@article {pmid31411686,
year = {2019},
author = {Tang, Z and Chen, S and Chen, A and He, B and Zhou, Y and Chai, G and Guo, F and Huang, J},
title = {CasPDB: an integrated and annotated database for Cas proteins from bacteria and archaea.},
journal = {Database : the journal of biological databases and curation},
volume = {2019},
number = {},
pages = {},
doi = {10.1093/database/baz093},
pmid = {31411686},
issn = {1758-0463},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) constitute CRISPR-Cas systems, which are antiphage immune systems present in numerous bacterial and most archaeal species. In recent years, CRISPR-Cas systems have been developed into reliable and powerful genome editing tools. Nevertheless, finding similar or better tools from bacteria or archaea remains crucial. This requires the exploration of different CRISPR systems, identification and characterization new Cas proteins. Archives tailored for Cas proteins are urgently needed and necessitate the prediction and grouping of Cas proteins into an information center with all available experimental evidence. Here, we constructed Cas Protein Data Bank (CasPDB), an integrated and annotated online database for Cas proteins from bacteria and archaea. The CasPDB database contains 287 reviewed Cas proteins, 257 745 putative Cas proteins and 3593 Cas operons from 32 023 bacteria species and 1802 archaea species. The database can be freely browsed and searched. The CasPDB web interface also represents all the 3593 putative Cas operons and its components. Among these operons, 328 are members of the type II CRISPR-Cas system.},
}

@article {pmid31409685,
year = {2019},
author = {Hullahalli, K and Rodrigues, M and Nguyen, UT and Palmer, K},
title = {Erratum for Hullahalli et al., "An Attenuated CRISPR-Cas System in Enterococcus faecalis Permits DNA Acquisition".},
journal = {mBio},
volume = {10},
number = {4},
pages = {},
doi = {10.1128/mBio.01775-19},
pmid = {31409685},
issn = {2150-7511},
}

@article {pmid31409682,
year = {2019},
author = {Gloag, ES and Marshall, CW and Snyder, D and Lewin, GR and Harris, JS and Santos-Lopez, A and Chaney, SB and Whiteley, M and Cooper, VS and Wozniak, DJ},
title = {Pseudomonas aeruginosa Interstrain Dynamics and Selection of Hyperbiofilm Mutants during a Chronic Infection.},
journal = {mBio},
volume = {10},
number = {4},
pages = {},
doi = {10.1128/mBio.01698-19},
pmid = {31409682},
issn = {2150-7511},
abstract = {Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Capturing this initial dynamic is critical for identifying the first adaptations that drive pathogenesis. Here we used a porcine full-thickness burn wound model of chronic infection to study the evolutionary dynamics of diverse Pseudomonas aeruginosa infections. Wounds were infected with a mixed community of six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days postinfection. Hyperbiofilm-forming rugose small-colony variants (RSCVs) were the earliest and predominant phenotypic variant. These variants were detected on day 3 and persisted, with the majority evolved from PA14-1. Whole-genome sequencing of PA14-1 RSCV isolates revealed driver mutations exclusively in the wsp pathway, conferring hyperbiofilm phenotypes. Several of the wsp mutant RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) that was also present in the inoculum. These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. Rather than being a side effect of chronicity, the rapid rise of RSCVs in wounds is evidence of positive selection on the Wsp chemosensory system to produce mutants with elevated biofilm formation capacity. We predict that RSCVs provide a level of phenotypic diversity to the infecting bacterial community and are common, early adaptations during infections. This would likely have significant consequences for clinical outcomes.IMPORTANCE Bacteria adapt to infections by evolving variants that are more fit and persistent. These recalcitrant variants are typically observed in chronic infections. However, it is unclear when and why these variants evolve. To address these questions, we used a porcine chronic wound model to study the evolutionary dynamics of Pseudomonas aeruginosa in a mixed-strain infection. We isolated hyperbiofilm variants that persisted early in the infection. Interstrain interactions were also observed, where adapted variants acquired CRISPR-mediated immunity to phages. We show that when initiating infection, P. aeruginosa experiences strong positive selection for hyperbiofilm phenotypes produced by mutants of a single chemosensory system, the Wsp pathway. We predict that hyperbiofilm variants are early adaptations to infection and that interstrain interactions may influence bacterial burden and infection outcomes.},
}

@article {pmid31307968,
year = {2019},
author = {Niu, XR and Yin, SM and Chen, X and Shao, TT and Li, DL},
title = {[Gene editing technology and its recent progress in disease therapy].},
journal = {Yi chuan = Hereditas},
volume = {41},
number = {7},
pages = {582-598},
doi = {10.16288/j.yczz.19-102},
pmid = {31307968},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems ; Disease ; Epigenesis, Genetic ; Gene Editing/*trends ; *Genetic Therapy ; Humans ; },
abstract = {Gene editing is a genetic manipulation technology which utilizes bacterial nucleases to accurately and efficiently modify DNA or RNA. Gene editing has broad applications in basic research, breeding, and drug screening, and it is gaining validity and applicability to the therapy of many diseases especially genetic-based disease. In this review, we summarize the development of gene editing technology, its different strategies and applications in the treatment of disease, and the research of gene editing therapy for genetic diseases (including base editor and epigenetic regulation) in the treatment of disorders and diseases of the blood system, liver, muscle and nervous system. Finally, we discuss the future development prospects of gene editing therapy.},
}

*CRISPR-Cas Systems
Disease
Epigenesis, Genetic
Gene Editing/*trends
*Genetic Therapy
Humans

@article {pmid31106778,
year = {2019},
author = {Wang, J and Huang, J and Xu, R},
title = {[Seamless genome editing in Drosophila by combining CRISPR/Cas9 and piggyBac technologies].},
journal = {Yi chuan = Hereditas},
volume = {41},
number = {5},
pages = {422-429},
doi = {10.16288/j.yczz.18-345},
pmid = {31106778},
issn = {0253-9772},
mesh = {Animals ; *CRISPR-Cas Systems ; Drosophila/*genetics ; *Gene Editing ; *Genome, Insect ; Transposases/*genetics ; },
abstract = {The type2 CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated protein 9) is an efficient RNA-guided genome-editing technique. Guided by sgRNA, the Cas9 endonuclease generates site-specific double-stranded breaks (DSB) at specific site, which is amenable to repair by homology-directed repair (HDR) to generate a designed knock-out or knock-in transgene. In combination with CRISPR/Cas9 and Cre/loxP or FLP/FRT system, efficient gene targeting can be achieved, and meanwhile screening markers introduced can be readily removed except a 34-base pair residual fragment. Thus, difficulties remain in accurate editing of the genome without introducing any extraneous sequences. In human induced pluripotent stem cells (iPSCs), a two-step strategy has been developed using CRISPR/Cas9 and the piggyBac system to establish a seamless genomic editing, in which CRISPR/Cas9 is initially used to introduce mutations along with screening markers by HDR, then the markers are precisely excised by piggyBac transposase. Using this strategy, we have successfully transformed the tyrosine to cysteine at position 21 within the 18th exon of the CG4894 gene in the Drosophila genome without introducing any extraneous sequence. Hence, this strategy provides more options for precise and seamless editing of the Drosophila genome.},
}

Animals
*CRISPR-Cas Systems
Drosophila/*genetics
*Gene Editing
*Genome, Insect
Transposases/*genetics

@article {pmid30205875,
year = {2018},
author = {Bellin, M},
title = {Crispr/Cas9 homologous recombination (HR).},
journal = {Drug discovery today. Technologies},
volume = {28},
number = {},
pages = {1-2},
doi = {10.1016/j.ddtec.2018.08.006},
pmid = {30205875},
issn = {1740-6749},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; *Homologous Recombination ; Humans ; },
}

*CRISPR-Cas Systems
Gene Editing/*methods
*Homologous Recombination
Humans

@article {pmid30005866,
year = {2018},
author = {Cromer, MK and Vaidyanathan, S and Ryan, DE and Curry, B and Lucas, AB and Camarena, J and Kaushik, M and Hay, SR and Martin, RM and Steinfeld, I and Bak, RO and Dever, DP and Hendel, A and Bruhn, L and Porteus, MH},
title = {Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {26},
number = {10},
pages = {2431-2442},
pmid = {30005866},
issn = {1525-0024},
support = {R01 AI097320/AI/NIAID NIH HHS/United States ; R01 AI120766/AI/NIAID NIH HHS/United States ; T32 HL120824/HL/NHLBI NIH HHS/United States ; },
mesh = {Antigens, CD34/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/genetics ; Electroporation ; Gene Editing/methods ; Gene Expression Regulation/genetics ; *Genetic Therapy ; Genetic Vectors/*genetics/therapeutic use ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells/drug effects ; Humans ; Microarray Analysis/*methods ; Parvovirinae/*genetics ; Stem Cells/drug effects ; },
abstract = {Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34+ hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.},
}

Antigens, CD34/genetics
CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems/genetics
Electroporation
Gene Editing/methods
Gene Expression Regulation/genetics
*Genetic Therapy
Genetic Vectors/*genetics/therapeutic use
Hematopoietic Stem Cell Transplantation
Hematopoietic Stem Cells/drug effects
Humans
Microarray Analysis/*methods
Parvovirinae/*genetics
Stem Cells/drug effects

@article {pmid29673923,
year = {2019},
author = {Zhan, T and Rindtorff, N and Betge, J and Ebert, MP and Boutros, M},
title = {CRISPR/Cas9 for cancer research and therapy.},
journal = {Seminars in cancer biology},
volume = {55},
number = {},
pages = {106-119},
doi = {10.1016/j.semcancer.2018.04.001},
pmid = {29673923},
issn = {1096-3650},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/trends ; Genome, Human/genetics ; Humans ; Neoplasms/*genetics/therapy ; Research/*trends ; },
abstract = {CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review current applications of the CRISPR/Cas9 technology for cancer research and therapy. We describe novel Cas9 variants and how they are used in functional genomics to discover novel cancer-specific vulnerabilities. Furthermore, we highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials that apply CRISPR/Cas9 as a therapeutic approach against cancer.},
}

CRISPR-Cas Systems/*genetics
Gene Editing/trends
Genome, Human/genetics
Humans
Neoplasms/*genetics/therapy
Research/*trends

@article {pmid31407519,
year = {2019},
author = {Li, QV and Rosen, BP and Huangfu, D},
title = {Decoding pluripotency: Genetic screens to interrogate the acquisition, maintenance, and exit of pluripotency.},
journal = {Wiley interdisciplinary reviews. Systems biology and medicine},
volume = {},
number = {},
pages = {e1464},
doi = {10.1002/wsbm.1464},
pmid = {31407519},
issn = {1939-005X},
support = {R01DK096239/DK/NIDDK NIH HHS/United States ; T32GM008539//NIH T32 Training Grant/ ; P30 CA008748/CA/NCI NIH HHS/United States ; 1-19-IBS-125//American Diabetes Association/ ; 3-SRA-2017-364-S-B//Juvenile Diabetes Research Foundation International/ ; #2016-004//Tri-Institutional Stem Cell Initiative/ ; 2016-032//Tri-Institutional Stem Cell Initiative/ ; C32593GG//New York State Stem Cell Science/ ; C029156//New York State Stem Cell Science/ ; C029567//New York State Stem Cell Science/ ; },
abstract = {Pluripotent stem cells have the ability to unlimitedly self-renew and differentiate to any somatic cell lineage. A number of systems biology approaches have been used to define this pluripotent state. Complementary to systems level characterization, genetic screens offer a unique avenue to functionally interrogate the pluripotent state and identify the key players in pluripotency acquisition and maintenance, exit of pluripotency, and lineage differentiation. Here we review how genetic screens have helped us decode pluripotency regulation. We will summarize results from RNA interference (RNAi) based screens, discuss recent advances in CRISPR/Cas-based genetic perturbation methods, and how these advances have made it possible to more comprehensively interrogate pluripotency and differentiation through genetic screens. Such investigations will not only provide a better understanding of this unique developmental state, but may enhance our ability to use pluripotent stem cells as an experimental model to study human development and disease progression. Functional interrogation of pluripotency also provides a valuable roadmap for utilizing genetic perturbation to gain systems level understanding of additional cellular states, from later stages of development to pathological disease states. This article is categorized under: Developmental Biology > Stem Cell Biology and Regeneration Developmental Biology > Developmental Processes in Health and Disease Biological Mechanisms > Cell Fates.},
}

@article {pmid31403072,
year = {2019},
author = {Hanewich-Hollatz, MH and Chen, Z and Hochrein, LM and Huang, J and Pierce, NA},
title = {Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology.},
journal = {ACS central science},
volume = {5},
number = {7},
pages = {1241-1249},
doi = {10.1021/acscentsci.9b00340},
pmid = {31403072},
issn = {2374-7943},
abstract = {A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF "terminator switch" mechanism, ≈15-fold for an ON → OFF "splinted switch" mechanism, and ≈3-fold for an OFF → ON "toehold switch" mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.},
}

@article {pmid31203891,
year = {2019},
author = {Huang, Y and Xuan, H and Yang, C and Guo, N and Wang, H and Zhao, J and Xing, H},
title = {GmHsp90A2 is involved in soybean heat stress as a positive regulator.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {285},
number = {},
pages = {26-33},
doi = {10.1016/j.plantsci.2019.04.016},
pmid = {31203891},
issn = {1873-2259},
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Chlorophyll/metabolism ; Gene Editing ; Gene Expression Regulation, Plant ; Heat-Shock Proteins/*physiology ; Heat-Shock Response ; Malondialdehyde/metabolism ; Plant Proteins/*physiology ; Plants, Genetically Modified ; Promoter Regions, Genetic ; Real-Time Polymerase Chain Reaction ; Soybeans/*metabolism/physiology ; Two-Hybrid System Techniques ; },
abstract = {Heat shock protein 90 s (Hsp90s), one of the most conserved and abundant molecular chaperones, is an essential component of the protective stress response. A previous study reported at least 12 genes in the GmHsp90s family in soybean and that GmHsp90A2 overexpression enhanced thermotolerance in Arabidopsis thaliana. Here, we investigate the roles of GmHsp90A2 in soybean by utilizing stable transgenic soybean lines overexpressing GmHsp90A2 and mutant lines generated by the CRISPR/Cas9 system. The results showed that compared with wild-type plants (WT) and empty vector control plants (VC), T3 transgenic soybean plants overexpressing GmHsp90A2 exhibited increased tolerance to heat stress through higher chlorophyll and lower malondialdehyde (MDA) contents in plants. Conversely, reduced chlorophyll and increased MDA contents in T2 homozygous GmHsp90A2-knockout mutants indicated decreased tolerance to heat stress. GmHsp90A2 was found to interact with GmHsp90A1 in yeast two-hybrid assays. Furthermore, subcellular localization analyses revealed that GmHsp90A2 was localized to the cytoplasm and cell membrane; as shown by bimolecular fluorescence complementation (BiFC) assays, GmHsp90A2 interacted with GmHsp90A1 in the nucleus and cytoplasm and cell membrane. Hence, we conclude that GmHsp90A1 is able to bind to GmHsp90A2 to form a complex and that this complex enters the nucleus. In summary, GmHsp90A2 might respond to heat stress and positively regulate thermotolerance by interacting with GmHsp90A1.},
}

CRISPR-Associated Protein 9
CRISPR-Cas Systems
Chlorophyll/metabolism
Gene Editing
Gene Expression Regulation, Plant
Heat-Shock Proteins/*physiology
Heat-Shock Response
Malondialdehyde/metabolism
Plant Proteins/*physiology
Plants, Genetically Modified
Promoter Regions, Genetic
Real-Time Polymerase Chain Reaction
Soybeans/*metabolism/physiology
Two-Hybrid System Techniques

@article {pmid31013576,
year = {2019},
author = {Albrechtsen, R and Wewer Albrechtsen, NJ and Gnosa, S and Schwarz, J and Dyrskjøt, L and Kveiborg, M},
title = {Identification of ADAM12 as a Novel Basigin Sheddase.},
journal = {International journal of molecular sciences},
volume = {20},
number = {8},
pages = {},
doi = {10.3390/ijms20081957},
pmid = {31013576},
issn = {1422-0067},
support = {R146-A9211-16-S2//Danish Cancer Society/International ; },
mesh = {ADAM12 Protein/chemistry/genetics/*metabolism ; Amino Acid Sequence ; Basigin/chemistry/genetics/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Gene Expression ; Gene Knockdown Techniques ; Genes, Reporter ; Humans ; Mutation ; Substrate Specificity ; },
abstract = {The transmembrane glycoprotein basigin, a member of the immunoglobulin superfamily, stimulates matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) degradation and thereby drives cancer cell invasion. Basigin is proteolytically shed from the cell surface and high concentrations of soluble basigin in the blood dictates poor prognosis in cancer patients. A positive correlation between basigin and a disintegrin and metalloproteinase (ADAM)-12 in serum from prostate cancer patients has been reported. Yet, the functional relevance of this correlation is unknown. Here, we show that ADAM12 interacts with basigin and cleaves it in the juxtamembrane region. Specifically, overexpression of ADAM12 increases ectodomain shedding of an alkaline phosphatase-tagged basigin reporter protein from the cell surface. Moreover, CRISPR/Cas9-mediated knockout of ADAM12 in human HeLa carcinoma cells results in reduced shedding of the basigin reporter, which can be rescued by ADAM12 re-expression. We detected endogenous basigin fragments, corresponding to the expected size of the ADAM12-generated ectodomain, in conditioned media from ADAM12 expressing cancer cell-lines, as well as serum samples from a healthy pregnant donor and five bladder cancer patients, known to contain high ADAM12 levels. Supporting the cancer relevance of our findings, we identified several cancer-associated mutations in the basigin membrane proximal region. Subsequent in vitro expression showed that some of these mutants are more prone to ADAM12-mediated shedding and that the shed ectodomain can enhance gelatin degradation by cancer cells. In conclusion, we identified ADAM12 as a novel basigin sheddase with a potential implication in cancer.},
}

ADAM12 Protein/chemistry/genetics/*metabolism
Amino Acid Sequence
Basigin/chemistry/genetics/*metabolism
CRISPR-Cas Systems
Cell Line
Gene Expression
Gene Knockdown Techniques
Genes, Reporter
Humans
Mutation
Substrate Specificity

@article {pmid30980103,
year = {2019},
author = {Zhai, Y and Cai, S and Hu, L and Yang, Y and Amoo, O and Fan, C and Zhou, Y},
title = {CRISPR/Cas9-mediated genome editing reveals differences in the contribution of INDEHISCENT homologues to pod shatter resistance in Brassica napus L.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {132},
number = {7},
pages = {2111-2123},
doi = {10.1007/s00122-019-03341-0},
pmid = {30980103},
issn = {1432-2242},
support = {31371240//National Natural Science Foundation of China/ ; 31671279//National Natural Science Foundation of China/ ; 2015CB150202//National Key Basic Research Program of China/ ; 2662015PY172//the Fundamental Research Funds for the Central Universities/ ; },
mesh = {Alleles ; Basic Helix-Loop-Helix Transcription Factors/*genetics ; Brassica napus/*genetics ; *CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; Genes, Plant ; Phenotype ; Plant Proteins/*genetics ; Plants, Genetically Modified ; Seeds/*physiology ; },
abstract = {The INDEHISCENT (IND) and ALCATRAZ (ALC) gene homologues have been reported to be essential for dehiscence of fruits in Brassica species. But their functions for pod shatter resistance in Brassica napus, an important oil crops, are not well understood. Here, we assessed the functions of these two genes in rapeseed using CRISPR/Cas9 technology. The induced mutations were stably transmitted to successive generations, and a variety of homozygous mutants with loss-of-function alleles of the target genes were obtained for phenotyping. The results showed that the function of BnIND gene is essential for pod shatter and highly conserved in Brassica species, whereas the BnALC gene appears to have limited potential for rapeseed shatter resistance. The homoeologous copies of the BnIND gene have partially redundant roles in rapeseed pod shatter, with BnA03.IND exhibiting higher contributions than BnC03.IND. Analysis of data obtained from the gene expression and sequence variations of gene copies revealed that cis-regulatory divergences alter gene expression and underlie the functional differentiation of BnIND homologues. Collectively, our results generate valuable resources for rapeseed breeding programs, and more importantly provide a strategy to improve polyploid crops.},
}

Alleles
Basic Helix-Loop-Helix Transcription Factors/*genetics
Brassica napus/*genetics
*CRISPR-Cas Systems
Gene Editing
Gene Knockout Techniques
Genes, Plant
Phenotype
Plant Proteins/*genetics
Plants, Genetically Modified
Seeds/*physiology

@article {pmid30809070,
year = {2019},
author = {Cyranoski, D},
title = {The CRISPR-baby scandal: what's next for human gene-editing.},
journal = {Nature},
volume = {566},
number = {7745},
pages = {440-442},
doi = {10.1038/d41586-019-00673-1},
pmid = {30809070},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems/*genetics ; China ; Dissent and Disputes ; Embryo Research/ethics ; Embryonic Development/genetics ; *Ethics, Research ; Female ; Gene Editing/*ethics/*legislation & jurisprudence ; Genetic Diseases, Inborn/genetics ; Germ-Line Mutation/*genetics ; HIV Infections/blood/virology ; Humans ; Infant, Newborn ; International Cooperation/legislation & jurisprudence ; Patient Safety ; Receptors, CCR5/deficiency/genetics ; Research Personnel/*ethics/legislation & jurisprudence ; Scientific Misconduct/*legislation & jurisprudence ; Twins/*genetics ; },
}

CRISPR-Cas Systems/*genetics
China
Dissent and Disputes
Embryo Research/ethics
Embryonic Development/genetics
*Ethics, Research
Female
Gene Editing/*ethics/*legislation & jurisprudence
Genetic Diseases, Inborn/genetics
Germ-Line Mutation/*genetics
HIV Infections/blood/virology
Humans
Infant, Newborn
International Cooperation/legislation & jurisprudence
Patient Safety
Receptors, CCR5/deficiency/genetics
Research Personnel/*ethics/legislation & jurisprudence
Scientific Misconduct/*legislation & jurisprudence
Twins/*genetics

@article {pmid30765415,
year = {2019},
author = {Kuil, LE and Oosterhof, N and Geurts, SN and van der Linde, HC and Meijering, E and van Ham, TJ},
title = {Reverse genetic screen reveals that Il34 facilitates yolk sac macrophage distribution and seeding of the brain.},
journal = {Disease models & mechanisms},
volume = {12},
number = {3},
pages = {},
doi = {10.1242/dmm.037762},
pmid = {30765415},
issn = {1754-8411},
mesh = {Animals ; Animals, Genetically Modified ; Base Sequence ; Brain/growth & development/*metabolism ; CRISPR-Cas Systems/genetics ; Cell Count ; Cell Proliferation ; *Genetic Testing ; Interleukins/*metabolism ; Macrophages/*metabolism ; Microglia/metabolism ; Mutation/genetics ; *Reverse Genetics ; Yolk Sac/*metabolism ; Zebrafish/*metabolism ; },
abstract = {Microglia are brain-resident macrophages, which have specialized functions important in brain development and in disease. They colonize the brain in early embryonic stages, but few factors that drive the migration of yolk sac macrophages (YSMs) into the embryonic brain, or regulate their acquisition of specialized properties, are currently known. Here, we present a CRISPR/Cas9-based in vivo reverse genetic screening pipeline to identify new microglia regulators using zebrafish. Zebrafish larvae are particularly suitable due to their external development, transparency and conserved microglia features. We targeted putative microglia regulators, by Cas9/gRNA complex injections, followed by Neutral-Red-based visualization of microglia. Microglia were quantified automatically in 3-day-old larvae using a software tool we called SpotNGlia. We identified that loss of zebrafish colony-stimulating factor 1 receptor (Csf1r) ligand, Il34, caused reduced microglia numbers. Previous studies on the role of IL34 in microglia development in vivo were ambiguous. Our data, and a concurrent paper, show that, in zebrafish, il34 is required during the earliest seeding of the brain by microglia. Our data also indicate that Il34 is required for YSM distribution to other organs. Disruption of the other Csf1r ligand, Csf1, did not reduce microglia numbers in mutants, whereas overexpression increased the number of microglia. This shows that Csf1 can influence microglia numbers, but might not be essential for the early seeding of the brain. In all, we identified il34 as a modifier of microglia colonization, by affecting distribution of YSMs to target organs, validating our reverse genetic screening pipeline in zebrafish.This article has an associated First Person interview with the joint first authors of the paper.},
}

Animals
Animals, Genetically Modified
Base Sequence
Brain/growth & development/*metabolism
CRISPR-Cas Systems/genetics
Cell Count
Cell Proliferation
*Genetic Testing
Interleukins/*metabolism
Macrophages/*metabolism
Microglia/metabolism
Mutation/genetics
*Reverse Genetics
Yolk Sac/*metabolism
Zebrafish/*metabolism

@article {pmid30755408,
year = {2019},
author = {Hayashi, A and Tanaka, K},
title = {Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {4},
pages = {1153-1163},
doi = {10.1534/g3.118.200976},
pmid = {30755408},
issn = {2160-1836},
mesh = {CRISPR-Cas Systems ; Cloning, Molecular ; Gene Editing/*methods ; Genetic Vectors ; Mutagenesis, Site-Directed ; Point Mutation ; Schizosaccharomyces/*genetics ; Schizosaccharomyces pombe Proteins/chemistry/*genetics ; },
abstract = {The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the noncanonical homology-directed DNA repair (HDR) based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the short-homology-mediated genome editing and found that the HDR pathway provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing short homology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.},
}

CRISPR-Cas Systems
Cloning, Molecular
Gene Editing/*methods
Genetic Vectors
Mutagenesis, Site-Directed
Point Mutation
Schizosaccharomyces/*genetics
Schizosaccharomyces pombe Proteins/chemistry/*genetics

@article {pmid30723103,
year = {2019},
author = {Hollerer, I and Barker, JC and Jorgensen, V and Tresenrider, A and Dugast-Darzacq, C and Chan, LY and Darzacq, X and Tjian, R and Ünal, E and Brar, GA},
title = {Evidence for an Integrated Gene Repression Mechanism Based on mRNA Isoform Toggling in Human Cells.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {4},
pages = {1045-1053},
doi = {10.1534/g3.118.200802},
pmid = {30723103},
issn = {2160-1836},
support = {DP2 AG055946/AG/NIA NIH HHS/United States ; DP2 GM119138/GM/NIGMS NIH HHS/United States ; U01 DA047729/DA/NIDA NIH HHS/United States ; 003061/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {CRISPR-Cas Systems ; Chromatin Immunoprecipitation ; *Gene Expression Regulation ; Gene Knockdown Techniques ; Histones/metabolism ; Humans ; MCF-7 Cells ; *Models, Genetic ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-mdm2/*genetics ; },
abstract = {We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference collaborate to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that prevented translation of the main ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2 We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs.},
}

CRISPR-Cas Systems
Chromatin Immunoprecipitation
*Gene Expression Regulation
Gene Knockdown Techniques
Histones/metabolism
Humans
MCF-7 Cells
*Models, Genetic
Promoter Regions, Genetic
Proto-Oncogene Proteins c-mdm2/*genetics

@article {pmid30696702,
year = {2019},
author = {Voutev, R and Mann, RS},
title = {TP901-1 Phage Recombinase Facilitates Genome Engineering in Drosophila melanogaster.},
journal = {G3 (Bethesda, Md.)},
volume = {9},
number = {4},
pages = {983-986},
doi = {10.1534/g3.119.0002},
pmid = {30696702},
issn = {2160-1836},
support = {R35 GM118336/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Bacteriophages/*genetics ; CRISPR-Cas Systems ; Drosophila melanogaster/*genetics ; Genetic Engineering/*methods ; Genetic Vectors ; Genome, Insect ; Recombinases/genetics/*physiology ; Recombination, Genetic ; },
abstract = {Molecular biology techniques have a large impact on biomedical research and the availability of diverse tools to perform genome manipulations advances the ease of executing complicated genetic research. Here, we introduce in the fruit fly another such tool by harnessing the phage recombinase TP901-1 to perform site-directed recombination that leads to recombinase-mediated cassette exchange (RMCE). The TP901-1 system complements already existing recombination systems and enhances genome engineering in the fruit fly and other organisms.},
}

Animals
Bacteriophages/*genetics
CRISPR-Cas Systems
Drosophila melanogaster/*genetics
Genetic Engineering/*methods
Genetic Vectors
Genome, Insect
Recombinases/genetics/*physiology
Recombination, Genetic

@article {pmid30686786,
year = {2019},
author = {Liu, H and Li, DM and Zhu, LY and Lai, LJ and Yan, WY and Lu, YH and Wei, Y and Huang, YQ and Fang, M and Su, YG and Yang, F and Shu, W},
title = {[Research on the knockout of LMNA gene by CRISPR/Cas9 system in human cell lines].},
journal = {Yi chuan = Hereditas},
volume = {41},
number = {1},
pages = {66-75},
doi = {10.16288/j.yczz.18-146},
pmid = {30686786},
issn = {0253-9772},
mesh = {*CRISPR-Cas Systems ; Cell Nucleus ; *Gene Knockout Techniques ; HEK293 Cells ; Hep G2 Cells ; Humans ; Lamin Type A/*genetics ; Mutation ; },
abstract = {The LMNA gene encodes the nuclear Lamin A and Lamin C proteins, and is related to nuclear membrane organization, genome stability and cell differentiation. Abnormal expression of LMNA is ubiquitous in human tumors, and its mutation leads to various forms of laminopathies, including Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), and Hutchinson-Gliford progeria syndrome (HGPS). To further determine the functions of the LMNA gene in cellular physiology, the present study used the CRISPR/Cas9 technique to edit the LMNA gene of 293T and HepG2 cells in vitro, which resulted in two stable LMNA gene knockout (LMNA KO) cell lines. Compared to the respective wild type cells, the LMNA KO cell lines showed decrease in proliferation ability, increase in apoptosis, alteration in cellular morphology and uneven structures in the nucleus membrane. In this study, we report for the first time the results on the construction of LMNA KO immortalized cell lines and characterization of their morphological changes, thereby laying the foundation for the further studies of the LMNA gene functions and pathogenic mutations.},
}

*CRISPR-Cas Systems
Cell Nucleus
*Gene Knockout Techniques
HEK293 Cells
Hep G2 Cells
Humans
Lamin Type A/*genetics
Mutation

@article {pmid30472287,
year = {2019},
author = {Durand, GA and Raoult, D and Dubourg, G},
title = {Antibiotic discovery: history, methods and perspectives.},
journal = {International journal of antimicrobial agents},
volume = {53},
number = {4},
pages = {371-382},
doi = {10.1016/j.ijantimicag.2018.11.010},
pmid = {30472287},
issn = {1872-7913},
mesh = {Actinobacteria/metabolism ; Anti-Bacterial Agents/*isolation & purification ; CRISPR-Cas Systems ; Chromosome Mapping ; Drug Discovery/*methods ; Drug Resistance, Multiple, Bacterial/genetics ; Fungi/metabolism ; Gastrointestinal Microbiome/*drug effects ; Humans ; },
abstract = {Antimicrobial resistance is considered a major public-health issue. Policies recommended by the World Health Organization (WHO) include research on new antibiotics. No new class has been discovered since daptomycin and linezolid in the 1980s, and only optimisation or combination of already known compounds has been recently commercialised. Antibiotics are natural products of soil-living organisms. Actinobacteria and fungi are the source of approximately two-thirds of the antimicrobial agents currently used in human medicine; they were mainly discovered during the golden age of antibiotic discovery. This era declined after the 1970s owing to the difficulty of cultivating fastidious bacterial species under laboratory conditions. Various strategies, such as rational drug design, to date have not led to the discovery of new antimicrobial agents. However, new promising approaches, e.g. genome mining or CRISPR-Cas9, are now being developed. The recent rebirth of culture methods from complex samples has, as a matter of fact, permitted the discovery of teixobactin from a new species isolated from soil. Recently, many biosynthetic gene clusters were identified from human-associated microbiota, especially from the gut and oral cavity. For example, the antimicrobial lugdunin was recently discovered in the oral cavity. The repertoire of human gut microbiota has recently substantially increased, with the discovery of hundreds of new species. Exploration of the repertoire of prokaryotes associated with humans using genome mining or newer culture approaches could be promising strategies for discovering new classes of antibiotics.},
}

Actinobacteria/metabolism
Anti-Bacterial Agents/*isolation & purification
CRISPR-Cas Systems
Chromosome Mapping
Drug Discovery/*methods
Drug Resistance, Multiple, Bacterial/genetics
Fungi/metabolism
Gastrointestinal Microbiome/*drug effects
Humans

@article {pmid30030848,
year = {2018},
author = {Lavender, P and Kelly, A and Hendy, E and McErlean, P},
title = {CRISPR-based reagents to study the influence of the epigenome on gene expression.},
journal = {Clinical and experimental immunology},
volume = {194},
number = {1},
pages = {9-16},
pmid = {30030848},
issn = {1365-2249},
mesh = {CRISPR-Cas Systems/*genetics ; Chromatin/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Epigenesis, Genetic/*genetics ; Gene Editing/*methods ; Gene Expression/genetics ; Gene Expression Regulation/*genetics ; Histone Code/genetics ; Humans ; },
abstract = {The use of epigenome editing is set to expand our knowledge of how epigenetic landscapes facilitate gene expression capacity within a given cell. As epigenetic landscape profiling in health and disease becomes more commonplace, so does the requirement to assess the functional impact that particular regulatory domains and DNA methylation profiles have upon gene expression capacity. That functional assessment is particularly pertinent when analysing epigenomes in disease states where the reversible nature of histone and DNA modification might yield plausible therapeutic targets. In this review we discuss first the nature of the epigenetic landscape, secondly the types of factors that deposit and erase the various modifications, consider how modifications transduce their signals, and lastly address current tools for experimental epigenome editing with particular emphasis on the immune system.},
}

CRISPR-Cas Systems/*genetics
Chromatin/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Epigenesis, Genetic/*genetics
Gene Editing/*methods
Gene Expression/genetics
Gene Expression Regulation/*genetics
Histone Code/genetics
Humans

@article {pmid29947794,
year = {2018},
author = {Dastidar, S and Ardui, S and Singh, K and Majumdar, D and Nair, N and Fu, Y and Reyon, D and Samara, E and Gerli, MFM and Klein, AF and De Schrijver, W and Tipanee, J and Seneca, S and Tulalamba, W and Wang, H and Chai, YC and In't Veld, P and Furling, D and Tedesco, FS and Vermeesch, JR and Joung, JK and Chuah, MK and VandenDriessche, T},
title = {Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.},
journal = {Nucleic acids research},
volume = {46},
number = {16},
pages = {8275-8298},
pmid = {29947794},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems ; Cells, Cultured ; Child ; Female ; Gene Editing/*methods ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Middle Aged ; Muscle Development/genetics ; Myoblasts/*metabolism ; Myotonic Dystrophy/*genetics/metabolism/pathology ; Trinucleotide Repeat Expansion/*genetics ; },
abstract = {CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.},
}

*CRISPR-Cas Systems
Cells, Cultured
Child
Female
Gene Editing/*methods
Humans
Induced Pluripotent Stem Cells/*metabolism
Middle Aged
Muscle Development/genetics
Myoblasts/*metabolism
Myotonic Dystrophy/*genetics/metabolism/pathology
Trinucleotide Repeat Expansion/*genetics

@article {pmid29939295,
year = {2018},
author = {Takahashi, T and Nakano, Y and Onomoto, K and Murakami, F and Komori, C and Suzuki, Y and Yoneyama, M and Ui-Tei, K},
title = {LGP2 virus sensor regulates gene expression network mediated by TRBP-bound microRNAs.},
journal = {Nucleic acids research},
volume = {46},
number = {17},
pages = {9134-9147},
pmid = {29939295},
issn = {1362-4962},
mesh = {CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation/genetics ; Gene Knockdown Techniques ; Gene Regulatory Networks/*genetics ; HeLa Cells ; Humans ; MicroRNAs/*metabolism/physiology ; RNA Helicases/*physiology ; RNA Interference ; RNA Viruses/genetics/metabolism ; RNA-Binding Proteins/*metabolism/physiology ; Signal Transduction ; },
abstract = {Here we show that laboratory of genetics and physiology 2 (LGP2) virus sensor protein regulates gene expression network of endogenous genes mediated by TAR-RNA binding protein (TRBP)-bound microRNAs (miRNAs). TRBP is an enhancer of RNA silencing, and functions to recruit precursor-miRNAs (pre-miRNAs) to Dicer that processes pre-miRNA into mature miRNA. Viral infection activates the antiviral innate immune response in mammalian cells. Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I, melanoma-differentiation-associated gene 5 (MDA5), and LGP2, function as cytoplasmic virus sensor proteins during viral infection. RIG-I and MDA5 can distinguish between different types of RNA viruses to produce antiviral cytokines, including type I interferon. However, the role of LGP2 is controversial. We found that LGP2 bound to the double-stranded RNA binding sites of TRBP, resulting in inhibition of pre-miRNA binding and recruitment by TRBP. Furthermore, although it is unclear whether TRBP binds to specific pre-miRNA, we found that TRBP bound to particular pre-miRNAs with common structural characteristics. Thus, LGP2 represses specific miRNA activities by interacting with TRBP, resulting in selective regulation of target genes. Our findings show that a novel function of LGP2 is to modulate RNA silencing, indicating the crosstalk between RNA silencing and RLR signaling in mammalian cells.},
}

CRISPR-Cas Systems
Gene Editing
Gene Expression Regulation/genetics
Gene Knockdown Techniques
Gene Regulatory Networks/*genetics
HeLa Cells
Humans
MicroRNAs/*metabolism/physiology
RNA Helicases/*physiology
RNA Interference
RNA Viruses/genetics/metabolism
RNA-Binding Proteins/*metabolism/physiology
Signal Transduction

@article {pmid29924347,
year = {2018},
author = {Sturm, Á and Saskoi, É and Tibor, K and Weinhardt, N and Vellai, T},
title = {Highly efficient RNAi and Cas9-based auto-cloning systems for C. elegans research.},
journal = {Nucleic acids research},
volume = {46},
number = {17},
pages = {e105},
pmid = {29924347},
issn = {1362-4962},
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/*physiology ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/genetics ; Cloning, Molecular/*methods ; Forkhead Transcription Factors/genetics ; Gene Expression Profiling/methods ; Gene Knockdown Techniques/methods ; *Genetic Vectors/chemical synthesis/chemistry/genetics ; Green Fluorescent Proteins/genetics ; Homeostasis/genetics ; RNA Interference/*physiology ; Real-Time Polymerase Chain Reaction/methods ; Research ; },
abstract = {RNA interference (RNAi) technology used for the functional analysis of Caenorhabditis elegans genes frequently leads to phenotypes with low penetrance or even proves completely ineffective. The methods previously developed to solve this problem were built on mutant genetic backgrounds, such as those defective for rrf-3, in which endogenous RNAi pathways are overexpressed. These mutations, however, interferes with many other genetic pathways so that the detected phenotype cannot always be clearly linked to the RNAi-exposed gene. In addition, using RNAi-overexpressing mutant backgrounds requires time-consuming genetic crossing. Here, we present an improved RNAi vector that produces specific double-stranded RNA species only, and thereby significantly stronger phenotypes than the standard gene knockdown vector. The further advantage of the new RNAi vector is that the detected phenotype can be specifically linked to the gene silenced. We also created a new all-in-one C. elegans Cas9 vector whose spacer sequence is much easier to replace. Both new vectors include a novel CRISPR/Cas9-based auto-cloning vector system rendering needless the use of restriction and ligase enzymes in generating DNA constructs. This novel, efficient RNAi and auto-cloning Cas9 systems can be easily adapted to any other genetic model.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems/*physiology
Caenorhabditis elegans/*genetics
Caenorhabditis elegans Proteins/genetics
Cloning, Molecular/*methods
Forkhead Transcription Factors/genetics
Gene Expression Profiling/methods
Gene Knockdown Techniques/methods
*Genetic Vectors/chemical synthesis/chemistry/genetics
Green Fluorescent Proteins/genetics
Homeostasis/genetics
RNA Interference/*physiology
Real-Time Polymerase Chain Reaction/methods
Research

@article {pmid29912475,
year = {2018},
author = {Tasan, I and Sustackova, G and Zhang, L and Kim, J and Sivaguru, M and HamediRad, M and Wang, Y and Genova, J and Ma, J and Belmont, AS and Zhao, H},
title = {CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci.},
journal = {Nucleic acids research},
volume = {46},
number = {17},
pages = {e100},
pmid = {29912475},
issn = {1362-4962},
support = {R01 GM058460/GM/NIGMS NIH HHS/United States ; R01 HG007352/HG/NHGRI NIH HHS/United States ; U54 DK107965/DK/NIDDK NIH HHS/United States ; },
mesh = {Bacterial Proteins/*genetics ; *CRISPR-Cas Systems/genetics ; Carrier Proteins/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA End-Joining Repair/genetics ; DNA Repeat Expansion/genetics ; Gene Knock-In Techniques/*methods ; HCT116 Cells ; HEK293 Cells ; Humans ; In Situ Hybridization, Fluorescence/*methods ; K562 Cells ; Organisms, Genetically Modified ; Sequence Homology, Nucleic Acid ; Single Molecule Imaging/*methods ; },
abstract = {Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.},
}

Bacterial Proteins/*genetics
*CRISPR-Cas Systems/genetics
Carrier Proteins/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
DNA End-Joining Repair/genetics
DNA Repeat Expansion/genetics
Gene Knock-In Techniques/*methods
HCT116 Cells
HEK293 Cells
Humans
In Situ Hybridization, Fluorescence/*methods
K562 Cells
Organisms, Genetically Modified
Sequence Homology, Nucleic Acid
Single Molecule Imaging/*methods

@article {pmid29912461,
year = {2018},
author = {Vasquez, JJ and Wedel, C and Cosentino, RO and Siegel, TN},
title = {Exploiting CRISPR-Cas9 technology to investigate individual histone modifications.},
journal = {Nucleic acids research},
volume = {46},
number = {18},
pages = {e106},
pmid = {29912461},
issn = {1362-4962},
mesh = {*CRISPR-Cas Systems/genetics ; Cell Line ; Gene Editing/*methods ; Genome, Protozoan/genetics ; Green Fluorescent Proteins/genetics ; Histone Code/*genetics ; Histones/*metabolism ; Mutagenesis, Site-Directed/methods ; Organisms, Genetically Modified ; Plasmids/genetics ; Protein Processing, Post-Translational ; RNA, Guide/genetics ; Trypanosoma brucei brucei/cytology/genetics/metabolism ; },
abstract = {Despite their importance for most DNA-templated processes, the function of individual histone modifications has remained largely unknown because in vivo mutational analyses are lacking. The reason for this is that histone genes are encoded by multigene families and that tools to simultaneously edit multiple genomic loci with high efficiency are only now becoming available. To overcome these challenges, we have taken advantage of the power of CRISPR-Cas9 for precise genome editing and of the fact that most DNA repair in the protozoan parasite Trypanosoma brucei occurs via homologous recombination. By establishing an episome-based CRISPR-Cas9 system for T. brucei, we have edited wild type cells without inserting selectable markers, inserted a GFP tag between an ORF and its 3'UTR, deleted both alleles of a gene in a single transfection, and performed precise editing of genes that exist in multicopy arrays, replacing histone H4K4 with H4R4 in the absence of detectable off-target effects. The newly established genome editing toolbox allows for the generation of precise mutants without needing to change other regions of the genome, opening up opportunities to study the role of individual histone modifications, catalytic sites of enzymes or the regulatory potential of UTRs in their endogenous environments.},
}

*CRISPR-Cas Systems/genetics
Cell Line
Gene Editing/*methods
Genome, Protozoan/genetics
Green Fluorescent Proteins/genetics
Histone Code/*genetics
Histones/*metabolism
Mutagenesis, Site-Directed/methods
Organisms, Genetically Modified
Plasmids/genetics
Protein Processing, Post-Translational
RNA, Guide/genetics
Trypanosoma brucei brucei/cytology/genetics/metabolism

@article {pmid29905858,
year = {2018},
author = {Prykhozhij, SV and Fuller, C and Steele, SL and Veinotte, CJ and Razaghi, B and Robitaille, JM and McMaster, CR and Shlien, A and Malkin, D and Berman, JN},
title = {Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.},
journal = {Nucleic acids research},
volume = {46},
number = {17},
pages = {e102},
pmid = {29905858},
issn = {1362-4962},
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Embryo, Nonmammalian ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Microinjections ; Mutagenesis, Site-Directed/methods ; Point Mutation/*genetics ; Zebrafish/embryology/*genetics ; },
abstract = {We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.},
}

Animals
Animals, Genetically Modified
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Embryo, Nonmammalian
Gene Editing/*methods
Gene Knock-In Techniques/*methods
Microinjections
Mutagenesis, Site-Directed/methods
Point Mutation/*genetics
Zebrafish/embryology/*genetics

@article {pmid29790974,
year = {2018},
author = {Couvin, D and Bernheim, A and Toffano-Nioche, C and Touchon, M and Michalik, J and Néron, B and Rocha, EPC and Vergnaud, G and Gautheret, D and Pourcel, C},
title = {CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins.},
journal = {Nucleic acids research},
volume = {46},
number = {W1},
pages = {W246-W251},
pmid = {29790974},
issn = {1362-4962},
mesh = {CRISPR-Associated Proteins/chemistry/*genetics ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Internet ; *Software ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) arrays and their associated (Cas) proteins confer bacteria and archaea adaptive immunity against exogenous mobile genetic elements, such as phages or plasmids. CRISPRCasFinder allows the identification of both CRISPR arrays and Cas proteins. The program includes: (i) an improved CRISPR array detection tool facilitating expert validation based on a rating system, (ii) prediction of CRISPR orientation and (iii) a Cas protein detection and typing tool updated to match the latest classification scheme of these systems. CRISPRCasFinder can either be used online or as a standalone tool compatible with Linux operating system. All third-party software packages employed by the program are freely available. CRISPRCasFinder is available at https://crisprcas.i2bc.paris-saclay.fr.},
}

CRISPR-Associated Proteins/chemistry/*genetics
CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Internet
*Software

@article {pmid29762716,
year = {2018},
author = {Concordet, JP and Haeussler, M},
title = {CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens.},
journal = {Nucleic acids research},
volume = {46},
number = {W1},
pages = {W242-W245},
pmid = {29762716},
issn = {1362-4962},
support = {U41 HG002371/HG/NHGRI NIH HHS/United States ; },
mesh = {Base Sequence ; CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques ; Genome ; Internet ; Mutagenesis ; Oligonucleotides ; RNA/*chemistry ; *Software ; Workflow ; },
abstract = {CRISPOR.org is a web tool for genome editing experiments with the CRISPR-Cas9 system. It finds guide RNAs in an input sequence and ranks them according to different scores that evaluate potential off-targets in the genome of interest and predict on-target activity. The list of genomes is continuously expanded, with more 150 genomes added in the last two years. CRISPOR tries to provide a comprehensive solution from selection, cloning and expression of guide RNA as well as providing primers needed for testing guide activity and potential off-targets. Recent developments include batch design for genome-wide CRISPR and saturation screens, creating custom oligonucleotides for guide cloning and the design of next generation sequencing primers to test for off-target mutations. CRISPOR is available from http://crispor.org, including the full source code of the website and a stand-alone, command-line version.},
}

Base Sequence
CRISPR-Associated Protein 9
*CRISPR-Cas Systems
*Gene Editing
Gene Knockout Techniques
Genome
Internet
Mutagenesis
Oligonucleotides
RNA/*chemistry
*Software
Workflow

@article {pmid29662218,
year = {2018},
author = {Lieberman, J},
title = {Tapping the RNA world for therapeutics.},
journal = {Nature structural & molecular biology},
volume = {25},
number = {5},
pages = {357-364},
pmid = {29662218},
issn = {1545-9985},
support = {R01 CA184718/CA/NCI NIH HHS/United States ; },
mesh = {Alternative Splicing/genetics ; CRISPR-Cas Systems/genetics ; Drug Design ; Humans ; Nucleic Acid Conformation ; RNA/*therapeutic use ; RNA, Long Noncoding/*genetics ; RNA, Small Interfering/*genetics ; },
abstract = {A recent revolution in RNA biology has led to the identification of new RNA classes with unanticipated functions, new types of RNA modifications, an unexpected multiplicity of alternative transcripts and widespread transcription of extragenic regions. This development in basic RNA biology has spawned a corresponding revolution in RNA-based strategies to generate new types of therapeutics. Here, I review RNA-based drug design and discuss barriers to broader applications and possible ways to overcome them. Because they target nucleic acids rather than proteins, RNA-based drugs promise to greatly extend the domain of 'druggable' targets beyond what can be achieved with small molecules and biologics.},
}

Alternative Splicing/genetics
CRISPR-Cas Systems/genetics
Drug Design
Humans
Nucleic Acid Conformation
RNA/*therapeutic use
RNA, Long Noncoding/*genetics
RNA, Small Interfering/*genetics

@article {pmid29632374,
year = {2018},
author = {Joseph, B and Kondo, S and Lai, EC},
title = {Short cryptic exons mediate recursive splicing in Drosophila.},
journal = {Nature structural & molecular biology},
volume = {25},
number = {5},
pages = {365-371},
doi = {10.1038/s41594-018-0052-6},
pmid = {29632374},
issn = {1545-9985},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 GM083300/GM/NIGMS NIH HHS/United States ; R01 NS083833/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Disintegrins/genetics ; Drosophila Proteins/genetics ; Drosophila melanogaster/*genetics ; Homeodomain Proteins/genetics ; Loss of Function Mutation/genetics ; Metalloendopeptidases/genetics ; RNA Splice Sites/*genetics ; RNA Splicing/*genetics ; Transcription Factors/genetics ; },
abstract = {Many long Drosophila introns are processed by an unusual recursive strategy. The presence of ~200 adjacent splice acceptor and splice donor sites, termed ratchet points (RPs), were inferred to reflect 'zero-nucleotide exons', whose sequential processing subdivides removal of long host introns. We used CRISPR-Cas9 to disrupt several intronic RPs in Drosophila melanogaster, some of which recapitulated characteristic loss-of-function phenotypes. Unexpectedly, selective disruption of RP splice donors revealed constitutive retention of unannotated short exons. Assays using functional minigenes confirm that unannotated cryptic splice donor sites are critical for recognition of intronic RPs, demonstrating that recursive splicing involves the recognition of cryptic RP exons. This appears to be a general mechanism, because canonical, conserved splice donors are specifically enriched in a 40-80-nt window downstream of known and newly annotated intronic RPs and exhibit similar properties to a broadly expanded class of expressed RP exons. Overall, these studies unify the mechanism of Drosophila recursive splicing with that in mammals.},
}

Animals
CRISPR-Cas Systems/genetics
Disintegrins/genetics
Drosophila Proteins/genetics
Drosophila melanogaster/*genetics
Homeodomain Proteins/genetics
Loss of Function Mutation/genetics
Metalloendopeptidases/genetics
RNA Splice Sites/*genetics
RNA Splicing/*genetics
Transcription Factors/genetics

@article {pmid29624775,
year = {2018},
author = {Tang, L and Zeng, Y and Zhou, X and Du, H and Li, C and Liu, J and Zhang, P},
title = {Highly efficient ssODN-mediated homology-directed repair of DSBs generated by CRISPR/Cas9 in human 3PN zygotes.},
journal = {Molecular reproduction and development},
volume = {85},
number = {6},
pages = {461-463},
doi = {10.1002/mrd.22983},
pmid = {29624775},
issn = {1098-2795},
mesh = {*CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; Humans ; Oligonucleotides/*pharmacology ; *Recombinational DNA Repair ; Zygote/cytology/*metabolism ; },
}

*CRISPR-Cas Systems
*DNA Breaks, Double-Stranded
Humans
Oligonucleotides/*pharmacology
*Recombinational DNA Repair
Zygote/cytology/*metabolism

@article {pmid29565710,
year = {2018},
author = {Cheng, L and Tang, Y and Chen, X and Zhao, L and Liu, S and Ma, Y and Wang, N and Zhou, K and Zhou, J and Zhou, M},
title = {Deletion of MBD2 inhibits proliferation of chronic myeloid leukaemia blast phase cells.},
journal = {Cancer biology & therapy},
volume = {19},
number = {8},
pages = {676-686},
pmid = {29565710},
issn = {1555-8576},
mesh = {Animals ; Blast Crisis/*genetics ; CRISPR-Cas Systems ; Cell Cycle Checkpoints/genetics ; Cell Line, Tumor ; Cell Proliferation ; CpG Islands ; DNA Methylation ; DNA-Binding Proteins/*genetics ; Disease Models, Animal ; *Gene Deletion ; Gene Knockdown Techniques ; Humans ; Janus Kinase 2/metabolism ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*genetics/metabolism ; Mice ; RNA, Small Interfering/genetics ; STAT3 Transcription Factor/metabolism ; Signal Transduction ; Xenograft Model Antitumor Assays ; },
abstract = {Aberrant methylation of tumour suppressor genes is associated with the progression to a blast crisis in chronic myeloid leukaemia (CML). Methyl-CpG-binding domain protein 2 (MBD2) has been studied as a "reader" of DNA methylation in many cancers, but its role in CML is unclear. We constructed cell models of a homozygous deletion mutation of MBD2 using gene-editing technology in K562 cells and BV173 cells. Here, we demonstrated that the deletion of MBD2 inhibited cell proliferation capacity in vitro. MBD2 deletion also significantly inhibited K562 cell proliferation in a xenograft tumour model in vivo. Additionally, the JAK2/STAT3 signalling pathway, which is abnormally active in CML, was inhibited by MBD2 deletion, and MBD2 deletion could up-regulate the expression of SHP1. In conclusion, our findings suggest that MBD2 is a candidate therapeutic strategy for the CML blast phase.},
}

Animals
Blast Crisis/*genetics
CRISPR-Cas Systems
Cell Cycle Checkpoints/genetics
Cell Line, Tumor
Cell Proliferation
CpG Islands
DNA Methylation
DNA-Binding Proteins/*genetics
Disease Models, Animal
*Gene Deletion
Gene Knockdown Techniques
Humans
Janus Kinase 2/metabolism
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*genetics/metabolism
Mice
RNA, Small Interfering/genetics
STAT3 Transcription Factor/metabolism
Signal Transduction
Xenograft Model Antitumor Assays

@article {pmid29490999,
year = {2018},
author = {Baylis, F},
title = {Counterpoint: The Potential Harms of Human Gene Editing Using CRISPR-Cas9.},
journal = {Clinical chemistry},
volume = {64},
number = {3},
pages = {489-491},
doi = {10.1373/clinchem.2017.278317},
pmid = {29490999},
issn = {1530-8561},
mesh = {*CRISPR-Cas Systems ; Clinical Trials as Topic ; Gene Editing/ethics/*methods ; Gene Expression Regulation ; Genetic Therapy/adverse effects/*methods ; Human Genetics/*ethics ; Humans ; Neoplasms/genetics/therapy ; Translational Medical Research ; },
}

*CRISPR-Cas Systems
Clinical Trials as Topic
Gene Editing/ethics/*methods
Gene Expression Regulation
Genetic Therapy/adverse effects/*methods
Human Genetics/*ethics
Humans
Neoplasms/genetics/therapy
Translational Medical Research

@article {pmid29490998,
year = {2018},
author = {Katsanis, N},
title = {Point: Treating Human Genetic Disease One Base Pair at a Time: The Benefits of Gene Editing.},
journal = {Clinical chemistry},
volume = {64},
number = {3},
pages = {486-488},
doi = {10.1373/clinchem.2017.278309},
pmid = {29490998},
issn = {1530-8561},
mesh = {CRISPR-Cas Systems ; Gene Editing/*methods ; Genetic Diseases, Inborn/genetics/*therapy ; Genetic Therapy/*methods ; Human Genetics/methods ; Humans ; },
}

CRISPR-Cas Systems
Gene Editing/*methods
Genetic Diseases, Inborn/genetics/*therapy
Genetic Therapy/*methods
Human Genetics/methods
Humans

@article {pmid29427479,
year = {2018},
author = {Schinazi, RF and Ehteshami, M and Bassit, L and Asselah, T},
title = {Towards HBV curative therapies.},
journal = {Liver international : official journal of the International Association for the Study of the Liver},
volume = {38 Suppl 1},
number = {},
pages = {102-114},
doi = {10.1111/liv.13656},
pmid = {29427479},
issn = {1478-3231},
support = {P30 AI050409/AI/NIAID NIH HHS/United States ; R01 AI132833/AI/NIAID NIH HHS/United States ; },
mesh = {Antiviral Agents/*therapeutic use ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; DNA, Circular/blood ; DNA, Viral/blood ; Hepatitis B/*therapy ; Hepatitis B virus/physiology ; Humans ; *Immunotherapy ; RNA, Small Interfering/genetics ; Transcription Activator-Like Effector Nucleases ; Virus Replication/drug effects ; },
abstract = {Tremendous progress has been made over the last 2 decades to discover and develop approaches to control hepatitis B virus (HBV) infections and to prevent the development of hepatocellular carcinoma using various interferons and small molecules as antiviral agents. However, none of these agents have significant impact on eliminating HBV from infected cells. Currently the emphasis is on silencing or eliminating cccDNA, which could lead to a cure for HBV. Various approaches are being developed including the development of capsid effectors, CRISPR/Cas9, TALENS, siRNA, entry and secretion inhibitors, as well as immunological approaches. It is very likely that a combination of these modalities will need to be employed to successfully eliminate HBV or prevent virus rebound on discontinuation of therapy. In the next 5 years clinical data will emerge which will provide insight on the safety and feasibility of these approaches and if they can be applied to eradicate HBV infections globally. In this review, we summarize current treatments and we highlight and examine recent therapeutic strategies that are currently being evaluated at the preclinical and clinical stage.},
}

Antiviral Agents/*therapeutic use
CRISPR-Associated Protein 9
CRISPR-Cas Systems
DNA, Circular/blood
DNA, Viral/blood
Hepatitis B/*therapy
Hepatitis B virus/physiology
Humans
*Immunotherapy
RNA, Small Interfering/genetics
Transcription Activator-Like Effector Nucleases
Virus Replication/drug effects

@article {pmid29234093,
year = {2017},
author = {Vilarino, M and Rashid, ST and Suchy, FP and McNabb, BR and van der Meulen, T and Fine, EJ and Ahsan, S and Mursaliyev, N and Sebastiano, V and Diab, SS and Huising, MO and Nakauchi, H and Ross, PJ},
title = {CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {17472},
pmid = {29234093},
issn = {2045-2322},
support = {MR/L006537/1//Medical Research Council/United Kingdom ; },
mesh = {Animals ; Animals, Genetically Modified ; *CRISPR-Cas Systems ; Coumarins ; Gene Editing/methods ; Gene Knockdown Techniques/methods ; Homeodomain Proteins/*genetics/*metabolism ; Microinjections ; Oocytes/*metabolism ; Pancreas/*embryology/*metabolism/pathology ; RNA, Guide/administration & dosage ; Reproductive Techniques, Assisted ; Sequence Analysis, DNA ; Sheep ; Trans-Activators/*genetics/*metabolism ; },
abstract = {One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1-/- fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.},
}

Animals
Animals, Genetically Modified
*CRISPR-Cas Systems
Coumarins
Gene Editing/methods
Gene Knockdown Techniques/methods
Homeodomain Proteins/*genetics/*metabolism
Microinjections
Oocytes/*metabolism
Pancreas/*embryology/*metabolism/pathology
RNA, Guide/administration & dosage
Reproductive Techniques, Assisted
Sequence Analysis, DNA
Sheep
Trans-Activators/*genetics/*metabolism

@article {pmid29089503,
year = {2017},
author = {Dampier, W and Sullivan, NT and Chung, CH and Mell, JC and Nonnemacher, MR and Wigdahl, B},
title = {Designing broad-spectrum anti-HIV-1 gRNAs to target patient-derived variants.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {14413},
pmid = {29089503},
issn = {2045-2322},
support = {P30 MH092177/MH/NIMH NIH HHS/United States ; R01 MH110360/MH/NIMH NIH HHS/United States ; T32 MH079785/MH/NIMH NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology ; Computer Simulation ; Gene Editing ; Genome, Viral ; HIV Infections/genetics ; HIV-1/*genetics ; Humans ; *RNA, Guide ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9), including specific guide RNAs (gRNAs), can excise integrated human immunodeficiency virus type 1 (HIV-1) provirus from host chromosomes. To date, anti-HIV-1 gRNAs have been designed to account for off-target activity, however, they seldom account for genetic variation in the HIV-1 genome within and between patients, which will be crucial for therapeutic application of this technology. This analysis tests the ability of published anti-HIV-1 gRNAs to cleave publicly available patient-derived HIV-1 sequences to inform gRNA design and provides basic computational tools to researchers in the field.},
}

*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Computational Biology
Computer Simulation
Gene Editing
Genome, Viral
HIV Infections/genetics
HIV-1/*genetics
Humans
*RNA, Guide

@article {pmid31015611,
year = {2017},
author = {Bashir, S and Kühn, R},
title = {Enhanced precision and efficiency.},
journal = {Nature biomedical engineering},
volume = {1},
number = {11},
pages = {856-857},
doi = {10.1038/s41551-017-0159-9},
pmid = {31015611},
issn = {2157-846X},
mesh = {*CRISPR-Cas Systems ; *DNA Repair ; *Gene Editing ; Oligodeoxyribonucleotides/genetics ; },
}

*CRISPR-Cas Systems
*DNA Repair
*Gene Editing
Oligodeoxyribonucleotides/genetics

@article {pmid31015610,
year = {2017},
author = {},
title = {The expanding toolbox for genome engineering.},
journal = {Nature biomedical engineering},
volume = {1},
number = {11},
pages = {853},
doi = {10.1038/s41551-017-0163-0},
pmid = {31015610},
issn = {2157-846X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; Genome/genetics ; Humans ; Translational Medical Research ; },
}

Animals
CRISPR-Cas Systems/genetics
Gene Editing/*methods
Genome/genetics
Humans
Translational Medical Research

@article {pmid31401729,
year = {2019},
author = {Hsu, CT and Cheng, YJ and Yuan, YH and Hung, WF and Cheng, QW and Wu, FH and Lee, LY and Gelvin, SB and Lin, CS},
title = {Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco.},
journal = {Plant molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s11103-019-00907-w},
pmid = {31401729},
issn = {1573-5028},
support = {105-2313-B-001 -007 -MY3//Ministry of Science and Technology, Taiwan/ ; AS-KPQ-107-ITAR-10//Innovative Translational Agricultural Research Administrative Office/ ; },
abstract = {KEY MESSAGE: Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants. Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.},
}

@article {pmid31400605,
year = {2019},
author = {Liu, X and Li, G and Zhou, X and Qiao, Y and Wang, R and Tang, S and Liu, J and Wang, L and Huang, X},
title = {Improving Editing Efficiency for the Sequences with NGH PAM Using xCas9-Derived Base Editors.},
journal = {Molecular therapy. Nucleic acids},
volume = {17},
number = {},
pages = {626-635},
doi = {10.1016/j.omtn.2019.06.024},
pmid = {31400605},
issn = {2162-2531},
abstract = {The development of CRISPR/Cas9-mediated base editors (BEs) provided a versatile tool for precise genome editing. The recently developed xCas9-derived base editors (xBEs) that recognize the NG PAM substantially expand the targeting scope in the genome, while their editing efficiency needs to be improved. Here, we described an improved version of xBEs by fusing the BPNLS and Gam to the N terminus of xBEs (BPNLS-Gam-xBE3 and BPNLS-xABE), and this version of base editor displayed higher targeting efficiency for the majority of detected sites. By using this improved version of xBEs, we successfully created and corrected pathogenic mutations at genomic sites with the NGN protospacer-adjacent motif in human cells. Lastly, we used BPNLS-Gam-xBE3 to model pathogenic mutations in discarded human tripronuclear (3PN) zygotes, and no obvious off-targets and indels were detected. Taken together, the data in our study offer an efficient tool for precise genome editing and, thus, an enriched base editing toolkit.},
}

@article {pmid31399410,
year = {2019},
author = {Maikova, A and Kreis, V and Boutserin, A and Severinov, K and Soutourina, O},
title = {Using endogenous CRISPR-Cas system for genome editing in the human pathogen Clostridium difficile.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1128/AEM.01416-19},
pmid = {31399410},
issn = {1098-5336},
abstract = {Human enteropathogen Clostridium difficile constitutes a key public health issue in industrialized countries. Many aspects of C. difficile pathophysiology and adaptation inside the host remain poorly understood. We have recently reported that this bacterium possesses an active CRISPR-Cas system of subtype I-B for defence against phages and other mobile genetic elements that could contribute to its success during infection. In this paper, we demonstrate that redirecting this endogenous CRISPR-Cas system towards autoimmunity allows efficient genome editing in C. difficile We provide detailed description of this newly developed approach and show, as a proof of principle, its efficient application for deletion of a specific gene in reference 630Δerm and in epidemic R20291 C. difficile strains. The new method expands the arsenal of the currently limiting set of gene engineering tools available for investigation of C. difficile and may serve as the basis for new strategies to control C. difficile infections.ImportanceClostridium difficile represents today a real danger for human and animal health. It is the leading cause of diarrhoea associated with healthcare in adults in industrialized countries. The incidence of these infections continues to increase and this trend is accentuated by the general aging of the population. Many questions remain unanswered on the mechanisms contributing to C. difficile success inside the host. The set of genetic tools available for this pathogen is limited and new developments are badly needed. C. difficile has developed efficient defence systems that are directed against foreign DNA and could contribute to its survival in phage-rich gut communities. We show how one of such defence systems, named CRISPR-Cas, can be hijacked for C. difficile genome editing. Our results also show a great potential of CRISPR-Cas system for development of new therapeutic strategies against C. difficile infections.},
}

@article {pmid31399023,
year = {2019},
author = {Kruse, T and Ratnadevi, CM and Erikstad, HA and Birkeland, NK},
title = {Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph "Candidatus Methylacidiphilum kamchatkense" strain Kam1 and comparison with its closest relatives.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {642},
doi = {10.1186/s12864-019-5995-4},
pmid = {31399023},
issn = {1471-2164},
support = {261923//Norges Forskningsråd/ ; },
abstract = {BACKGROUND: The candidate genus "Methylacidiphilum" comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the "Ca. Methylacidiphilum" and the sister genus "Ca. Methylacidimicrobium" represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms.

RESULTS: Here we present the closed genome of "Ca. Methylacidiphilum kamchatkense" strain Kam1 and a comparison with the genomes of its two closest relatives "Ca. Methylacidiphilum fumariolicum" strain SolV and "Ca. Methylacidiphilum infernorum" strain V4. The genome consists of a single 2,2 Mbp chromosome with 2119 predicted protein coding sequences. Genome analysis showed that the majority of the genes connected with metabolic traits described for one member of "Ca. Methylacidiphilum" is conserved between all three genomes. All three strains encode class I CRISPR-cas systems. The average nucleotide identity between "Ca. M. kamchatkense" strain Kam1 and strains SolV and V4 is ≤95% showing that they should be regarded as separate species. Whole genome comparison revealed a high degree of synteny between the genomes of strains Kam1 and SolV. In contrast, comparison of the genomes of strains Kam1 and V4 revealed a number of rearrangements. There are large differences in the numbers of transposable elements found in the genomes of the three strains with 12, 37 and 80 transposable elements in the genomes of strains Kam1, V4 and SolV respectively. Genomic rearrangements and the activity of transposable elements explain much of the genomic differences between strains. For example, a type 1h uptake hydrogenase is conserved between strains Kam1 and SolV but seems to have been lost from strain V4 due to genomic rearrangements.

CONCLUSIONS: Comparing three closed genomes of "Ca. Methylacidiphilum" spp. has given new insights into the evolution of these organisms and revealed large differences in numbers of transposable elements between strains, the activity of these explains much of the genomic differences between strains.},
}

@article {pmid31397669,
year = {2019},
author = {Knott, GJ and Cress, BF and Liu, JJ and Thornton, BW and Lew, RJ and Al-Shayeb, B and Rosenberg, DJ and Hammel, M and Adler, BA and Lobba, MJ and Xu, M and Arkin, AP and Fellmann, C and Doudna, JA},
title = {Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a.},
journal = {eLife},
volume = {8},
number = {},
pages = {},
doi = {10.7554/eLife.49110},
pmid = {31397669},
issn = {2050-084X},
support = {HR0011-17-2-0043//Defense Advanced Research Projects Agency/ ; MCB-1244557//National Science Foundation/ ; P50GM082250/NH/NIH HHS/United States ; Graduate Research Fellowship,DGE 1752814//National Science Foundation/ ; RM1HG009490/NH/NIH HHS/United States ; R00GM118909/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The A. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.},
}

@article {pmid29910815,
year = {2018},
author = {Wang, C and Cheng, W and Yu, Q and Xing, T and Chen, S and Liu, L and Yu, L and Du, J and Luo, Q and Shen, J and Xu, Y},
title = {Toxoplasma Chinese 1 Strain of WH3Δrop16I/III /gra15II Genetic Background Contributes to Abnormal Pregnant Outcomes in Murine Model.},
journal = {Frontiers in immunology},
volume = {9},
number = {},
pages = {1222},
pmid = {29910815},
issn = {1664-3224},
mesh = {Animals ; Apoptosis ; CRISPR-Cas Systems ; Disease Models, Animal ; Female ; Gene Targeting ; *Genetic Background ; *Genetic Predisposition to Disease ; Macrophages, Peritoneal/immunology/metabolism/parasitology ; Male ; Mice ; Placenta ; Pregnancy ; Pregnancy Complications, Infectious/immunology/*parasitology ; *Pregnancy Outcome ; Spleen/immunology/metabolism ; Th1 Cells/immunology/metabolism ; Th17 Cells/immunology/metabolism ; Toxoplasma/*classification/*genetics ; Toxoplasmosis, Animal/immunology/*parasitology ; Trophoblasts/metabolism ; },
abstract = {Toxoplasma gondii infection evokes a strong Th1-type response with interleukin (IL)-12 and interferon (IFN)-γ secretion. Recent studies suggest that the infection of pregnant mice with T. gondii may lead to adverse pregnancy results caused by subversion of physiological immune tolerance at maternofetal interface rather than direct invasion of the parasite. Genotype-associated dense granule protein GRA15II tends to induce classically activated macrophage (M1) differentiation and subsequently activating NK, Th1, and Th17 cells whereas rhoptry protein ROP16I/III drives macrophages to alternatively activated macrophage (M2) polarization and elicits Th2 immune response. Unlike the archetypal strains of types I, II, and III, type Chinese 1 strains possess both GRA15II and ROP16I/III, suggesting a distinct pathogenesis of Toxoplasma-involved adverse pregnancies. We constructed T. gondii type Chinese 1 strain of WH3Δrop16 based on CRISPR/Cas9 technology to explore the ROP16I/III-deficient/GRA15II-dominant parasites in induction of trophoblast apoptosis in vitro and abnormal pregnant outcomes of mice in vivo. Our study showed that Toxoplasma WH3Δrop16 remarkably induced apoptosis of trophoblasts. C57BL/6 pregnant mice injected with the tachyzoites of WH3Δrop16 presented increased absorptivity of fetuses in comparison with the mice infected with WH3 wild type (WH3 WT) parasites although no remarkable difference of virulence to mice was seen between the two strains. Additionally, the mice inoculated with WH3Δrop16 tachyzoites exhibited a notable expression of both IL-17A and IFN-γ, while the percentage of CD4+CD25+FoxP3 [T regulatory cells (Tregs)] were diminished in splenocytes and placenta tissues compared to those infected with WH3 WT parasites. Accordingly, expressions of IL-4, IL-10, and transforming growth factor beta 1, the pivotal cytokines of Th2 and Tregs response, were significantly dampened whereas IFN-γ and IL-12 expressions were upregulated in WH3Δrop16-infected mice, which gave rise to more prominent outcomes of abnormal pregnancies. Our results indicated that the WH3Δrop16 parasites with gra15II background of T. gondii type Chinese 1 strains may cause miscarriage and stillbirth due to subversion of the maternal immune tolerance and system immunity of the animals and the GRA15II effector contributes to the process of adverse pregnant consequences.},
}

Animals
Apoptosis
CRISPR-Cas Systems
Disease Models, Animal
Female
Gene Targeting
*Genetic Background
*Genetic Predisposition to Disease
Macrophages, Peritoneal/immunology/metabolism/parasitology
Male
Mice
Placenta
Pregnancy
Pregnancy Complications, Infectious/immunology/*parasitology
*Pregnancy Outcome
Spleen/immunology/metabolism
Th1 Cells/immunology/metabolism
Th17 Cells/immunology/metabolism
Toxoplasma/*classification/*genetics
Toxoplasmosis, Animal/immunology/*parasitology
Trophoblasts/metabolism

@article {pmid29370725,
year = {2018},
author = {Lane-Reticker, SK and Manguso, RT and Haining, WN},
title = {Pooled in vivo screens for cancer immunotherapy target discovery.},
journal = {Immunotherapy},
volume = {10},
number = {3},
pages = {167-170},
doi = {10.2217/imt-2017-0164},
pmid = {29370725},
issn = {1750-7448},
mesh = {Animals ; CRISPR-Cas Systems ; Drug Resistance, Neoplasm/genetics ; Genes, Essential/genetics ; Genome/genetics ; Humans ; *Immunotherapy ; Mutation ; Neoplasms/*genetics/immunology/*therapy ; Tumor Escape/genetics/immunology ; },
}

Animals
CRISPR-Cas Systems
Drug Resistance, Neoplasm/genetics
Genes, Essential/genetics
Genome/genetics
Humans
*Immunotherapy
Mutation
Neoplasms/*genetics/immunology/*therapy
Tumor Escape/genetics/immunology

@article {pmid31392984,
year = {2019},
author = {Kim, JG and Garrett, S and Wei, Y and Graveley, BR and Terns, MP},
title = {CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkz677},
pmid = {31392984},
issn = {1362-4962},
abstract = {CRISPR-Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of Streptococcus thermophilus. We determined that purified Cas1 and Cas2 proteins catalyze spacer integration with high specificity for CRISPR repeat junctions. We show that 10 bp of the CRISPR leader sequence is critical for stimulating polarized integration preferentially at the repeat proximal to the leader. Spacer integration proceeds through a two-step transesterification reaction where the 3' hydroxyl groups of the spacer target both repeat borders on opposite strands. The leader-proximal end of the repeat is preferentially targeted for the first site of integration through recognition of sequences spanning the leader-repeat junction. Subsequently, second-site integration at the leader-distal end of the repeat is specified by multiple determinants including a length-defining mechanism relying on a repeat element proximal to the second site of integration. Our results highlight the intrinsic ability of type II Cas1/Cas2 proteins to coordinate directional and site-specific spacer integration into the CRISPR locus to ensure precise duplication of the repeat required for CRISPR immunity.},
}

@article {pmid31392501,
year = {2019},
author = {Ai, L and Guo, W and Chen, W and Teng, Y and Bai, L},
title = {The gal80 Deletion by CRISPR-Cas9 in Engineered Saccharomyces cerevisiae Produces Artemisinic Acid Without Galactose Induction.},
journal = {Current microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00284-019-01752-2},
pmid = {31392501},
issn = {1432-0991},
support = {2017ZX09101002-003-003//The Drug Innovation Major Project/ ; 31870059//National Natural Science Foundation of China/ ; 2018ZX09711001-007-003//Drug Innovation Major Project/ ; 2018YFA0902000//National Key Research and Development Program of China/ ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has emerged as the dominating tool for genome engineering, while also changes the speed and efficiency of metabolic engineering in conventional and non-conventional yeasts. Among these CRISPR-Cas systems, CRISPR-Cas9 technology has usually been applied for removing unfavorable target genes. Here, we used CRISPR-Cas9 technology to delete the gal80 gene in uracil-deficient strain and had successfully remolded the engineered Saccharomyces cerevisiae that can produce artemisinic acid without galactose induction. An L9(34) orthogonal test was adopted to investigate the effects of different factors on artemisinic acid production. Fermentation medium III with sucrose as carbon sources, 1% inoculum level, and 84-h culture time were identified as the optimal fermentation conditions. Under this condition, the maximum artemisinic acid production by engineered S. cerevisiae 1211-2 was 740 mg/L in shake-flask cultivation level. This study provided an effective approach to reform metabolic pathway of artemisinic acid-producing strain. The engineered S. cerevisiae 1211-2 may be applied to artemisinic acid production by industrial fermentation in the future.},
}

@article {pmid31392299,
year = {2019},
author = {Zhang, F and Song, G and Tian, Y},
title = {Anti-CRISPRs: The natural inhibitors for CRISPR-Cas systems.},
journal = {Animal models and experimental medicine},
volume = {2},
number = {2},
pages = {69-75},
doi = {10.1002/ame2.12069},
pmid = {31392299},
issn = {2576-2095},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated protein) systems serve as the adaptive immune system by which prokaryotes defend themselves against phages. It has also been developed into a series of powerful gene-editing tools. As the natural inhibitors of CRISPR-Cas systems, anti-CRISPRs (Acrs) can be used as the "off-switch" for CRISPR-Cas systems to limit the off-target effects caused by Cas9. Since the discovery of CRISPR-Cas systems, much research has focused on the identification, mechanisms and applications of Acrs. In light of the rapid development and scientific significance of this field, this review summarizes the history and research status of Acrs, and considers future applications.},
}

@article {pmid31391532,
year = {2019},
author = {Martens, KJA and van Beljouw, SPB and van der Els, S and Vink, JNA and Baas, S and Vogelaar, GA and Brouns, SJJ and van Baarlen, P and Kleerebezem, M and Hohlbein, J},
title = {Visualisation of dCas9 target search in vivo using an open-microscopy framework.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {3552},
doi = {10.1038/s41467-019-11514-0},
pmid = {31391532},
issn = {2041-1723},
abstract = {CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.},
}

@article {pmid31307375,
year = {2019},
author = {Do, PT and Nguyen, CX and Bui, HT and Tran, LTN and Stacey, G and Gillman, JD and Zhang, ZJ and Stacey, MG},
title = {Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and α-linolenic acid phenotype in soybean.},
journal = {BMC plant biology},
volume = {19},
number = {1},
pages = {311},
doi = {10.1186/s12870-019-1906-8},
pmid = {31307375},
issn = {1471-2229},
support = {R01 DA007250/DA/NIDA NIH HHS/United States ; T32 DA007250/DA/NIDA NIH HHS/United States ; 1444581//National Science Foundation-Plant Genome Research Program/ ; },
mesh = {Agrobacterium/genetics ; CRISPR-Cas Systems ; Fatty Acid Desaturases/*genetics ; Gene Editing/*methods ; *Genes, Plant ; Genetic Markers ; Genetic Vectors ; Genotyping Techniques ; Inheritance Patterns ; Plants, Genetically Modified ; *RNA, Guide ; Soybeans/*genetics ; alpha-Linolenic Acid/*genetics ; },
abstract = {BACKGROUND: CRISPR/Cas9 gene editing is now revolutionizing the ability to effectively modify plant genomes in the absence of efficient homologous recombination mechanisms that exist in other organisms. However, soybean is allotetraploid and is commonly viewed as difficult and inefficient to transform. In this study, we demonstrate the utility of CRISPR/Cas9 gene editing in soybean at relatively high efficiency. This was shown by specifically targeting the Fatty Acid Desaturase 2 (GmFAD2) that converts the monounsaturated oleic acid (C18:1) to the polyunsaturated linoleic acid (C18:2), therefore, regulating the content of monounsaturated fats in soybean seeds.

RESULTS: We designed two gRNAs to guide Cas9 to simultaneously cleave two sites, spaced 1Kb apart, within the second exons of GmFAD2-1A and GmFAD2-1B. In order to test whether the Cas9 and gRNAs would perform properly in transgenic soybean plants, we first tested the CRISPR construct we developed by transient hairy root transformation using Agrobacterium rhizogenesis strain K599. Once confirmed, we performed stable soybean transformation and characterized ten, randomly selected T0 events. Genotyping of CRISPR/Cas9 T0 transgenic lines detected a variety of mutations including large and small DNA deletions, insertions and inversions in the GmFAD2 genes. We detected CRISPR- edited DNA in all the tested T0 plants and 77.8% of the events transmitted the GmFAD2 mutant alleles to T1 progenies. More importantly, null mutants for both GmFAD2 genes were obtained in 40% of the T0 plants we genotyped. The fatty acid profile analysis of T1 seeds derived from CRISPR-edited plants homozygous for both GmFAD2 genes showed dramatic increases in oleic acid content to over 80%, whereas linoleic acid decreased to 1.3-1.7%. In addition, transgene-free high oleic soybean homozygous genotypes were created as early as the T1 generation.

CONCLUSIONS: Overall, our data showed that dual gRNA CRISPR/Cas9 system offers a rapid and highly efficient method to simultaneously edit homeologous soybean genes, which can greatly facilitate breeding and gene discovery in this important crop plant.},
}

Agrobacterium/genetics
CRISPR-Cas Systems
Fatty Acid Desaturases/*genetics
Gene Editing/*methods
*Genes, Plant
Genetic Markers
Genetic Vectors
Genotyping Techniques
Inheritance Patterns
Plants, Genetically Modified
*RNA, Guide
Soybeans/*genetics
alpha-Linolenic Acid/*genetics

@article {pmid31064825,
year = {2019},
author = {Bertolini, MS and Chiurillo, MA and Lander, N and Vercesi, AE and Docampo, R},
title = {MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca2+ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi.},
journal = {mBio},
volume = {10},
number = {3},
pages = {},
doi = {10.1128/mBio.00348-19},
pmid = {31064825},
issn = {2150-7511},
support = {R01 AI107663/AI/NIAID NIH HHS/United States ; R56 AI107663/AI/NIAID NIH HHS/United States ; },
mesh = {Adaptation, Physiological ; Biological Transport ; CRISPR-Cas Systems ; Calcium/*metabolism ; Calcium-Binding Proteins/genetics/*metabolism ; Cation Transport Proteins ; Cytosol/chemistry/metabolism ; Gene Knockout Techniques ; Humans ; Mitochondria/*metabolism ; Mitochondrial Membrane Transport Proteins/genetics/*metabolism ; Protozoan Proteins/genetics/*metabolism ; Trypanosoma cruzi/*genetics/pathogenicity ; },
abstract = {The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.IMPORTANCETrypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.},
}

Adaptation, Physiological
Biological Transport
CRISPR-Cas Systems
Calcium/*metabolism
Calcium-Binding Proteins/genetics/*metabolism
Cation Transport Proteins
Cytosol/chemistry/metabolism
Gene Knockout Techniques
Humans
Mitochondria/*metabolism
Mitochondrial Membrane Transport Proteins/genetics/*metabolism
Protozoan Proteins/genetics/*metabolism
Trypanosoma cruzi/*genetics/pathogenicity

@article {pmid30632384,
year = {2019},
author = {Gulei, D and Raduly, L and Berindan-Neagoe, I and Calin, GA},
title = {CRISPR-based RNA editing: diagnostic applications and therapeutic options.},
journal = {Expert review of molecular diagnostics},
volume = {19},
number = {2},
pages = {83-88},
doi = {10.1080/14737159.2019.1568242},
pmid = {30632384},
issn = {1744-8352},
support = {UH3 TR000943/TR/NCATS NIH HHS/United States ; R01 CA182905/CA/NCI NIH HHS/United States ; R01 CA222007/CA/NCI NIH HHS/United States ; R01 GM122775/GM/NIGMS NIH HHS/United States ; U54 CA096297/CA/NCI NIH HHS/United States ; U54 CA096300/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *RNA Editing ; },
}

*CRISPR-Cas Systems
Humans
*RNA Editing

@article {pmid30578646,
year = {2019},
author = {Cornelissen, LAM and Blanas, A and van der Horst, JC and Kruijssen, L and Zaal, A and O'Toole, T and Wiercx, L and van Kooyk, Y and van Vliet, SJ},
title = {Disruption of sialic acid metabolism drives tumor growth by augmenting CD8+ T cell apoptosis.},
journal = {International journal of cancer},
volume = {144},
number = {9},
pages = {2290-2302},
doi = {10.1002/ijc.32084},
pmid = {30578646},
issn = {1097-0215},
mesh = {Animals ; Apoptosis/*immunology ; CD8-Positive T-Lymphocytes/*immunology ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Colorectal Neoplasms/genetics/*pathology ; Disease-Free Survival ; Glycosylation ; Humans ; Lymphocyte Count ; Mice ; Mice, Inbred C57BL ; N-Acylneuraminate Cytidylyltransferase/*genetics ; Sialic Acids/*metabolism ; Tumor Escape/*immunology ; Tumor Microenvironment/genetics/immunology ; },
abstract = {Sialylated glycan structures are known for their immunomodulatory capacities and their contribution to tumor immune evasion. However, the role of aberrant sialylation in colorectal cancer and the consequences of complete tumor desialylation on anti-tumor immunity remain unstudied. Here, we report that CRISPR/Cas9-mediated knock out of the CMAS gene, encoding a key enzyme in the sialylation pathway, in the mouse colorectal cancer MC38 cell line completely abrogated cell surface expression of sialic acids (MC38-Sianull) and, unexpectedly, significantly increased in vivo tumor growth compared to the control MC38-MOCK cells. This enhanced tumor growth of MC38-Sianull cells could be attributed to decreased CD8+ T cell frequencies in the tumor microenvironment only, as immune cell frequencies in tumor-draining lymph nodes remained unaffected. In addition, MC38-Sianull cells were able to induce CD8+ T cell apoptosis in an antigen-independent manner. Moreover, low CMAS gene expression correlated with reduced recurrence-free survival in a human colorectal cancer cohort, supporting the clinical relevance of our work. Together, these results demonstrate for the first time a detrimental effect of complete tumor desialylation on colorectal cancer tumor growth, which greatly impacts the design of novel cancer therapeutics aimed at altering the tumor glycosylation profile.},
}

Animals
Apoptosis/*immunology
CD8-Positive T-Lymphocytes/*immunology
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Colorectal Neoplasms/genetics/*pathology
Disease-Free Survival
Glycosylation
Humans
Lymphocyte Count
Mice
Mice, Inbred C57BL
N-Acylneuraminate Cytidylyltransferase/*genetics
Sialic Acids/*metabolism
Tumor Escape/*immunology
Tumor Microenvironment/genetics/immunology

@article {pmid30562583,
year = {2019},
author = {Bruni, GO and Zhong, K and Lee, SC and Wang, P},
title = {CRISPR-Cas9 induces point mutation in the mucormycosis fungus Rhizopus delemar.},
journal = {Fungal genetics and biology : FG & B},
volume = {124},
number = {},
pages = {1-7},
doi = {10.1016/j.fgb.2018.12.002},
pmid = {30562583},
issn = {1096-0937},
support = {R21 AI121451/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; Genes, Fungal ; Genetic Vectors ; Orotate Phosphoribosyltransferase/genetics ; Orotic Acid/analogs & derivatives/pharmacology ; *Point Mutation ; Rhizopus/drug effects/enzymology/*genetics ; Uracil ; },
abstract = {Rhizopus delemar causes devastating mucormycosis in immunodeficient individuals. Despite its medical importance, R. delemar remains understudied largely due to the lack of available genetic markers, the presence of multiple gene copies due to genome duplication, and mitotically unstable transformants resulting from conventional and limited genetic approaches. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system induces efficient homologous and non-homologous break points and generates individual and multiple mutant alleles without requiring selective marker genes in a wide variety of organisms including fungi. Here, we have successfully adapted this technology for inducing gene-specific single nucleotide (nt) deletions in two clinical strains of R. delemar: FGSC-9543 and CDC-8219. For comparative reasons, we first screened for spontaneous uracil auxotrophic mutants resistant to 5-fluoroorotic acid (5-FOA) and obtained one substitution (f1) mutationin the FGSC-9543 strain and one deletion (f2) mutation in the CDC-8219 strain. The f2 mutant was then successfully complemented with a pyrF-dpl200 marker gene. We then introduced a vector pmCas9:tRNA-gRNA that expresses both Cas9 endonuclease and pyrF-specific gRNA into FGSC-9543 and CDC-8219 strains and obtained 34 and 42 5-FOA resistant isolates, respectively. Candidate transformants were successively transferred eight times by propagating hyphal tips prior to genotype characterization. Sequencing of the amplified pyrF allele in all transformants tested revealed a single nucleotide (nt) deletion at the 4th nucleotide before the protospacer adjacent motif (PAM) sequence, which is consistent with CRISPR-Cas9 induced gene mutation through non-homologous end joining (NHEJ). Our study provides a new research tool for investigating molecular pathogenesis mechanisms of R. delemar while also highlighting the utilization of CRISPR-Cas9 technology for generating specific mutants of Mucorales fungi.},
}

CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems
Genes, Fungal
Genetic Vectors
Orotate Phosphoribosyltransferase/genetics
Orotic Acid/analogs & derivatives/pharmacology
*Point Mutation
Rhizopus/drug effects/enzymology/*genetics
Uracil

@article {pmid30528226,
year = {2019},
author = {Przybilla, MJ and Ou, L and Tăbăran, AF and Jiang, X and Sidhu, R and Kell, PJ and Ory, DS and O'Sullivan, MG and Whitley, CB},
title = {Comprehensive behavioral and biochemical outcomes of novel murine models of GM1-gangliosidosis and Morquio syndrome type B.},
journal = {Molecular genetics and metabolism},
volume = {126},
number = {2},
pages = {139-150},
doi = {10.1016/j.ymgme.2018.11.002},
pmid = {30528226},
issn = {1096-7206},
mesh = {Animals ; CRISPR-Cas Systems ; *Disease Models, Animal ; Female ; Fluorometry ; Gangliosidosis, GM1/genetics/*pathology ; Gene Editing ; Mental Status and Dementia Tests ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Mucopolysaccharidosis IV/genetics/*pathology ; Mutation ; Mutation, Missense ; Phenotype ; beta-Galactosidase/genetics/*metabolism ; },
abstract = {Deficiencies in the lysosomal hydrolase β-galactosidase (β-gal) lead to two distinct diseases: the skeletal disease Morquio syndrome type B, and the neurodegenerative disease GM1-gangliosidosis. Utilizing CRISPR-Cas9 genome editing, the mouse β-gal encoding gene, Glb1, was targeted to generate both models of β-gal deficiency in a single experiment. For Morquio syndrome type B, the common human missense mutation W273L (position 274 in mice) was introduced into the Glb1 gene (Glb1W274L), while for GM1-gangliosidosis, a 20 bp mutation was generated to remove the catalytic nucleophile of β-gal (β-gal-/-). Glb1W274L mice showed a significant reduction in β-gal enzyme activity (8.4-13.3% of wildtype), but displayed no marked phenotype after one year. In contrast, β-gal-/- mice were devoid of β-gal enzyme activity (≤1% of wildtype), resulting in ganglioside accumulation and severe cellular vacuolation throughout the central nervous system (CNS). β-gal-/- mice also displayed severe neuromotor and neurocognitive dysfunction, and as the disease progressed, the mice became emaciated and succumbed to the disease by 10 months of age. Overall, in addition to generating a novel murine model that phenotypically resembles GM1-gangliosidosis, the first model of β-galactosidase deficiency with residual enzyme activity has been developed.},
}

Animals
CRISPR-Cas Systems
*Disease Models, Animal
Female
Fluorometry
Gangliosidosis, GM1/genetics/*pathology
Gene Editing
Mental Status and Dementia Tests
Mice
Mice, Inbred C57BL
Mice, Knockout
Mucopolysaccharidosis IV/genetics/*pathology
Mutation
Mutation, Missense
Phenotype
beta-Galactosidase/genetics/*metabolism

@article {pmid30340581,
year = {2018},
author = {Liu, X and Wang, M and Qin, Y and Shi, X and Cong, P and Chen, Y and He, Z},
title = {Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9.},
journal = {BMC biotechnology},
volume = {18},
number = {1},
pages = {66},
pmid = {30340581},
issn = {1472-6750},
mesh = {*CRISPR-Cas Systems ; Crossing Over, Genetic ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; Endodeoxyribonucleases/genetics/metabolism ; Gene Targeting/*methods ; Genome ; Humans ; Plasmids/genetics ; Receptors, CCR5/genetics ; },
abstract = {BACKGROUND: Targeted DNA integration is widely used in basic research and commercial applications because it eliminates positional effects on transgene expression. Targeted integration in mammalian cells is generally achieved through a double crossover event between the genome and a linear donor containing two homology arms flanking the gene of interest. However, this strategy is generally less efficient at introducing larger DNA fragments. Using the homology-independent NHEJ mechanism has recently been shown to improve efficiency of integrating larger DNA fragments at targeted sites, but integration through this mechanism is direction-independent. Therefore, developing new methods for direction-dependent integration with improved efficiency is desired.

RESULTS: We generated site-specific double-strand breaks using ZFNs or CRISPR/Cas9 in the human CCR5 gene and a donor plasmid containing a 1.6-kb fragment homologous to the CCR5 gene in the genome. These DSBs efficiently drove the direction-dependent integration of 6.4-kb plasmids into the genomes of two human cell lines through single-crossover recombination. The integration was direction-dependent and resulted in the duplication of the homology region in the genome, allowing the integration of another copy of the donor plasmid. The CRISPR/Cas9 system tended to disrupt the sgRNA-binding site within the duplicated homology region, preventing the integration of another plasmid donor. In contrast, ZFNs were less likely to completely disrupt their binding sites, allowing the successive integration of additional plasmid donor copies. This could be useful in promoting multi-copy integration for high-level expression of recombinant proteins. Targeted integration through single crossover recombination was highly efficient (frequency: 33%) as revealed by Southern blot analysis of clonal cells. This is more efficient than a previously described NHEJ-based method (0.17-0.45%) that was used to knock in an approximately 5-kb long DNA fragment.

CONCLUSION: We developed a method for the direction-dependent integration of large DNA fragments through single crossover recombination. We compared and contrasted our method to a previously reported technique for the direction-independent integration of DNA cassettes into the genomes of cultured cells via NHEJ. Our method, due to its directionality and ability to efficiently integrate large fragments, is an attractive strategy for both basic research and industrial application.},
}

*CRISPR-Cas Systems
Crossing Over, Genetic
DNA Breaks, Double-Stranded
DNA End-Joining Repair
Endodeoxyribonucleases/genetics/metabolism
Gene Targeting/*methods
Genome
Humans
Plasmids/genetics
Receptors, CCR5/genetics

@article {pmid30285700,
year = {2018},
author = {Tang, JX and Chen, D and Deng, SL and Li, J and Li, Y and Fu, Z and Wang, XX and Zhang, Y and Chen, SR and Liu, YX},
title = {CRISPR/Cas9-mediated genome editing induces gene knockdown by altering the pre-mRNA splicing in mice.},
journal = {BMC biotechnology},
volume = {18},
number = {1},
pages = {61},
pmid = {30285700},
issn = {1472-6750},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Exons ; *Gene Editing ; Gene Knockdown Techniques/*methods ; INDEL Mutation ; Introns ; Mice/*genetics ; Mice, Inbred C57BL ; RNA Precursors/*genetics ; *RNA Splicing ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) has been wildly used to generate gene knockout models through inducing indels causing frame-shift. However, there are few studies concerning the post-transcript effects caused by CRISPR-mediated genome editing.

RESULTS: In the present study, we showed that gene knockdown model also could be generated using CRISPR-mediated gene editing by disrupting the boundary of exon and intron in mice (C57BL/6 J). CRISPR induced indel at the boundary of exon and intron (5' splice site) caused alternative splicing and produced multiple different mRNAs, most of these mRNAs introduced premature termination codon causing down expression of the gene.

CONCLUSIONS: These results showed that alternative splicing mutants were able to generate through CRISPR-mediated genome editing by deleting the boundary of exon and intron causing disruption of 5' splice site. Although alternative splicing was an unexpected outcome, this finding could be developed as a technology to generate gene knockdown models or to investigate pre-mRNA splicing.},
}

Animals
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Exons
*Gene Editing
Gene Knockdown Techniques/*methods
INDEL Mutation
Introns
Mice/*genetics
Mice, Inbred C57BL
RNA Precursors/*genetics
*RNA Splicing

@article {pmid30253761,
year = {2018},
author = {Chen, D and Tang, JX and Li, B and Hou, L and Wang, X and Kang, L},
title = {CRISPR/Cas9-mediated genome editing induces exon skipping by complete or stochastic altering splicing in the migratory locust.},
journal = {BMC biotechnology},
volume = {18},
number = {1},
pages = {60},
pmid = {30253761},
issn = {1472-6750},
mesh = {Animal Migration ; Animals ; *CRISPR-Cas Systems ; Exons ; Frameshift Mutation ; Gene Editing/*methods ; Grasshoppers/*genetics/physiology ; INDEL Mutation ; Insect Proteins/genetics/metabolism ; *RNA Splicing ; Receptors, Odorant/genetics/metabolism ; Sequence Deletion ; },
abstract = {BACKGROUND: The CRISPR/Cas9 system has been widely used to generate gene knockout/knockin models by inducing frameshift mutants in cell lines and organisms. Several recent studies have reported that such mutants can lead to in-frame exon skipping in cell lines. However, there was little research about post-transcriptional effect of CRISPR-mediated gene editing in vivo.

RESULTS: We showed that frameshift indels also induced complete or stochastic exon skipping by deleting different regions to influence pre-mRNA splicing in vivo. In the migratory locust, the missing 55 bp at the boundary of intron 3 and exon 4 of an olfactory receptor gene, LmigOr35, resulted in complete exon 4 skipping, whereas the lacking 22 bp in exon 4 of LmigOr35 only resulted in stochastic exon 4 skipping. A single sgRNA induced small insertions or deletions at the boundary of intron and exon to disrupt the 3' splicing site causing completely exon skipping, or alternatively induce small insertions or deletions in the exon to stochastic alter splicing causing the stochastic exon skipping.

CONCLUSIONS: These results indicated that complete or stochastic exon skipping could result from the CRISPR-mediated genome editing by deleting different regions of the gene. Although exon skipping caused by CRISPR-mediated editing was an unexpected outcome, this finding could be developed as a technology to investigate pre-mRNA splicing or to cure several human diseases caused by splicing mutations.},
}

Animal Migration
Animals
*CRISPR-Cas Systems
Exons
Frameshift Mutation
Gene Editing/*methods
Grasshoppers/*genetics/physiology
INDEL Mutation
Insect Proteins/genetics/metabolism
*RNA Splicing
Receptors, Odorant/genetics/metabolism
Sequence Deletion

@article {pmid30056335,
year = {2018},
author = {de Buhr, H and Lebbink, RJ},
title = {Harnessing CRISPR to combat human viral infections.},
journal = {Current opinion in immunology},
volume = {54},
number = {},
pages = {123-129},
doi = {10.1016/j.coi.2018.06.002},
pmid = {30056335},
issn = {1879-0372},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Humans ; Virus Diseases/*genetics/*therapy ; },
abstract = {CRISPR/Cas9 is a technology that allows for targeted and precise genome editing in eukaryotic cells. The technique has changed the landscape of molecular biology and may be applied to repair genetic disorders in future therapies. Besides targeting the human genome, it can be used to cleave and edit viral DNA present in infected cells, and as such provides a promising new strategy for anti-viral therapy. Here, we discuss recent studies on the use of anti-viral CRISPRs to target pathogenic human viruses, with a focus on in vivo studies, challenges, and potential for future clinical applications.},
}

CRISPR-Cas Systems/*genetics
Gene Editing/*methods
Humans
Virus Diseases/*genetics/*therapy

@article {pmid29292368,
year = {2018},
author = {Allison, SJ},
title = {Genetic engineering: Trans-epigenetic modulation of target genes in acute kidney injury.},
journal = {Nature reviews. Nephrology},
volume = {14},
number = {2},
pages = {72},
pmid = {29292368},
issn = {1759-507X},
mesh = {Acute Kidney Injury/*genetics ; *CRISPR-Cas Systems ; Epigenesis, Genetic ; Genetic Engineering ; Humans ; Transcriptional Activation ; },
}

Acute Kidney Injury/*genetics
*CRISPR-Cas Systems
Epigenesis, Genetic
Genetic Engineering
Humans
Transcriptional Activation

@article {pmid31389773,
year = {2019},
author = {Young, CS and Pyle, AD and Spencer, MJ},
title = {CRISPR for Neuromuscular Disorders: Gene Editing and Beyond.},
journal = {Physiology (Bethesda, Md.)},
volume = {34},
number = {5},
pages = {341-353},
doi = {10.1152/physiol.00012.2019},
pmid = {31389773},
issn = {1548-9221},
abstract = {This is a review describing advances in CRISPR/Cas-mediated therapies for neuromuscular disorders (NMDs). We explore both CRISPR-mediated editing and dead Cas approaches as potential therapeutic strategies for multiple NMDs. Last, therapeutic considerations, including delivery and off-target effects, are also discussed.},
}

@article {pmid31389128,
year = {2019},
author = {Kim, Y and Lee, SJ and Yoon, HJ and Kim, NK and Lee, BJ and Suh, JY},
title = {Anti-CRISPR AcrIIC3 discriminates between Cas9 orthologs via targeting the variable surface of the HNH nuclease domain.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.15037},
pmid = {31389128},
issn = {1742-4658},
abstract = {CRISPR-Cas systems constitute the adaptive immunity of bacteria and archaea, degrading nucleic acids of invading phages and plasmids. In response, phages employ anti-CRISPR (Acr) proteins as a counter-defense mechanism to neutralize the host immunity. AcrIIC3 directly inhibits target DNA cleavage of type II-C Cas9 of Neisseria meningitidis. Here we show that AcrIIC3 interacts with the HNH nuclease domain of N. meningitidis Cas9 to inhibit its nuclease activity in an allosteric manner. The crystal structure of the AcrIIC3-HNH complex reveals that AcrIIC3 binds opposite the active site on the HNH nuclease domain. AcrIIC3 employs a unique interface for HNH, allowing it to discriminate between Cas9 orthologs, which contrasts with the broad spectrum of Cas9 inhibition by AcrIIC1. Interface residues of HNH provide key electrostatic and hydrophobic interactions that determine the host specificity of AcrIIC3. Mutations that replace HNH interfaces of N. meningitidis Cas9 with those of Geobacillus stearothermophilus Cas9 or Campylobacter jejuni Cas9 significantly attenuate AcrIIC3 binding, illustrating that the divergent interaction surface confers the host specificity of AcrIIC3. Our study demonstrates that the variable sequences of binding interface can define the target specificity of Acr proteins, suggesting potential applications in Cas9 control for gene editing. This article is protected by copyright. All rights reserved.},
}

@article {pmid30809786,
year = {2019},
author = {Chen, G and Chu, J},
title = {Characterization of Two Polyketide Synthases Involved in Sorbicillinoid Biosynthesis by Acremonium chrysogenum Using the CRISPR/Cas9 System.},
journal = {Applied biochemistry and biotechnology},
volume = {188},
number = {4},
pages = {1134-1144},
doi = {10.1007/s12010-019-02960-z},
pmid = {30809786},
issn = {1559-0291},
support = {2013DFG32630//NOW-MoST Joint Program/ ; 2012CB721006//National Basic Research Program/ ; },
mesh = {Acremonium/*metabolism ; CRISPR-Cas Systems/genetics/physiology ; Gene Editing ; Polyketide Synthases/*metabolism ; RNA, Guide/*economics ; },
abstract = {Acremonium chrysogenum is an important fungal strain used for cephalosporin C production. Many efforts have been made to develop versatile genome-editing tools to better understand the mechanism of A. chrysogenum. Here, we developed a feasible and efficient CRISPR/Cas9 system. Two genes responsible for the synthesis of yellow pigments (sorbicillinoids) were chosen as targets, and plasmids expressing both the Cas9 protein and single-guide RNAs were constructed. After introducing the plasmids into the protoplasts of A. chrysogenum, 83 to 93% albino mutants harboring the expected genomic alteration, on average, were obtained. We have generated two mutant strains that respectively disrupt sorA and sorB by flexible CRISPR/Cas9 system. We further confirmed that the sorbicillinoid biosynthetic gene cluster is regulated by an autoinduction mechanism. This work will lay a solid foundation for gene function research and regulation in the sorbicillinoid biosynthetic pathway.},
}

Acremonium/*metabolism
CRISPR-Cas Systems/genetics/physiology
Gene Editing
Polyketide Synthases/*metabolism
RNA, Guide/*economics

@article {pmid29939484,
year = {2018},
author = {Zhou, W and Cui, H and Ying, L and Yu, XF},
title = {Enhanced Cytosolic Delivery and Release of CRISPR/Cas9 by Black Phosphorus Nanosheets for Genome Editing.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {57},
number = {32},
pages = {10268-10272},
doi = {10.1002/anie.201806941},
pmid = {29939484},
issn = {1521-3773},
mesh = {CRISPR-Cas Systems/*genetics ; Cytosol/chemistry/*metabolism ; *Gene Editing ; *Gene Transfer Techniques ; Nanoparticles/*chemistry ; Phosphorus/*chemistry ; },
abstract = {A biodegradable two-dimensional (2D) delivery platform based on loading black phosphorus nanosheets (BPs) with Cas9 ribonucleoprotein engineered with three nuclear localization signals (NLSs) at C terminus (Cas9N3) is successfully established. The Cas9N3-BPs enter cells effectively via membrane penetration and endocytosis pathways, followed by a BPs biodegradation-associated endosomal escape and cytosolic releases of the loaded Cas9N3 complexes. The Cas9N3-BPs thus provide efficient genome editing and gene silencing in vitro and in vivo at a relatively low dose as compared with other nanoparticle-based delivery platforms. This biodegradable 2D delivery platform offers a versatile cytosolic delivery approach for CRISPR/Cas9 ribonucleoprotein and other bioactive macromolecules for biomedical applications.},
}

CRISPR-Cas Systems/*genetics
Cytosol/chemistry/*metabolism
*Gene Editing
*Gene Transfer Techniques
Nanoparticles/*chemistry
Phosphorus/*chemistry

@article {pmid29473342,
year = {2018},
author = {Huang, M and Inukai, T and Miyake, K and Tanaka, Y and Kagami, K and Abe, M and Goto, H and Minegishi, M and Iwamoto, S and Sugihara, E and Watanabe, A and Somazu, S and Shinohara, T and Oshiro, H and Akahane, K and Goi, K and Sugita, K},
title = {Clofarabine exerts antileukemic activity against cytarabine-resistant B-cell precursor acute lymphoblastic leukemia with low deoxycytidine kinase expression.},
journal = {Cancer medicine},
volume = {7},
number = {4},
pages = {1297-1316},
pmid = {29473342},
issn = {2045-7634},
mesh = {Amino Acid Sequence ; Antimetabolites, Antineoplastic/*pharmacology ; Apoptosis/drug effects/genetics ; Base Sequence ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Survival/drug effects/genetics ; Clofarabine/*pharmacology ; DNA Methylation ; Deoxycytidine Kinase/*genetics ; Dose-Response Relationship, Drug ; Drug Resistance, Neoplasm/*drug effects/*genetics ; Exons ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; Mutation ; Polymorphism, Genetic ; Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy/*genetics/pathology ; Promoter Regions, Genetic ; },
abstract = {Cytosine arabinoside (Ara-C) is one of the key drugs for the treatment of acute myeloid leukemia. It is also used for consolidation therapy of acute lymphoblastic leukemia (ALL). Ara-C is a deoxyadenosine analog and is phosphorylated to form cytosine arabinoside triphosphate (Ara-CTP) as an active form. In the first step of the metabolic pathway, Ara-C is phosphorylated to Ara-CMP by deoxycytidine kinase (DCK). However, the current cumulative evidence in the association of the Ara-C sensitivity in ALL appears inconclusive. We analyzed various cell lines for the possible involvement of DCK in the sensitivities of B-cell precursor ALL (BCP-ALL) to Ara-C. Higher DCK expression was associated with higher Ara-C sensitivity. DCK knockout by genome editing with a CRISPR-Cas9 system in an Ara-C-sensitive-ALL cell line induced marked resistance to Ara-C, but not to vincristine and daunorubicin, indicating the involvement of DCK expression in the Ara-C sensitivity of BCP-ALL. DCK gene silencing due to the hypermethylation of a CpG island and reduced DCK activity due to a nonsynonymous variant allele were not associated with Ara-C sensitivity. Clofarabine is a second-generation deoxyadenosine analog rationally synthesized to improve stability and reduce toxicity. The IC50 of clofarabine in 79 BCP-ALL cell lines was approximately 20 times lower than that of Ara-C. In contrast to Ara-C, although the knockout of DCK induced marked resistance to clofarabine, sensitivity to clofarabine was only marginally associated with DCK gene expression level, suggesting a possible efficacy of clofarabine for BCP-ALL that shows relative Ara-C resistance due to low DCK expression.},
}

Amino Acid Sequence
Antimetabolites, Antineoplastic/*pharmacology
Apoptosis/drug effects/genetics
Base Sequence
CRISPR-Cas Systems
Cell Line, Tumor
Cell Survival/drug effects/genetics
Clofarabine/*pharmacology
DNA Methylation
Deoxycytidine Kinase/*genetics
Dose-Response Relationship, Drug
Drug Resistance, Neoplasm/*drug effects/*genetics
Exons
Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
Humans
Mutation
Polymorphism, Genetic
Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy/*genetics/pathology
Promoter Regions, Genetic

@article {pmid29116155,
year = {2017},
author = {McAllister, KN and Bouillaut, L and Kahn, JN and Self, WT and Sorg, JA},
title = {Using CRISPR-Cas9-mediated genome editing to generate C. difficile mutants defective in selenoproteins synthesis.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {14672},
pmid = {29116155},
issn = {2045-2322},
support = {R01 AI116895/AI/NIAID NIH HHS/United States ; R01 GM042219/GM/NIGMS NIH HHS/United States ; U01 AI124290/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Clostridium difficile/genetics/*metabolism ; Electrophoresis, Polyacrylamide Gel ; Gene Editing/*methods ; Genes, Bacterial/genetics ; Phosphotransferases/genetics/metabolism ; Selenium/metabolism ; Selenoproteins/genetics/*metabolism ; },
abstract = {Clostridium difficile is a significant concern as a nosocomial pathogen, and genetic tools are important when analyzing the physiology of such organisms so that the underlying physiology/pathogenesis of the organisms can be studied. Here, we used TargeTron to investigate the role of selenoproteins in C. difficile Stickland metabolism and found that a TargeTron insertion into selD, encoding the selenophosphate synthetase that is essential for the specific incorporation of selenium into selenoproteins, results in a significant growth defect and a global loss of selenium incorporation. However, because of potential polar effects of the TargeTron insertion, we developed a CRISPR-Cas9 mutagenesis system for C. difficile. This system rapidly and efficiently introduces site-specific mutations into the C. difficile genome (20-50% mutation frequency). The selD CRISPR deletion mutant had a growth defect in protein-rich medium and mimicked the phenotype of a generated TargeTron selD mutation. Our findings suggest that Stickland metabolism could be a target for future antibiotic therapies and that the CRISPR-Cas9 system can introduce rapid and efficient modifications into the C. difficile genome.},
}

CRISPR-Associated Protein 9
CRISPR-Cas Systems
Clostridium difficile/genetics/*metabolism
Electrophoresis, Polyacrylamide Gel
Gene Editing/*methods
Genes, Bacterial/genetics
Phosphotransferases/genetics/metabolism
Selenium/metabolism
Selenoproteins/genetics/*metabolism

@article {pmid29106570,
year = {2018},
author = {Casper, J and Zweig, AS and Villarreal, C and Tyner, C and Speir, ML and Rosenbloom, KR and Raney, BJ and Lee, CM and Lee, BT and Karolchik, D and Hinrichs, AS and Haeussler, M and Guruvadoo, L and Navarro Gonzalez, J and Gibson, D and Fiddes, IT and Eisenhart, C and Diekhans, M and Clawson, H and Barber, GP and Armstrong, J and Haussler, D and Kuhn, RM and Kent, WJ},
title = {The UCSC Genome Browser database: 2018 update.},
journal = {Nucleic acids research},
volume = {46},
number = {D1},
pages = {D762-D769},
pmid = {29106570},
issn = {1362-4962},
support = {U41 HG002371/HG/NHGRI NIH HHS/United States ; U41 HG007234/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Data Display ; *Databases, Genetic ; Gene Regulatory Networks ; *Genome ; Genome, Human ; Humans ; Molecular Sequence Annotation ; Terminology as Topic ; User-Computer Interface ; *Web Browser ; },
abstract = {The UCSC Genome Browser (https://genome.ucsc.edu) provides a web interface for exploring annotated genome assemblies. The assemblies and annotation tracks are updated on an ongoing basis-12 assemblies and more than 28 tracks were added in the past year. Two recent additions are a display of CRISPR/Cas9 guide sequences and an interactive navigator for gene interactions. Other upgrades from the past year include a command-line version of the Variant Annotation Integrator, support for Human Genome Variation Society variant nomenclature input and output, and a revised highlighting tool that now supports multiple simultaneous regions and colors.},
}

CRISPR-Cas Systems
Data Display
*Databases, Genetic
Gene Regulatory Networks
*Genome
Genome, Human
Humans
Molecular Sequence Annotation
Terminology as Topic
User-Computer Interface
*Web Browser

@article {pmid31385197,
year = {2019},
author = {Hajizadeh Dastjerdi, A and Newman, A and Burgio, G},
title = {The Expanding Class 2 CRISPR Toolbox: Diversity, Applicability, and Targeting Drawbacks.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.1007/s40259-019-00369-y},
pmid = {31385197},
issn = {1179-190X},
abstract = {The class 2 clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, one of the prokaryotic adaptive immune systems, has sparked a lot of attention for its use as a gene editing tool. Currently, type II, V, and VI effector modules of this class have been characterized and extensively tested for nucleic acid editing, imaging, and disease diagnostics. Due to the unique composition of their nuclease catalytic center, the effector modules substantially vary in their function and possible biotechnology applications. In this review, we discuss the structural and functional diversity in class 2 CRISPR effectors, and debate their suitability for nucleic acid targeting and their shortcomings as gene editing tools.},
}

@article {pmid31381206,
year = {2019},
author = {Wolter, F and Puchta, H},
title = {In planta gene targeting can be enhanced by the use of CRISPR/Cas12a.},
journal = {The Plant journal : for cell and molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/tpj.14488},
pmid = {31381206},
issn = {1365-313X},
abstract = {The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a DSB and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell specific expression of the potent but less broadly applicable SaCas9 nuclease. We now tested whether we can improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20 to 80 fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for GT and chromosomal repeat-induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower NHEJ induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT-rich PAM broadens the range of ipGT drastically, particularly when targeting in CG-deserts like promoters and introns. This article is protected by copyright. All rights reserved.},
}

@article {pmid31287004,
year = {2019},
author = {Lalonde, S and Codina-Fauteux, VA and de Bellefon, SM and Leblanc, F and Beaudoin, M and Simon, MM and Dali, R and Kwan, T and Lo, KS and Pastinen, T and Lettre, G},
title = {Integrative analysis of vascular endothelial cell genomic features identifies AIDA as a coronary artery disease candidate gene.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {133},
doi = {10.1186/s13059-019-1749-5},
pmid = {31287004},
issn = {1474-760X},
support = {MOP #136979//CIHR/Canada ; NTC-154083//CIHR/Canada ; U01CA200147/NH/NIH HHS/United States ; },
abstract = {BACKGROUND: Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD) and blood pressure (BP) or hypertension. Many of these loci are not linked to traditional risk factors, nor do they include obvious candidate genes, complicating their functional characterization. We hypothesize that many GWAS loci associated with vascular diseases modulate endothelial functions. Endothelial cells play critical roles in regulating vascular homeostasis, such as roles in forming a selective barrier, inflammation, hemostasis, and vascular tone, and endothelial dysfunction is a hallmark of atherosclerosis and hypertension. To test this hypothesis, we generate an integrated map of gene expression, open chromatin region, and 3D interactions in resting and TNFα-treated human endothelial cells.

RESULTS: We show that genetic variants associated with CAD and BP are enriched in open chromatin regions identified in endothelial cells. We identify physical loops by Hi-C and link open chromatin peaks that include CAD or BP SNPs with the promoters of genes expressed in endothelial cells. This analysis highlights 991 combinations of open chromatin regions and gene promoters that map to 38 CAD and 92 BP GWAS loci. We validate one CAD locus, by engineering a deletion of the TNFα-sensitive regulatory element using CRISPR/Cas9 and measure the effect on the expression of the novel CAD candidate gene AIDA.

CONCLUSIONS: Our data support an important role played by genetic variants acting in the vascular endothelium to modulate inter-individual risk in CAD and hypertension.},
}

@article {pmid31285598,
year = {2019},
author = {Urnov, FD},
title = {Hijack of CRISPR defences by selfish genes holds clinical promise.},
journal = {Nature},
volume = {571},
number = {7764},
pages = {180-181},
doi = {10.1038/d41586-019-01824-0},
pmid = {31285598},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Gene Editing ; RNA ; },
}

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

@article {pmid31262344,
year = {2019},
author = {Teng, F and Guo, L and Cui, T and Wang, XG and Xu, K and Gao, Q and Zhou, Q and Li, W},
title = {CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity.},
journal = {Genome biology},
volume = {20},
number = {1},
pages = {132},
doi = {10.1186/s13059-019-1742-z},
pmid = {31262344},
issn = {1474-760X},
abstract = {CRISPR-based nucleic acid detection methods are reported to facilitate rapid and sensitive DNA detection. However, precise DNA detection at the single-base resolution and its wide applications including high-fidelity SNP genotyping remain to be explored. Here we develop a Cas12b-mediated DNA detection (CDetection) strategy, which shows higher sensitivity on examined targets compared with the previously reported Cas12a-based detection platform. Moreover, we show that CDetection can distinguish differences at the single-base level upon combining the optimized tuned guide RNA (tgRNA). Therefore, our findings highlight the high sensitivity and accuracy of CDetection, which provides an efficient and highly practical platform for DNA detection.},
}

@article {pmid31136000,
year = {2019},
author = {Nishiki, I and Yoshida, T and Fujiwara, A},
title = {Complete genome sequence and characterization of virulence genes in Lancefield group C Streptococcus dysgalactiae isolated from farmed amberjack (Seriola dumerili).},
journal = {Microbiology and immunology},
volume = {63},
number = {7},
pages = {243-250},
doi = {10.1111/1348-0421.12716},
pmid = {31136000},
issn = {1348-0421},
support = {//Ministry of Agriculture, Forestry and Fisheries of Japan/ ; },
abstract = {Lancefield group C Streptococcus dysgalactiae causes infections in farmed fish. Here, the genome of S. dysgalactiae strain kdys0611, isolated from farmed amberjack (Seriola dumerili) was sequenced. The complete genome sequence of kdys0611 consists of a single chromosome and five plasmids. The chromosome is 2,142,780 bp long and has a GC content of 40%. It possesses 2061 coding sequences and 67 tRNA and 6 rRNA operons. One clustered regularly interspaced short palindromic repeat, 125 insertion sequences, and four predicted prophage elements were identified. Phylogenetic analysis based on 126 core genes suggested that the kdys0611 strain is more closely related to S. dysgalactiae subsp. dysgalactiae than to S. dysgalactiae subsp. equisimilis. The genome of kdys0611 harbors 87 genes with sequence similarity to putative virulence-associated genes identified in other bacteria, of which 57 exhibit amino acid identity (>52%) to genes of the S. dysgalactiae subsp. equisimilis GGS124 human clinical isolate. Four putative virulence genes, emm5 (FGCSD_0256), spg_2 (FGCSD_1961), skc (FGCSD_1012), and cna (FGCSD_0159), in kdys0611 did not show significant homology with any deposited S. dysgalactiae genes. The chromosomal sequence of kdys0611 has been deposited in GenBank under Accession No. AP018726. This is the first report of the complete genome sequence of S. dysgalactiae isolated from fish.},
}

@article {pmid31065786,
year = {2019},
author = {Matsuo, K and Atsumi, G},
title = {CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins.},
journal = {Planta},
volume = {250},
number = {2},
pages = {463-473},
doi = {10.1007/s00425-019-03180-9},
pmid = {31065786},
issn = {1432-2048},
support = {16K14833//Japan Society for the Promotion of Science/ ; },
mesh = {Arabidopsis/genetics/metabolism ; Arabidopsis Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Gene Expression ; Green Fluorescent Proteins ; Plant Proteins/genetics/metabolism ; *Plants, Genetically Modified ; RNA Interference ; RNA Replicase/genetics/*metabolism ; Recombinant Proteins ; Tobacco/*genetics/metabolism ; },
abstract = {MAIN CONCLUSION: RDR6 gene knockout Nicotiana benthamiana plant was successfully produced using CRISPR/Cas9 technology. The production of recombinant proteins in plants has many advantages, such as safety and reduced costs. However, there are several problems with this technology, especially low levels of protein production. The dysfunction of the RNA silencing mechanism in plant cells would be effective to improve recombinant protein production because the RNA silencing mechanism efficiently degrades transgene-derived mRNAs. Therefore, to overcome this problem, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was used to develop RNA silencing-related gene knockout transgenic Nicotiana benthamiana. We successfully produced RNA-dependent RNA polymerase 6 (RDR6), one of the most important components of the RNA silencing mechanism-knockout N. benthamiana (ΔRDR6 plants). The ΔRDR6 plants had abnormal flowers and were sterile, as with the Arabidopsis RDR6 mutants. However, a transient gene expression assay showed that the ΔRDR6 plants accumulated larger amounts of green fluorescent protein (GFP) and GFP mRNA than the wild-type (WT) plants. Small RNA sequencing analysis revealed that levels of small interfering RNA against the GFP gene were greatly reduced in the ΔRDR6 plants, as compared to that of the WT plants. These findings demonstrate that the ΔRDR6 plants can express larger amounts of recombinant proteins than WT plants and, therefore, would be useful for recombinant protein production and understanding the contributions of RDR6 to genetic and physiological events in plants.},
}

Arabidopsis/genetics/metabolism
Arabidopsis Proteins/genetics/metabolism
*CRISPR-Cas Systems
Gene Editing
Gene Expression
Green Fluorescent Proteins
Plant Proteins/genetics/metabolism
*Plants, Genetically Modified
RNA Interference
RNA Replicase/genetics/*metabolism
Recombinant Proteins
Tobacco/*genetics/metabolism

@article {pmid30980304,
year = {2019},
author = {Beneke, T and Gluenz, E},
title = {LeishGEdit: A Method for Rapid Gene Knockout and Tagging Using CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1971},
number = {},
pages = {189-210},
doi = {10.1007/978-1-4939-9210-2_9},
pmid = {30980304},
issn = {1940-6029},
support = {15/16_MSD_836338//Medical Research Council/United Kingdom ; },
abstract = {Postgenomic analyses of Leishmania biology benefit from rapid and precise methods for gene manipulation. Traditional methods of gene knockout or tagging by homologous recombination have limitations: they tend to be slow and require successive transfection and selection rounds to knock out multiple alleles of a gene. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems overcome these limitations. We describe here in detail a simple, rapid, and scalable method for CRISPR-Cas9-mediated gene knockout and tagging in Leishmania. This method details how to use simple PCR to generate (1) templates for single guide RNA (sgRNA) transcription in cells expressing Cas9 and T7 RNA polymerase and (2) drug-selectable editing cassettes, using a modular set of plasmids as templates. pT plasmids allow for amplification of drug resistance genes for knockouts and pPLOT plasmids provide a choice of different tags to generate N- or C-terminally tagged proteins. We describe how to use an online platform (LeishGEdit.net) for automated primer design and how to perform PCRs and transfections in small batches or on 96-well plates for large-scale knockout or tagging screens. This method allows generation of knockout mutants or tagged cell lines within 1 week.},
}

@article {pmid30929208,
year = {2019},
author = {Kjos, M},
title = {Transcriptional Knockdown in Pneumococci Using CRISPR Interference.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1968},
number = {},
pages = {89-98},
doi = {10.1007/978-1-4939-9199-0_8},
pmid = {30929208},
issn = {1940-6029},
mesh = {Bacterial Proteins/genetics ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Expression Regulation, Bacterial/genetics ; Polymerase Chain Reaction ; RNA, Guide/genetics ; Streptococcus pneumoniae/*genetics ; },
abstract = {Sequence-specific knockdown of gene expression using CRISPR interference (CRISPRi) has recently been developed for Streptococcus pneumoniae. By coexpression of a catalytically inactive Cas9-protein (dCas9) and a single guide RNA (sgRNA), CRISPRi can be used to knock down transcription of any gene of interest. Gene specificity is mediated by a 20 bp sequence on the sgRNA, and new genes can be targeted by replacing this 20 bp sequence. Here, a protocol is provided for design of sgRNAs and construction of CRIPSRi strains in S. pneumoniae, based on the vectors published by Liu et al. (Mol Syst Biol 13:931, 2017).},
}

Bacterial Proteins/genetics
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Expression Regulation, Bacterial/genetics
Polymerase Chain Reaction
RNA, Guide/genetics
Streptococcus pneumoniae/*genetics

@article {pmid30890798,
year = {2019},
author = {Ledford, H},
title = {Bull 'super dads' are being engineered to produce sperm from another father.},
journal = {Nature},
volume = {567},
number = {7748},
pages = {292-293},
doi = {10.1038/d41586-019-00718-5},
pmid = {30890798},
issn = {1476-4687},
}

@article {pmid30819013,
year = {2019},
author = {Dong, Y and Peng, T and Wu, W and Tan, D and Liu, X and Xie, D},
title = {Efficient introduction of an isogenic homozygous mutation to induced pluripotent stem cells from a hereditary hearing loss family using CRISPR/Cas9 and single-stranded donor oligonucleotides.},
journal = {The Journal of international medical research},
volume = {47},
number = {4},
pages = {1717-1730},
doi = {10.1177/0300060519829990},
pmid = {30819013},
issn = {1473-2300},
mesh = {Adult ; *CRISPR-Cas Systems ; Child ; Female ; *Gene Editing ; Hearing Loss/*genetics/pathology ; Heterozygote ; *Homozygote ; Humans ; Induced Pluripotent Stem Cells/metabolism/*pathology ; Male ; Middle Aged ; *Mutation ; Oligonucleotides/administration & dosage ; Pedigree ; Phenotype ; Prognosis ; Receptors, Purinergic P2X2/*genetics ; },
abstract = {BACKGROUND: Heterozygous purinergic receptor p2x gene (P2RX2) c.178G>T (p.V60L) mutations can lead to progressive hearing loss (HL) and increased susceptibility to noise. However, the underlying mechanisms remain unclear. A combination of human induced pluripotent stem cell (hiPSC) technology with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9-mediated gene editing may provide a promising tool to study gene function and treat hereditary deafness in humans.

METHODS: hiPSC technology and CRISPR/Cas9-mediated gene editing were used to generate heterozygous and homozygous P2RX2 c.178G>T (p.V60L) cell models.

RESULTS: We generated non-integrative hiPSCs from urine samples derived from three members of a large Chinese family carrying heterozygous P2RX2 c.178G>T mutations (designated P2RX2+/-) as a model to study P2RX2-mediated hereditary HL. Furthermore, we used CRISPR/Cas9 and single-stranded donor oligonucleotides to genetically establish homozygous P2RX2 c.178G>T hiPSCs (designated P2RX2-/-) from heterozygous patient-specific hiPSCs as a control to further study the pathological gene function.

CONCLUSIONS: Heterozygous and homozygous P2RX2-mutated hiPSC lines are good models to investigate the pathological mechanisms of P2RX2 mutations in HL pathogenesis. Our findings confirmed our hypothesis that it is feasible and convenient to introduce precise point mutations into genomic loci of interest to generate gene-mutated hiPSC models.},
}

Adult
*CRISPR-Cas Systems
Child
Female
*Gene Editing
Hearing Loss/*genetics/pathology
Heterozygote
*Homozygote
Humans
Induced Pluripotent Stem Cells/metabolism/*pathology
Male
Middle Aged
*Mutation
Oligonucleotides/administration & dosage
Pedigree
Phenotype
Prognosis
Receptors, Purinergic P2X2/*genetics

@article {pmid30787439,
year = {2019},
author = {Karakus, U and Thamamongood, T and Ciminski, K and Ran, W and Günther, SC and Pohl, MO and Eletto, D and Jeney, C and Hoffmann, D and Reiche, S and Schinköthe, J and Ulrich, R and Wiener, J and Hayes, MGB and Chang, MW and Hunziker, A and Yángüez, E and Aydillo, T and Krammer, F and Oderbolz, J and Meier, M and Oxenius, A and Halenius, A and Zimmer, G and Benner, C and Hale, BG and García-Sastre, A and Beer, M and Schwemmle, M and Stertz, S},
title = {MHC class II proteins mediate cross-species entry of bat influenza viruses.},
journal = {Nature},
volume = {567},
number = {7746},
pages = {109-112},
doi = {10.1038/s41586-019-0955-3},
pmid = {30787439},
issn = {1476-4687},
support = {HHSN272201400008C/AI/NIAID NIH HHS/United States ; U19 AI135972/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Chickens/genetics/immunology ; Chiroptera/genetics/immunology/metabolism/*virology ; Female ; Gene Expression Profiling ; HLA-DR Antigens/genetics/immunology/metabolism ; Histocompatibility Antigens Class II/genetics/immunology/*metabolism ; *Host Specificity/genetics/immunology ; Humans ; Influenza A virus/*immunology/*physiology ; Male ; Mice ; Mice, Knockout ; Respiratory System/virology ; Swine/genetics/immunology ; Viral Tropism/genetics/immunology ; Virus Replication ; Zoonoses/genetics/*immunology/metabolism/*virology ; },
abstract = {Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.},
}

Animals
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Chickens/genetics/immunology
Chiroptera/genetics/immunology/metabolism/*virology
Female
Gene Expression Profiling
HLA-DR Antigens/genetics/immunology/metabolism
Histocompatibility Antigens Class II/genetics/immunology/*metabolism
*Host Specificity/genetics/immunology
Humans
Influenza A virus/*immunology/*physiology
Male
Mice
Mice, Knockout
Respiratory System/virology
Swine/genetics/immunology
Viral Tropism/genetics/immunology
Virus Replication
Zoonoses/genetics/*immunology/metabolism/*virology

@article {pmid30726867,
year = {2019},
author = {Li, Z and Zhou, L and Jiang, T and Fan, L and Liu, X and Qiu, X},
title = {Proteasomal deubiquitinase UCH37 inhibits degradation of β-catenin and promotes cell proliferation and motility.},
journal = {Acta biochimica et biophysica Sinica},
volume = {51},
number = {3},
pages = {277-284},
doi = {10.1093/abbs/gmy176},
pmid = {30726867},
issn = {1745-7270},
mesh = {CRISPR-Cas Systems ; Cell Movement ; Cell Proliferation ; HEK293 Cells ; HeLa Cells ; Humans ; Membrane Glycoproteins/physiology ; Proteasome Endopeptidase Complex/metabolism ; Ubiquitin Thiolesterase/*physiology ; Ubiquitination ; beta Catenin/*metabolism ; },
abstract = {The ubiquitin-proteasome system degrades most cellular proteins in eukaryotes. UCH37, also known as UCH-L5, is a deubiquitinase binding to Rpn13, a receptor for ubiquitinated substrates in the 26 S proteasome. But, it remains unclear how UCH37 influences the proteasomal degradation of the ubiquitinated substrates. Because deletion of UCH37 is embryonically lethal in mice, this study aims to investigate the role of UCH37 in proteasomal degradation by constructing the UCH37-deficient cell lines using CRISPR/Cas9 technology. Our results demonstrated that deletion of UCH37 decreased the levels of proteasomal Rpn13, implying that UCH37 might facilitate incorporation of Rpn13 into the proteasome. Meanwhile, deletion of UCH37 decreased the levels of β-catenin and the early endosomal protein Rab8. β-Catenin interacts with TCF/LEF to control transcription, and is involved in development, tissue homeostasis and tumorigenesis. We further found that deletion of UCH37 increased the levels of the ubiquitinated β-catenin and accelerated the hydrogen peroxide-stimulated degradation of β-catenin. Deletion of UCH37 also down-regulated the transcription of c-Myc, a downstream effector of β-catenin, and inhibited cell proliferation and motility. These results raise the possibility that UCH37 maintains the homeostasis of proteasomal degradation reciprocally by assisting the recruitment of the ubiquitin receptor Rpn13 into the proteasome and by reversing ubiquitination of certain critical substrates of the 26 S proteasome.},
}

CRISPR-Cas Systems
Cell Movement
Cell Proliferation
HEK293 Cells
HeLa Cells
Humans
Membrane Glycoproteins/physiology
Proteasome Endopeptidase Complex/metabolism
Ubiquitin Thiolesterase/*physiology
Ubiquitination
beta Catenin/*metabolism

@article {pmid30688002,
year = {2019},
author = {Bi, HL and Xu, J and He, L and Zhang, Y and Li, K and Huang, YP},
title = {CRISPR/Cas9-mediated ebony knockout results in puparium melanism in Spodoptera litura.},
journal = {Insect science},
volume = {26},
number = {6},
pages = {1011-1019},
doi = {10.1111/1744-7917.12663},
pmid = {30688002},
issn = {1744-7917},
support = {XDB11010500//Strategic Priority Research Program of Chinese Academy of Sciences/ ; 31420103918//National Natural Science Foundation of China/ ; 31530072//National Natural Science Foundation of China/ ; 31572330//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Female ; Gene Expression ; Genotype ; Melanins/*metabolism ; Phylogeny ; Pigmentation/*genetics ; Pupa/*metabolism ; Spodoptera/*genetics/metabolism ; },
abstract = {Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.},
}

Animals
Base Sequence
CRISPR-Cas Systems
Female
Gene Expression
Genotype
Melanins/*metabolism
Phylogeny
Pigmentation/*genetics
Pupa/*metabolism
Spodoptera/*genetics/metabolism

@article {pmid30658254,
year = {2019},
author = {Li, Y and Huang, C and Zha, L and Kong, M and Yang, Q and Zhu, Y and Peng, Y and Ouyang, Q and Lu, G and Lin, G and Zhou, D},
title = {Generation of NERCe003-A-3, a p53 compound heterozygous mutation human embryonic stem cell line, by CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {34},
number = {},
pages = {101371},
doi = {10.1016/j.scr.2018.101371},
pmid = {30658254},
issn = {1876-7753},
abstract = {p53 is a tumor suppressor gene involved mainly in the regulation of the G1/S cell cycle phase, DNA repair, and senescence. Although p53 is frequently altered in human cancer, the consequences of its depletion in human embryonic stem cells (hESCs) are unknown. We generated NERCe003-A-3, a p53 knockout hESC line, from the normal NERCe003-A hESC line by using CRISPR/Cas9 editing. This cell line maintained a normal 46, XY karyotype. Further analysis suggested that the cells expressed pluripotency-related markers and had the capacity to differentiate in vitro into derivatives of all three germ layers.},
}

@article {pmid30605842,
year = {2019},
author = {Schuster, S and Saravanakumar, S and Schöls, L and Hauser, S},
title = {Generation of a homozygous CRISPR/Cas9-mediated knockout human iPSC line for the STUB1 locus.},
journal = {Stem cell research},
volume = {34},
number = {},
pages = {101378},
doi = {10.1016/j.scr.2018.101378},
pmid = {30605842},
issn = {1876-7753},
abstract = {STUB1/CHIP is a central component of cellular protein homeostasis and interacts with key proteins involved in the pathogenesis of many neurodegenerative diseases. Missense and truncating mutations in STUB1 lead to SCAR16. For ideal in vitro disease modelling with isogenic controls, we generated a CHIP knockout cell line from a healthy control with no CHIP functionality, but remaining genomic integrity and verified pluripotency.},
}

@article {pmid30605838,
year = {2019},
author = {Xue, Y and Liao, B and Xie, Y and Li, S and Ma, X and Sun, X},
title = {Establishment of an ectodermal dysplasia related gene EDA Knockout human embryonic stem cell line (WAe001-A-22) by CRISPR-Cas9 technology.},
journal = {Stem cell research},
volume = {34},
number = {},
pages = {101379},
doi = {10.1016/j.scr.2018.101379},
pmid = {30605838},
issn = {1876-7753},
abstract = {EDA is a gene located at Xq13.1. It encodes different isoforms of tumor necrosis factor (TNF) superfamily member ectodysplasin A. Ectodysplasin A is a transmembrane protein which can be cleaved to form a secreted form and interact with EDA receptor to mediate the development of ectoderm. Mutations of the EDA gene are related to ectodermal dysplasia and tooth agenesis. Here, we report the establishment of the EDA gene knockout human embryonic stem (hES) cell line by CRISPR-Cas9 technology. This cell line provides good materials for further studies of the roles ectodysplasin A plays in ectoderm differentiation and tooth development.},
}

@article {pmid30549429,
year = {2019},
author = {Xu, J and Chen, RM and Chen, SQ and Chen, K and Tang, LM and Yang, DH and Yang, X and Zhang, Y and Song, HS and Huang, YP},
title = {Identification of a germline-expression promoter for genome editing in Bombyx mori.},
journal = {Insect science},
volume = {26},
number = {6},
pages = {991-999},
doi = {10.1111/1744-7917.12657},
pmid = {30549429},
issn = {1744-7917},
support = {31420103918//National Science Foundation of China/ ; 31530072//National Science Foundation of China/ ; 31802005//National Science Foundation of China/ ; XDB11010600//Strategic Priority Research Program of Chinese Academy of Sciences/ ; BX201700268//National Postdoctoral Program for Innovative Talents/ ; 2017M621548//China Postdoctoral Science Foundation/ ; },
mesh = {Animals ; Animals, Genetically Modified ; Bombyx/*genetics ; CRISPR-Cas Systems ; Cell Line ; Embryo, Nonmammalian ; *Gene Editing ; Genes, Reporter ; Green Fluorescent Proteins ; *Promoter Regions, Genetic ; Transformation, Genetic ; },
abstract = {Identification of stage- and tissue-specific cis-regulatory elements will enable more precise genomic editing. In previous studies of the silkworm Bombyx mori, we identified and characterized several tissue- and sex-specific cis-regulatory elements using transgenic technology, including a female- and fat body-specific promoter, vitellogenin, testis-specific promoters, Radial spoke head 1 (BmR1) and beta-tubulin 4 (Bmβ4). Here we report a cis-regulatory element specific for a somatic and germ cell-expressed promoter, nanos (Bmnos). We investigated activities of three truncated promoter sequences upstream of the transcriptional initiation site sequences of Bmnos in vitro (nos-0.6kb, nos-1kb and nos-2kb) and in vivo (nos-2kb). In BmN cultured cells, all three lengths drove expression of the gene encoding enhanced green fluorescence protein (EGFP), although nos-2kb had the highest fluorescence activity. In transgenic silkworms, nos-2kb drove EGFP expression at the early embryonic stage, and fluorescence was concentrated in the gonads at later embryonic stages. In addition, this cis-regulatory element was not sex differentiated. The fluorescence intensity gradually weakened following the larval developmental stage in the gonads and were broadly expressed in the whole body. The nos-2kb promoter drove the Cas9 system with efficiency comparable to that of the broad-spectrum strong IE1 promoter. These results indicate that Bmnos is an effective endogenous cis-regulatory element in the early embryo and in the gonad that can be used in applications involving the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system.},
}

Animals
Animals, Genetically Modified
Bombyx/*genetics
CRISPR-Cas Systems
Cell Line
Embryo, Nonmammalian
*Gene Editing
Genes, Reporter
Green Fluorescent Proteins
*Promoter Regions, Genetic
Transformation, Genetic

@article {pmid30311353,
year = {2019},
author = {Liu, T and Yang, WQ and Xie, YG and Liu, PW and Xie, LH and Lin, F and Li, CY and Gu, JB and Wu, K and Yan, GY and Chen, XG},
title = {Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus.},
journal = {Insect science},
volume = {26},
number = {6},
pages = {1045-1054},
doi = {10.1111/1744-7917.12645},
pmid = {30311353},
issn = {1744-7917},
support = {AI136850//National Institute of Allergy and Infectious Diseases Grants of USA/ ; 201508020263//Guangzhou Synergy Innovation Key Program for Health/ ; 201605030010//Guangzhou Synergy Innovation Key Program for Health/ ; 81420108024//National Natural Science Foundation of China/ ; 81528013//National Natural Science Foundation of China/ ; 2014A030312016//Natural Science Foundation of Guangdong Province/ ; 2016A020251001//Natural Science Foundation of Guangdong Province/ ; 2016YFC1200500//National Key R&D Program of China/ ; },
mesh = {Aedes/*genetics ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; Insect Vectors/*genetics ; Kynurenine 3-Monooxygenase/genetics ; Male ; Mutation ; },
abstract = {Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in G1 pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used in Ae. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.},
}

Aedes/*genetics
Animals
Base Sequence
CRISPR-Cas Systems
Female
Gene Editing/*methods
Insect Vectors/*genetics
Kynurenine 3-Monooxygenase/genetics
Male
Mutation

@article {pmid30144341,
year = {2018},
author = {Damodharan, S and Corem, S and Gupta, SK and Arazi, T},
title = {Tuning of SlARF10A dosage by sly-miR160a is critical for auxin-mediated compound leaf and flower development.},
journal = {The Plant journal : for cell and molecular biology},
volume = {96},
number = {4},
pages = {855-868},
doi = {10.1111/tpj.14073},
pmid = {30144341},
issn = {1365-313X},
abstract = {miR160 adjusts auxin-mediated development by post-transcriptional regulation of the auxin response factors ARF10/16/17. In tomato, knockdown of miR160 (sly-miR160) suggested that it is required for auxin-driven leaf blade outgrowth, but whether additional developmental events are adjusted by sly-miR160 is not clear. Here, the SlMIR160 genes and the genes of its SlARFs targets were edited by CRISPR/Cas9 resulting in the isolation of loss-of-function mutants. In addition, hypomorphic mutants that accumulate variable reduced levels of sly-miR160a were isolated. We found that the loss-of-function mutants in SlMIR160a (CR-slmir160a-6/7) produced only four wiry leaves, whereas the hypomorphic mutants developed leaves and flowers with graded developmental abnormalities. Phenotypic severity correlated with the upregulation of SlARF10A. Consistent with that, double mutants in SlMIR160a and SlARF10A restored leaf and flower development indicating that over-accumulation of SlARF10A underlay the developmental abnormalities exhibited in the CR-slmir160a mutants. Phenotype severity also correlated with the upregulation of the SHOOT MERISTEMLESS homolog Tomato Knotted 2, which in turn activated the transcription of the cytokinin biosynthesis genes SlIPT2 and SlIPT4. However, no change in Tomato Knotted 2 was detected in the absence of SlARF10A, suggesting that it is upregulated due to auxin signaling suppression by SlARF10A. Knockout of sly-miR160a-targeted SlARFs showed that whereas SlARF10A is indispensable for leaf blade outgrowth and floral organ patterning, the functions of SlARF16A and SlARF17 are redundant. Taken together our results suggest that sly-miR160a promotes blade outgrowth as well as leaf and leaflet initiation and floral organ development through the quantitative regulation of its major target SlARF10A.},
}

@article {pmid30088858,
year = {2019},
author = {Wang, YH and Chen, XE and Yang, Y and Xu, J and Fang, GQ and Niu, CY and Huang, YP and Zhan, S},
title = {The Masc gene product controls masculinization in the black cutworm, Agrotis ipsilon.},
journal = {Insect science},
volume = {26},
number = {6},
pages = {1037-1044},
doi = {10.1111/1744-7917.12635},
pmid = {30088858},
issn = {1744-7917},
support = {31522053//National Science Foundation of China/ ; 31501648//National Science Foundation of China/ ; 31420103918//National Science Foundation of China/ ; 91631103//National Science Foundation of China/ ; XDB11010600//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 2015CB755703//National Basic Research Program of China/ ; },
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; CRISPR-Cas Systems ; Female ; Insect Proteins/*physiology ; Male ; Moths/*genetics ; Mutation ; Phenotype ; *Sex Differentiation ; },
abstract = {Sex determination has been studied in the model lepidopteran species Bombyx mori, but it remains poorly understood in lepidopteran pests. In the present study, we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species, Agrotis ipsilon. Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280). We assessed the masculinizing activity of AiMasc in BmN cells and found that AiMasc induced expression of the male-specific doublesex isoform. Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A. ipsilon caused abnormalities in abdominal segments and external genitalia, resulting in male-specific sterility. These results suggest that Masc participates in the process of sex determination in A. ipsilon. Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control.},
}

Amino Acid Sequence
Animals
Base Sequence
CRISPR-Cas Systems
Female
Insect Proteins/*physiology
Male
Moths/*genetics
Mutation
Phenotype
*Sex Differentiation

@article {pmid30088341,
year = {2019},
author = {Wang, XG and Ma, SY and Chang, JS and Shi, R and Wang, RL and Zhao, P and Xia, QY},
title = {Programmable activation of Bombyx gene expression using CRISPR/dCas9 fusion systems.},
journal = {Insect science},
volume = {26},
number = {6},
pages = {983-990},
doi = {10.1111/1744-7917.12634},
pmid = {30088341},
issn = {1744-7917},
support = {Xm2016030//Chongqing Postdoctoral Science Foundation/ ; cstc2017jcyjAX0349//Chongqing Research program of basic Research and Frontier Technology/ ; 31530071//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Bombyx/*metabolism ; *CRISPR-Cas Systems ; *Genetic Techniques ; *Transcriptional Activation ; Up-Regulation ; },
abstract = {The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing genomic sequence in mammalian cells and several multicellular organisms. We previously showed that CRISPR-associated protein 9 (Cas9) and CRISPR from Prevotella and Francisella 1 (Cpf1) could induce target mutations, deletions, inversions, and duplications both singly and multiplex in silkworm, Bombyx mori. However, it remains unknown whether the CRISPR activation (CRISPRa) system can be used in B. mori. In this study, we investigated the CRISPRa system, in which a nuclease dead Streptococcus pyogenes Cas9 (SpCas9) is fused to two transcription activation domains, including VP64 (a tetramer of the herpes simplex VP16 transcriptional activator domain), and VPR (a tripartite activator, composed of VP64, p65, and Rta). The results showed that both dCas9-VP64 and dCas9-VPR systems could be used in B. mori cells, of which the latter showed significantly higher activity. The dCas9-VPR system showed considerable activity on all five tested target genes, and further analysis revealed that the up-regulation of genes was negatively correlated to their basal expression level. We also observed that this system could be used to upregulate a range of target genes. Taken together, our findings demonstrate that CRISPRa can be a powerful tool to study gene functions in B. mori and perhaps other non-drosophila insects.},
}

Animals
Bombyx/*metabolism
*CRISPR-Cas Systems
*Genetic Techniques
*Transcriptional Activation
Up-Regulation

@article {pmid30084531,
year = {2019},
author = {Jia, L and Ma, SY and Zhang, T and Xing, WQ and Liu, Y and Li, YF and Chen, XX and Xia, QY},
title = {Enhanced Bombyx genome editing via Cas9 ribonucleoprotein injection.},
journal = {Insect science},
volume = {26},
number = {6},
pages = {1059-1062},
doi = {10.1111/1744-7917.12633},
pmid = {30084531},
issn = {1744-7917},
support = {31530071//National Natural Science Foundation of China/International ; Xm2016030//Chongqing Postdoctoral Science Foundation/International ; cstc2017jcyjAX0349//Chongqing Research program of basic Research and Frontier Technology/International ; },
mesh = {Animals ; Base Sequence ; Bombyx/*genetics ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; Gene Editing/*methods ; },
}

Animals
Base Sequence
Bombyx/*genetics
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Gene Editing/*methods

@article {pmid29955892,
year = {2018},
author = {Bollen, Y and Post, J and Koo, BK and Snippert, HJG},
title = {How to create state-of-the-art genetic model systems: strategies for optimal CRISPR-mediated genome editing.},
journal = {Nucleic acids research},
volume = {46},
number = {13},
pages = {6435-6454},
pmid = {29955892},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9 ; *CRISPR-Cas Systems ; Endodeoxyribonucleases ; *Gene Editing ; *Models, Genetic ; Polymerase Chain Reaction ; Recombinational DNA Repair ; },
abstract = {Model systems with defined genetic modifications are powerful tools for basic research and translational disease modelling. Fortunately, generating state-of-the-art genetic model systems is becoming more accessible to non-geneticists due to advances in genome editing technologies. As a consequence, solely relying on (transient) overexpression of (mutant) effector proteins is no longer recommended since scientific standards increasingly demand genetic modification of endogenous loci. In this review, we provide up-to-date guidelines with respect to homology-directed repair (HDR)-mediated editing of mammalian model systems, aimed at assisting researchers in designing an efficient genome editing strategy.},
}

CRISPR-Associated Protein 9
*CRISPR-Cas Systems
Endodeoxyribonucleases
*Gene Editing
*Models, Genetic
Polymerase Chain Reaction
Recombinational DNA Repair

@article {pmid29938905,
year = {2019},
author = {You, L and Bi, HL and Wang, YH and Li, XW and Chen, XE and Li, ZQ},
title = {CRISPR/Cas9-based mutation reveals Argonaute 1 is essential for pigmentation in Ostrinia furnacalis.},
journal = {Insect science},
volume = {26},
number = {6},
pages = {1020-1028},
doi = {10.1111/1744-7917.12628},
pmid = {29938905},
issn = {1744-7917},
support = {31372257//National Natural Science Foundation of China/ ; 31420103918//National Natural Science Foundation of China/ ; 31601903//National Natural Science Foundation of China/ ; },
mesh = {Amino Acid Sequence ; Animals ; Argonaute Proteins/*genetics ; Base Sequence ; CRISPR-Cas Systems ; Cloning, Molecular ; Moths/*genetics ; Mutation ; Pigmentation/*genetics ; },
abstract = {Ostrinia furnacalis (Lepidoptera: Pyralidae) is one of the most destructive agricultural pests in Asia. Traditional pest-management methods include sex pheromone capture, transgenic crops that produce Bacillus thuringiensis toxin, and pesticides. Although these strategies control pest populations effectively, they also cause negative side effects, including dramatically increased pesticide resistance, severe pollution, and hazards for human health. Recently developed genome editing tools provide new prospects for pest management and have been successfully used in several species. However, few examples have been reported in the agricultural pest O. furnacalis due to a lack in genomic information. In this report, we identified only one transcript of O. furnacalis Argonaute 1 (OfAgo1) gene from the genome and cloned the open reading frame. OfAgo1 presented the maximum expression at the embryo stage or in the fat body during the larval stages. To understand its function, an OfAgo1 mutant was constructed using the Clustered Regularly Interspaced Short Palindromic Repeat/RNA-guided Cas9 nuclease (CRISPR/Cas9). Mutagenesis of OfAgo1 disrupted cuticle pigmentation by down-regulating micro RNAs and pigmentation-related genes. This is the first report for the cloning and functional analysis of OfAgo1, revealing a role of OfAgo1 in cuticle pigmentation. The current report also established a CRISPR/Cas9 system in O. furnacalis, providing a new insight for pest management.},
}

Amino Acid Sequence
Animals
Argonaute Proteins/*genetics
Base Sequence
CRISPR-Cas Systems
Cloning, Molecular
Moths/*genetics
Mutation
Pigmentation/*genetics

@article {pmid29906414,
year = {2018},
author = {Ribeiro, JM and Garriga, M and Potchen, N and Crater, AK and Gupta, A and Ito, D and Desai, SA},
title = {Guide RNA selection for CRISPR-Cas9 transfections in Plasmodium falciparum.},
journal = {International journal for parasitology},
volume = {48},
number = {11},
pages = {825-832},
doi = {10.1016/j.ijpara.2018.03.009},
pmid = {29906414},
issn = {1879-0135},
mesh = {CRISPR-Cas Systems/*genetics ; Computer Simulation ; Gene Editing ; Genetic Markers ; Genetic Vectors ; Genome, Protozoan ; Genome-Wide Association Study ; Models, Genetic ; Plasmodium falciparum/*genetics ; RNA, Guide/*genetics ; *Transfection ; },
abstract = {CRISPR-Cas9 mediated genome editing is addressing key limitations in the transfection of malaria parasites. While this method has already simplified the needed molecular cloning and reduced the time required to generate mutants in the human pathogen Plasmodium falciparum, optimal selection of required guide RNAs and guidelines for successful transfections have not been well characterised, leading workers to use time-consuming trial and error approaches. We used a genome-wide computational approach to create a comprehensive and publicly accessible database of possible guide RNA sequences in the P. falciparum genome. For each guide, we report on-target efficiency and specificity scores as well as information about the genomic site relevant to optimal design of CRISPR-Cas9 transfections to modify, disrupt, or conditionally knockdown any gene. As many antimalarial drug and vaccine targets are encoded by multigene families, we also developed a new paralog specificity score that should facilitate modification of either a single family member of interest or multiple paralogs that serve overlapping roles. Finally, we tabulated features of successful transfections in our laboratory, providing broadly useful guidelines for parasite transfections. Molecular studies aimed at understanding parasite biology or characterising drug and vaccine targets in P. falciparum should be facilitated by this comprehensive database.},
}

CRISPR-Cas Systems/*genetics
Computer Simulation
Gene Editing
Genetic Markers
Genetic Vectors
Genome, Protozoan
Genome-Wide Association Study
Models, Genetic
Plasmodium falciparum/*genetics
RNA, Guide/*genetics
*Transfection

@article {pmid29861287,
year = {2018},
author = {Lin, YT and Seo, J and Gao, F and Feldman, HM and Wen, HL and Penney, J and Cam, HP and Gjoneska, E and Raja, WK and Cheng, J and Rueda, R and Kritskiy, O and Abdurrob, F and Peng, Z and Milo, B and Yu, CJ and Elmsaouri, S and Dey, D and Ko, T and Yankner, BA and Tsai, LH},
title = {APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types.},
journal = {Neuron},
volume = {98},
number = {6},
pages = {1141-1154.e7},
pmid = {29861287},
issn = {1097-4199},
support = {R01 MH113279/MH/NIMH NIH HHS/United States ; RC1 AG036106/AG/NIA NIH HHS/United States ; RF1 AG048029/AG/NIA NIH HHS/United States ; RF1 AG048056/AG/NIA NIH HHS/United States ; },
mesh = {Alzheimer Disease/*genetics/metabolism ; Amyloid beta-Peptides/*metabolism ; Apolipoprotein E3/metabolism ; Apolipoprotein E4/*genetics/metabolism ; Astrocytes/metabolism ; Brain/cytology/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; Humans ; Induced Pluripotent Stem Cells/*metabolism ; Lipid Metabolism ; Microglia/immunology/metabolism ; Neuroglia/*metabolism ; Neurons/*metabolism ; Organoids/metabolism ; Peptide Fragments/*metabolism ; Phosphoproteins/metabolism ; Synaptic Transmission ; Transcriptome ; tau Proteins/*metabolism ; },
abstract = {The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimer's disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function (neurons), lipid metabolism (astrocytes), and immune response (microglia-like cells). APOE4 neurons exhibited increased synapse number and elevated Aβ42 secretion relative to isogenic APOE3 cells while APOE4 astrocytes displayed impaired Aβ uptake and cholesterol accumulation. Notably, APOE4 microglia-like cells exhibited altered morphologies, which correlated with reduced Aβ phagocytosis. Consistently, converting APOE4 to APOE3 in brain cell types from sAD iPSCs was sufficient to attenuate multiple AD-related pathologies. Our study establishes a reference for human cell-type-specific changes associated with the APOE4 variant. VIDEO ABSTRACT.},
}

Alzheimer Disease/*genetics/metabolism
Amyloid beta-Peptides/*metabolism
Apolipoprotein E3/metabolism
Apolipoprotein E4/*genetics/metabolism
Astrocytes/metabolism
Brain/cytology/metabolism
CRISPR-Cas Systems
Cell Differentiation
Humans
Induced Pluripotent Stem Cells/*metabolism
Lipid Metabolism
Microglia/immunology/metabolism
Neuroglia/*metabolism
Neurons/*metabolism
Organoids/metabolism
Peptide Fragments/*metabolism
Phosphoproteins/metabolism
Synaptic Transmission
Transcriptome
tau Proteins/*metabolism

@article {pmid29808584,
year = {2019},
author = {Du, Q and Wen, L and Zheng, SC and Bi, HL and Huang, YP and Feng, QL and Liu, L},
title = {Identification and functional characterization of doublesex gene in the testis of Spodoptera litura.},
journal = {Insect science},
volume = {26},
number = {6},
pages = {1000-1010},
doi = {10.1111/1744-7917.12608},
pmid = {29808584},
issn = {1744-7917},
support = {31330071//National Natural Science Foundation of China/ ; 31772519//National Natural Science Foundation of China/ ; S2012010010185//Natural Science Foundation of Guangdong Province/ ; 2017A030313210//Natural Science Foundation of Guangdong Province/ ; },
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Insect Proteins/*genetics/metabolism ; Male ; Phenotype ; Spodoptera/*genetics/growth & development/metabolism ; Testis/growth & development/metabolism ; },
abstract = {Fusion of the testis occurs in most Lepidoptera insects, including Spodoptera litura, an important polyphagous pest. Testicular fusion in S. litura is advantageous for male reproduction, and the molecular mechanism of fusion remains unknown. Doublesex influences the formation of genitalia, the behavior of courtship, and sexually dimorphic traits in fruit-fly and silkworm, and is essential for sexual differentiation. However, its purpose in the testis of S. litura remains unknown. The doublesex gene of S. litura (Sldsx) has male-specific SldsxM and female-specific SldsxF isoforms, and exhibits a higher expression level in the male testis. At the testicular fusion stage (L6D6), Sldsx attained the highest expression compared to the pre-fusion and post-fusion periods. Moreover, Sldsx had a higher expression in the peritoneal sheaths of testis than that of germ cells in the follicle. CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/Cas9) was applied to S. litura to determine the role of Sldsx. A mixture of single guide RNA messenger RNA and Cas9 protein (300 ng/μL each) was injected into eggs within 2 h following oviposition. CRISPR/Cas9 successfully induced genomic mutagenesis of Sldsx at G0 generation. The mutant males had smaller testis surrounded by less tracheae. Moreover, the mutant males had abnormal external genitalia and could not finish mating with wild-type females. Additionally, testes were fused for almost all mutant males. The results showed that Sldsx was not related to testicular fusion, and is required for both testis development and the formation and function of external genitalia in S. litura. The main roles of doublesex on the male are similar to other insects.},
}

Animals
Base Sequence
CRISPR-Cas Systems
Insect Proteins/*genetics/metabolism
Male
Phenotype
Spodoptera/*genetics/growth & development/metabolism
Testis/growth & development/metabolism

@article {pmid29735544,
year = {2018},
author = {Luo, XL and Deng, CC and Su, XD and Wang, F and Chen, Z and Wu, XP and Liang, SB and Liu, JH and Fu, LW},
title = {Loss of MED12 Induces Tumor Dormancy in Human Epithelial Ovarian Cancer via Downregulation of EGFR.},
journal = {Cancer research},
volume = {78},
number = {13},
pages = {3532-3543},
doi = {10.1158/0008-5472.CAN-18-0134},
pmid = {29735544},
issn = {1538-7445},
mesh = {Adult ; Aged ; Animals ; Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Carcinoma, Ovarian Epithelial/*genetics/pathology/therapy ; Cell Line, Tumor ; Cell Proliferation/genetics ; Cohort Studies ; Cytoreduction Surgical Procedures ; Down-Regulation ; Drug Resistance, Neoplasm/genetics ; ErbB Receptors/genetics/metabolism ; Female ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; Kaplan-Meier Estimate ; Mediator Complex/genetics/*metabolism ; Mice ; Mice, Nude ; Middle Aged ; Neoplasm Recurrence, Local/*genetics/pathology ; Ovarian Neoplasms/*genetics/pathology/therapy ; Promoter Regions, Genetic/genetics ; Xenograft Model Antitumor Assays ; Young Adult ; },
abstract = {A high rate of disease relapse makes epithelial ovarian cancer (EOC) the leading cause of death among all gynecologic malignancies. These relapses are often due to tumor dormancy. Here we identify the RNA polymerase II transcriptional mediator subunit 12 (MED12) as an important molecular regulator of tumor dormancy. MED12 knockout (KO) induced dormancy of EOC cells in vitro and in vivo, and microarray analysis showed that MED12 KO decreased expression of EGFR. Restoration of EGFR expression in MED12 KO cells restored proliferation. Additionally, MED12 bound to the promoter of EGFR, and correlation studies showed that MED12 expression positively correlated with EGFR expression in EOC patient samples. Clinical data demonstrated that chemotherapy-resistant patients expressed lower levels of MED12 compared with responsive patients. Overall, our data show that MED12 plays an important role in regulating dormancy of EOC through regulation of EGFR.Significance: MED12 is identified as a novel, important regulator of tumor dormancy in human ovarian cancer. Cancer Res; 78(13); 3532-43. ©2018 AACR.},
}

Adult
Aged
Animals
Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use
CRISPR-Cas Systems/genetics
Carcinoma, Ovarian Epithelial/*genetics/pathology/therapy
Cell Line, Tumor
Cell Proliferation/genetics
Cohort Studies
Cytoreduction Surgical Procedures
Down-Regulation
Drug Resistance, Neoplasm/genetics
ErbB Receptors/genetics/metabolism
Female
*Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
Humans
Kaplan-Meier Estimate
Mediator Complex/genetics/*metabolism
Mice
Mice, Nude
Middle Aged
Neoplasm Recurrence, Local/*genetics/pathology
Ovarian Neoplasms/*genetics/pathology/therapy
Promoter Regions, Genetic/genetics
Xenograft Model Antitumor Assays
Young Adult

@article {pmid29718399,
year = {2018},
author = {Wu, X and Mao, S and Yang, Y and Rushdi, MN and Krueger, CJ and Chen, AK},
title = {A CRISPR/molecular beacon hybrid system for live-cell genomic imaging.},
journal = {Nucleic acids research},
volume = {46},
number = {13},
pages = {e80},
pmid = {29718399},
issn = {1362-4962},
mesh = {CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems ; Chromatin/metabolism ; *Fluorescent Dyes ; Genomics ; HEK293 Cells ; HeLa Cells ; Humans ; *Microscopy, Fluorescence ; *Oligonucleotide Probes ; },
abstract = {The clustered regularly interspersed short palindromic repeat (CRISPR) gene-editing system has been repurposed for live-cell genomic imaging, but existing approaches rely on fluorescent protein reporters, making sensitive and continuous imaging difficult. Here, we present a fluorophore-based live-cell genomic imaging system that consists of a nuclease-deactivated mutant of the Cas9 protein (dCas9), a molecular beacon (MB), and an engineered single-guide RNA (sgRNA) harboring a unique MB target sequence (sgRNA-MTS), termed CRISPR/MB. Specifically, dCas9 and sgRNA-MTS are first co-expressed to target a specific locus in cells, followed by delivery of MBs that can then hybridize to MTS to illuminate the target locus. We demonstrated the feasibility of this approach for quantifying genomic loci, for monitoring chromatin dynamics, and for dual-color imaging when using two orthogonal MB/MTS pairs. With flexibility in selecting different combinations of fluorophore/quencher pairs and MB/MTS sequences, our CRISPR/MB hybrid system could be a promising platform for investigating chromatin activities.},
}

CRISPR-Associated Protein 9/genetics/metabolism
*CRISPR-Cas Systems
Chromatin/metabolism
*Fluorescent Dyes
Genomics
HEK293 Cells
HeLa Cells
Humans
*Microscopy, Fluorescence
*Oligonucleotide Probes

@article {pmid29617873,
year = {2018},
author = {Gaidukov, L and Wroblewska, L and Teague, B and Nelson, T and Zhang, X and Liu, Y and Jagtap, K and Mamo, S and Tseng, WA and Lowe, A and Das, J and Bandara, K and Baijuraj, S and Summers, NM and Lu, TK and Zhang, L and Weiss, R},
title = {A multi-landing pad DNA integration platform for mammalian cell engineering.},
journal = {Nucleic acids research},
volume = {46},
number = {8},
pages = {4072-4086},
pmid = {29617873},
issn = {1362-4962},
mesh = {Animals ; CHO Cells ; CRISPR-Associated Protein 9 ; CRISPR-Cas Systems ; *Cell Engineering ; Cricetulus ; Genetic Loci ; Genome ; Homologous Recombination ; Recombinant Proteins/biosynthesis/*genetics ; Transgenes ; },
abstract = {Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three 'landing pad' recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection. We used this method to controllably integrate up to nine copies of a monoclonal antibody, representing about 100 kb of heterologous DNA in 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload resulted in a linear increase in antibody expression. Overall, this multi-copy site-specific integration platform allows for controllable and reproducible insertion of large amounts of DNA into stable genomic sites, which has broad applications for mammalian synthetic biology, recombinant protein production and biomanufacturing.},
}

Animals
CHO Cells
CRISPR-Associated Protein 9
CRISPR-Cas Systems
*Cell Engineering
Cricetulus
Genetic Loci
Genome
Homologous Recombination
Recombinant Proteins/biosynthesis/*genetics
Transgenes