZD1775 robustly inhibited the growth of several ATRX-deficient HCC cell lines in vitro, as well as xenografts in vivo. The increased sensitivity to the WEE1 inhibitor was caused by accumulated DNA damage-induced apoptosis. AZD1775 also selectively inhibited the proliferation of patient-derived primary cell lines from gliomas with naturally occurring ATRX mutations, indicating that the synthetic lethal relationship between WEE1 and ATRX could be exploited in a broader spectrum of human tumors. As WEE1 inhibitors have been investigated in several phase II clinical trials, our discovery provides the basis for an easily clinically testable therapeutic strategy specific for cancers deficient in ATRX. SIGNIFICANCE: ATRX-mutant cancer cells depend on WEE1, which provides a basis for therapeutically targeting WEE1 in ATRX-deficient cancers.See related commentary by Cole, p. 375.},
}

CRISPR-Cas Systems
Carcinoma, Hepatocellular/*genetics
Cell Cycle Proteins/genetics
Humans
Liver Neoplasms/*genetics
Nuclear Proteins/genetics
Protein-Tyrosine Kinases
Pyrimidinones
X-linked Nuclear Protein

@article {pmid31199589,
year = {2019},
author = {Gao, Q and Dong, X and Xu, Q and Zhu, L and Wang, F and Hou, Y and Chao, CC},
title = {Therapeutic potential of CRISPR/Cas9 gene editing in engineered T-cell therapy.},
journal = {Cancer medicine},
volume = {8},
number = {9},
pages = {4254-4264},
pmid = {31199589},
issn = {2045-7634},
mesh = {Animals ; CRISPR-Cas Systems ; Cell- and Tissue-Based Therapy ; Gene Editing/*methods ; Humans ; Immunotherapy ; Immunotherapy, Adoptive ; Neoplasms/immunology/*therapy ; Receptors, Antigen, T-Cell/*genetics/metabolism ; Receptors, Chimeric Antigen/*genetics/metabolism ; T-Lymphocytes/immunology/*transplantation ; Tumor Microenvironment ; },
abstract = {Cancer patients have been treated with various types of therapies, including conventional strategies like chemo-, radio-, and targeted therapy, as well as immunotherapy like checkpoint inhibitors, vaccine and cell therapy etc. Among the therapeutic alternatives, T-cell therapy like CAR-T (Chimeric Antigen Receptor Engineered T cell) and TCR-T (T Cell Receptor Engineered T cell), has emerged as the most promising therapeutics due to its impressive clinical efficacy. However, there are many challenges and obstacles, such as immunosuppressive tumor microenvironment, manufacturing complexity, and poor infiltration of engrafted cells, etc still, need to be overcome for further treatment with different forms of cancer. Recently, the antitumor activities of CAR-T and TCR-T cells have shown great improvement with the utilization of CRISPR/Cas9 gene editing technology. Thus, the genome editing system could be a powerful genetic tool to use for manipulating T cells and enhancing the efficacy of cell immunotherapy. This review focuses on pros and cons of various gene delivery methods, challenges, and safety issues of CRISPR/Cas9 gene editing application in T-cell-based immunotherapy.},
}

Animals
CRISPR-Cas Systems
Cell- and Tissue-Based Therapy
Gene Editing/*methods
Humans
Immunotherapy
Immunotherapy, Adoptive
Neoplasms/immunology/*therapy
Receptors, Antigen, T-Cell/*genetics/metabolism
Receptors, Chimeric Antigen/*genetics/metabolism
T-Lymphocytes/immunology/*transplantation
Tumor Microenvironment

@article {pmid32737398,
year = {2020},
author = {Tng, PYL and Carabajal Paladino, L and Verkuijl, SAN and Purcell, J and Merits, A and Leftwich, PT and Fragkoudis, R and Noad, R and Alphey, L},
title = {Cas13b-dependent and Cas13b-independent RNA knockdown of viral sequences in mosquito cells following guide RNA expression.},
journal = {Communications biology},
volume = {3},
number = {1},
pages = {413},
doi = {10.1038/s42003-020-01142-6},
pmid = {32737398},
issn = {2399-3642},
support = {110117/Z/15/Z//Wellcome Trust (Wellcome)/ ; 200171/Z/15/7//Wellcome Trust (Wellcome)/ ; BB/M011224/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BBS/E/I/00001985//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BBS/E/I/00007033//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BBS/E/I/00007038//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BBS/E/I/00007039//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
abstract = {Aedes aegypti and Aedes albopictus mosquitoes are vectors of the RNA viruses chikungunya (CHIKV) and dengue that currently have no specific therapeutic treatments. The development of new methods to generate virus-refractory mosquitoes would be beneficial. Cas13b is an enzyme that uses RNA guides to target and cleave RNA molecules and has been reported to suppress RNA viruses in mammalian and plant cells. We investigated the potential use of the Prevotella sp. P5-125 Cas13b system to provide viral refractoriness in mosquito cells, using a virus-derived reporter and a CHIKV split replication system. Cas13b in combination with suitable guide RNAs could induce strong suppression of virus-derived reporter RNAs in insect cells. Surprisingly, the RNA guides alone (without Cas13b) also gave substantial suppression. Our study provides support for the potential use of Cas13b in mosquitoes, but also caution in interpreting CRISPR/Cas data as we show that guide RNAs can have Cas-independent effects.},
}

@article {pmid32735657,
year = {2020},
author = {Makarova, KS and Timinskas, A and Wolf, YI and Gussow, AB and Siksnys, V and Venclovas, Č and Koonin, EV},
title = {Evolutionary and functional classification of the CARF domain superfamily, key sensors in prokaryotic antivirus defense.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaa635},
pmid = {32735657},
issn = {1362-4962},
abstract = {CRISPR-associated Rossmann Fold (CARF) and SMODS-associated and fused to various effector domains (SAVED) are key components of cyclic oligonucleotide-based antiphage signaling systems (CBASS) that sense cyclic oligonucleotides and transmit the signal to an effector inducing cell dormancy or death. Most of the CARFs are components of a CBASS built into type III CRISPR-Cas systems, where the CARF domain binds cyclic oligoA (cOA) synthesized by Cas10 polymerase-cyclase and allosterically activates the effector, typically a promiscuous ribonuclease. Additionally, this signaling pathway includes a ring nuclease, often also a CARF domain (either the sensor itself or a specialized enzyme) that cleaves cOA and mitigates dormancy or death induction. We present a comprehensive census of CARF and SAVED domains in bacteria and archaea, and their sequence- and structure-based classification. There are 10 major families of CARF domains and multiple smaller groups that differ in structural features, association with distinct effectors, and presence or absence of the ring nuclease activity. By comparative genome analysis, we predict specific functions of CARF and SAVED domains and partition the CARF domains into those with both sensor and ring nuclease functions, and sensor-only ones. Several families of ring nucleases functionally associated with sensor-only CARF domains are also predicted.},
}

@article {pmid32734287,
year = {2020},
author = {Khumsupan, P and Kozlowska, MA and Orr, DJ and Andreou, AI and Nakayama, N and Patron, N and Carmo-Silva, E and McCormick, AJ},
title = {Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis.},
journal = {Journal of experimental botany},
volume = {},
number = {},
pages = {},
doi = {10.1093/jxb/eraa316},
pmid = {32734287},
issn = {1460-2431},
abstract = {The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.},
}

@article {pmid32732424,
year = {2020},
author = {Lapinaite, A and Knott, GJ and Palumbo, CM and Lin-Shiao, E and Richter, MF and Zhao, KT and Beal, PA and Liu, DR and Doudna, JA},
title = {DNA capture by a CRISPR-Cas9-guided adenine base editor.},
journal = {Science (New York, N.Y.)},
volume = {369},
number = {6503},
pages = {566-571},
doi = {10.1126/science.abb1390},
pmid = {32732424},
issn = {1095-9203},
abstract = {CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.},
}

@article {pmid32732286,
year = {2020},
author = {Nakamura, K and Noguchi, T and Takahara, M and Omori, Y and Furukawa, T and Katoh, Y and Nakayama, K},
title = {Anterograde trafficking of ciliary MAP kinase-like ICK/CILK1 by the IFT machinery is required for intraciliary retrograde protein trafficking.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA120.014142},
pmid = {32732286},
issn = {1083-351X},
abstract = {ICK (also known as CILK1) is a MAPK-like kinase localized at the ciliary tip. Its deficiency is known to result in the elongation of cilia, and causes ciliopathies in humans. However, little is known about how ICK is transported to the ciliary tip. We here show that the C-terminal noncatalytic region of ICK interacts with the intraflagellar transport (IFT)-B complex of the IFT machinery, and participates in its transport to the ciliary tip. Furthermore, total internal reflection fluorescence microscopy demonstrated that ICK undergoes bidirectional movement within cilia, similarly to IFT particles. Analysis of ICK-knockout cells demonstrated that ICK deficiency severely impairs the retrograde trafficking of IFT particles and ciliary GPCRs. In addition, we found that in ICK-knockout cells, ciliary proteins are accumulated at the bulged ciliary tip, which appeared to be torn off and release into the environment as an extracellular vesicle. The exogenous expression of various ICK constructs in ICK-knockout cells indicated that the IFT-dependent transport of ICK, as well as its kinase activity and phosphorylation at the canonical TDY motif, is essential for ICK function. Thus, we unequivocally show that ICK transported to the ciliary tip is required for retrograde ciliary protein trafficking and consequently for normal ciliary function.},
}

@article {pmid32470504,
year = {2020},
author = {Ebrahimi, V and Hashemi, A},
title = {Challenges of in vitro genome editing with CRISPR/Cas9 and possible solutions: A review.},
journal = {Gene},
volume = {753},
number = {},
pages = {144813},
doi = {10.1016/j.gene.2020.144813},
pmid = {32470504},
issn = {1879-0038},
mesh = {Animals ; Bacteria/genetics ; Bacterial Proteins/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/*methods/*trends ; Genome, Bacterial/genetics ; Humans ; Metabolic Engineering/methods/*trends ; },
abstract = {Microbial production of bio-based ingredients often requires metabolically engineered bacterial strains with the edited genome. Genome editing tools are also essential for gene identification and investigating genotype-phenotype connections. Currently, one of the most common tools of genome editing is based on a natural bacterial adaptive immune system known as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) due to its simple, rapid, and efficient activities. Although successful in some in vitro systems, its application as an approach of metabolic engineering and genome editing is still not so extensive. Here, we discuss existing barriers and challenges of the CRISPR/Cas9 editing tool for in vitro systems. Firstly, we aim to briefly introduce the CRISPR/Cas9 method as an in vitro gene editing tool. Next, we discuss existing obstacles to CRISPR-based editing in bacterial and in vitro model systems and offer guidelines to help achieve editing in an expanded range of in vitro systems.},
}

Animals
Bacteria/genetics
Bacterial Proteins/genetics
CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Editing/*methods/*trends
Genome, Bacterial/genetics
Humans
Metabolic Engineering/methods/*trends

@article {pmid32450202,
year = {2020},
author = {Dheer, P and Rautela, I and Sharma, V and Dhiman, M and Sharma, A and Sharma, N and Sharma, MD},
title = {Evolution in crop improvement approaches and future prospects of molecular markers to CRISPR/Cas9 system.},
journal = {Gene},
volume = {753},
number = {},
pages = {144795},
doi = {10.1016/j.gene.2020.144795},
pmid = {32450202},
issn = {1879-0038},
mesh = {Agriculture/*methods ; Biomarkers ; Biotechnology ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Crops, Agricultural/*genetics ; Disease Resistance/genetics ; Gene Editing/methods ; Gene Targeting/methods ; Genetic Engineering/*methods ; Genome, Plant/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {The advent of genetic selection and genome modification method assure about a real novel reformation in biotechnology and genetic engineering. With the extensive capabilities of molecular markers of them being stable, cost-effective and easy to use, they ultimately become a potent tool for variety of applications such a gene targeting, selection, editing, functional genomics; mainly for the improvisation of commercially important crops. Three main benefits of molecular marker in the field of agriculture and crop improvement programmes first, reduction of the duration of breeding programmes, second, they allow creation of new genetic variation and genetic diversity of plants and third most promising benefit is help in production of engineered plant for disease resistance, or resistance from pathogen and herbicides. This review is anticipated to present an outline how the techniques have been evolved from the simple conventional applications of DNA based molecular markers to highly throughput CRISPR technology and geared the crop yield. Techniques like using Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) systems have revolutionised in the field of genome editing. These have been promptly accepted in both the research and commercial industry. On the whole, the widespread use of molecular markers with their types, their appliance in plant breeding along with the advances in genetic selection and genome editing together being a novel strategy to boost crop yield has been reviewed.},
}

Agriculture/*methods
Biomarkers
Biotechnology
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Crops, Agricultural/*genetics
Disease Resistance/genetics
Gene Editing/methods
Gene Targeting/methods
Genetic Engineering/*methods
Genome, Plant/genetics
Plant Breeding/methods
Plants, Genetically Modified/genetics

@article {pmid32374791,
year = {2020},
author = {Nadipuram, SM and Thind, AC and Rayatpisheh, S and Wohlschlegel, JA and Bradley, PJ},
title = {Proximity biotinylation reveals novel secreted dense granule proteins of Toxoplasma gondii bradyzoites.},
journal = {PloS one},
volume = {15},
number = {5},
pages = {e0232552},
pmid = {32374791},
issn = {1932-6203},
support = {R01 AI123360/AI/NIAID NIH HHS/United States ; T32 AI007323/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Biotinylation ; Brain/metabolism/parasitology ; CRISPR-Cas Systems ; Female ; Gene Knockout Techniques ; Genes, Protozoan ; Humans ; Life Cycle Stages ; Mice ; Mice, Inbred C57BL ; Proteome/metabolism ; Protozoan Proteins/genetics/*metabolism ; Toxoplasma/genetics/growth & development/*physiology ; Toxoplasmosis, Animal/metabolism/parasitology ; Toxoplasmosis, Cerebral/metabolism/parasitology ; Vacuoles/metabolism ; Virulence ; },
abstract = {Toxoplasma gondii is an obligate intracellular parasite which is capable of establishing life-long chronic infection in any mammalian host. During the intracellular life cycle, the parasite secretes an array of proteins into the parasitophorous vacuole (PV) where it resides. Specialized organelles called the dense granules secrete GRA proteins that are known to participate in nutrient acquisition, immune evasion, and host cell-cycle manipulation. Although many GRAs have been discovered which are expressed during the acute infection mediated by tachyzoites, little is known about those that participate in the chronic infection mediated by the bradyzoite form of the parasite. In this study, we sought to uncover novel bradyzoite-upregulated GRA proteins using proximity biotinylation, which we previously used to examine the secreted proteome of the tachyzoites. Using a fusion of the bradyzoite upregulated protein MAG1 to BirA* as bait and a strain with improved switch efficiency, we identified a number of novel GRA proteins which are expressed in bradyzoites. After using the CRISPR/Cas9 system to characterize these proteins by gene knockout, we focused on one of these GRAs (GRA55) and found it was important for the establishment or maintenance of cysts in the mouse brain. These findings highlight new components of the GRA proteome of the tissue-cyst life stage of T. gondii and identify potential targets that are important for maintenance of parasite persistence in vivo.},
}

Animals
Biotinylation
Brain/metabolism/parasitology
CRISPR-Cas Systems
Female
Gene Knockout Techniques
Genes, Protozoan
Humans
Life Cycle Stages
Mice
Mice, Inbred C57BL
Proteome/metabolism
Protozoan Proteins/genetics/*metabolism
Toxoplasma/genetics/growth & development/*physiology
Toxoplasmosis, Animal/metabolism/parasitology
Toxoplasmosis, Cerebral/metabolism/parasitology
Vacuoles/metabolism
Virulence

@article {pmid32245970,
year = {2020},
author = {Yao, L and Shabestary, K and Björk, SM and Asplund-Samuelsson, J and Joensson, HN and Jahn, M and Hudson, EP},
title = {Pooled CRISPRi screening of the cyanobacterium Synechocystis sp PCC 6803 for enhanced industrial phenotypes.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1666},
pmid = {32245970},
issn = {2041-1723},
mesh = {Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Bacterial ; Lactic Acid/metabolism ; Peroxiredoxins/*genetics/metabolism ; Photobioreactors/*microbiology ; Synechocystis/*genetics/metabolism ; },
abstract = {Cyanobacteria are model organisms for photosynthesis and are attractive for biotechnology applications. To aid investigation of genotype-phenotype relationships in cyanobacteria, we develop an inducible CRISPRi gene repression library in Synechocystis sp. PCC 6803, where we aim to target all genes for repression. We track the growth of all library members in multiple conditions and estimate gene fitness. The library reveals several clones with increased growth rates, and these have a common upregulation of genes related to cyclic electron flow. We challenge the library with 0.1 M L-lactate and find that repression of peroxiredoxin bcp2 increases growth rate by 49%. Transforming the library into an L-lactate-secreting Synechocystis strain and sorting top lactate producers enriches clones with sgRNAs targeting nutrient assimilation, central carbon metabolism, and cyclic electron flow. In many examples, productivity can be enhanced by repression of essential genes, which are difficult to access by transposon insertion.},
}

Bacterial Proteins/*genetics/metabolism
CRISPR-Cas Systems/genetics
*Gene Expression Regulation, Bacterial
Lactic Acid/metabolism
Peroxiredoxins/*genetics/metabolism
Photobioreactors/*microbiology
Synechocystis/*genetics/metabolism

@article {pmid31167143,
year = {2019},
author = {Lopez-Martinez, D and Kupculak, M and Yang, D and Yoshikawa, Y and Liang, CC and Wu, R and Gygi, SP and Cohn, MA},
title = {Phosphorylation of FANCD2 Inhibits the FANCD2/FANCI Complex and Suppresses the Fanconi Anemia Pathway in the Absence of DNA Damage.},
journal = {Cell reports},
volume = {27},
number = {10},
pages = {2990-3005.e5},
pmid = {31167143},
issn = {2211-1247},
support = {210640/Z/18/Z/WT_/Wellcome Trust/United Kingdom ; MR/N021002/1/MRC_/Medical Research Council/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; R01 GM067945/GM/NIGMS NIH HHS/United States ; /CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; Casein Kinase II/metabolism ; DNA/metabolism ; DNA Damage ; DNA Repair ; Fanconi Anemia/metabolism/pathology ; Fanconi Anemia Complementation Group D2 Protein/chemistry/genetics/*metabolism ; Fanconi Anemia Complementation Group Proteins/*metabolism ; HeLa Cells ; Humans ; Phosphorylation ; Protein Binding ; Protein Structure, Quaternary ; RNA, Guide/metabolism ; Sequence Alignment ; Ubiquitination ; },
abstract = {Interstrand crosslinks (ICLs) of the DNA helix are a deleterious form of DNA damage. ICLs can be repaired by the Fanconi anemia pathway. At the center of the pathway is the FANCD2/FANCI complex, recruitment of which to DNA is a critical step for repair. After recruitment, monoubiquitination of both FANCD2 and FANCI leads to their retention on chromatin, ensuring subsequent repair. However, regulation of recruitment is poorly understood. Here, we report a cluster of phosphosites on FANCD2 whose phosphorylation by CK2 inhibits both FANCD2 recruitment to ICLs and its monoubiquitination in vitro and in vivo. We have found that phosphorylated FANCD2 possesses reduced DNA binding activity, explaining the previous observations. Thus, we describe a regulatory mechanism operating as a molecular switch, where in the absence of DNA damage, the FANCD2/FANCI complex is prevented from loading onto DNA, effectively suppressing the FA pathway.},
}

Amino Acid Sequence
Animals
CRISPR-Cas Systems/genetics
Casein Kinase II/metabolism
DNA/metabolism
DNA Damage
DNA Repair
Fanconi Anemia/metabolism/pathology
Fanconi Anemia Complementation Group D2 Protein/chemistry/genetics/*metabolism
Fanconi Anemia Complementation Group Proteins/*metabolism
HeLa Cells
Humans
Phosphorylation
Protein Binding
Protein Structure, Quaternary
RNA, Guide/metabolism
Sequence Alignment
Ubiquitination

@article {pmid32731256,
year = {2020},
author = {Kuzmenko, A and Oguienko, A and Esyunina, D and Yudin, D and Petrova, M and Kudinova, A and Maslova, O and Ninova, M and Ryazansky, S and Leach, D and Aravin, AA and Kulbachinskiy, A},
title = {DNA targeting and interference by a bacterial Argonaute nuclease.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-020-2605-1},
pmid = {32731256},
issn = {1476-4687},
abstract = {Members of the conserved Argonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes1,2. Argonautes are also present in many bacterial and archaeal species3-5. Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference6-10. However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here, we studied the in vivo activities of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo). We demonstrated that CbAgo targets multicopy genetic elements and suppresses propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. Loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interplay was reported for acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes encoding pAgo nucleases are enriched in CRISPR-Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest common principles of recognition of foreign nucleic acids by prokaryotic defense systems.},
}

@article {pmid32730741,
year = {2020},
author = {Sofos, N and Feng, M and Stella, S and Pape, T and Fuglsang, A and Lin, J and Huang, Q and Li, Y and She, Q and Montoya, G},
title = {Structures of the Cmr-β Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2020.07.008},
pmid = {32730741},
issn = {1097-4164},
abstract = {Cmr-β is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-β, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-β and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.},
}

@article {pmid32729545,
year = {2020},
author = {Wang, M and Zhang, R and Li, J},
title = {CRISPR/cas systems redefine nucleic acid detection: Principles and methods.},
journal = {Biosensors & bioelectronics},
volume = {165},
number = {},
pages = {112430},
doi = {10.1016/j.bios.2020.112430},
pmid = {32729545},
issn = {1873-4235},
abstract = {Methods that enable rapid, sensitive and specific analyses of nucleic acid sequences have positive effects on precise disease diagnostics and effective clinical treatments by providing direct insight into clinically relevant genetic information. Thus far, many CRISPR/Cas systems have been repurposed for diagnostic functions and are revolutionizing the accessibility of robust diagnostic tools due to their high flexibility, sensitivity and specificity. As RNA-guided targeted recognition effectors, Cas9 variants have been utilized for a variety of diagnostic applications, including biosensing assays, imaging assays and target enrichment for next-generation sequencing (NGS), thereby enabling the development of flexible and cost-effective tests. In addition, the ensuing discovery of Cas proteins (Cas12 and Cas13) with collateral cleavage activities has facilitated the development of numerous diagnostic tools for rapid and portable detection, and these tools have great potential for point-of-care settings. However, representative reviews proposed on this topic are mainly confined to classical biosensing applications; thus, a comprehensive and systematic description of this fast-developing field is required. In this review, based on the detection principle, we provide a detailed classification and comprehensive discussion of recent works that harness these CRISPR-based diagnostic tools from a new perspective. Furthermore, current challenges and future perspectives of CRISPR-based diagnostics are outlined.},
}

@article {pmid32728052,
year = {2020},
author = {Gussow, AB and Park, AE and Borges, AL and Shmakov, SA and Makarova, KS and Wolf, YI and Bondy-Denomy, J and Koonin, EV},
title = {Machine-learning approach expands the repertoire of anti-CRISPR protein families.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {3784},
doi = {10.1038/s41467-020-17652-0},
pmid = {32728052},
issn = {2041-1723},
abstract = {The CRISPR-Cas are adaptive bacterial and archaeal immunity systems that have been harnessed for the development of powerful genome editing and engineering tools. In the incessant host-parasite arms race, viruses evolved multiple anti-defense mechanisms including diverse anti-CRISPR proteins (Acrs) that specifically inhibit CRISPR-Cas and therefore have enormous potential for application as modulators of genome editing tools. Most Acrs are small and highly variable proteins which makes their bioinformatic prediction a formidable task. We present a machine-learning approach for comprehensive Acr prediction. The model shows high predictive power when tested against an unseen test set and was employed to predict 2,500 candidate Acr families. Experimental validation of top candidates revealed two unknown Acrs (AcrIC9, IC10) and three other top candidates were coincidentally identified and found to possess anti-CRISPR activity. These results substantially expand the repertoire of predicted Acrs and provide a resource for experimental Acr discovery.},
}

@article {pmid32727858,
year = {2020},
author = {Repizo, GD and Espariz, M and Seravalle, JL and Díaz Miloslavich, JI and Steimbrüch, BA and Shuman, HA and Viale, AM},
title = {Acinetobacter baumannii NCIMB8209: a Rare Environmental Strain Displaying Extensive Insertion Sequence-Mediated Genome Remodeling Resulting in the Loss of Exposed Cell Structures and Defensive Mechanisms.},
journal = {mSphere},
volume = {5},
number = {4},
pages = {},
doi = {10.1128/mSphere.00404-20},
pmid = {32727858},
issn = {2379-5042},
abstract = {Acinetobacter baumannii represents nowadays an important nosocomial pathogen of poorly defined reservoirs outside the clinical setting. Here, we conducted whole-genome sequencing analysis of the Acinetobacter sp. NCIMB8209 collection strain, isolated in 1943 from the aerobic degradation (retting) of desert guayule shrubs. Strain NCIMB8209 contained a 3.75-Mb chromosome and a plasmid of 134 kb. Phylogenetic analysis based on core genes indicated NCIMB8209 affiliation to A. baumannii, a result supported by the identification of a chromosomal blaOXA-51-like gene. Seven genomic islands lacking antimicrobial resistance determinants, 5 regions encompassing phage-related genes, and notably, 93 insertion sequences (IS) were found in this genome. NCIMB8209 harbors most genes linked to persistence and virulence described in contemporary A. baumannii clinical strains, but many of the genes encoding components of surface structures are interrupted by IS. Moreover, defense genetic islands against biological aggressors such as type 6 secretion systems or CRISPR-cas are absent from this genome. These findings correlate with a low capacity of NCIMB8209 to form biofilm and pellicle, low motility on semisolid medium, and low virulence toward Galleria mellonella and Caenorhabditis elegans Searching for catabolic genes and concomitant metabolic assays revealed the ability of NCIMB8209 to grow on a wide range of substances produced by plants, including aromatic acids and defense compounds against external aggressors. All the above features strongly suggest that NCIMB8209 has evolved specific adaptive features to a particular environmental niche. Moreover, they also revealed that the remarkable genetic plasticity identified in contemporary A. baumannii clinical strains represents an intrinsic characteristic of the species.IMPORTANCEAcinetobacter baumannii is an ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) opportunistic pathogen, with poorly defined natural habitats/reservoirs outside the clinical setting. A. baumannii arose from the Acinetobacter calcoaceticus-A. baumannii complex as the result of a population bottleneck, followed by a recent population expansion from a few clinically relevant clones endowed with an arsenal of resistance and virulence genes. Still, the identification of virulence traits and the evolutionary paths leading to a pathogenic lifestyle has remained elusive, and thus, the study of nonclinical ("environmental") A. baumannii isolates is necessary. We conducted here comparative genomic and virulence studies on A. baumannii NCMBI8209 isolated in 1943 from the microbiota responsible for the decomposition of guayule, and therefore well differentiated both temporally and epidemiologically from the multidrug-resistant strains that are predominant nowadays. Our work provides insights on the adaptive strategies used by A. baumannii to escape from host defenses and may help the adoption of measures aimed to limit its further dissemination.},
}

@article {pmid32401787,
year = {2020},
author = {Picariello, T and Hou, Y and Kubo, T and McNeill, NA and Yanagisawa, HA and Oda, T and Witman, GB},
title = {TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii.},
journal = {PloS one},
volume = {15},
number = {5},
pages = {e0232594},
pmid = {32401787},
issn = {1932-6203},
support = {R35 GM122574/GM/NIGMS NIH HHS/United States ; R37 GM030626/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Chlamydomonas reinhardtii/*genetics ; DNA/genetics ; Gene Editing/*methods ; Mutagenesis, Insertional/*methods ; RNA, Guide/genetics ; },
abstract = {Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA. The donor DNA contains gene-specific homology arms and an integral antibiotic-resistance gene that inserts at the double-stranded break generated by Cas9. After optimizing multiple parameters of this method, we were able to generate mutants for six out of six different genes in two different cell-walled strains with mutation efficiencies ranging from 40% to 95%. Furthermore, these high efficiencies allowed simultaneous targeting of two separate genes in a single experiment. TIM is flexible with regard to many parameters and can be carried out using either electroporation or the glass-bead method for delivery of the RNP and donor DNA. TIM achieves a far higher mutation rate than any previously reported for CRISPR-based methods in C. reinhardtii and promises to be effective for many, if not all, non-essential nuclear genes.},
}

*CRISPR-Cas Systems
Chlamydomonas reinhardtii/*genetics
DNA/genetics
Gene Editing/*methods
Mutagenesis, Insertional/*methods
RNA, Guide/genetics

@article {pmid32328608,
year = {2020},
author = {Zhang, NN and Wang, CN and Ni, X},
title = {[Construction of transgenic mice with specific Cre recombinase expression in the zona fasciculata in adrenal cortex].},
journal = {Sheng li xue bao : [Acta physiologica Sinica]},
volume = {72},
number = {2},
pages = {148-156},
pmid = {32328608},
issn = {0371-0874},
mesh = {Adrenal Cortex/*enzymology ; Animals ; CRISPR-Cas Systems ; Cystathionine gamma-Lyase/genetics ; Integrases/genetics/*metabolism ; Mice ; *Mice, Transgenic ; Zona Fasciculata/*enzymology ; },
abstract = {The adrenal gland is an important endocrine organ of human body. CYP11B1 gene was specifically expressed in the zona fasciculata in adrenal cortex. In order to better study the function of genes specifically expressed in the zona fasciculata in adrenal cortex, the mice with Cre recombinase specifically expressed in the zona fasciculata in adrenal cortex were constructed. It was then confirmed that CYP11B1 was specifically expressed in adrenal glands. Then, using CRISPR/Cas9 technique, CYP11B1-2A-GfpCre recombinant vector was constructed and subsequently injected into the fertilized eggs of mice. It was confirmed that the Cre gene was mainly expressed in the zona fasciculata in adrenal cortex of CYP11B1Cre mice by using mTmG and LacZ staining. The CYP11B1Cre mice were then mated with cystathionine γ-lyase (CTH)f/f mice, thereby generating CTHf/f/CYP11B1Cre mice. It was also confirmed that CTH gene in the zona fasciculata in adrenal cortex was specifically knocked out in these mice. These results suggest that transgenic mice with specific Cre recombinase expression in the zona fasciculata in adrenal cortex were constructed successfully. This animal model can be a powerful tool for the study of the function of genes expressed in the zona fasciculata in adrenal cortex.},
}

Adrenal Cortex/*enzymology
Animals
CRISPR-Cas Systems
Cystathionine gamma-Lyase/genetics
Integrases/genetics/*metabolism
Mice
*Mice, Transgenic
Zona Fasciculata/*enzymology

@article {pmid31633477,
year = {2019},
author = {Liu, W and Yang, C and Liu, Y and Jiang, G},
title = {CRISPR/Cas9 System and its Research Progress in Gene Therapy.},
journal = {Anti-cancer agents in medicinal chemistry},
volume = {19},
number = {16},
pages = {1912-1919},
doi = {10.2174/1871520619666191014103711},
pmid = {31633477},
issn = {1875-5992},
mesh = {Animals ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Mutation ; Skin Diseases/genetics/*therapy ; },
abstract = {Genome editing refers to changing the genome sequence of an organism by knockout, insertion, and site mutation, resulting in changes in the genetic information of the organism. The clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein-9 nuclease (Cas9) system is a genome editing technique developed by the acquired immune system in the microbes, such as bacteria and archaebacteria, which targets and edits genome sequences according to the principle of complementary base pairing. This technique can be used to edit endogenous genomic DNA sequences in organisms accurately and has been widely used in fields, such as biotechnology, cancer gene therapy, and dermatology. In this review, we summarize the history, structure, mechanism, and application of CRISPR/Cas9 in gene therapy and dermatological diseases.},
}

Animals
CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/*methods
Genetic Therapy/*methods
Humans
Mutation
Skin Diseases/genetics/*therapy

@article {pmid32724054,
year = {2020},
author = {Jin, YH and Liao, B and Migaud, H and Davie, A},
title = {Physiological impact and comparison of mutant screening methods in piwil2 KO founder Nile tilapia produced by CRISPR/Cas9 system.},
journal = {Scientific reports},
volume = {10},
number = {1},
pages = {12600},
doi = {10.1038/s41598-020-69421-0},
pmid = {32724054},
issn = {2045-2322},
abstract = {The application of genome engineering techniques to understand the mechanisms that regulate germ cell development opens promising new avenues to develop methods to control sexual maturation and mitigate associated detrimental effects in fish. In this study, the functional role of piwil2 in primordial germ cells (PGCs) was investigated in Nile tilapia using CRISPR/Cas9 and the resultant genotypes were further explored. piwil2 is a gonad-specific and maternally deposited gene in Nile tilapia eggs which is known to play a role in repression of transposon elements and is therefore thought to be important for maintaining germline cell fate. A functional domain of piwil2, PIWI domain, was targeted by injecting Cas9 mRNA and sgRNAs into Nile tilapia embryos at 1 cell stage. Results showed 54% of injected mutant larvae had no or less putative PGCs compared to control fish, suggesting an essential role of piwil2 in survival of PGCs. The genotypic features of the different phenotypic groups were explored by next generation sequencing (NGS) and other mutant screening methods including T7 endonuclease 1 (T7E1), CRISPR/Cas-derived RNA-guided engineered nuclease (RGEN), high resolution melt curve analysis (HRMA) and fragment analysis. Linking phenotypes to genotypes in F0 was hindered by the complex mosacism and wide indel spectrum revealed by NGS and fragment analysis. This study strongly suggests the functional importance of piwil2 in PGCs survival. Further studies should focus on reducing mosaicism when using CRISPR/Cas9 system to facilitate direct functional analysis in F0.},
}

@article {pmid32723866,
year = {2020},
author = {Stachler, AE and Wörtz, J and Alkhnbashi, OS and Turgeman-Grott, I and Smith, R and Allers, T and Backofen, R and Gophna, U and Marchfelder, A},
title = {Adaptation induced by self-targeting in a type I-B CRISPR-Cas system.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA120.014030},
pmid = {32723866},
issn = {1083-351X},
abstract = {Haloferax volcanii is to our knowledge the only prokaryote known to tolerate CRISPR-Cas mediated damage to its genome in the wild type background; the resulting cleavage of the genome is repaired by homologous recombination restoring the wild type version. In mutant Haloferax strains with enhanced self-targeting, cell fitness decreases and microhomology-mediated end joining becomes active, generating deletions in the targeted gene. Here we use self-targeting to investigate adaptation in H. volcanii CRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalysed by active transposases, can generate DNA fragments that are used by the CRISPR-Cas adaptation machinery for integration into the CRISPR loci. Low cellular concentrations of self-targeting crRNAs resulted in acquisition of large numbers of spacers originating from the entire genomic DNA. In contrast, high concentrations of self-targeting crRNAs resulted in lower acquisition that was mostly centred around the targeting site. Furthermore, we observed naïve spacer acquisition at a low level in wild type Haloferax cells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2 and Cas4. Taken together these findings indicate that naïve adaptation is a regulated process in H. volcanii that operates at low basal levels and is induced by DNA breaks.},
}

@article {pmid32723863,
year = {2020},
author = {Van Eck, J},
title = {Applying gene editing to tailor precise genetic modifications in plants.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.REV120.010850},
pmid = {32723863},
issn = {1083-351X},
abstract = {The ability to tailor alterations in genomes, including plant genomes, in a site-specific manner has been greatly advanced through approaches that reduced the complexity and time of genome sequencing along with development of gene editing technologies. These technologies provide a valuable foundation for studies of gene function, metabolic engineering and trait modification for crop improvement. Development of genome editing methodologies began approximately 20 years ago first with meganucleases followed by Zinc Finger Nucleases, Transcriptional Activator-Like Effector Nucleases and, most recently, Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR)-Associated Protein (Cas) (CRISPR/Cas), which is by far the most utilized method. The premise of CRISPR/Cas centers on the cleaving of one or both DNA strands by a Cas protein, an endonuclease, followed by mending of the DNA by repair mechanisms inherent in cells. Its user-friendly construct design, greater flexibility in targeting genomic regions, and the cost-effective attributes have resulted in it being widely adopted and revolutionizing precise modification of the genomes of many organisms. Indeed, the CRISPR/Cas system has been utilized for gene editing in many plant species including important food crops such as maize, wheat, rice, and potatoes. This review summarizes the various approaches including the most recent designs being used to make modifications from as small as a single base pair change to insertion of DNA fragments. On the gene expression level, strategies are presented that make it possible to knock-out or modulate through activation and repression. Also discussed are pre-requisites necessary for CRISPR/Cas-mediated editing as well as the current challenges.},
}

@article {pmid31421397,
year = {2019},
author = {Irion, U and Nüsslein-Volhard, C},
title = {The identification of genes involved in the evolution of color patterns in fish.},
journal = {Current opinion in genetics & development},
volume = {57},
number = {},
pages = {31-38},
pmid = {31421397},
issn = {1879-0380},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cichlids/*genetics/physiology ; *Evolution, Molecular ; Gene Expression Regulation, Developmental/genetics ; Pigmentation/*genetics/physiology ; Transcriptome/*genetics ; Zebrafish/genetics/physiology ; },
abstract = {The genetic basis of morphological variation, both within and between species, provides a major topic in evolutionary biology. Teleost fish produce most elaborate color patterns, and among the more than 20000 species a number have been chosen for more detailed analyses because they are suitable to study particular aspects of color pattern evolution. In several fish species, color variants and pattern variants have been collected, transcriptome analyses have been carried out, and the recent advent of gene editing tools, such as CRISPR/Cas9, has allowed the production of mutants. Covering mostly the literature from the last three years, we discuss the cellular basis of coloration and the identification of loci involved in color pattern differences between sister species in cichlids and Danio species, in which cis-regulatory changes seem to prevail.},
}

Animals
CRISPR-Cas Systems/genetics
Cichlids/*genetics/physiology
*Evolution, Molecular
Gene Expression Regulation, Developmental/genetics
Pigmentation/*genetics/physiology
Transcriptome/*genetics
Zebrafish/genetics/physiology

@article {pmid31087560,
year = {2019},
author = {Li, SW and Yu, B and Byrne, G and Wright, M and O'Rourke, S and Mesa, K and Berman, PW},
title = {Identification and CRISPR/Cas9 Inactivation of the C1s Protease Responsible for Proteolysis of Recombinant Proteins Produced in CHO Cells.},
journal = {Biotechnology and bioengineering},
volume = {116},
number = {9},
pages = {2130-2145},
pmid = {31087560},
issn = {1097-0290},
support = {R01 DA036335/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; CHO Cells ; *CRISPR-Cas Systems ; Complement C1s/*genetics/*metabolism ; Cricetulus ; *Gene Knockout Techniques ; HIV Envelope Protein gp120/*biosynthesis/genetics ; *HIV-1 ; *Proteolysis ; Recombinant Proteins/biosynthesis/genetics ; },
abstract = {Proteolysis associated with recombinant protein expression in Chinese Hamster Ovary (CHO) cells has hindered the development of biologics including HIV vaccines. When expressed in CHO cells, the recombinant HIV envelope protein, gp120, undergoes proteolytic clipping by a serine protease at a key epitope recognized by neutralizing antibodies. The problem is particularly acute for envelope proteins from clade B viruses that represent the major genetic subtype circulating in much of the developed world, including the US and Europe. In this paper, we have identified complement Component 1's (C1s), a serine protease from the complement cascade, as the protease responsible for the proteolysis of gp120 in CHO cells. CRISPR/Cas9 knockout of the C1s protease in a CHO cell line was shown to eliminate the proteolytic activity against the recombinantly expressed gp120. In addition, the C1s-/- MGAT1- CHO cell line, with the C1s protease and the MGAT1 glycosyltransferase knocked out, enabled the production of unclipped gp120 from a clade B isolate (BaL-rgp120) and enriched for mannose-5 glycans on gp120 that are required for the binding of multiple broadly neutralizing monoclonal antibodies (bN-mAbs). The availability of this technology will allow for the scale-up and testing of multiple vaccine concepts in regions of the world where clade B viruses are in circulation. Furthermore, the proteolysis issues caused by the C1s protease suggests a broader need for a C1s-deficient CHO cell line to express other recombinant proteins that are susceptible to serine protease activity in CHO cells. Similarly, the workflow described here to identify and knockout C1s in a CHO cell line can be applied to remedy the proteolysis of biologics by other CHO proteases.},
}

Animals
CHO Cells
*CRISPR-Cas Systems
Complement C1s/*genetics/*metabolism
Cricetulus
*Gene Knockout Techniques
HIV Envelope Protein gp120/*biosynthesis/genetics
*HIV-1
*Proteolysis
Recombinant Proteins/biosynthesis/genetics

@article {pmid30561900,
year = {2019},
author = {Luhur, A and Klueg, KM and Zelhof, AC},
title = {Generating and working with Drosophila cell cultures: Current challenges and opportunities.},
journal = {Wiley interdisciplinary reviews. Developmental biology},
volume = {8},
number = {3},
pages = {e339},
pmid = {30561900},
issn = {1759-7692},
support = {P40 OD010949/OD/NIH HHS/United States ; P40OD010949/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Cell Culture Techniques/*methods ; Drosophila/*cytology/*genetics/metabolism ; Drosophila Proteins/*genetics/metabolism ; *Gene Editing ; *Gene Expression Regulation ; Phenotype ; },
abstract = {The use of Drosophila cell cultures has positively impacted both fundamental and biomedical research. The most widely used cell lines: Schneider, Kc, the CNS and imaginal disc lines continue to be the choice for many applications. Drosophila cell lines provide a homogenous source of cells suitable for biochemical experimentations, transcriptomics, functional genomics, and biomedical applications. They are amenable to RNA interference and serve as a platform for high-throughput screens to identify relevant candidate genes or drugs for any biological process. Currently, CRISPR-based functional genomics are also being developed for Drosophila cell lines. Even though many uniquely derived cell lines exist, cell genetic techniques such the transgenic UAS-GAL4-based RasV12 oncogene expression, CRISPR-Cas9 editing and recombination mediated cassette exchange are likely to drive the establishment of many more lines from specific tissues, cells, or genotypes. However, the pace of creating new lines is hindered by several factors inherent to working with Drosophila cell cultures: single cell cloning, optimal media formulations and culture conditions capable of supporting lines from novel tissue sources or genotypes. Moreover, even though many Drosophila cell lines are morphologically and transcriptionally distinct it may be necessary to implement a standard for Drosophila cell line authentication, ensuring the identity and purity of each cell line. Altogether, recent advances and a standardized authentication effort should improve the utility of Drosophila cell cultures as a relevant model for fundamental and biomedical research. This article is categorized under: Technologies > Analysis of Cell, Tissue, and Animal Phenotypes.},
}

Animals
CRISPR-Cas Systems
Cell Culture Techniques/*methods
Drosophila/*cytology/*genetics/metabolism
Drosophila Proteins/*genetics/metabolism
*Gene Editing
*Gene Expression Regulation
Phenotype

@article {pmid32718240,
year = {2020},
author = {Rocha, LFM and Braga, LAM and Mota, FB},
title = {Gene editing for treatment and prevention of human diseases: a global survey of gene editing-related researchers.},
journal = {Human gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1089/hum.2020.136},
pmid = {32718240},
issn = {1557-7422},
abstract = {In the next decades, gene editing technologies are expected to be used in the treatment and prevention of human diseases. Yet, the future uses of gene editing in medicine are still unknown, including its applicability and effectiveness to the treatment and prevention of infectious diseases, cancer, monogenic, and polygenic hereditary diseases. This study aims to address this gap by analyzing the views of over one thousand gene editing-related researchers from all over the world. Some of our survey results show that, in the next ten years, DNA double-strand breaks are expected to be the main method for gene editing and CRISPR-Cas Systems to be the mainstream programmable nuclease. In the same period, gene editing is expected to have more applicability and effectiveness to treat and prevent infectious diseases and cancer. Off targeting mutations, reaching therapeutic levels of editing efficiency, difficulties in targeting specific tissues in vivo, regulatory, and ethical challenges are among the most relevant factors that might hamper the use of gene editing in humans. In conclusion, our results suggest that gene editing might become a reality to the treatment and prevention of a variety of human diseases in the coming ten years. If the future confirms these researchers' expectations, gene editing could change the way medicine, health systems, and public health deal with the treatment and prevention of human diseases.},
}

@article {pmid32004439,
year = {2020},
author = {Wertz, MH and Mitchem, MR and Pineda, SS and Hachigian, LJ and Lee, H and Lau, V and Powers, A and Kulicke, R and Madan, GK and Colic, M and Therrien, M and Vernon, A and Beja-Glasser, VF and Hegde, M and Gao, F and Kellis, M and Hart, T and Doench, JG and Heiman, M},
title = {Genome-wide In Vivo CNS Screening Identifies Genes that Modify CNS Neuronal Survival and mHTT Toxicity.},
journal = {Neuron},
volume = {106},
number = {1},
pages = {76-89.e8},
doi = {10.1016/j.neuron.2020.01.004},
pmid = {32004439},
issn = {1097-4199},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R01 NS085880/NS/NINDS NIH HHS/United States ; R35 GM130119/GM/NIGMS NIH HHS/United States ; T32 EB019940/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; Behavior, Animal ; CRISPR-Cas Systems ; Cell Survival/*genetics ; Gene Knockdown Techniques ; Gene Library ; Genes, Essential/genetics ; Huntingtin Protein/*genetics ; Huntington Disease/*genetics ; Mice ; Mice, Transgenic ; NM23 Nucleoside Diphosphate Kinases/genetics ; Neostriatum/*metabolism ; Neurons/*metabolism ; Nucleoside Diphosphate Kinase D/genetics ; Protein Aggregates ; RNA Interference ; RNA, Guide ; RNA, Small Interfering ; Receptors, Dopamine D2/genetics ; Sequence Analysis, RNA ; },
abstract = {Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms, but such screening has not been possible to perform in the mammalian central nervous system (CNS). Here, we report the results of the first genome-wide genetic screens in the CNS using both short hairpin RNA (shRNA) and CRISPR libraries. Our screens identify many classes of CNS neuronal essential genes and demonstrate that CNS neurons are particularly sensitive not only to perturbations to synaptic processes but also autophagy, proteostasis, mRNA processing, and mitochondrial function. These results reveal a molecular logic for the common implication of these pathways across multiple neurodegenerative diseases. To further identify disease-relevant genetic modifiers, we applied our screening approach to two mouse models of Huntington's disease (HD). Top mutant huntingtin toxicity modifier genes included several Nme genes and several genes involved in methylation-dependent chromatin silencing and dopamine signaling, results that reveal new HD therapeutic target pathways.},
}

Animals
Behavior, Animal
CRISPR-Cas Systems
Cell Survival/*genetics
Gene Knockdown Techniques
Gene Library
Genes, Essential/genetics
Huntingtin Protein/*genetics
Huntington Disease/*genetics
Mice
Mice, Transgenic
NM23 Nucleoside Diphosphate Kinases/genetics
Neostriatum/*metabolism
Neurons/*metabolism
Nucleoside Diphosphate Kinase D/genetics
Protein Aggregates
RNA Interference
RNA, Guide
RNA, Small Interfering
Receptors, Dopamine D2/genetics
Sequence Analysis, RNA

@article {pmid31280681,
year = {2020},
author = {Hameed, A and Mehmood, MA and Shahid, M and Fatma, S and Khan, A and Ali, S},
title = {Prospects for potato genome editing to engineer resistance against viruses and cold-induced sweetening.},
journal = {GM crops & food},
volume = {11},
number = {4},
pages = {185-205},
doi = {10.1080/21645698.2019.1631115},
pmid = {31280681},
issn = {2164-5701},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Genetic Engineering ; *Plant Viruses ; Solanum tuberosum/*genetics ; },
abstract = {Crop improvement through transgenic technologies is commonly tagged with GMO (genetically-modified-organisms) where the presence of transgene becomes a big question for the society and the legislation authorities. However, new plant breeding techniques like CRISPR/Cas9 system [clustered regularly interspaced palindromic repeats (CRISPR)-associated 9] can overcome these limitations through transgene-free products. Potato (Solanum tuberosum L.) being a major food crop has the potential to feed the rising world population. Unfortunately, the cultivated potato suffers considerable production losses due to several pre- and post-harvest stresses such as plant viruses (majorly RNA viruses) and cold-induced sweetening (CIS; the conversion of sucrose to glucose and fructose inside cell vacuole). A number of strategies, ranging from crop breeding to genetic engineering, have been employed so far in potato for trait improvement. Recently, new breeding techniques have been utilized to knock-out potato genes/factors like eukaryotic translation initiation factors [elF4E and isoform elF(iso)4E)], that interact with viruses to assist viral infection, and vacuolar invertase, a core enzyme in CIS. In this context, CRISPR technology is predicted to reduce the cost of potato production and is likely to pass through the regulatory process being marker and transgene-free. The current review summarizes the potential application of the CRISPR/Cas9 system for traits improvement in potato. Moreover, the prospects for engineering resistance against potato fungal pathogens and current limitations/challenges are discussed.},
}

CRISPR-Cas Systems
Gene Editing
Genetic Engineering
*Plant Viruses
Solanum tuberosum/*genetics

@article {pmid30679547,
year = {2019},
author = {Shimada, R and Kiso, M and Saga, Y},
title = {ES-mediated chimera analysis revealed requirement of DDX6 for NANOS2 localization and function in mouse germ cells.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {515},
pmid = {30679547},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Chimera/genetics/metabolism ; DEAD-box RNA Helicases/analysis/genetics/*metabolism ; Embryonic Stem Cells/cytology/metabolism ; Female ; Gene Deletion ; Gene Expression Regulation, Developmental ; Germ Cells/cytology/*metabolism ; Male ; Mice ; Proto-Oncogene Proteins/analysis/genetics/*metabolism ; RNA-Binding Proteins/analysis/genetics/*metabolism ; Spermatogenesis ; Transcriptome ; },
abstract = {In embryonic male germ cells, the RNA-binding protein NANOS2 recruits its target RNAs to processing bodies (P-bodies), where they are repressed. This process is necessary to promote male-type germ cell differentiation. However, it remains unclear whether all NANOS2 functions depend on P-bodies. To address this question, we established ES cell lines containing a germ cell-specific inducible Cre and reporter together with the floxed Ddx6 allele. We deleted the Ddx6 gene by administering tamoxifen to chimeric embryos containing germ cells derived from recombinant ES cells. DDX6-null germ cells exhibited both similar and distinct defects from those observed in NANOS2-null germ cells. These results demonstrate that NANOS2 function is carried out via both P-body-dependent and -independent mechanisms. RNA-seq analyses further supported the phenotypic differences between DDX6-null and NANOS2-null germ cells, and indicated distinct molecular cascades involved in NANOS2-mediated gene regulation.},
}

Animals
CRISPR-Cas Systems
Cell Line
Chimera/genetics/metabolism
DEAD-box RNA Helicases/analysis/genetics/*metabolism
Embryonic Stem Cells/cytology/metabolism
Female
Gene Deletion
Gene Expression Regulation, Developmental
Germ Cells/cytology/*metabolism
Male
Mice
Proto-Oncogene Proteins/analysis/genetics/*metabolism
RNA-Binding Proteins/analysis/genetics/*metabolism
Spermatogenesis
Transcriptome

@article {pmid30439390,
year = {2019},
author = {Yamaguchi, H and de Lecea, L},
title = {In vivo cell type-specific CRISPR gene editing for sleep research.},
journal = {Journal of neuroscience methods},
volume = {316},
number = {},
pages = {99-102},
pmid = {30439390},
issn = {1872-678X},
support = {R01 AG047671/AG/NIA NIH HHS/United States ; R01 MH087592/MH/NIMH NIH HHS/United States ; R01 MH102638/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; Brain/metabolism/*physiology ; *CRISPR-Cas Systems ; *Gene Editing ; Sleep/*genetics ; },
abstract = {Sleep is an innate behavior conserved in all animals and, in vertebrates, is regulated by neuronal circuits in the brain. The conventional techniques of forward and reverse genetics have enabled researchers to investigate the molecular mechanisms that regulate sleep and arousal. However, functional interrogation of genes in specific cell subtypes in the brain remains a challenge. Here, we review the background of newly developed gene-editing technologies using engineered CRISPR/Cas9 system and describe the application to interrogate gene functions within genetically-defined brain cell populations in sleep research.},
}

Animals
Brain/metabolism/*physiology
*CRISPR-Cas Systems
*Gene Editing
Sleep/*genetics

@article {pmid32713893,
year = {2020},
author = {Wake, Y and Kaneko, T},
title = {Production of genome-edited mice by visualization of nucleases introduced into the embryos using electroporation.},
journal = {The Journal of reproduction and development},
volume = {},
number = {},
pages = {},
doi = {10.1262/jrd.2020-068},
pmid = {32713893},
issn = {1348-4400},
abstract = {Genome editing technology contributes to the quick and highly efficient production of genetically engineered animals. These animals are helpful in clarifying the mechanism of human disease. Recently, a new electroporation technique (TAKE: Technique for animal knockout system by electroporation) was developed to produce genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. The aim of this study was to increase the efficiency of production of genome-edited animals using the TAKE method. In the conventional protocol, it was difficult to confirm the introduction of nucleases into embryos and energization during operation. Using only embryos that introduced nucleases for embryo transfer, it will lead to increased efficiency in the production of genome-edited animals. This study examined the visualization in the introduction of nucleases into the embryos by using nucleases fluorescent labeled with ATTO-550. The embryos were transfected with Cas9 protein and fluorescent labeled dual guide RNA (mixture with crRNA and tracrRNA with ATTO-550) targeted tyrosinase gene by the TAKE method. All embryos that survived after electroporation showed fluorescence. Of these embryos with fluorescence, 43.7% developed to morphologically normal offspring. In addition, 91.7% of offspring were edited by the tyrosinase gene. This study is the first to demonstrate that the introduction of nucleases into embryos by the TAKE method could be visualized using fluorescent-labeled nucleases. This improved TAKE method can be used to produce genome-edited animals and confirm energization during operation.},
}

@article {pmid32712922,
year = {2021},
author = {Berkhout, B and Gao, Z and Herrera-Carrillo, E},
title = {Design and Evaluation of Guide RNA Transcripts with a 3'-Terminal HDV Ribozyme to Enhance CRISPR-Based Gene Inactivation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2167},
number = {},
pages = {205-224},
doi = {10.1007/978-1-0716-0716-9_12},
pmid = {32712922},
issn = {1940-6029},
abstract = {The recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)-Cpf1 system, now reclassified as Cas12a, is a DNA-editing platform analogous to the widely used CRISPR-Cas9 system. The Cas12a system exhibits several distinct features over the CRISPR-Cas9 system, such as increased specificity and a smaller gene size to encode the nuclease and the matching CRISPR guide RNA (crRNA), which could mitigate off-target and delivery problems, respectively, described for the Cas9 system. However, the Cas12a system exhibits reduced gene editing efficiency compared to Cas9. A closer inspection of the crRNA sequence raised some uncertainty about the actual 5' and 3'-ends. RNA Polymerase (Pol) III promoters are generally used for the production of small RNAs with a precise 5' terminus, but the Pol III enzyme generates small RNAs with 3' U-tails of variable length. To optimize the CRISPR-Cas12a system, we describe the inclusion of a self-cleaving ribozyme in the vector design to facilitate accurate 3'-end processing of the crRNA transcript to produce precise molecules. This optimized design enhanced not only the gene editing efficiency, but also the activity of the catalytically inactive Cas12a-based CRISPR gene activation platform. We thus generated an improved CRISPR-Cas12a system for more efficient gene editing and gene regulation purposes.},
}

@article {pmid32712502,
year = {2020},
author = {Molina, R and Sofos, N and Montoya, G},
title = {Structural basis of CRISPR-Cas Type III prokaryotic defence systems.},
journal = {Current opinion in structural biology},
volume = {65},
number = {},
pages = {119-129},
doi = {10.1016/j.sbi.2020.06.010},
pmid = {32712502},
issn = {1879-033X},
abstract = {CRISPR loci and CRISPR-associated (Cas) genes encode an adaptive immune system that protects many bacterial and almost all archaea against invasive genetic elements from bacteriophages and plasmids. Several classes of CRISPR systems have been characterized, of which the type III CRISPR systems exhibit the most unique functions. Members of type III cleave both RNA and DNA not only through their corresponding effector complexes but also by CRISPR-Cas associated proteins activated by second messengers produced by those effector complexes. Furthermore, the recent discovery of second messenger degrading proteins called ring nucleases adds an extra regulatory layer to fine-tune these immunity systems. Here, we review the defense mechanisms that govern type III CRISPR interference immunity systems focusing on the structural information available.},
}

@article {pmid32707148,
year = {2020},
author = {Huang, D and Miller, M and Ashok, B and Jain, S and Peppas, NA},
title = {CRISPR/Cas systems to overcome challenges in developing the next generation of T cells for cancer therapy.},
journal = {Advanced drug delivery reviews},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.addr.2020.07.015},
pmid = {32707148},
issn = {1872-8294},
abstract = {Genetically engineered immune cells with chimeric antigen receptors (CAR) or modified T cell receptors (TCR) have demonstrated their potential as a potent class of new cancer therapeutic strategy. Despite the clinical success of autologous CD19 CAR T cells in hematological malignancies, allogeneic T cells exhibit many advantages over their autologous counterparts and have recently gathered widespread attention due to the emergence of multiplex genome editing techniques, particularly CRISPR/Cas systems. Furthermore, genetically engineered T cells face a host of major challenges in solid tumors that are not as significant for blood cancers such as T cell targeted delivery, target specificity, proliferation, persistence, and the immunosuppressive tumor microenvironment. We take this opportunity to analyze recent strategies to develop allogeneic T cells, specifically in consideration of CRISPR/Cas and its delivery systems for multiplex gene editing. Additionally, we discuss the current methods used to delivery CRISPR/Cas systems for immunotherapeutic applications, and the challenges to continued development of novel delivery systems. We also provide a comprehensive analysis of the major challenges that genetically engineered T cells face in solid tumors along with the most recent strategies to overcome these barriers, with an emphasis on CRISPR-based approaches. We illustrate the synergistic prospects for how the combination of synthetic biology and immune-oncology could pave the way for designing the next generation of precision cancer therapy.},
}

@article {pmid32703438,
year = {2020},
author = {Shirai, Y and Daimon, T},
title = {Mutations in cardinal are responsible for the red-1 and peach eye color mutants of the red flour beetle Tribolium castaneum.},
journal = {Biochemical and biophysical research communications},
volume = {529},
number = {2},
pages = {372-378},
doi = {10.1016/j.bbrc.2020.05.214},
pmid = {32703438},
issn = {1090-2104},
abstract = {Ommochromes are the major pigments found in the eyes, eggs, wings and epidermis of insects. Here, we report the identification and characterization of the gene responsible for red-1 locus of Tribolium, whose mutants have white eyes due to lack of ommochrome pigments in the eyes. Using a candidate gene approach, we demonstrated that red-1 and peach mutants have molecular defects in the cardinal gene, which encodes a haem peroxidase that is considered to convert 3-hydroxykynurenine into ommochromes in pigment granules. Our experiments showed that the expression pattern of cardinal correlates well with the progression of eye pigmentation during pupal stages. We performed gene editing experiments using the Receptor-Mediated Ovary Transduction of Cargo (ReMOT) Control technique to disrupt the cardinal gene by adult injection, and were able to establish a novel cardinal mutant line. Our complementation test provided definitive genetic evidence that cardinal is located at the red-1 locus. The present study will lead to a greater understanding of the function and diversity of ommochrome pathway genes in insects. Our successful use of ReMOT Control in beetles will facilitate the development of more efficient and versatile systems for insect genome editing by simple adult injection.},
}

@article {pmid32275651,
year = {2020},
author = {Willems, J and de Jong, APH and Scheefhals, N and Mertens, E and Catsburg, LAE and Poorthuis, RB and de Winter, F and Verhaagen, J and Meye, FJ and MacGillavry, HD},
title = {ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons.},
journal = {PLoS biology},
volume = {18},
number = {4},
pages = {e3000665},
pmid = {32275651},
issn = {1545-7885},
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; Dependovirus/genetics ; Epitopes/*genetics ; Female ; Gene Editing/*methods ; Gene Knock-In Techniques ; Genes, Reporter ; Genetic Vectors ; Genome ; Green Fluorescent Proteins/genetics/metabolism ; Male ; Mice, Transgenic ; Microscopy, Fluorescence ; Molecular Imaging/methods ; Neurons/*immunology/physiology ; Organ Culture Techniques ; Proteins/*genetics/immunology/metabolism ; Rats, Wistar ; Receptors, N-Methyl-D-Aspartate/genetics/metabolism ; Spatio-Temporal Analysis ; },
abstract = {The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.},
}

Animals
*CRISPR-Cas Systems
Cells, Cultured
Dependovirus/genetics
Epitopes/*genetics
Female
Gene Editing/*methods
Gene Knock-In Techniques
Genes, Reporter
Genetic Vectors
Genome
Green Fluorescent Proteins/genetics/metabolism
Male
Mice, Transgenic
Microscopy, Fluorescence
Molecular Imaging/methods
Neurons/*immunology/physiology
Organ Culture Techniques
Proteins/*genetics/immunology/metabolism
Rats, Wistar
Receptors, N-Methyl-D-Aspartate/genetics/metabolism
Spatio-Temporal Analysis

@article {pmid32273496,
year = {2020},
author = {Jiang, Y and Wei, J and Cui, H and Liu, C and Zhi, Y and Jiang, Z and Li, Z and Li, S and Yang, Z and Wang, X and Qian, P and Zhang, C and Zhong, C and Su, XZ and Yuan, J},
title = {An intracellular membrane protein GEP1 regulates xanthurenic acid induced gametogenesis of malaria parasites.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1764},
pmid = {32273496},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Calcium/metabolism ; Culicidae/*metabolism/parasitology ; Cyclic GMP/metabolism ; Cyclic GMP-Dependent Protein Kinases/metabolism ; Gametogenesis/*drug effects ; Gene Editing/methods ; Intracellular Membranes/*metabolism ; Malaria/parasitology ; Mosquito Vectors/metabolism/parasitology ; Plasmodium/genetics/metabolism/physiology ; Protozoan Proteins/genetics/*metabolism ; Xanthurenates/metabolism/*pharmacology ; },
abstract = {Gametocytes differentiation to gametes (gametogenesis) within mosquitos is essential for malaria parasite transmission. Both reduction in temperature and mosquito-derived XA or elevated pH are required for triggering cGMP/PKG dependent gametogenesis. However, the parasite molecule for sensing or transducing these environmental signals to initiate gametogenesis remains unknown. Here we perform a CRISPR/Cas9-based functional screening of 59 membrane proteins expressed in the gametocytes of Plasmodium yoelii and identify that GEP1 is required for XA-stimulated gametogenesis. GEP1 disruption abolishes XA-stimulated cGMP synthesis and the subsequent signaling and cellular events, such as Ca2+ mobilization, gamete formation, and gametes egress out of erythrocytes. GEP1 interacts with GCα, a cGMP synthesizing enzyme in gametocytes. Both GEP1 and GCα are expressed in cytoplasmic puncta of both male and female gametocytes. Depletion of GCα impairs XA-stimulated gametogenesis, mimicking the defect of GEP1 disruption. The identification of GEP1 being essential for gametogenesis provides a potential new target for intervention of parasite transmission.},
}

Animals
CRISPR-Cas Systems/genetics
Calcium/metabolism
Culicidae/*metabolism/parasitology
Cyclic GMP/metabolism
Cyclic GMP-Dependent Protein Kinases/metabolism
Gametogenesis/*drug effects
Gene Editing/methods
Intracellular Membranes/*metabolism
Malaria/parasitology
Mosquito Vectors/metabolism/parasitology
Plasmodium/genetics/metabolism/physiology
Protozoan Proteins/genetics/*metabolism
Xanthurenates/metabolism/*pharmacology

@article {pmid32188851,
year = {2020},
author = {Schmidt, H and Collier, TC and Hanemaaijer, MJ and Houston, PD and Lee, Y and Lanzaro, GC},
title = {Abundance of conserved CRISPR-Cas9 target sites within the highly polymorphic genomes of Anopheles and Aedes mosquitoes.},
journal = {Nature communications},
volume = {11},
number = {1},
pages = {1425},
pmid = {32188851},
issn = {2041-1723},
support = {U01 CK000516/CK/NCEZID CDC HHS/United States ; R56 AI130277/AI/NIAID NIH HHS/United States ; },
mesh = {Aedes/*genetics ; Alleles ; Animals ; Anopheles/*genetics ; CRISPR-Cas Systems ; Female ; Gene Frequency ; *Genome, Insect ; Insect Proteins/genetics ; Mosquito Vectors/genetics ; },
abstract = {A number of recent papers report that standing genetic variation in natural populations includes ubiquitous polymorphisms within target sites for Cas9-based gene drive (CGD) and that these "drive resistant alleles" (DRA) preclude the successful application of CGD for managing these populations. Here we report the results of a survey of 1280 genomes of the mosquitoes Anopheles gambiae, An. coluzzii, and Aedes aegypti in which we determine that ~90% of all protein-encoding CGD target genes in natural populations include at least one target site with no DRAs at a frequency of ≥1.0%. We conclude that the abundance of conserved target sites in mosquito genomes and the inherent flexibility in CGD design obviates the concern that DRAs present in the standing genetic variation of mosquito populations will be detrimental to the deployment of this technology for population modification strategies.},
}

Aedes/*genetics
Alleles
Animals
Anopheles/*genetics
CRISPR-Cas Systems
Female
Gene Frequency
*Genome, Insect
Insect Proteins/genetics
Mosquito Vectors/genetics

@article {pmid32187363,
year = {2020},
author = {Chen, HM and Marques, JG and Sugino, K and Wei, D and Miyares, RL and Lee, T},
title = {CAMIO: a transgenic CRISPR pipeline to create diverse targeted genome deletions in Drosophila.},
journal = {Nucleic acids research},
volume = {48},
number = {8},
pages = {4344-4356},
pmid = {32187363},
issn = {1362-4962},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {5' Untranslated Regions ; Animals ; Animals, Genetically Modified ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Drosophila/*genetics ; Drosophila Proteins/genetics ; *Gene Editing ; Genome, Insect ; INDEL Mutation ; Male ; *Mutagenesis ; Nerve Tissue Proteins/genetics ; Spermatozoa/metabolism ; },
abstract = {The genome is the blueprint for an organism. Interrogating the genome, especially locating critical cis-regulatory elements, requires deletion analysis. This is conventionally performed using synthetic constructs, making it cumbersome and non-physiological. Thus, we created Cas9-mediated Arrayed Mutagenesis of Individual Offspring (CAMIO) to achieve comprehensive analysis of a targeted region of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate independent deletions in the intact Drosophila genome. Controlled by recombination, a single guide RNA is stochastically chosen from a set targeting a specific DNA region. Combining two sets increases variability, leading to either indels at 1-2 target sites or inter-target deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-break repair, consequently creating offspring with diverse mutations. Thus, from a single population cross, we can obtain a deletion matrix covering a large expanse of DNA at both coarse and fine resolution. We demonstrate the ease and power of CAMIO by mapping 5'UTR sequences crucial for chinmo's post-transcriptional regulation.},
}

5' Untranslated Regions
Animals
Animals, Genetically Modified
CRISPR-Associated Protein 9
*CRISPR-Cas Systems
Drosophila/*genetics
Drosophila Proteins/genetics
*Gene Editing
Genome, Insect
INDEL Mutation
Male
*Mutagenesis
Nerve Tissue Proteins/genetics
Spermatozoa/metabolism

@article {pmid31932482,
year = {2020},
author = {Gustafson, EA and Seymour, KA and Sigrist, K and Rooij, DGDE and Freiman, RN},
title = {ZFP628 Is a TAF4b-Interacting Transcription Factor Required for Mouse Spermiogenesis.},
journal = {Molecular and cellular biology},
volume = {40},
number = {7},
pages = {},
pmid = {31932482},
issn = {1098-5549},
mesh = {Animals ; Apoptosis/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Chromosomal Proteins, Non-Histone/genetics/metabolism ; DNA-Binding Proteins/genetics/*metabolism ; Female ; HEK293 Cells ; Humans ; Infertility, Male/*genetics ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Ovary/metabolism ; Protamines/genetics/metabolism ; Protein Domains/genetics ; Spermatogenesis/*genetics ; TATA-Binding Protein Associated Factors/*genetics ; Testis/metabolism ; Transcription Factor TFIID/*genetics ; Transcription Factors/genetics/*metabolism ; Transcriptional Activation/genetics ; Two-Hybrid System Techniques ; },
abstract = {TAF4b is a subunit of the TFIID complex that is highly expressed in the ovary and testis and required for mouse fertility. TAF4b-deficient male mice undergo a complex series of developmental defects that result in the inability to maintain long-term spermatogenesis. To decipher the transcriptional mechanisms upon which TAF4b functions in spermatogenesis, we used two-hybrid screening to identify a novel TAF4b-interacting transcriptional cofactor, ZFP628. Deletion analysis of both proteins reveals discrete and novel domains of ZFP628 and TAF4b protein that function to bridge their direct interaction in vitro Moreover, coimmunoprecipitation of ZFP628 and TAF4b proteins in testis-derived protein extracts supports their endogenous association. Using CRISPR-Cas9, we disrupted the expression of ZFP628 in the mouse and uncovered a postmeiotic germ cell arrest at the round spermatid stage in the seminiferous tubules of the testis in ZFP628-deficient mice that results in male infertility. Coincident with round spermatid arrest, we find reduced mRNA expression of transition protein (Tnp1 and Tnp2) and protamine (Prm1 and Prm2) genes, which are critical for the specialized maturation of haploid male germ cells called spermiogenesis. These data delineate a novel association of two transcription factors, TAF4b and ZFP628, and identify ZFP628 as a novel transcriptional regulator of stage-specific spermiogenesis.},
}

Animals
Apoptosis/genetics
CRISPR-Cas Systems/genetics
Cell Line
Chromosomal Proteins, Non-Histone/genetics/metabolism
DNA-Binding Proteins/genetics/*metabolism
Female
HEK293 Cells
Humans
Infertility, Male/*genetics
Male
Mice
Mice, Inbred C57BL
Mice, Knockout
Ovary/metabolism
Protamines/genetics/metabolism
Protein Domains/genetics
Spermatogenesis/*genetics
TATA-Binding Protein Associated Factors/*genetics
Testis/metabolism
Transcription Factor TFIID/*genetics
Transcription Factors/genetics/*metabolism
Transcriptional Activation/genetics
Two-Hybrid System Techniques

@article {pmid31730408,
year = {2020},
author = {Schoger, E and Carroll, KJ and Iyer, LM and McAnally, JR and Tan, W and Liu, N and Noack, C and Shomroni, O and Salinas, G and Groß, J and Herzog, N and Doroudgar, S and Bassel-Duby, R and Zimmermann, WH and Zelarayán, LC},
title = {CRISPR-Mediated Activation of Endogenous Gene Expression in the Postnatal Heart.},
journal = {Circulation research},
volume = {126},
number = {1},
pages = {6-24},
doi = {10.1161/CIRCRESAHA.118.314522},
pmid = {31730408},
issn = {1524-4571},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Dependovirus/genetics ; Fibroblasts/metabolism ; *Gene Expression Regulation/genetics ; Genes, Synthetic ; Genetic Vectors/genetics ; Heart/growth & development ; Kruppel-Like Transcription Factors/biosynthesis/genetics ; MEF2 Transcription Factors/biosynthesis/genetics ; Mice ; Mice, Transgenic ; Myocardium/*metabolism ; Myocytes, Cardiac/metabolism ; Myosin Heavy Chains/genetics ; Promoter Regions, Genetic ; Protein Domains ; RNA Polymerase III/genetics ; RNA, Guide/genetics ; *Transcriptional Activation ; },
abstract = {RATIONALE: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far.

OBJECTIVE: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription.

METHODS AND RESULTS: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects.

CONCLUSIONS: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.},
}

Animals
*CRISPR-Cas Systems
Cell Line
Dependovirus/genetics
Fibroblasts/metabolism
*Gene Expression Regulation/genetics
Genes, Synthetic
Genetic Vectors/genetics
Heart/growth & development
Kruppel-Like Transcription Factors/biosynthesis/genetics
MEF2 Transcription Factors/biosynthesis/genetics
Mice
Mice, Transgenic
Myocardium/*metabolism
Myocytes, Cardiac/metabolism
Myosin Heavy Chains/genetics
Promoter Regions, Genetic
Protein Domains
RNA Polymerase III/genetics
RNA, Guide/genetics
*Transcriptional Activation

@article {pmid31596609,
year = {2020},
author = {Koh, KD and Siddiqui, S and Cheng, D and Bonser, LR and Sun, DI and Zlock, LT and Finkbeiner, WE and Woodruff, PG and Erle, DJ},
title = {Efficient RNP-directed Human Gene Targeting Reveals SPDEF Is Required for IL-13-induced Mucostasis.},
journal = {American journal of respiratory cell and molecular biology},
volume = {62},
number = {3},
pages = {373-381},
pmid = {31596609},
issn = {1535-4989},
support = {R35 HL145235/HL/NHLBI NIH HHS/United States ; U19 AI077439/AI/NIAID NIH HHS/United States ; },
mesh = {Bronchi/cytology ; CRISPR-Cas Systems ; Cells, Cultured ; Down-Regulation ; Epithelial Cells/*drug effects/metabolism ; Gene Expression Regulation ; Gene Targeting/*methods ; Goblet Cells/metabolism ; Humans ; Interleukin-13/*physiology ; Metaplasia ; Mucin 5AC/biosynthesis/genetics ; Mucociliary Clearance/*physiology ; Primary Cell Culture ; Proto-Oncogene Proteins c-ets/deficiency/genetics/*physiology ; RNA, Guide/genetics ; Ribonucleoproteins/administration & dosage/*genetics ; Transcriptome ; },
abstract = {Primary human bronchial epithelial cell (HBEC) cultures are a useful model for studies of lung health and major airway diseases. However, mechanistic studies have been limited by our ability to selectively disrupt specific genes in these cells. Here we optimize methods for gene targeting in HBECs by direct delivery of single guide RNA (sgRNA) and rCas9 (recombinant Cas9) complexes by electroporation, without a requirement for plasmids, viruses, or antibiotic selection. Variations in the method of delivery, sgRNA and rCas9 concentrations, and sgRNA sequences all had effects on targeting efficiency, allowing for predictable control of the extent of gene targeting and for near-complete disruption of gene expression. To demonstrate the value of this system, we targeted SPDEF, which encodes a transcription factor previously shown to be essential for the differentiation of MUC5AC-producing goblet cells in mouse models of asthma. Targeting SPDEF led to proportional decreases in MUC5AC expression in HBECs stimulated with IL-13, a central mediator of allergic asthma. Near-complete targeting of SPDEF abolished IL-13-induced MUC5AC expression and goblet cell differentiation. In addition, targeting of SPDEF prevented IL-13-induced impairment of mucociliary clearance, which is likely to be an important contributor to airway obstruction, morbidity, and mortality in asthma. We conclude that direct delivery of sgRNA and rCas9 complexes allows for predictable and efficient gene targeting and enables mechanistic studies of disease-relevant pathways in primary HBECs.},
}

Bronchi/cytology
CRISPR-Cas Systems
Cells, Cultured
Down-Regulation
Epithelial Cells/*drug effects/metabolism
Gene Expression Regulation
Gene Targeting/*methods
Goblet Cells/metabolism
Humans
Interleukin-13/*physiology
Metaplasia
Mucin 5AC/biosynthesis/genetics
Mucociliary Clearance/*physiology
Primary Cell Culture
Proto-Oncogene Proteins c-ets/deficiency/genetics/*physiology
RNA, Guide/genetics
Ribonucleoproteins/administration & dosage/*genetics
Transcriptome

@article {pmid31593471,
year = {2019},
author = {Liu, Q and Cai, J and Zheng, Y and Tan, Y and Wang, Y and Zhang, Z and Zheng, C and Zhao, Y and Liu, C and An, Y and Jiang, C and Shi, L and Kang, C and Liu, Y},
title = {NanoRNP Overcomes Tumor Heterogeneity in Cancer Treatment.},
journal = {Nano letters},
volume = {19},
number = {11},
pages = {7662-7672},
doi = {10.1021/acs.nanolett.9b02501},
pmid = {31593471},
issn = {1530-6992},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Core Binding Factor Alpha 2 Subunit/genetics ; Gene Editing/*methods ; Genetic Therapy/methods ; Humans ; Mice ; Mice, Nude ; Nanomedicine/methods ; Neoplasms/genetics/pathology/*therapy ; STAT3 Transcription Factor/genetics ; },
abstract = {Tumor heterogeneity has been one of the most important factors leading to the failure of conventional cancer therapies due to the accumulation of genetically distinct tumor-cell subpopulations during the tumor development process. Due to the diversity of genetic mutations during tumor growth, combining the use of multiple drugs has only achieved limited success in combating heterogeneous tumors. Herein, we report a novel antitumor strategy that effectively addresses tumor heterogeneity by using a CRISPR/Cas9-based nanoRNP carrying a combination of sgRNAs. Such nanoRNP is synthesized from Cas9 ribonucleoprotein, any combinations of required sgRNAs, and a rationally designed responsive polymer that endows nanoRNP with high circulating stability, enhanced tumor accumulation, and the efficient gene editing in targeted tumor cells eventually. By carrying a combination of sgRNAs that targets STAT3 and RUNX1, the nanoRNP exhibited efficient gene expression disruptions on a heterogeneous tumor model with two subsets of cells whose proliferations were sensitive to the reduced expression of STAT3 and RUNX1, respectively, leading to the effective growth inhibition of the heterogeneous tumor. Considering the close relationship between tumor heterogeneity and cancer progression, resistance to therapy, and recurrences, nanoRNP provides a feasible strategy to overcome tumor heterogeneity in the development of more advanced cancer therapy against malignant tumors.},
}

Animals
*CRISPR-Cas Systems
Cell Line, Tumor
Core Binding Factor Alpha 2 Subunit/genetics
Gene Editing/*methods
Genetic Therapy/methods
Humans
Mice
Mice, Nude
Nanomedicine/methods
Neoplasms/genetics/pathology/*therapy
STAT3 Transcription Factor/genetics

@article {pmid31408165,
year = {2019},
author = {Liu, L and Lu, JY and Li, F and Xing, X and Li, T and Yang, X and Shen, X},
title = {IDH1 fine-tunes cap-dependent translation initiation.},
journal = {Journal of molecular cell biology},
volume = {11},
number = {10},
pages = {816-828},
pmid = {31408165},
issn = {1759-4685},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Embryonic Stem Cells/metabolism ; Isocitrate Dehydrogenase/genetics/*metabolism ; Ketoglutaric Acids/metabolism ; Mice ; Polyribosomes/metabolism ; RNA, Messenger/metabolism ; Ribosomes/metabolism ; },
abstract = {The metabolic enzyme isocitrate dehydrogenase 1 (IDH1) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG). Its mutation often leads to aberrant gene expression in cancer. IDH1 was reported to bind thousands of RNA transcripts in a sequence-dependent manner; yet, the functional significance of this RNA-binding activity remains elusive. Here, we report that IDH1 promotes mRNA translation via direct associations with polysome mRNA and translation machinery. Comprehensive proteomic analysis in embryonic stem cells (ESCs) revealed striking enrichment of ribosomal proteins and translation regulators in IDH1-bound protein interactomes. We performed ribosomal profiling and analyzed mRNA transcripts that are associated with actively translating polysomes. Interestingly, knockout of IDH1 in ESCs led to significant downregulation of polysome-bound mRNA in IDH1 targets and subtle upregulation of ribosome densities at the start codon, indicating inefficient translation initiation upon loss of IDH1. Tethering IDH1 to a luciferase mRNA via the MS2-MBP system promotes luciferase translation, independently of the catalytic activity of IDH1. Intriguingly, IDH1 fails to enhance luciferase translation driven by an internal ribosome entry site. Together, these results reveal an unforeseen role of IDH1 in fine-tuning cap-dependent translation via the initiation step.},
}

Animals
CRISPR-Cas Systems/genetics
Embryonic Stem Cells/metabolism
Isocitrate Dehydrogenase/genetics/*metabolism
Ketoglutaric Acids/metabolism
Mice
Polyribosomes/metabolism
RNA, Messenger/metabolism
Ribosomes/metabolism

@article {pmid31366580,
year = {2019},
author = {Humbert, O and Radtke, S and Samuelson, C and Carrillo, RR and Perez, AM and Reddy, SS and Lux, C and Pattabhi, S and Schefter, LE and Negre, O and Lee, CM and Bao, G and Adair, JE and Peterson, CW and Rawlings, DJ and Scharenberg, AM and Kiem, HP},
title = {Therapeutically relevant engraftment of a CRISPR-Cas9-edited HSC-enriched population with HbF reactivation in nonhuman primates.},
journal = {Science translational medicine},
volume = {11},
number = {503},
pages = {},
doi = {10.1126/scitranslmed.aaw3768},
pmid = {31366580},
issn = {1946-6242},
mesh = {Animals ; Antigens, CD34/metabolism ; CRISPR-Cas Systems/*genetics ; Fetal Hemoglobin/genetics/*metabolism ; Gene Editing ; Genotype ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/*cytology ; Humans ; Macaca mulatta ; Primates ; Thy-1 Antigens/metabolism ; },
abstract = {Reactivation of fetal hemoglobin (HbF) is being pursued as a treatment strategy for hemoglobinopathies. Here, we evaluated the therapeutic potential of hematopoietic stem and progenitor cells (HSPCs) edited with the CRISPR-Cas9 nuclease platform to recapitulate naturally occurring mutations identified in individuals who express increased amounts of HbF, a condition known as hereditary persistence of HbF. CRISPR-Cas9 treatment and transplantation of HSPCs purified on the basis of surface expression of the CD34 receptor in a nonhuman primate (NHP) autologous transplantation model resulted in up to 30% engraftment of gene-edited cells for >1 year. Edited cells effectively and stably reactivated HbF, as evidenced by up to 18% HbF-expressing erythrocytes in peripheral blood. Similar results were obtained by editing highly enriched stem cells, defined by the markers CD34+CD90+CD45RA-, allowing for a 10-fold reduction in the number of transplanted target cells, thus considerably reducing the need for editing reagents. The frequency of engrafted, gene-edited cells persisting in vivo using this approach may be sufficient to ameliorate the phenotype for a number of genetic diseases.},
}

Animals
Antigens, CD34/metabolism
CRISPR-Cas Systems/*genetics
Fetal Hemoglobin/genetics/*metabolism
Gene Editing
Genotype
Hematopoietic Stem Cell Transplantation
Hematopoietic Stem Cells/*cytology
Humans
Macaca mulatta
Primates
Thy-1 Antigens/metabolism

@article {pmid31344492,
year = {2019},
author = {Janakiraman, U and Yu, J and Moutal, A and Chinnasamy, D and Boinon, L and Batchelor, SN and Anandhan, A and Khanna, R and Nelson, MA},
title = {TAF1-gene editing alters the morphology and function of the cerebellum and cerebral cortex.},
journal = {Neurobiology of disease},
volume = {132},
number = {},
pages = {104539},
doi = {10.1016/j.nbd.2019.104539},
pmid = {31344492},
issn = {1095-953X},
support = {R01 DA042852/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; Animals, Newborn ; CRISPR-Cas Systems/*genetics ; Cerebellum/abnormalities/*pathology/physiology ; Cerebral Cortex/abnormalities/*pathology/physiology ; Female ; Gene Editing/*methods ; Histone Acetyltransferases/*genetics ; Injections, Intraventricular ; Locomotion/physiology ; Organ Culture Techniques ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; TATA-Binding Protein Associated Factors/*genetics ; Transcription Factor TFIID/*genetics ; },
abstract = {TAF1/MRSX33 intellectual disability syndrome is an X-linked disorder caused by loss-of-function mutations in the TAF1 gene. How these mutations cause dysmorphology, hypotonia, intellectual and motor defects is unknown. Mouse models which have embryonically targeted TAF1 have failed, possibly due to TAF1 being essential for viability, preferentially expressed in early brain development, and intolerant of mutation. Novel animal models are valuable tools for understanding neuronal pathology. Here, we report the development and characterization of a novel animal model for TAF1 ID syndrome in which the TAF1 gene is deleted in embryonic rats using clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) technology and somatic brain transgenesis mediated by lentiviral transduction. Rat pups, post-natal day 3, were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 vectors. Rats were subjected to a battery of behavioral tests followed by histopathological analyses of brains at post-natal day 14 and day 35. TAF1-edited rats exhibited behavioral deficits at both the neonatal and juvenile stages of development. Deletion of TAF1 lead to a hypoplasia and loss of the Purkinje cells. We also observed a decreased in GFAP positive astrocytes and an increase in Iba1 positive microglia within the granular layer of the cerebellum in TAF1-edited animals. Immunostaining revealed a reduction in the expression of the CaV3.1 T-type calcium channel. Abnormal motor symptoms in TAF1-edited rats were associated with irregular cerebellar output caused by changes in the intrinsic activity of the Purkinje cells due to loss of pre-synaptic CaV3.1. This animal model provides a powerful new tool for studies of neuronal dysfunction in conditions associated with TAF1 abnormalities and should prove useful for developing therapeutic strategies to treat TAF1 ID syndrome.},
}

Animals
Animals, Newborn
CRISPR-Cas Systems/*genetics
Cerebellum/abnormalities/*pathology/physiology
Cerebral Cortex/abnormalities/*pathology/physiology
Female
Gene Editing/*methods
Histone Acetyltransferases/*genetics
Injections, Intraventricular
Locomotion/physiology
Organ Culture Techniques
Pregnancy
Rats
Rats, Sprague-Dawley
TATA-Binding Protein Associated Factors/*genetics
Transcription Factor TFIID/*genetics

@article {pmid31303577,
year = {2019},
author = {Povedano, JM and Liou, J and Wei, D and Srivatsav, A and Kim, J and Xie, Y and Nijhawan, D and McFadden, DG},
title = {Engineering Forward Genetics into Cultured Cancer Cells for Chemical Target Identification.},
journal = {Cell chemical biology},
volume = {26},
number = {9},
pages = {1315-1321.e3},
pmid = {31303577},
issn = {2451-9448},
support = {R01 CA217333/CA/NCI NIH HHS/United States ; R37 CA226771/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Mismatch Repair/genetics ; Drug Discovery/*methods ; Genetic Engineering/*methods ; Genetic Testing/*methods ; Humans ; Mice ; Neoplasms/genetics ; },
abstract = {Target identification for biologically active small molecules remains a major barrier for drug discovery. Cancer cells exhibiting defective DNA mismatch repair (dMMR) have been used as a forward genetics system to uncover compound targets. However, this approach has been limited by the dearth of cancer cell lines that harbor naturally arising dMMR. Here, we establish a platform for forward genetic screening using CRISPR/Cas9 to engineer dMMR into mammalian cells. We demonstrate the utility of this approach to identify mechanisms of drug action in mouse and human cancer cell lines using in vitro selections against three cellular toxins. In each screen, compound-resistant alleles emerged in drug-resistant clones, supporting the notion that engineered dMMR enables forward genetic screening in mammalian cells.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
DNA Mismatch Repair/genetics
Drug Discovery/*methods
Genetic Engineering/*methods
Genetic Testing/*methods
Humans
Mice
Neoplasms/genetics

@article {pmid31219803,
year = {2019},
author = {Liu, X and Wei, L and Dong, Q and Liu, L and Zhang, MQ and Xie, Z and Wang, X},
title = {A large-scale CRISPR screen and identification of essential genes in cellular senescence bypass.},
journal = {Aging},
volume = {11},
number = {12},
pages = {4011-4031},
pmid = {31219803},
issn = {1945-4589},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Cell Line ; Cellular Senescence/*genetics/*physiology ; Fibroblasts/*physiology ; Gene Expression Regulation/*physiology ; Humans ; Lentivirus ; },
abstract = {Cellular senescence is an important mechanism of autonomous tumor suppression, while its consequence such as the senescence-associated secretory phenotype (SASP) may drive tumorigenesis and age-related diseases. Therefore, controlling the cell fate optimally when encountering senescence stress is helpful for anti-cancer or anti-aging treatments. To identify genes essential for senescence establishment or maintenance, we carried out a CRISPR-based screen with a deliberately designed single-guide RNA (sgRNA) library. The library comprised of about 12,000 kinds of sgRNAs targeting 1378 senescence-associated genes selected by integrating the information of literature mining, protein-protein interaction network, and differential gene expression. We successfully detected a dozen gene deficiencies potentially causing senescence bypass, and their phenotypes were further validated with a high true positive rate. RNA-seq analysis showed distinct transcriptome patterns of these bypass cells. Interestingly, in the bypass cells, the expression of SASP genes was maintained or elevated with CHEK2, HAS1, or MDK deficiency; but neutralized with MTOR, CRISPLD2, or MORF4L1 deficiency. Pathways of some age-related neurodegenerative disorders were also downregulated with MTOR, CRISPLD2, or MORF4L1 deficiency. The results demonstrated that disturbing these genes could lead to distinct cell fates as a consequence of senescence bypass, suggesting that they may play essential roles in cellular senescence.},
}

CRISPR-Associated Protein 9
CRISPR-Cas Systems
Cell Line
Cellular Senescence/*genetics/*physiology
Fibroblasts/*physiology
Gene Expression Regulation/*physiology
Humans
Lentivirus

@article {pmid30679690,
year = {2019},
author = {Bricogne, C and Fine, M and Pereira, PM and Sung, J and Tijani, M and Wang, Y and Henriques, R and Collins, MK and Hilgemann, DW},
title = {TMEM16F activation by Ca2+ triggers plasma membrane expansion and directs PD-1 trafficking.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {619},
pmid = {30679690},
issn = {2045-2322},
support = {T32 DK007257/DK/NIDDK NIH HHS/United States ; MR/K015826/1/MRC_/Medical Research Council/United Kingdom ; BB/M022374/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 1132770/MRC_/Medical Research Council/United Kingdom ; R01 HL119843/HL/NHLBI NIH HHS/United States ; },
mesh = {Anoctamins/genetics/*metabolism ; CRISPR-Cas Systems/genetics ; Calcium/*metabolism ; Cell Line ; Cell Membrane/*metabolism ; Flow Cytometry ; Humans ; Jurkat Cells ; Lentivirus/genetics ; Microscopy, Confocal ; Phospholipid Transfer Proteins/genetics/*metabolism ; Programmed Cell Death 1 Receptor/genetics/*metabolism ; },
abstract = {TMEM16F is a Ca2+ -gated ion channel that is required for Ca2+ -activated phosphatidylserine exposure on the surface of many eukaryotic cells. TMEM16F is widely expressed and has roles in platelet activation during blood clotting, bone formation and T cell activation. By combining microscopy and patch clamp recording we demonstrate that activation of TMEM16F by Ca2+ ionophores in Jurkat T cells triggers large-scale surface membrane expansion in parallel with phospholipid scrambling. With continued ionophore application,TMEM16F-expressing cells then undergo extensive shedding of ectosomes. The T cell co-receptor PD-1 is selectively incorporated into ectosomes. This selectivity depends on its transmembrane sequence. Surprisingly, cells lacking TMEM16F not only fail to expand surface membrane in response to elevated cytoplasmic Ca2+, but instead undergo rapid massive endocytosis with PD-1 internalisation. These results establish a new role for TMEM16F as a regulator of Ca2+ activated membrane trafficking.},
}

Anoctamins/genetics/*metabolism
CRISPR-Cas Systems/genetics
Calcium/*metabolism
Cell Line
Cell Membrane/*metabolism
Flow Cytometry
Humans
Jurkat Cells
Lentivirus/genetics
Microscopy, Confocal
Phospholipid Transfer Proteins/genetics/*metabolism
Programmed Cell Death 1 Receptor/genetics/*metabolism

@article {pmid30679624,
year = {2019},
author = {Adams, S and Pathak, P and Shao, H and Lok, JB and Pires-daSilva, A},
title = {Liposome-based transfection enhances RNAi and CRISPR-mediated mutagenesis in non-model nematode systems.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {483},
pmid = {30679624},
issn = {2045-2322},
support = {BB/L019884/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/International ; R21 AI105856/AI/NIAID NIH HHS/United States ; R33 AI105856/AI/NIAID NIH HHS/United States ; OD P40-10939//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/International ; R01 AI050668/AI/NIAID NIH HHS/United States ; AI50688//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression ; *Liposomes ; *Mutagenesis ; Nematoda/genetics ; *RNA Interference ; RNA, Double-Stranded/genetics ; *Transfection ; },
abstract = {Nematodes belong to one of the most diverse animal phyla. However, functional genomic studies in nematodes, other than in a few species, have often been limited in their reliability and success. Here we report that by combining liposome-based technology with microinjection, we were able to establish a wide range of genomic techniques in the newly described nematode genus Auanema. The method also allowed heritable changes in dauer larvae of Auanema, despite the immaturity of the gonad at the time of the microinjection. As proof of concept for potential functional studies in other nematode species, we also induced RNAi in the free-living nematode Pristionchus pacificus and targeted the human parasite Strongyloides stercoralis.},
}

Animals
CRISPR-Cas Systems
Caenorhabditis elegans/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Expression
*Liposomes
*Mutagenesis
Nematoda/genetics
*RNA Interference
RNA, Double-Stranded/genetics
*Transfection

@article {pmid32697075,
year = {2020},
author = {Wei, T and Cheng, Q and Farbiak, L and Anderson, DG and Langer, R and Siegwart, DJ},
title = {Delivery of Tissue-Targeted Scalpels: Opportunities and Challenges for In Vivo CRISPR/Cas-Based Genome Editing.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.0c04707},
pmid = {32697075},
issn = {1936-086X},
abstract = {CRISPR/Cas9-based genome editing has quickly emerged as a powerful breakthrough technology for use in diverse settings across biomedical research and therapeutic development. Recent efforts toward understanding gene modification methods in vitro have led to substantial improvements in ex vivo genome editing efficiency. Because disease targets for genomic correction are often localized in specific organs, realization of the full potential of genomic medicines will require delivery of CRISPR/Cas9 systems targeting specific tissues and cells directly in vivo. In this Perspective, we focus on progress toward in vivo delivery of CRISPR/Cas components. Viral and nonviral delivery systems are both promising for gene editing in diverse tissues via local injection and systemic injection. We describe the various viral vectors and synthetic nonviral materials used for in vivo gene editing and applications to research and therapeutic models, and summarize opportunities and progress to date for both methods. We also discuss challenges for viral delivery, including overcoming limited packaging capacity, immunogenicity associated with multiple dosing, and the potential for off-target effects, and nonviral delivery, including efforts to increase efficacy and to expand utility of nonviral carriers for use in extrahepatic tissues and cancer. Looking ahead, additional advances in the safety and efficiency of viral and nonviral delivery systems for tissue- and cell-type-specific gene editing will be required to enable broad clinical translation. We provide a summary of current delivery systems used for in vivo genome editing, organized with respect to route of administration, and highlight immediate opportunities for biomedical research and applications. Furthermore, we discuss current challenges for in vivo delivery of CRISPR/Cas9 systems to guide the development of future therapies.},
}

@article {pmid32695773,
year = {2020},
author = {Zhang, J and Zhang, D and Zhu, J and Liu, H and Liang, S and Luo, Y},
title = {Efficient Multiplex Genome Editing in Streptomyces via Engineered CRISPR-Cas12a Systems.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {726},
pmid = {32695773},
issn = {2296-4185},
abstract = {Streptomyces strains produce a great number of valuable natural products. With the development of genome sequencing, a vast number of biosynthetic gene clusters with high potential for use in the discovery of valuable clinical drugs have been revealed. Therefore, emerging needs for tools to manipulate these biosynthetic pathways are presented. Although the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas 9) system has exhibited great capabilities for gene editing in multiple Streptomyces strains, it has failed to work in some newly discovered strains and some important industrial strains. Additionally, the protospacer adjacent motif (PAM) recognition scope of this system sometimes limits its applications for generating precise site mutations and insertions. Here, we developed three efficient CRISPR-FnCas12a systems for multiplex genome editing in several Streptomyces strains. Each system exhibited advantages for different applications. The CRISPR-FnCas12a1 system was efficiently applied in the industrial strain Streptomyces hygroscopicus, in which SpCas9 does not work well. The CRISPR-FnCas12a2 system was used to delete large fragments ranging from 21.4 to 128 kb. Additionally, the CRISPR-FnCas12a3 system employing the engineered FnCas12a mutant EP16, which recognizes a broad spectrum of PAM sequences, was used to precisely perform site mutations and insertions. The CRISPR-FnCas12a3 system addressed the limitation of TTN PAM recognition in Streptomyces strains with high GC contents. In summary, all the CRISPR-FnCas12a systems developed in this study are powerful tools for precise and multiplex genome editing in Streptomyces strains.},
}

@article {pmid32695770,
year = {2020},
author = {Ding, W and Zhang, Y and Shi, S},
title = {Development and Application of CRISPR/Cas in Microbial Biotechnology.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {8},
number = {},
pages = {711},
pmid = {32695770},
issn = {2296-4185},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has been rapidly developed as versatile genomic engineering tools with high efficiency, accuracy and flexibility, and has revolutionized traditional methods for applications in microbial biotechnology. Here, key points of building reliable CRISPR/Cas system for genome engineering are discussed, including the Cas protein, the guide RNA and the donor DNA. Following an overview of various CRISPR/Cas tools for genome engineering, including gene activation, gene interference, orthogonal CRISPR systems and precise single base editing, we highlighted the application of CRISPR/Cas toolbox for multiplexed engineering and high throughput screening. We then summarize recent applications of CRISPR/Cas systems in metabolic engineering toward production of chemicals and natural compounds, and end with perspectives of future advancements.},
}

@article {pmid32695284,
year = {2020},
author = {Dara, M and Talebzadeh, M},
title = {CRISPR/Cas as a Potential Diagnosis Technique for COVID-19.},
journal = {Avicenna journal of medical biotechnology},
volume = {12},
number = {3},
pages = {201-202},
pmid = {32695284},
issn = {2008-2835},
}

@article {pmid32694114,
year = {2020},
author = {Li, X and Shi, W and Geng, LZ and Xu, JP},
title = {[Genome editing in plants directed by CRISPR/Cas ribonucleoprotein complexes].},
journal = {Yi chuan = Hereditas},
volume = {42},
number = {6},
pages = {556-564},
doi = {10.16288/j.yczz.20-017},
pmid = {32694114},
issn = {0253-9772},
abstract = {The CRISPR/Cas system is the most popular genome editing technology in recent years and has been widely used in crop improvement. Compared with introducing the CRISPR/Cas system into plant cells with DNA constructs, introducing CRISPR/Cas ribonucleoprotein (RNP) to perform genome editing excels in rapid action, low off-target rates and is free of DNA insertions in editing plants. However, efficient delivery of CRISPR/Cas RNP into plant cells and achieving high editing frequency are still very challenging, which limits the extensive implementation of CRISPR/Cas RNP-mediated genome editing in plants. In this review, we summarize the progress of protein and RNP delivery methods in plant cells, and provide new perspectives of further development and future applications of the CRISPR/Cas RNP technology in plant genome editing.},
}

@article {pmid32669702,
year = {2020},
author = {},
title = {Mitochondrial genome editing: another win for curiosity-driven research.},
journal = {Nature},
volume = {583},
number = {7816},
pages = {332},
doi = {10.1038/d41586-020-02094-x},
pmid = {32669702},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; DNA, Mitochondrial ; Exploratory Behavior ; *Gene Editing ; *Genome, Mitochondrial ; },
}

CRISPR-Cas Systems
DNA, Mitochondrial
Exploratory Behavior
*Gene Editing
*Genome, Mitochondrial

@article {pmid32645350,
year = {2020},
author = {Johnson, WE},
title = {Not Your Typical Anti-CRISPR.},
journal = {Cell host & microbe},
volume = {28},
number = {1},
pages = {1-2},
doi = {10.1016/j.chom.2020.06.016},
pmid = {32645350},
issn = {1934-6069},
mesh = {Bacteriophages/*genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Listeria ; Lysogeny ; },
abstract = {In this issue of Cell Host & Microbe, two papers by Osuna et al. describe the characterization of AcrIIA1, an anti-CRISPR protein distributed widely among Listeria phages. AcrIIA1 functions as an anti-CRISPR and as a dynamic repressor of acr loci, suggesting it may play an important role in lysogeny.},
}

Bacteriophages/*genetics
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
*Listeria
Lysogeny

@article {pmid32374759,
year = {2020},
author = {Bare, DJ and Cherny, VV and DeCoursey, TE and Abukhdeir, AM and Morgan, D},
title = {Expression and function of voltage gated proton channels (Hv1) in MDA-MB-231 cells.},
journal = {PloS one},
volume = {15},
number = {5},
pages = {e0227522},
pmid = {32374759},
issn = {1932-6203},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Movement/genetics ; Cell Proliferation/*genetics ; Gene Expression Regulation, Neoplastic/drug effects ; Gene Knockdown Techniques ; Gene Knockout Techniques ; Heterografts ; Humans ; Hydrogen Peroxide/pharmacology ; Immunohistochemistry ; Ion Channels/*genetics ; Membrane Proteins/*genetics ; Mice ; NADPH Oxidases/genetics ; RNA, Small Interfering/genetics ; Triple Negative Breast Neoplasms/*genetics/pathology ; },
abstract = {Expression of the voltage gated proton channel (Hv1) as identified by immunocytochemistry has been reported previously in breast cancer tissue. Increased expression of HV1 was correlated with poor prognosis and decreased overall and disease-free survival but the mechanism of its involvement in the disease is unknown. Here we present electrophysiological recordings of HV1 channel activity, confirming its presence and function in the plasma membrane of a breast cancer cell line, MDA-MB-231. With western blotting we identify significant levels of HV1 expression in 3 out of 8 "triple negative" breast cancer cell lines (estrogen, progesterone, and HER2 receptor expression negative). We examine the function of HV1 in breast cancer using MDA-MB-231 cells as a model by suppressing the expression of HV1 using shRNA (knock-down; KD) and by eliminating HV1 using CRISPR/Cas9 gene editing (knock-out; KO). Surprisingly, these two approaches produced incongruous effects. Knock-down of HV1 using shRNA resulted in slower cell migration in a scratch assay and a significant reduction in H2O2 release. In contrast, HV1 Knock-out cells did not show reduced migration or H2O2 release. HV1 KO but not KD cells showed an increased glycolytic rate accompanied by an increase in p-AKT (phospho-AKT, Ser473) activity. The expression of CD171/LCAM-1, an adhesion molecule and prognostic indicator for breast cancer, was reduced in HV1 KO cells. When we compared MDA-MB-231 xenograft growth rates in immunocompromised mice, tumors from HV1 KO cells grew less than WT in mass, with lower staining for the Ki-67 marker for cell proliferation rate. Therefore, deletion of HV1 expression in MDA-MB-231 cells limits tumor growth rate. The limited growth thus appears to be independent of oxidant production by NADPH oxidase molecules and to be mediated by cell adhesion molecules. Although HV1 KO and KD affect certain cellular mechanisms differently, both implicate HV1-mediated pathways for control of tumor growth in the MDA-MB-231 cell line.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Movement/genetics
Cell Proliferation/*genetics
Gene Expression Regulation, Neoplastic/drug effects
Gene Knockdown Techniques
Gene Knockout Techniques
Heterografts
Humans
Hydrogen Peroxide/pharmacology
Immunohistochemistry
Ion Channels/*genetics
Membrane Proteins/*genetics
Mice
NADPH Oxidases/genetics
RNA, Small Interfering/genetics
Triple Negative Breast Neoplasms/*genetics/pathology

@article {pmid32284612,
year = {2020},
author = {Zeng, J and Wu, Y and Ren, C and Bonanno, J and Shen, AH and Shea, D and Gehrke, JM and Clement, K and Luk, K and Yao, Q and Kim, R and Wolfe, SA and Manis, JP and Pinello, L and Joung, JK and Bauer, DE},
title = {Therapeutic base editing of human hematopoietic stem cells.},
journal = {Nature medicine},
volume = {26},
number = {4},
pages = {535-541},
doi = {10.1038/s41591-020-0790-y},
pmid = {32284612},
issn = {1546-170X},
support = {P01 HL053749/HL/NHLBI NIH HHS/United States ; R01 AI117839/AI/NIAID NIH HHS/United States ; R01 GM115911/GM/NIGMS NIH HHS/United States ; R35 GM118158/GM/NIGMS NIH HHS/United States ; R00 HG008399/HG/NHGRI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {Anemia, Sickle Cell/*pathology/therapy ; Animals ; Antigens, CD34/metabolism ; CRISPR-Cas Systems ; Cells, Cultured ; Feasibility Studies ; Female ; *Gene Editing/methods ; Gene Targeting/methods ; Genetic Therapy/*methods ; Hematopoietic Stem Cell Transplantation/methods ; Hematopoietic Stem Cells/*metabolism/pathology ; Heterografts ; Humans ; Mice ; Mice, Inbred NOD ; Mice, Transgenic ; Primary Cell Culture ; Repressor Proteins/*genetics/metabolism ; beta-Thalassemia/pathology/therapy ; gamma-Globins/*genetics/metabolism ; },
abstract = {Base editing by nucleotide deaminases linked to programmable DNA-binding proteins represents a promising approach to permanently remedy blood disorders, although its application in engrafting hematopoietic stem cells (HSCs) remains unexplored. In this study, we purified A3A (N57Q)-BE3 base editor for ribonucleoprotein (RNP) electroporation of human-peripheral-blood-mobilized CD34+ hematopoietic stem and progenitor cells (HSPCs). We observed frequent on-target cytosine base edits at the BCL11A erythroid enhancer at +58 with few indels. Fetal hemoglobin (HbF) induction in erythroid progeny after base editing or nuclease editing was similar. A single therapeutic base edit of the BCL11A enhancer prevented sickling and ameliorated globin chain imbalance in erythroid progeny from sickle cell disease and β-thalassemia patient-derived HSPCs, respectively. Moreover, efficient multiplex editing could be achieved with combined disruption of the BCL11A erythroid enhancer and correction of the HBB -28A>G promoter mutation. Finally, base edits could be produced in multilineage-repopulating self-renewing human HSCs with high frequency as assayed in primary and secondary recipient animals resulting in potent HbF induction in vivo. Together, these results demonstrate the potential of RNP base editing of human HSPCs as a feasible alternative to nuclease editing for HSC-targeted therapeutic genome modification.},
}

Anemia, Sickle Cell/*pathology/therapy
Animals
Antigens, CD34/metabolism
CRISPR-Cas Systems
Cells, Cultured
Feasibility Studies
Female
*Gene Editing/methods
Gene Targeting/methods
Genetic Therapy/*methods
Hematopoietic Stem Cell Transplantation/methods
Hematopoietic Stem Cells/*metabolism/pathology
Heterografts
Humans
Mice
Mice, Inbred NOD
Mice, Transgenic
Primary Cell Culture
Repressor Proteins/*genetics/metabolism
beta-Thalassemia/pathology/therapy
gamma-Globins/*genetics/metabolism

@article {pmid31826245,
year = {2020},
author = {Azouzi, S and Mikdar, M and Hermand, P and Gautier, EF and Salnot, V and Willemetz, A and Nicolas, G and Vrignaud, C and Raneri, A and Mayeux, P and Bole-Feysot, C and Nitschké, P and Cartron, JP and Colin, Y and Hermine, O and Jedlitschky, G and Cloutier, M and Constanzo-Yanez, J and Ethier, C and Robitaille, N and St-Louis, M and Le Van Kim, C and Peyrard, T},
title = {Lack of the multidrug transporter MRP4/ABCC4 defines the PEL-negative blood group and impairs platelet aggregation.},
journal = {Blood},
volume = {135},
number = {6},
pages = {441-448},
pmid = {31826245},
issn = {1528-0020},
mesh = {Blood Group Antigens/*genetics ; Blood Platelets/cytology/metabolism ; CRISPR-Cas Systems ; Erythroid Cells/cytology/metabolism ; Gene Deletion ; Humans ; Multidrug Resistance-Associated Proteins/*genetics ; Phenotype ; *Platelet Aggregation ; },
abstract = {The rare PEL-negative phenotype is one of the last blood groups with an unknown genetic basis. By combining whole-exome sequencing and comparative global proteomic investigations, we found a large deletion in the ABCC4/MRP4 gene encoding an ATP-binding cassette (ABC) transporter in PEL-negative individuals. The loss of PEL expression on ABCC4-CRISPR-Cas9 K562 cells and its overexpression in ABCC4-transfected cells provided evidence that ABCC4 is the gene underlying the PEL blood group antigen. Although ABCC4 is an important cyclic nucleotide exporter, red blood cells from ABCC4null/PEL-negative individuals exhibited a normal guanosine 3',5'-cyclic monophosphate level, suggesting a compensatory mechanism by other erythroid ABC transporters. Interestingly, PEL-negative individuals showed an impaired platelet aggregation, confirming a role for ABCC4 in platelet function. Finally, we showed that loss-of-function mutations in the ABCC4 gene, associated with leukemia outcome, altered the expression of the PEL antigen. In addition to ABCC4 genotyping, PEL phenotyping could open a new way toward drug dose adjustment for leukemia treatment.},
}

Blood Group Antigens/*genetics
Blood Platelets/cytology/metabolism
CRISPR-Cas Systems
Erythroid Cells/cytology/metabolism
Gene Deletion
Humans
Multidrug Resistance-Associated Proteins/*genetics
Phenotype
*Platelet Aggregation

@article {pmid31756390,
year = {2020},
author = {Holding, AN and Cook, HV and Markowetz, F},
title = {Data generation and network reconstruction strategies for single cell transcriptomic profiles of CRISPR-mediated gene perturbations.},
journal = {Biochimica et biophysica acta. Gene regulatory mechanisms},
volume = {1863},
number = {6},
pages = {194441},
doi = {10.1016/j.bbagrm.2019.194441},
pmid = {31756390},
issn = {1876-4320},
support = {BB/R006563/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*CRISPR-Cas Systems ; Cell Cycle ; Gene Regulatory Networks ; RNA-Seq/*methods ; Single-Cell Analysis/*methods ; },
abstract = {Recent advances in single-cell RNA-sequencing (scRNA-seq) in combination with CRISPR/Cas9 technologies have enabled the development of methods for large-scale perturbation studies with transcriptional readouts. These methods are highly scalable and have the potential to provide a wealth of information on the biological networks that underlie cellular response. Here we discuss how to overcome several key challenges to generate and analyse data for the confident reconstruction of models of the underlying cellular network. Some challenges are generic, and apply to analysing any single-cell transcriptomic data, while others are specific to combined single-cell CRISPR/Cas9 data, in particular barcode swapping, knockdown efficiency, multiplicity of infection and potential confounding factors. We also provide a curated collection of published data sets to aid the development of analysis strategies. Finally, we discuss several network reconstruction approaches, including co-expression networks and Bayesian networks, as well as their limitations, and highlight the potential of Nested Effects Models for network reconstruction from scRNA-seq data. This article is part of a Special Issue entitled: Transcriptional Profiles and Regulatory Gene Networks edited by Dr. Dr. Federico Manuel Giorgi and Dr. Shaun Mahony.},
}

*CRISPR-Cas Systems
Cell Cycle
Gene Regulatory Networks
RNA-Seq/*methods
Single-Cell Analysis/*methods

@article {pmid31694954,
year = {2020},
author = {Henderson, LJ and Reoma, LB and Kovacs, JA and Nath, A},
title = {Advances toward Curing HIV-1 Infection in Tissue Reservoirs.},
journal = {Journal of virology},
volume = {94},
number = {3},
pages = {},
pmid = {31694954},
issn = {1098-5514},
mesh = {Brain ; CRISPR-Cas Systems ; *Disease Reservoirs ; Gene Editing/methods ; Genetic Therapy/methods ; HIV Infections/immunology/*therapy/virology ; HIV-1/genetics/immunology/*physiology ; Humans ; Virus Latency ; },
abstract = {A disease of more than 39.6 million people worldwide, HIV-1 infection has no curative therapy. To date, one man has achieved a sterile cure, with millions more hoping to avoid the potential pitfalls of lifelong antiretroviral therapy and other HIV-related disorders, including neurocognitive decline. Recent developments in immunotherapies and gene therapies provide renewed hope in advancing efforts toward a sterilizing or functional cure. On the horizon is research concentrated in multiple separate but potentially complementary domains: vaccine research, viral transcript editing, T-cell effector response targeting including checkpoint inhibitors, and gene editing. Here, we review the concept of targeting the HIV-1 tissue reservoirs, with an emphasis on the central nervous system, and describe relevant new work in functional cure research and strategies for HIV-1 eradication.},
}

Brain
CRISPR-Cas Systems
*Disease Reservoirs
Gene Editing/methods
Genetic Therapy/methods
HIV Infections/immunology/*therapy/virology
HIV-1/genetics/immunology/*physiology
Humans
Virus Latency

@article {pmid31509794,
year = {2019},
author = {Verdikt, R and Darcis, G and Ait-Ammar, A and Van Lint, C},
title = {Applications of CRISPR/Cas9 tools in deciphering the mechanisms of HIV-1 persistence.},
journal = {Current opinion in virology},
volume = {38},
number = {},
pages = {63-69},
doi = {10.1016/j.coviro.2019.07.004},
pmid = {31509794},
issn = {1879-6265},
mesh = {*CRISPR-Cas Systems ; Disease Reservoirs ; Gene Editing ; HIV Infections/drug therapy/metabolism/*virology ; HIV-1/*physiology ; Humans ; Proteasome Endopeptidase Complex/metabolism ; Virus Integration ; Virus Latency ; *Virus Replication ; },
abstract = {HIV-1 infection can be controlled but not cured by combination antiretroviral therapy. Indeed, the virus persists in treated individuals in viral reservoirs, the best described of which consisting in latently infected central memory CD4+ T cells. However, other cell types in other body compartments than in the peripheral blood contribute to HIV-1 persistence. Addressing the molecular mechanisms of HIV-1 persistence and their cell-specific and tissue-specific variations is thus crucial to develop HIV-1 curative strategies. CRISPR/Cas9 editing technologies have revolutionized genetic engineering by their high specificity and their versatility. Multiple applications now allow to investigate the molecular mechanisms of HIV-1 persistence. Here, we review recent advances in CRISPR-based technologies in deciphering HIV-1 gene expression regulation during persistence.},
}

*CRISPR-Cas Systems
Disease Reservoirs
Gene Editing
HIV Infections/drug therapy/metabolism/*virology
HIV-1/*physiology
Humans
Proteasome Endopeptidase Complex/metabolism
Virus Integration
Virus Latency
*Virus Replication

@article {pmid31450074,
year = {2019},
author = {Das, AT and Binda, CS and Berkhout, B},
title = {Elimination of infectious HIV DNA by CRISPR-Cas9.},
journal = {Current opinion in virology},
volume = {38},
number = {},
pages = {81-88},
pmid = {31450074},
issn = {1879-6265},
support = {R01 AI145045/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA, Viral ; *Gene Editing ; Genetic Therapy ; *Genome, Viral ; HIV Infections/therapy/*virology ; HIV-1/*genetics ; Humans ; Mutation ; Proviruses/*genetics ; Virus Replication/genetics ; },
abstract = {Current antiretroviral drugs can efficiently block HIV replication and prevent transmission, but do not target the HIV provirus residing in cells that constitute the viral reservoir. Because drug therapy interruption will cause viral rebound from this reservoir, HIV-infected individuals face lifelong treatment. Therefore, novel therapeutic strategies are being investigated that aim to permanently inactivate the proviral DNA, which may lead to a cure. Multiple studies showed that CRISPR-Cas9 genome editing can be used to attack HIV DNA. Here, we will focus on not only how this endonuclease attack can trigger HIV provirus inactivation, but also how virus escape occurs and this can be prevented.},
}

*CRISPR-Cas Systems
DNA, Viral
*Gene Editing
Genetic Therapy
*Genome, Viral
HIV Infections/therapy/*virology
HIV-1/*genetics
Humans
Mutation
Proviruses/*genetics
Virus Replication/genetics

@article {pmid31392916,
year = {2019},
author = {Guo, G and Wang, K and Hu, SS and Tian, T and Liu, P and Mori, T and Chen, P and Johnson, CH and Qin, X},
title = {Autokinase Activity of Casein Kinase 1 δ/ε Governs the Period of Mammalian Circadian Rhythms.},
journal = {Journal of biological rhythms},
volume = {34},
number = {5},
pages = {482-496},
pmid = {31392916},
issn = {1552-4531},
support = {R01 GM067152/GM/NIGMS NIH HHS/United States ; R01 GM107434/GM/NIGMS NIH HHS/United States ; R37 GM067152/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Casein Kinase II/genetics ; Casein Kinase Idelta/*genetics/*metabolism ; *Circadian Clocks ; *Circadian Rhythm ; Gene Knockout Techniques ; Mice ; NIH 3T3 Cells ; Phosphorylation ; },
abstract = {Circadian rhythms exist in nearly all organisms. In mammals, transcriptional and translational feedback loops (TTFLs) are believed to underlie the mechanism of the circadian clock. Casein kinase 1δ/ε (CK1δ/ε) are key kinases that phosphorylate clock components such as PER proteins, determining the pace of the clock. Most previous studies of the biochemical properties of the key kinases CK1ε and CK1δ in vitro have focused on the properties of the catalytic domains from which the autoinhibitory C-terminus has been deleted (ΔC); those studies ignored the significance of self-inhibition by autophosphorylation. By comparing the properties of the catalytic domain of CK1δ/ε with the full-length kinase that can undergo autoinhibition, we found that recombinant full-length CK1 showed a sequential autophosphorylation process that induces conformational changes to affect the overall kinase activity. Furthermore, a direct relationship between the period change and the autokinase activity among CK1δ, CK1ε, and CK1ε-R178C was observed. These data implicate the autophosphorylation activity of CK1δ and CK1ε kinases in setting the pace of mammalian circadian rhythms and indicate that the circadian period can be modulated by tuning the autophosphorylation rates of CK1δ/ε.},
}

Animals
CRISPR-Cas Systems
Casein Kinase II/genetics
Casein Kinase Idelta/*genetics/*metabolism
*Circadian Clocks
*Circadian Rhythm
Gene Knockout Techniques
Mice
NIH 3T3 Cells
Phosphorylation

@article {pmid31237055,
year = {2019},
author = {Xu, X and Wan, T and Xin, H and Li, D and Pan, H and Wu, J and Ping, Y},
title = {Delivery of CRISPR/Cas9 for therapeutic genome editing.},
journal = {The journal of gene medicine},
volume = {21},
number = {7},
pages = {e3107},
doi = {10.1002/jgm.3107},
pmid = {31237055},
issn = {1521-2254},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/*methods/trends ; *Gene Transfer Techniques/adverse effects/trends ; Genetic Vectors ; Humans ; Liposomes/chemistry ; Metal Nanoparticles/chemistry ; Peptides/chemistry/genetics ; Polymers/chemistry ; },
abstract = {The clustered, regularly-interspaced, short palindromic repeat (CRISPR)-associated nuclease 9 (CRISPR/Cas9) is emerging as a promising genome-editing tool for treating diseases in a precise way, and has been applied to a wide range of research in the areas of biology, genetics, and medicine. Delivery of therapeutic genome-editing agents provides a promising platform for the treatment of genetic disorders. Although viral vectors are widely used to deliver CRISPR/Cas9 elements with high efficiency, they suffer from several drawbacks, such as mutagenesis, immunogenicity, and off-target effects. Recently, non-viral vectors have emerged as another class of delivery carriers in terms of their safety, simplicity, and flexibility. In this review, we discuss the modes of CRISPR/Cas9 delivery, the barriers to the delivery process and the application of CRISPR/Cas9 system for the treatment of genetic disorders. We also highlight several representative types of non-viral vectors, including polymers, liposomes, cell-penetrating peptides, and other synthetic vectors, for the therapeutic delivery of CRISPR/Cas9 system. The applications of CRISPR/Cas9 in treating genetic disorders mediated by the non-viral vectors are also discussed.},
}

*CRISPR-Cas Systems/genetics
Gene Editing/*methods/trends
*Gene Transfer Techniques/adverse effects/trends
Genetic Vectors
Humans
Liposomes/chemistry
Metal Nanoparticles/chemistry
Peptides/chemistry/genetics
Polymers/chemistry

@article {pmid30763088,
year = {2019},
author = {Palermo, G},
title = {Structure and Dynamics of the CRISPR-Cas9 Catalytic Complex.},
journal = {Journal of chemical information and modeling},
volume = {59},
number = {5},
pages = {2394-2406},
doi = {10.1021/acs.jcim.8b00988},
pmid = {30763088},
issn = {1549-960X},
mesh = {Biocatalysis ; *CRISPR-Cas Systems ; Gene Editing ; *Molecular Dynamics Simulation ; Protein Domains ; *Quantum Theory ; },
abstract = {CRISPR-Cas9 is a bacterial immune system with exciting applications for genome editing. In spite of extensive experimental characterization, the active site chemistry of the RuvC domain-which performs DNA cleavages-has remained elusive. Its knowledge is key for structure-based engineering aimed at improving DNA cleavages. Here, we deliver an in-depth characterization by using quantum-classical (QM/MM) molecular dynamics (MD) simulations and a Gaussian accelerated MD method, coupled with bioinformatics analysis. We disclose a two-metal aided architecture in the RuvC active site, which is poised to operate DNA cleavages, in analogy with other DNA/RNA processing enzymes. The conformational dynamics of the RuvC domain further reveals that an "arginine finger" stably contacts the scissile phosphate, with the function of stabilizing the active complex. Remarkably, the formation of a catalytically competent state of the RuvC domain is only observed upon the conformational activation of the other nuclease domain of CRISPR-Cas9-i.e., the HNH domain-such allowing concerted cleavages of double stranded DNA. This structure is in agreement with the available experimental data and remarkably differs from previous models based on classical mechanics, demonstrating also that only quantum mechanical simulations can accurately describe the metal-aided active site in CRISPR-Cas9. This fully catalytic structure-in which both the HNH and RuvC domains are prone to perform DNA cleavages-constitutes a stepping-stone for understanding DNA cleavage and specificity. It calls for novel experimental verifications and offers the structural foundations for engineering efforts aimed at improving the genome editing capability of CRISPR-Cas9.},
}

Biocatalysis
*CRISPR-Cas Systems
Gene Editing
*Molecular Dynamics Simulation
Protein Domains
*Quantum Theory

@article {pmid32168334,
year = {2020},
author = {Fasulo, B and Meccariello, A and Morgan, M and Borufka, C and Papathanos, PA and Windbichler, N},
title = {A fly model establishes distinct mechanisms for synthetic CRISPR/Cas9 sex distorters.},
journal = {PLoS genetics},
volume = {16},
number = {3},
pages = {e1008647},
pmid = {32168334},
issn = {1553-7404},
support = {BB/P000843/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/genetics ; Drosophila Proteins/genetics ; Drosophila melanogaster/genetics ; Endonucleases/genetics ; Female ; Gene Editing/*methods ; Male ; Models, Animal ; Pest Control, Biological/methods ; Sex Determination Processes/*genetics ; Sex Preselection/*methods ; Sex Ratio ; Spermatogenesis/genetics ; X Chromosome/genetics ; },
abstract = {Synthetic sex distorters have recently been developed in the malaria mosquito, relying on endonucleases that target the X-chromosome during spermatogenesis. Although inspired by naturally-occurring traits, it has remained unclear how they function and, given their potential for genetic control, how portable this strategy is across species. We established Drosophila models for two distinct mechanisms for CRISPR/Cas9 sex-ratio distortion-"X-shredding" and "X-poisoning"-and dissected their target-site requirements and repair dynamics. X-shredding resulted in sex distortion when Cas9 endonuclease activity occurred during the meiotic stages of spermatogenesis but not when Cas9 was expressed from the stem cell stages onwards. Our results suggest that X-shredding is counteracted by the NHEJ DNA repair pathway and can operate on a single repeat cluster of non-essential sequences, although the targeting of a number of such repeats had no effect on the sex ratio. X-poisoning by contrast, i.e. targeting putative haplolethal genes on the X chromosome, induced a high bias towards males (>92%) when we directed Cas9 cleavage to the X-linked ribosomal target gene RpS6. In the case of X-poisoning sex distortion was coupled to a loss in reproductive output, although a dominant-negative effect appeared to drive the mechanism of female lethality. These model systems will guide the study and the application of sex distorters to medically or agriculturally important insect target species.},
}

Animals
CRISPR-Cas Systems/genetics
DNA End-Joining Repair/genetics
Drosophila Proteins/genetics
Drosophila melanogaster/genetics
Endonucleases/genetics
Female
Gene Editing/*methods
Male
Models, Animal
Pest Control, Biological/methods
Sex Determination Processes/*genetics
Sex Preselection/*methods
Sex Ratio
Spermatogenesis/genetics
X Chromosome/genetics

@article {pmid31173713,
year = {2019},
author = {Roper, J and Yilmaz, ÖH},
title = {Breakthrough Moments: Genome Editing and Organoids.},
journal = {Cell stem cell},
volume = {24},
number = {6},
pages = {841-842},
doi = {10.1016/j.stem.2019.05.008},
pmid = {31173713},
issn = {1875-9777},
mesh = {Animals ; CRISPR-Cas Systems ; Disease Models, Animal ; Gene Editing/*methods ; Genetic Diseases, Inborn/genetics/*pathology ; Genetic Therapy/*methods ; Humans ; Organoids/*pathology/physiology ; },
abstract = {Six years ago, Schwank et al. (2013) adapted CRISPR-Cas9 and organoid technology to repair genetic diseases in patient-derived tissues. We shine a spotlight on how this work has inspired the development of tools to study and correct genetic diseases in experimental systems, with the ultimate goal of treating human disease.},
}

Animals
CRISPR-Cas Systems
Disease Models, Animal
Gene Editing/*methods
Genetic Diseases, Inborn/genetics/*pathology
Genetic Therapy/*methods
Humans
Organoids/*pathology/physiology

@article {pmid31165372,
year = {2019},
author = {Wang, W and Hou, J and Zheng, N and Wang, X and Zhang, J},
title = {Keeping our eyes on CRISPR: the "Atlas" of gene editing.},
journal = {Cell biology and toxicology},
volume = {35},
number = {4},
pages = {285-288},
doi = {10.1007/s10565-019-09480-w},
pmid = {31165372},
issn = {1573-6822},
mesh = {Acidaminococcus/genetics ; Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endodeoxyribonucleases/genetics ; Gene Editing/*ethics/methods ; Genome/genetics ; Humans ; Leptotrichia/genetics ; RNA, Guide/genetics ; },
}

Acidaminococcus/genetics
Bacterial Proteins/genetics
CRISPR-Associated Proteins/genetics
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Endodeoxyribonucleases/genetics
Gene Editing/*ethics/methods
Genome/genetics
Humans
Leptotrichia/genetics
RNA, Guide/genetics

@article {pmid30693970,
year = {2020},
author = {Phelps, MP and Seeb, LW and Seeb, JE},
title = {Transforming ecology and conservation biology through genome editing.},
journal = {Conservation biology : the journal of the Society for Conservation Biology},
volume = {34},
number = {1},
pages = {54-65},
doi = {10.1111/cobi.13292},
pmid = {30693970},
issn = {1523-1739},
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Conservation of Natural Resources ; Ecosystem ; *Gene Editing ; },
abstract = {As the conservation challenges increase, new approaches are needed to help combat losses in biodiversity and slow or reverse the decline of threatened species. Genome-editing technology is changing the face of modern biology, facilitating applications that were unimaginable only a decade ago. The technology has the potential to make significant contributions to the fields of evolutionary biology, ecology, and conservation, yet the fear of unintended consequences from designer ecosystems containing engineered organisms has stifled innovation. To overcome this gap in the understanding of what genome editing is and what its capabilities are, more research is needed to translate genome-editing discoveries into tools for ecological research. Emerging and future genome-editing technologies include new clustered regularly interspaced short palindromic repeats (CRISPR) targeted sequencing and nucleic acid detection approaches as well as species genetic barcoding and somatic genome-editing technologies. These genome-editing tools have the potential to transform the environmental sciences by providing new noninvasive methods for monitoring threatened species or for enhancing critical adaptive traits. A pioneering effort by the conservation community is required to apply these technologies to real-world conservation problems.},
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Conservation of Natural Resources
Ecosystem
*Gene Editing

@article {pmid30549232,
year = {2019},
author = {Schwefel, K and Spiegler, S and Ameling, S and Much, CD and Pilz, RA and Otto, O and Völker, U and Felbor, U and Rath, M},
title = {Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffening.},
journal = {Journal of cellular and molecular medicine},
volume = {23},
number = {3},
pages = {1771-1783},
pmid = {30549232},
issn = {1582-4934},
mesh = {Alleles ; Apoptosis ; Apoptosis Regulatory Proteins/antagonists & inhibitors/genetics/*metabolism ; CRISPR-Cas Systems ; *Clonal Evolution ; Endothelium, Vascular/metabolism/*pathology ; Gene Expression Profiling ; High-Throughput Nucleotide Sequencing ; Human Umbilical Vein Endothelial Cells ; Humans ; Membrane Proteins/antagonists & inhibitors/genetics/*metabolism ; MicroRNAs/genetics ; *Mutation ; Neovascularization, Pathologic/*etiology/metabolism/pathology ; Proto-Oncogene Proteins/antagonists & inhibitors/genetics/*metabolism ; },
abstract = {CCM3, originally described as PDCD10, regulates blood-brain barrier integrity and vascular maturation in vivo. CCM3 loss-of-function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral- and plasmid-free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss-of-function CCM3 variants into human ECs and studied the molecular and functional effects of long-term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real-time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3-deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long-term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3-deficient dysfunctional ECs contributes to CCM formation.},
}

Alleles
Apoptosis
Apoptosis Regulatory Proteins/antagonists & inhibitors/genetics/*metabolism
CRISPR-Cas Systems
*Clonal Evolution
Endothelium, Vascular/metabolism/*pathology
Gene Expression Profiling
High-Throughput Nucleotide Sequencing
Human Umbilical Vein Endothelial Cells
Humans
Membrane Proteins/antagonists & inhibitors/genetics/*metabolism
MicroRNAs/genetics
*Mutation
Neovascularization, Pathologic/*etiology/metabolism/pathology
Proto-Oncogene Proteins/antagonists & inhibitors/genetics/*metabolism

@article {pmid30328555,
year = {2019},
author = {Shin, HY and Hennighausen, L and Yoo, KH},
title = {STAT5-Driven Enhancers Tightly Control Temporal Expression of Mammary-Specific Genes.},
journal = {Journal of mammary gland biology and neoplasia},
volume = {24},
number = {1},
pages = {61-71},
pmid = {30328555},
issn = {1573-7039},
mesh = {Animals ; CCCTC-Binding Factor/metabolism ; CRISPR-Cas Systems/genetics ; Cytokines/metabolism ; *Enhancer Elements, Genetic ; Female ; Gene Editing/methods ; *Gene Expression Regulation, Developmental ; Gene Regulatory Networks ; Genetic Loci ; Lactation/*genetics ; Mammary Glands, Animal/*growth & development/metabolism ; Milk Proteins/metabolism ; Pregnancy ; Prolactin/metabolism ; RNA-Seq ; STAT5 Transcription Factor/genetics/*metabolism ; },
abstract = {The de novo formation of milk-secreting mammary epithelium during pregnancy is regulated by prolactin through activation of the transcription factor STAT5, which stimulates the expression of several hundred mammary-specific genes. In addition to its key role in activating gene expression in mammary tissue, STAT5, which is ubiquitously expressed in most cell types, implements T cell-specific programs controlled by interleukins. However, the mechanisms by which STAT5 controls cell-specific genetic programs activated by distinct cytokines remain relatively unknown. Integration of data from genome-wide surveys of chromatin markers and transcription factor binding at regulatory elements may shed light on the mechanisms that drive cell-specific programs. Here, we have illustrated how STAT5 controls cell-specific gene expression through its concentration and an auto-regulatory enhancer supporting its high levels in mammary tissue. The unique genomic features of STAT5-driven enhancers or super-enhancers that regulate mammary-specific genes and their dynamic remodeling in response to pregnancy hormone levels are described. We have further provided biological evidence supporting the in vivo function of a STAT5-driven super-enhancer with the aid of CRISPR/Cas9 genome editing. Finally, we discuss how the functions of mammary-specific super-enhancers are confined by the zinc finger protein, CTCF, to allow exclusive activation of mammary-specific genes without affecting common neighboring genes. This review comprehensively summarizes the molecular pathways underlying differential control of cell-specific gene sets by STAT5 and provides novel insights into STAT5-dependent mammary physiology.},
}

Animals
CCCTC-Binding Factor/metabolism
CRISPR-Cas Systems/genetics
Cytokines/metabolism
*Enhancer Elements, Genetic
Female
Gene Editing/methods
*Gene Expression Regulation, Developmental
Gene Regulatory Networks
Genetic Loci
Lactation/*genetics
Mammary Glands, Animal/*growth & development/metabolism
Milk Proteins/metabolism
Pregnancy
Prolactin/metabolism
RNA-Seq
STAT5 Transcription Factor/genetics/*metabolism

@article {pmid32682455,
year = {2020},
author = {Yoon, AR and Jung, BK and Choi, E and Chung, E and Hong, J and Kim, JS and Koo, T and Yun, CO},
title = {CRISPR-Cas12a with an oAd Induces Precise and Cancer-Specific Genomic Reprogramming of EGFR and Efficient Tumor Regression.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2020.07.003},
pmid = {32682455},
issn = {1525-0024},
abstract = {CRISPR-Cas12a represents a class 2/type V CRISPR RNA-guided endonuclease, holding promise as a precise genome-editing tool in vitro and in vivo. For efficient delivery of the CRISPR-Cas system into cancer, oncolytic adenovirus (oAd) has been recognized as a promising alternative vehicle to conventional cancer therapy, owing to its cancer specificity; however, to our knowledge, it has not been used for genome editing. In this study, we show that CRISPR-Cas12a mediated by oAd disrupts the oncogenic signaling pathway with excellent cancer specificity. The intratumoral delivery of a single oAd co-expressing a Cas12a and a CRISPR RNA (crRNA) targeting the epidermal growth factor receptor (EGFR) gene (oAd/Cas12a/crEGFR) induces efficient and precise editing of the targeted EGFR gene in a cancer-specific manner, without detectable off-target nuclease activity. Importantly, oAd/Cas12a/crEGFR elicits a potent antitumor effect via robust induction of apoptosis and inhibition of tumor cell proliferation, ultimately leading to complete tumor regression in a subset of treated mice. Collectively, in this study we show precise genomic reprogramming via a single oAd vector-mediated CRISPR-Cas system and the feasibility of such system as an alternative cancer therapy.},
}

@article {pmid32680590,
year = {2020},
author = {Wang, Y and Liu, Y and Zheng, P and Sun, J and Wang, M},
title = {Microbial Base Editing: A Powerful Emerging Technology for Microbial Genome Engineering.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2020.06.010},
pmid = {32680590},
issn = {1879-3096},
abstract = {Genome engineering is crucial for answering fundamental questions about, and exploring practical applications of, microorganisms. Various microbial genome-engineering tools, including CRISPR/Cas-enhanced homologous recombination (HR), have been developed, with ever-improving simplicity, efficiency, and applicability. Recently, a powerful emerging technology based on CRISPR/Cas-nucleobase deaminase fusions, known as base editing, opened new avenues for microbial genome engineering. Base editing enables nucleotide transition without inducing lethal double-stranded (ds)DNA cleavage, adding foreign donor DNA, or depending on inefficient HR. Here, we review ongoing efforts to develop and apply base editing to engineer industrially and clinically relevant microorganisms. We also summarize bioinformatics tools that would greatly facilitate guide (g)RNA design and sequencing data analysis and discuss the future challenges and prospects associated with this technology.},
}

@article {pmid32597755,
year = {2020},
author = {Athukoralage, JS and McQuarrie, S and Grüschow, S and Graham, S and Gloster, TM and White, MF},
title = {Tetramerisation of the CRISPR ring nuclease Crn3/Csx3 facilitates cyclic oligoadenylate cleavage.},
journal = {eLife},
volume = {9},
number = {},
pages = {},
doi = {10.7554/eLife.57627},
pmid = {32597755},
issn = {2050-084X},
support = {204821/Z/16/Z//Wellcome Trust Institutional Strategic Support Fund/ ; BB/S000313/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/T004789/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {Type III CRISPR systems detect foreign RNA and activate the cyclase domain of the Cas10 subunit, generating cyclic oligoadenylate (cOA) molecules that act as a second messenger to signal infection, activating nucleases that degrade the nucleic acid of both invader and host. This can lead to dormancy or cell death; to avoid this, cells need a way to remove cOA from the cell once a viral infection has been defeated. Enzymes specialised for this task are known as ring nucleases, but are limited in their distribution. Here, we demonstrate that the widespread CRISPR associated protein Csx3, previously described as an RNA deadenylase, is a ring nuclease that rapidly degrades cyclic tetra-adenylate (cA4). The enzyme has an unusual cooperative reaction mechanism involving an active site that spans the interface between two dimers, sandwiching the cA4 substrate. We propose the name Crn3 (CRISPR associated ring nuclease 3) for the Csx3 family.},
}

@article {pmid32507978,
year = {2020},
author = {Xiao, J and Deng, J and Zhang, Q and Ma, P and Lv, L and Zhang, Y and Li, C and Zhang, Y},
title = {Targeting human cytomegalovirus IE genes by CRISPR/Cas9 nuclease effectively inhibits viral replication and reactivation.},
journal = {Archives of virology},
volume = {165},
number = {8},
pages = {1827-1835},
doi = {10.1007/s00705-020-04687-3},
pmid = {32507978},
issn = {1432-8798},
support = {81902055//Natural Science for Youth Foundation/ ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cytomegalovirus/*genetics ; Cytomegalovirus Infections/virology ; DNA, Viral/genetics ; Endonucleases/*genetics ; Fibroblasts/virology ; Gene Editing/methods ; Gene Expression/genetics ; Genes, Viral/*genetics ; HEK293 Cells ; Humans ; RNA, Guide/genetics ; THP-1 Cells ; Virus Replication/*genetics ; },
abstract = {Human cytomegalovirus (HCMV) infection causes high morbidity and mortality among immunocompromised patients and can remain in a latent state in host cells. Expression of the immediate-early (IE) genes sustains HCMV replication and reactivation. As a novel genome-editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been extensively utilized to modify and edit genomic DNA. In the present study, the CRISPR/Cas9 system was used to target the IE region of the HCMV genome via specific single-guide RNAs (sgRNAs). Infection with CRISPR/Cas9/sgRNA lentiviral constructs significantly reduced viral gene expression and virion production in HFF primary fibroblasts and inhibited viral DNA production and reactivation in the THP-1 monocytic cell line. Thus, the CRISPR/Cas9/sgRNA system can accurately and efficiently target HCMV replication and reactivation and represents a novel therapeutic strategy against latent HCMV infection.},
}

CRISPR-Cas Systems/*genetics
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Cytomegalovirus/*genetics
Cytomegalovirus Infections/virology
DNA, Viral/genetics
Endonucleases/*genetics
Fibroblasts/virology
Gene Editing/methods
Gene Expression/genetics
Genes, Viral/*genetics
HEK293 Cells
Humans
RNA, Guide/genetics
THP-1 Cells
Virus Replication/*genetics

@article {pmid32371236,
year = {2020},
author = {van Wyk, N and Kroukamp, H and Espinosa, MI and von Wallbrunn, C and Wendland, J and Pretorius, IS},
title = {Blending wine yeast phenotypes with the aid of CRISPR DNA editing technologies.},
journal = {International journal of food microbiology},
volume = {324},
number = {},
pages = {108615},
doi = {10.1016/j.ijfoodmicro.2020.108615},
pmid = {32371236},
issn = {1879-3460},
mesh = {Acetic Acid/analysis/metabolism ; *CRISPR-Cas Systems ; Fermentation ; Gene Editing ; Glycerol/analysis/metabolism ; Glycerol-3-Phosphate Dehydrogenase (NAD+)/genetics/metabolism ; Phenotype ; Proteins/genetics/metabolism ; Saccharomyces cerevisiae/*genetics/metabolism ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Wine/analysis/*microbiology ; },
abstract = {In recent years, CRISPR/Cas9-based genetic editing has become a mainstay in many laboratories including manipulations done with yeast. We utilized this technique to generate a self-cloned wine yeast strain that overexpresses two genes of oenological relevance i.e. the glycerol-3-phosphate dehydrogenase 1 (GPD1) and the alcohol acetyltransferase 1 (ATF1) directly implicated in glycerol and acetate ester production respectively. Riesling wine made from the resulting strain showed increased glycerol and acetate ester levels compared to the parental strain. In addition, significantly less acetic acid levels were measured in wine made with yeast containing both genetic alterations compared to wine made with the strain that only overexpresses GPD1. Thus, this strain provides an alternative strategy for alleviating the accumulation of acetic acid once glycerol production is favoured during alcoholic fermentation with the addition of dramatically increasing acetate esters production.},
}

Acetic Acid/analysis/metabolism
*CRISPR-Cas Systems
Fermentation
Gene Editing
Glycerol/analysis/metabolism
Glycerol-3-Phosphate Dehydrogenase (NAD+)/genetics/metabolism
Phenotype
Proteins/genetics/metabolism
Saccharomyces cerevisiae/*genetics/metabolism
Saccharomyces cerevisiae Proteins/genetics/metabolism
Wine/analysis/*microbiology

@article {pmid32123387,
year = {2020},
author = {Bratovič, M and Fonfara, I and Chylinski, K and Gálvez, EJC and Sullivan, TJ and Boerno, S and Timmermann, B and Boettcher, M and Charpentier, E},
title = {Bridge helix arginines play a critical role in Cas9 sensitivity to mismatches.},
journal = {Nature chemical biology},
volume = {16},
number = {5},
pages = {587-595},
doi = {10.1038/s41589-020-0490-4},
pmid = {32123387},
issn = {1552-4469},
mesh = {Arginine/*chemistry ; CRISPR-Associated Protein 9/*chemistry/*metabolism ; CRISPR-Cas Systems ; DNA/metabolism ; DNA Mismatch Repair ; Escherichia coli/genetics ; HEK293 Cells ; Humans ; MCF-7 Cells ; Protein Conformation ; RNA/metabolism ; },
abstract = {The RNA-programmable DNA-endonuclease Cas9 is widely used for genome engineering, where a high degree of specificity is required. To investigate which features of Cas9 determine the sensitivity to mismatches along the target DNA, we performed in vitro biochemical assays and bacterial survival assays in Escherichia coli. We demonstrate that arginines in the Cas9 bridge helix influence guide RNA, and target DNA binding and cleavage. They cluster in two groups that either increase or decrease the Cas9 sensitivity to mismatches. We show that the bridge helix is essential for R-loop formation and that R63 and R66 reduce Cas9 specificity by stabilizing the R-loop in the presence of mismatches. Additionally, we identify Q768 that reduces sensitivity of Cas9 to protospacer adjacent motif-distal mismatches. The Cas9_R63A/Q768A variant showed increased specificity in human cells. Our results provide a firm basis for function- and structure-guided mutagenesis to increase Cas9 specificity for genome engineering.},
}

Arginine/*chemistry
CRISPR-Associated Protein 9/*chemistry/*metabolism
CRISPR-Cas Systems
DNA/metabolism
DNA Mismatch Repair
Escherichia coli/genetics
HEK293 Cells
Humans
MCF-7 Cells
Protein Conformation
RNA/metabolism

@article {pmid31914674,
year = {2020},
author = {Watanabe, H and Higashimoto, K and Miyake, N and Morita, S and Horii, T and Kimura, M and Suzuki, T and Maeda, T and Hidaka, H and Aoki, S and Yatsuki, H and Okamoto, N and Uemura, T and Hatada, I and Matsumoto, N and Soejima, H},
title = {DNA methylation analysis of multiple imprinted DMRs in Sotos syndrome reveals IGF2-DMR0 as a DNA methylation-dependent, P0 promoter-specific enhancer.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {34},
number = {1},
pages = {960-973},
pmid = {31914674},
issn = {1530-6860},
mesh = {CRISPR-Cas Systems ; Child ; Child, Preschool ; *DNA Methylation ; Enhancer Elements, Genetic ; Epigenome ; Female ; Gene Deletion ; Genomic Imprinting ; HEK293 Cells ; Histone-Lysine N-Methyltransferase/*genetics ; Histones/chemistry ; Humans ; Infant ; Infant, Newborn ; Insulin-Like Growth Factor II/*genetics ; Lysine/chemistry ; Male ; Phenotype ; Point Mutation ; Promoter Regions, Genetic ; Sotos Syndrome/*genetics ; },
abstract = {Haploinsufficiency of NSD1, which dimethylates histone H3 lysine 36 (H3K36), causes Sotos syndrome (SoS), an overgrowth syndrome. DNMT3A and DNMT3B recognizes H3K36 trimethylation (H3K36me3) through PWWP domain to exert de novo DNA methyltransferase activity and establish imprinted differentially methylated regions (DMRs). Since decrease of H3K36me3 and genome-wide DNA hypomethylation in SoS were observed, hypomethylation of imprinted DMRs in SoS was suggested. We explored DNA methylation status of 28 imprinted DMRs in 31 SoS patients with NSD1 defect and found that hypomethylation of IGF2-DMR0 and IG-DMR in a substantial proportion of SoS patients. Luciferase assay revealed that IGF2-DMR0 enhanced transcription from the IGF2 P0 promoter but not the P3 and P4 promoters. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) revealed active enhancer histone modifications at IGF2-DMR0, with high enrichment of H3K4me1 and H3 lysine 27 acetylation (H3K27ac). CRISPR-Cas9 epigenome editing revealed that specifically induced hypomethylation at IGF2-DMR0 increased transcription from the P0 promoter but not the P3 and P4 promoters. NSD1 knockdown suggested that NSD1 targeted IGF2-DMR0; however, IGF2-DMR0 DNA methylation and IGF2 expression were unaltered. This study could elucidate the function of IGF2-DMR0 as a DNA methylation dependent, P0 promoter-specific enhancer. NSD1 may play a role in the establishment or maintenance of IGF2-DMR0 methylation during the postimplantation period.},
}

CRISPR-Cas Systems
Child
Child, Preschool
*DNA Methylation
Enhancer Elements, Genetic
Epigenome
Female
Gene Deletion
Genomic Imprinting
HEK293 Cells
Histone-Lysine N-Methyltransferase/*genetics
Histones/chemistry
Humans
Infant
Infant, Newborn
Insulin-Like Growth Factor II/*genetics
Lysine/chemistry
Male
Phenotype
Point Mutation
Promoter Regions, Genetic
Sotos Syndrome/*genetics

@article {pmid31907374,
year = {2020},
author = {Lebar, T and Lainšček, D and Merljak, E and Aupič, J and Jerala, R},
title = {A tunable orthogonal coiled-coil interaction toolbox for engineering mammalian cells.},
journal = {Nature chemical biology},
volume = {16},
number = {5},
pages = {513-519},
pmid = {31907374},
issn = {1552-4469},
support = {787115/ERC_/European Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems ; Genetic Engineering/*methods ; HEK293 Cells ; Humans ; Mammals/genetics ; Mice ; NIH 3T3 Cells ; Peptides/*genetics/*metabolism ; Recombinant Proteins/genetics/metabolism ; Transcription, Genetic ; },
abstract = {Protein interactions guide most cellular processes. Orthogonal hetero-specific protein-protein interaction domains may facilitate better control of engineered biological systems. Here, we report a tunable de novo designed set of orthogonal coiled-coil (CC) peptide heterodimers (called the NICP set) and its application for the regulation of diverse cellular processes, from cellular localization to transcriptional regulation. We demonstrate the application of CC pairs for multiplex localization in single cells and exploit the interaction strength and variable stoichiometry of CC peptides for tuning of gene transcription strength. A concatenated CC peptide tag (CCC-tag) was used to construct highly potent CRISPR-dCas9-based transcriptional activators and to amplify the response of light and small molecule-inducible transcription in cell culture as well as in vivo. The NICP set and its implementations represent a valuable toolbox of minimally disruptive modules for the recruitment of versatile functional domains and regulation of cellular processes for synthetic biology.},
}

Animals
CRISPR-Cas Systems
Genetic Engineering/*methods
HEK293 Cells
Humans
Mammals/genetics
Mice
NIH 3T3 Cells
Peptides/*genetics/*metabolism
Recombinant Proteins/genetics/metabolism
Transcription, Genetic