@article {pmid30901150,
year = {2019},
author = {Mehnert, M and Li, W and Wu, C and Salovska, B and Liu, Y},
title = {Combining Rapid Data Independent Acquisition and CRISPR Gene Deletion for Studying Potential Protein Functions: A Case of HMGN1.},
journal = {Proteomics},
volume = {},
number = {},
pages = {e1800438},
doi = {10.1002/pmic.201800438},
pmid = {30901150},
issn = {1615-9861},
abstract = {CRISPR-Cas gene editing holds substantial promise in many biomedical disciplines and basic research. Due to the important functional implications of non-histone chromosomal protein HMG-14 (HMGN1) in regulating chromatin structure and tumor immunity, we performed gene knockout of HMGN1 by CRISPR in cancer cells and studied the following proteomic regulation events. In particular, we utilized DIA mass spectrometry (DIA-MS) and reproducibly measured more than 6200 proteins (protein- FDR 1%) and more than 82,000 peptide precursors in the single MS shots of two hours. HMGN1 protein deletion was confidently verified by DIA-MS in all of the clone- and dish- replicates following CRISPR. Statistical analysis revealed 147 proteins changed their expressions significantly after HMGN1 knockout. Functional annotation and enrichment analysis indicate the deletion of HMGN1 induces the histone inactivation, various stress pathways, remodeling of extracellular proteomes, cell proliferation, as well as immune regulation processes such as complement and coagulation cascade and interferon alpha/ gamma response in cancer cells. These results shed new lights on the cellular functions of HMGN1. We suggest that DIA-MS can be reliably used as a rapid, robust, and cost-effective proteomic-screening tool to assess the outcome of the CRISPR experiments. This article is protected by copyright. All rights reserved.},
}

@article {pmid30900787,
year = {2019},
author = {Schmidt, C and Pacher, M and Puchta, H},
title = {Efficient induction of heritable inversions in plant genomes using the CRISPR/Cas system.},
journal = {The Plant journal : for cell and molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/tpj.14322},
pmid = {30900787},
issn = {1365-313X},
abstract = {During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Thus, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from S. aureus (SaCas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana. Via deep sequencing, we defined the patterns of junction formation in wild-type and in the non-homologous end joining (NHEJ) mutant ku70-1. Surprisingly, in plants deficient of KU70, inversion induction is enhanced, indicating that KU70 is required for tethering the local broken ends together during repair. However, in contrast to wild-type, most junctions are formed by microhomology-mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg cell specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will. This article is protected by copyright. All rights reserved.},
}

@article {pmid30898877,
year = {2019},
author = {Choquet, K and Forget, D and Meloche, E and Dicaire, MJ and Bernard, G and Vanderver, A and Schiffmann, R and Fabian, MR and Teichmann, M and Coulombe, B and Brais, B and Kleinman, CL},
title = {Leukodystrophy-associated POLR3A mutations down-regulate the RNA polymerase III transcript and important regulatory RNA BC200.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA118.006271},
pmid = {30898877},
issn = {1083-351X},
abstract = {RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small non-coding RNAs, a number of which are involved in mRNA translation. Recessive mutations in POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3-HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has been so far elusive. Here, we used CRISPR-Cas9 to introduce the POLR3A mutation c.2554A>G (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. Transcriptomic profiling uncovered a subset of transcripts vulnerable to Pol III hypofunction, including a global reduction in tRNA levels. The brain cytoplasmic BC200 RNA (BCYRN1), involved in translation regulation, was consistently affected in all our cellular models, including patient-derived fibroblasts. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Upon differentiation, mRNA levels of the MBP gene, encoding myelin basic protein, were significantly decreased in POLR3A-mutant cells. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3-HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease.},
}

@article {pmid30644027,
year = {2019},
author = {Xu, ZS and Feng, K and Xiong, AS},
title = {CRISPR/Cas9-Mediated Multiply Targeted Mutagenesis in Orange and Purple Carrot Plants.},
journal = {Molecular biotechnology},
volume = {61},
number = {3},
pages = {191-199},
doi = {10.1007/s12033-018-00150-6},
pmid = {30644027},
issn = {1559-0305},
support = {31501775//National Natural Science Foundation of China/ ; 31872098//National Natural Science Foundation of China/ ; ZW201710//Open Project of State Key Laboratory of Crop Genetics and Germplasm Enhancement/ ; PAPD//Priority Academic Program Development of Jiangsu Higher Education Institutions/ ; },
mesh = {Agrobacterium ; CRISPR-Cas Systems ; Citrus sinensis/genetics/*growth & development ; Daucus carota/genetics/*growth & development ; Gene Editing/methods ; Mutagenesis, Site-Directed/*methods ; Mutation Rate ; Plant Proteins/*genetics ; RNA, Guide/genetics ; Sequence Analysis, DNA ; Transformation, Bacterial ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system has been successfully used for precise genome editing in many plant species, including in carrot cells, very recently. However, no stable gene-editing carrot plants were obtained with CRISPR/Cas9 system to date. In the present study, four sgRNA expression cassettes, individually driven by four different promoters and assembled in a single CRISPR/Cas9 vector, were transformed into carrots using Agrobacterium-mediated genetic transformation. Four sites of DcPDS and DcMYB113-like genes were chosen as targets. Knockout of DcPDS in orange carrot 'Kurodagosun' resulted in the generation of albino carrot plantlets, with about 35.3% editing efficiency. DcMYB113-like was also successfully edited in purple carrot 'Deep purple', resulting in purple depigmented carrot plants, with about 36.4% rate of mutation. Sequencing analyses showed that insertion, deletion, and substitution occurred in the target sites, generating heterozygous, biallelic, and chimeric mutations. The highest efficiency of mutagenesis was observed in the sites targeted by AtU6-29-driven sgRNAs in both DcPDS- and DcMYB113-like-knockout T0 plants, which always induced double-strand breaks in the target sites. Our results proved that CRISPR/Cas9 system could be for generating stable gene-editing carrot plants.},
}

Agrobacterium
CRISPR-Cas Systems
Citrus sinensis/genetics/*growth & development
Daucus carota/genetics/*growth & development
Gene Editing/methods
Mutagenesis, Site-Directed/*methods
Mutation Rate
Plant Proteins/*genetics
RNA, Guide/genetics
Sequence Analysis, DNA
Transformation, Bacterial

@article {pmid30247490,
year = {2018},
author = {Kyrou, K and Hammond, AM and Galizi, R and Kranjc, N and Burt, A and Beaghton, AK and Nolan, T and Crisanti, A},
title = {A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes.},
journal = {Nature biotechnology},
volume = {36},
number = {11},
pages = {1062-1066},
pmid = {30247490},
issn = {1546-1696},
mesh = {Animals ; Anopheles/*genetics ; CRISPR-Cas Systems/*genetics ; DNA-Binding Proteins ; Exons/genetics ; Female ; *Gene Drive Technology ; Gene Targeting ; Insect Proteins/genetics/*metabolism ; Introns/genetics ; Male ; Mosquito Vectors/*genetics ; Sex Determination Processes/genetics ; },
abstract = {In the human malaria vector Anopheles gambiae, the gene doublesex (Agdsx) encodes two alternatively spliced transcripts, dsx-female (AgdsxF) and dsx-male (AgdsxM), that control differentiation of the two sexes. The female transcript, unlike the male, contains an exon (exon 5) whose sequence is highly conserved in all Anopheles mosquitoes so far analyzed. We found that CRISPR-Cas9-targeted disruption of the intron 4-exon 5 boundary aimed at blocking the formation of functional AgdsxF did not affect male development or fertility, whereas females homozygous for the disrupted allele showed an intersex phenotype and complete sterility. A CRISPR-Cas9 gene drive construct targeting this same sequence spread rapidly in caged mosquitoes, reaching 100% prevalence within 7-11 generations while progressively reducing egg production to the point of total population collapse. Owing to functional constraint of the target sequence, no selection of alleles resistant to the gene drive occurred in these laboratory experiments. Cas9-resistant variants arose in each generation at the target site but did not block the spread of the drive.},
}

Animals
Anopheles/*genetics
CRISPR-Cas Systems/*genetics
DNA-Binding Proteins
Exons/genetics
Female
*Gene Drive Technology
Gene Targeting
Insect Proteins/genetics/*metabolism
Introns/genetics
Male
Mosquito Vectors/*genetics
Sex Determination Processes/genetics

@article {pmid30103756,
year = {2018},
author = {Cao, J and Wei, J and Yang, P and Zhang, T and Chen, Z and He, F and Wei, F and Chen, H and Hu, H and Zhong, J and Yang, Z and Cai, W and Li, W and Wang, Q},
title = {Genome-scale CRISPR-Cas9 knockout screening in gastrointestinal stromal tumor with Imatinib resistance.},
journal = {Molecular cancer},
volume = {17},
number = {1},
pages = {121},
pmid = {30103756},
issn = {1476-4598},
mesh = {CRISPR-Cas Systems ; Cell Line, Tumor ; *Drug Resistance, Neoplasm ; Gastrointestinal Neoplasms/*genetics ; Gastrointestinal Stromal Tumors/*genetics ; Gene Expression Regulation, Neoplastic ; *Gene Regulatory Networks ; Humans ; Imatinib Mesylate ; Signal Transduction ; },
abstract = {Genome-scale CRISPR-Cas9 Knockout Screening was applied to investigate novel targets in imatinib-resistant gastrointestinal stromal tumor (GIST). 20 genes and 2 miRNAs have been selected by total reads of sgRNA and sgRNA diversity, which has been further validated in imatinib-resistant GIST cells by CCK8 and qPCR analysis. Our study has finally revealed 9 genes (DBP, NR3C1, TCF12, TP53, ZNF12, SOCS6, ZFP36, ACYP1, and DRD1) involved in imatinib-resistant GIST-T1 cells. TP53 and SOCS6 may be the most promising candidate genes for imatinib-resistance due to the possible signaling pathway, such as apoptosis pathway and Wnt signaling pathway, JAK-STAT signaling pathway. It is necessary to perform more studies to discover novel targets in imatinib-resistant GIST, including DBP, NR3C1, TCF12, ZNF12, ZFP36, ACYP1 and DRD1.},
}

CRISPR-Cas Systems
Cell Line, Tumor
*Drug Resistance, Neoplasm
Gastrointestinal Neoplasms/*genetics
Gastrointestinal Stromal Tumors/*genetics
Gene Expression Regulation, Neoplastic
*Gene Regulatory Networks
Humans
Imatinib Mesylate
Signal Transduction

@article {pmid29932899,
year = {2018},
author = {Yamazaki, T and Souquere, S and Chujo, T and Kobelke, S and Chong, YS and Fox, AH and Bond, CS and Nakagawa, S and Pierron, G and Hirose, T},
title = {Functional Domains of NEAT1 Architectural lncRNA Induce Paraspeckle Assembly through Phase Separation.},
journal = {Molecular cell},
volume = {70},
number = {6},
pages = {1038-1053.e7},
doi = {10.1016/j.molcel.2018.05.019},
pmid = {29932899},
issn = {1097-4164},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cell Nucleus/metabolism ; HeLa Cells ; Humans ; Nuclear Proteins/genetics/metabolism ; Protein Domains ; RNA Recognition Motif Proteins/genetics/metabolism ; RNA Stability ; RNA, Long Noncoding/*genetics/*metabolism ; Transcription Factors/metabolism ; },
abstract = {A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.},
}

Base Sequence
CRISPR-Cas Systems
Cell Nucleus/metabolism
HeLa Cells
Humans
Nuclear Proteins/genetics/metabolism
Protein Domains
RNA Recognition Motif Proteins/genetics/metabolism
RNA Stability
RNA, Long Noncoding/*genetics/*metabolism
Transcription Factors/metabolism

@article {pmid29554589,
year = {2018},
author = {Wu, M and Liu, S and Gao, Y and Bai, H and Machairaki, V and Li, G and Chen, T and Cheng, L},
title = {Conditional gene knockout and reconstitution in human iPSCs with an inducible Cas9 system.},
journal = {Stem cell research},
volume = {29},
number = {},
pages = {6-14},
doi = {10.1016/j.scr.2018.03.003},
pmid = {29554589},
issn = {1876-7753},
support = {R01 HL130676/HL/NHLBI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Differentiation ; Cell Line ; Gene Knockout Techniques/*methods ; Humans ; Induced Pluripotent Stem Cells/cytology/*metabolism ; },
abstract = {Precise genome editing in human induced pluripotent stem cells (iPSCs) significantly enhances our capability to use human iPSCs for disease modeling, drug testing and screening as well as investigation of human cell biology. In this study, we seek to achieve conditional expression of the CD55 gene in order to interrogate its functions. We used two human iPSC lines that have unique genotypes, and constructed an inducible Cas9 gene expression system that is integrated at the AAVS1 safe harbor site in the human genome. Using paired guide RNAs, we observed efficient knock-out with an intended deletion in the coding region of several genes including CD55 and ETV6 genes. This paired guide RNA approach enabled us to efficiently identify homozygous iPSC clones with an intended deletion. Once an iPSC clone lacking CD55 expression was identified and characterized, we were able to use the same doxycycline system to induce expression of a CD55 transgene from a piggyBac vector, in both undifferentiated and differentiated iPSCs. This single cell line of gene knock-out complemented with an inducible transgene is sufficient to achieve conditional expression of the CD55 gene. The methodology described here is broadly applicable to other genes in order to interrogate their functions.},
}

CRISPR-Cas Systems/*genetics
Cell Differentiation
Cell Line
Gene Knockout Techniques/*methods
Humans
Induced Pluripotent Stem Cells/cytology/*metabolism

@article {pmid29121025,
year = {2017},
author = {Hyun, I and Osborn, C},
title = {Query the merits of embryo editing for reproductive research now.},
journal = {Nature biotechnology},
volume = {35},
number = {11},
pages = {1023-1025},
pmid = {29121025},
issn = {1546-1696},
mesh = {CRISPR-Cas Systems ; Embryo Research/*ethics ; Embryo, Mammalian ; Gene Editing/*ethics ; Humans ; },
}

CRISPR-Cas Systems
Embryo Research/*ethics
Embryo, Mammalian
Gene Editing/*ethics
Humans

@article {pmid30895956,
year = {2019},
author = {Karpov, DS and Karpov, VL and Klimova, RR and Demidova, NA and Kushch, AA},
title = {[A Plasmid-Expressed CRISPR/Cas9 System Suppresses Replication of HSV Type I in a Vero Cell Culture].},
journal = {Molekuliarnaia biologiia},
volume = {53},
number = {1},
pages = {91-100},
doi = {10.1134/S0026898419010051},
pmid = {30895956},
issn = {0026-8984},
abstract = {Herpesviruses are widespread in the human population. Herpes simplex virus type 1 (HSV1) alone infects more than 3.7 billion people. In most of these, the virus establishes a latent form resistant to the action of all antiviral drugs. Moreover, completely drug-resistant strains of herpesviruses are known, which has prompted the search for alternative approaches to the treatment of herpesviruses, including genome editing with prokaryotic CRISPR/Cas. The CRISPR/Cas9 system of Streptococcus pyogens effectively suppresses HSV1 infection when expressed from genome-integrated lentiviral vectors. However, there are concerns about the safety of this approach. Here we describe the system built upon the plasmid-encoded CRISPR/Cas9 targeted against UL52 and UL29 genes of the HSV1 primase-helicase complex. The construct was transfected into Vero cells with no significant cytotoxic effects detected. Complete suppression of HSV1 infection within two days was observed, raising the possibility that the proposed plasmid-expressed CRISPR/Cas9 system may be used for the screening of genes important for the HSV1 life cycle and for development of novel strategies for targeted therapy of herpesvirus infections.},
}

@article {pmid30895693,
year = {2019},
author = {Castelli, A and Susani, L and Menale, C and Muggeo, S and Caldana, E and Strina, D and Cassani, B and Recordati, C and Scanziani, E and Ficara, F and Villa, A and Vezzoni, P and Paulis, M},
title = {Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse iPSCs.},
journal = {Stem cells (Dayton, Ohio)},
volume = {},
number = {},
pages = {},
doi = {10.1002/stem.3006},
pmid = {30895693},
issn = {1549-4918},
abstract = {In spite of the progresses in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed Chromosomal Transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the Chronic Granulomatous Disease (CGD), an X-linked severe immunodeficiency due to abnormalities in Cybb (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the HAT selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer (MMCT). X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. SIGNIFICANCE STATEMENT: A lot of genetic diseases involving chromosome abnormalities can not be treated by conventional gene therapy approaches. Here we applied Chromosome Transplantation, a new genomic therapy method that we have recently developed, to induced pluripotent stem cells (iPSCs) derived from an X-linked mouse disease model. As a proof of principle we demonstrated that X-chromosome transplanted corrected cells restored the normal function to differentiated cells. Although all the strategies based on iPSCs are still far away from the clinic, we believe that our results establish an important step for the usefulness of this kind of genomic therapy in selected patients. © AlphaMed Press 2019.},
}

@article {pmid30895249,
year = {2018},
author = {Hendriks, S and Giesbertz, NAA and Bredenoord, AL and Repping, S},
title = {Reasons for being in favour of or against genome modification: a survey of the Dutch general public.},
journal = {Human reproduction open},
volume = {2018},
number = {3},
pages = {hoy008},
doi = {10.1093/hropen/hoy008},
pmid = {30895249},
issn = {2399-3529},
abstract = {STUDY QUESTION: What are the general public's reasons for being in favour of or against the use of genome modification for five potential applications?

SUMMARY ANSWER: Overall, 43 reasons for being in favour, 45 reasons for being against as well as 26 conditional reasons for the use of genome modification were identified.

WHAT IS KNOWN ALREADY: Various applications of somatic genome modification are progressing towards clinical introduction and several recent studies have reported on germline genome modification. This has incited a debate on ethical and legal implications and acceptability. There is a growing plea to involve the general public earlier on in the developmental process of science and (bio)technology including genome modification.

STUDY DESIGN SIZE DURATION: In April 2016, a cross-sectional survey was launched online among the Dutch general public. A documentary on genome modification on public television and calls in social media invited viewers and non-viewers, respectively, to participate.

The questionnaire introduced five potential future applications of genome modification: modified wheat for individuals with gluten intolerance; somatic modification for individuals with neuromuscular diseases; germline modification to prevent passing on a neuromuscular disease; germline modification to introduce resistance to HIV; and germline modification to increase intelligence. Participants were asked to indicate whether and why they would make use of genome modification in these scenarios. The reasons mentioned were analysed through content analysis by two researchers independently. The proportion of respondents that was willing to modify was described per scenario and associations with respondent characteristics were analysed.

The survey was completed by 1013 participants. Forty-three reasons for being in favour, 45 reasons for being against as well as 26 conditional reasons for the use of genome modification were identified. These could be categorized into 14 domains: safety of the individuals concerned; effectiveness; quality of life of the individuals concerned; existence of a clinical need or an alternative; biodiversity and ecosystems; animal homo sapiens (i.e. relating to effects on humans as a species); human life and dignity; trust in regulation; justice; costs; slippery slope; argument of nature; parental rights and duties; and (reproductive) autonomy. Participants' willingness to use genome modification was dependent on the application: most participants would eat modified wheat if gluten intolerant (74%), would use genome modification to cure his/her own neuromuscular disease (85%) and would apply germline modification to prevent passing on this neuromuscular disease (66%). A minority would apply germline modification to introduce resistance to HIV (30%) or increase intelligence (16%). Being young (odds ratio (OR) = 0.98 per year increase), being male (OR = 2.38), and having watched the documentary (OR = 1.82) were associated with being willing to apply genome modification in more scenarios.

Inquiring for reasons through open questions in a survey allowed for a larger sample size and intuitive responses but resulted in less depth than traditional face-to-face interviews. As the survey was disseminated through social media, the sample is not representative of the overall Dutch population, and hence the quantitative results should not be interpreted as such.

Further public consultation and a more in-depth ethical and societal debate on principles and conditions for responsible use of (germline) genome modification is required prior to future clinical introduction.

Funded by the University of Amsterdam and University Medical Centre Utrecht. No conflict of interest.

TRIAL REGISTRATION NUMBER: Not applicable.},
}

@article {pmid30894153,
year = {2019},
author = {Najah, S and Saulnier, C and Pernodet, JL and Bury-Moné, S},
title = {Design of a generic CRISPR-Cas9 approach using the same sgRNA to perform gene editing at distinct loci.},
journal = {BMC biotechnology},
volume = {19},
number = {1},
pages = {18},
doi = {10.1186/s12896-019-0509-7},
pmid = {30894153},
issn = {1472-6750},
support = {ANR-13-BSV6-0009//Agence Nationale de la Recherche/ ; },
abstract = {BACKGROUND: The CRISPR/Cas (clustered regularly interspaced short palindromic repeat and CRISPR-associated nucleases) based technologies have revolutionized genome engineering. While their use for prokaryotic genome editing is expanding, some limitations remain such as possible off-target effects and design constraints. These are compounded when performing systematic genome editing at distinct loci or when targeting repeated sequences (e.g. multicopy genes or mobile genetic elements). To overcome these limitations, we designed an approach using the same sgRNA and CRISPR-Cas9 system to independently perform gene editing at different loci.

RESULTS: We developed a two-step procedure based on the introduction by homologous recombination of 'bait' DNA at the vicinity of a gene copy of interest before inducing CRISPR-Cas9 activity. The introduction of a genetic tool encoding a CRISPR-Cas9 complex targeting this 'bait' DNA induces a double strand break near the copy of interest. Its repair by homologous recombination can lead either to reversion or gene copy-specific editing. The relative frequencies of these events are linked to the impact of gene editing on cell fitness. In our study, we used this technology to successfully delete the native copies of two xenogeneic silencers lsr2 paralogs in Streptomyces ambofaciens. We observed that one of these paralogs is a candidate-essential gene since its native locus can be deleted only in the presence of an extra copy.

CONCLUSION: By targeting 'bait' DNA, we designed a 'generic' CRISPR-Cas9 toolkit that can be used to edit different loci. The differential action of this CRISPR-Cas9 system is exclusively based on the specific recombination between regions surrounding the gene copy of interest. This approach is suitable to edit multicopy genes. One such particular example corresponds to the mutagenesis of candidate-essential genes that requires the presence of an extra copy of the gene before gene disruption. This opens new insights to explore gene essentiality in bacteria and to limit off-target effects during systematic CRISPR-Cas9 based approaches.},
}

@article {pmid30301855,
year = {2018},
author = {Bando, H and Lee, Y and Sakaguchi, N and Pradipta, A and Ma, JS and Tanaka, S and Cai, Y and Liu, J and Shen, J and Nishikawa, Y and Sasai, M and Yamamoto, M},
title = {Inducible Nitric Oxide Synthase Is a Key Host Factor for Toxoplasma GRA15-Dependent Disruption of the Gamma Interferon-Induced Antiparasitic Human Response.},
journal = {mBio},
volume = {9},
number = {5},
pages = {},
pmid = {30301855},
issn = {2150-7511},
mesh = {Animals ; Antigens, Protozoan/immunology ; CRISPR-Cas Systems ; Cell Line ; Coculture Techniques ; Hepatocytes/drug effects/immunology/parasitology ; Host-Parasite Interactions/immunology ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism ; Interferon-gamma/*pharmacology ; Interleukin-1beta/metabolism ; Mice ; Mice, Inbred C57BL ; Monocytes/immunology/parasitology ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism ; Nitric Oxide Synthase Type II/*immunology ; Protozoan Proteins/*immunology ; Toxoplasma/*immunology/pathogenicity ; Virulence Factors/*immunology ; },
abstract = {Although Toxoplasma virulence mechanisms targeting gamma interferon (IFN-γ)-induced cell-autonomous antiparasitic immunity have been extensively characterized in mice, the virulence mechanisms in humans remain uncertain, partly because cell-autonomous immune responses against Toxoplasma differ markedly between mice and humans. Despite the identification of inducible nitric oxide synthase (iNOS) as an anti-Toxoplasma host factor in mice, here we show that iNOS in humans is a pro-Toxoplasma host factor that promotes the growth of the parasite. The GRA15 Toxoplasma effector-dependent disarmament of IFN-γ-induced parasite growth inhibition was evident when parasite-infected monocytes were cocultured with hepatocytes. Interleukin-1β (IL-1β), produced from monocytes in a manner dependent on GRA15 and the host's NLRP3 inflammasome, combined with IFN-γ to strongly stimulate iNOS expression in hepatocytes; this dramatically reduced the levels of indole 2,3-dioxygenase 1 (IDO1), a critically important IFN-γ-inducible anti-Toxoplasma protein in humans, thus allowing parasite growth. Taking the data together, Toxoplasma utilizes human iNOS to antagonize IFN-γ-induced IDO1-mediated cell-autonomous immunity via its GRA15 virulence factor.IMPORTANCEToxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Gamma interferon (IFN-γ) is produced in the host to inhibit the proliferation of this parasite and eventually cause its death. Unlike mouse disease models, which involve well-characterized virulence strategies that are used by Toxoplasma to suppress IFN-γ-dependent immunity, the strategies used by Toxoplasma in humans remain unclear. Here, we show that GRA15, a Toxoplasma effector protein, suppresses the IFN-γ-induced indole-2,3-dioxygenase 1-dependent antiparasite immune response in human cells. Because NLRP3-dependent production of IL-1β and nitric oxide (NO) in Toxoplasma-infected human cells is involved in the GRA15-dependent virulence mechanism, blocking NO or IL-1β production in the host could represent a novel therapeutic approach for treating human toxoplasmosis.},
}

Animals
Antigens, Protozoan/immunology
CRISPR-Cas Systems
Cell Line
Coculture Techniques
Hepatocytes/drug effects/immunology/parasitology
Host-Parasite Interactions/immunology
Humans
Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism
Interferon-gamma/*pharmacology
Interleukin-1beta/metabolism
Mice
Mice, Inbred C57BL
Monocytes/immunology/parasitology
NLR Family, Pyrin Domain-Containing 3 Protein/metabolism
Nitric Oxide Synthase Type II/*immunology
Protozoan Proteins/*immunology
Toxoplasma/*immunology/pathogenicity
Virulence Factors/*immunology

@article {pmid30017531,
year = {2018},
author = {Foronda, M and Dow, LE},
title = {CRISPR: Stressed about p53?.},
journal = {Trends in molecular medicine},
volume = {24},
number = {9},
pages = {731-733},
doi = {10.1016/j.molmed.2018.06.010},
pmid = {30017531},
issn = {1471-499X},
support = {R01 CA195787/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Damage ; *Gene Editing ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Two recent reports show that, in some contexts, CRISPR-mediated genome editing can lead to a p53-mediated stress response and cell-cycle arrest. These findings may help to explain why CRISPR-mediated genetic manipulation in different cell types leads to dissimilar outcomes, and highlights the need for a better understanding of the factors that influence effective genome editing in vitro and in vivo.},
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA Damage
*Gene Editing
Tumor Suppressor Protein p53/genetics

@article {pmid29630899,
year = {2018},
author = {Slotta, C and Storm, J and Pfisterer, N and Henkel, E and Kleinwächter, S and Pieper, M and Ruiz-Perera, LM and Greiner, JFW and Kaltschmidt, B and Kaltschmidt, C},
title = {IKK1/2 protect human cells from TNF-mediated RIPK1-dependent apoptosis in an NF-κB-independent manner.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1865},
number = {8},
pages = {1025-1033},
doi = {10.1016/j.bbamcr.2018.04.003},
pmid = {29630899},
issn = {0167-4889},
mesh = {*Apoptosis ; CRISPR-Cas Systems ; HEK293 Cells ; Humans ; I-kappa B Kinase/genetics/*immunology ; NF-kappa B/*immunology ; Receptor-Interacting Protein Serine-Threonine Kinases/*immunology ; Signal Transduction ; Tumor Necrosis Factor-alpha/*immunology ; },
abstract = {TNF signaling is directly linked to cancer development and progression. A broad range of tumor cells is able to evade cell death induced by TNF impairing the potential anti-cancer value of TNF in therapy. Although sensitizing cells to TNF-induced death therefore has great clinical implications, detailed mechanistic insights into TNF-mediated human cell death still remain unknown. Here, we analyzed human cells by applying CRISPR/Cas9n to generate cells deficient of IKK1, IKK2, IKK1/2 and RELA. Despite stimulation with TNF resulted in impaired NF-κB activation in all genotypes compared to wildtype cells, increased cell death was observable only in IKK1/2-double-deficient cells. Cell death could be detected by Caspase-3 activation and binding of Annexin V. TNF-induced programmed cell death in IKK1/2-/- cells was further shown to be mediated via RIPK1 in a predominantly apoptotic manner. Our findings demonstrate the IKK complex to protect from TNF-induced cell death in human cells independently to NF-κB RelA suggesting IKK1/2 to be highly promising targets for cancer therapy.},
}

*Apoptosis
CRISPR-Cas Systems
HEK293 Cells
Humans
I-kappa B Kinase/genetics/*immunology
NF-kappa B/*immunology
Receptor-Interacting Protein Serine-Threonine Kinases/*immunology
Signal Transduction
Tumor Necrosis Factor-alpha/*immunology

@article {pmid28831158,
year = {2017},
author = {Sapio, RT and Nezdyur, AN and Krevetski, M and Anikin, L and Manna, VJ and Minkovsky, N and Pestov, DG},
title = {Inhibition of post-transcriptional steps in ribosome biogenesis confers cytoprotection against chemotherapeutic agents in a p53-dependent manner.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {9041},
pmid = {28831158},
issn = {2045-2322},
support = {R01 GM074091/GM/NIGMS NIH HHS/United States ; },
mesh = {3T3 Cells ; Animals ; Antineoplastic Agents/*pharmacology ; CRISPR-Cas Systems ; Cell Cycle/genetics ; Cytoprotection ; DNA Damage ; *Drug Resistance, Neoplasm/genetics ; Gene Editing ; Gene Targeting ; Mice ; Models, Biological ; Nuclear Proteins/genetics/metabolism ; *Protein Biosynthesis ; *RNA Processing, Post-Transcriptional ; Ribosomes/*metabolism ; Sequence Deletion ; Tumor Suppressor Protein p53/*genetics/metabolism ; },
abstract = {The p53-mediated nucleolar stress response associated with inhibition of ribosomal RNA transcription was previously shown to potentiate killing of tumor cells. Here, we asked whether targeting of ribosome biogenesis can be used as the basis for selective p53-dependent cytoprotection of nonmalignant cells. Temporary functional inactivation of the 60S ribosome assembly factor Bop1 in a 3T3 cell model markedly increased cell recovery after exposure to camptothecin or methotrexate. This was due, at least in part, to reversible pausing of the cell cycle preventing S phase associated DNA damage. Similar cytoprotective effects were observed after transient shRNA-mediated silencing of Rps19, but not several other tested ribosomal proteins, indicating distinct cellular responses to the inhibition of different steps in ribosome biogenesis. By temporarily inactivating Bop1 function, we further demonstrate selective killing of p53-deficient cells with camptothecin while sparing isogenic p53-positive cells. Thus, combining cytotoxic treatments with inhibition of select post-transcriptional steps of ribosome biogenesis holds potential for therapeutic targeting of cells that have lost p53.},
}

3T3 Cells
Animals
Antineoplastic Agents/*pharmacology
CRISPR-Cas Systems
Cell Cycle/genetics
Cytoprotection
DNA Damage
*Drug Resistance, Neoplasm/genetics
Gene Editing
Gene Targeting
Mice
Models, Biological
Nuclear Proteins/genetics/metabolism
*Protein Biosynthesis
*RNA Processing, Post-Transcriptional
Ribosomes/*metabolism
Sequence Deletion
Tumor Suppressor Protein p53/*genetics/metabolism

@article {pmid30891445,
year = {2019},
author = {Eckerstorfer, MF and Heissenberger, A and Reichenbecher, W and Steinbrecher, RA and Waßmann, F},
title = {An EU Perspective on Biosafety Considerations for Plants Developed by Genome Editing and Other New Genetic Modification Techniques (nGMs).},
journal = {Frontiers in bioengineering and biotechnology},
volume = {7},
number = {},
pages = {31},
doi = {10.3389/fbioe.2019.00031},
pmid = {30891445},
issn = {2296-4185},
abstract = {The question whether new genetic modification techniques (nGM) in plant development might result in non-negligible negative effects for the environment and/or health is significant for the discussion concerning their regulation. However, current knowledge to address this issue is limited for most nGMs, particularly for recently developed nGMs, like genome editing, and their newly emerging variations, e.g., base editing. This leads to uncertainties regarding the risk/safety-status of plants which are developed with a broad range of different nGMs, especially genome editing, and other nGMs such as cisgenesis, transgrafting, haploid induction or reverse breeding. A literature survey was conducted to identify plants developed by nGMs which are relevant for future agricultural use. Such nGM plants were analyzed for hazards associated either (i) with their developed traits and their use or (ii) with unintended changes resulting from the nGMs or other methods applied during breeding. Several traits are likely to become particularly relevant in the future for nGM plants, namely herbicide resistance (HR), resistance to different plant pathogens as well as modified composition, morphology, fitness (e.g., increased resistance to cold/frost, drought, or salinity) or modified reproductive characteristics. Some traits such as resistance to certain herbicides are already known from existing GM crops and their previous assessments identified issues of concern and/or risks, such as the development of herbicide resistant weeds. Other traits in nGM plants are novel; meaning they are not present in agricultural plants currently cultivated with a history of safe use, and their underlying physiological mechanisms are not yet sufficiently elucidated. Characteristics of some genome editing applications, e.g., the small extent of genomic sequence change and their higher targeting efficiency, i.e., precision, cannot be considered an indication of safety per se, especially in relation to novel traits created by such modifications. All nGMs considered here can result in unintended changes of different types and frequencies. However, the rapid development of nGM plants can compromise the detection and elimination of unintended effects. Thus, a case-specific premarket risk assessment should be conducted for nGM plants, including an appropriate molecular characterization to identify unintended changes and/or confirm the absence of unwanted transgenic sequences.},
}

@article {pmid30890613,
year = {2019},
author = {Gentile, GM and Wetzel, KS and Dedrick, RM and Montgomery, MT and Garlena, RA and Jacobs-Sera, D and Hatfull, GF},
title = {More Evidence of Collusion: a New Prophage-Mediated Viral Defense System Encoded by Mycobacteriophage Sbash.},
journal = {mBio},
volume = {10},
number = {2},
pages = {},
doi = {10.1128/mBio.00196-19},
pmid = {30890613},
issn = {2150-7511},
abstract = {The arms race between bacteria and their bacteriophages profoundly influences microbial evolution. With an estimated 1023 phage infections occurring per second, there is strong selection for both bacterial survival and phage coevolution for continued propagation. Many phage resistance systems, including restriction-modification systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) systems, a variety of abortive infection systems, and many others that are not yet mechanistically defined, have been described. Temperate bacteriophages are common and form stable lysogens that are immune to superinfection by the same or closely related phages. However, temperate phages collude with their hosts to confer defense against genomically distinct phages, to the mutual benefit of the bacterial host and the prophage. Prophage-mediated viral systems have been described in Mycobacterium phages and Pseudomonas phages but are predicted to be widespread throughout the microbial world. Here we describe a new viral defense system in which the mycobacteriophage Sbash prophage colludes with its Mycobacterium smegmatis host to confer highly specific defense against infection by the unrelated mycobacteriophage Crossroads. Sbash genes 30 and 31 are lysogenically expressed and are necessary and sufficient to confer defense against Crossroads but do not defend against any of the closely related phages grouped in subcluster L2. The mapping of Crossroads defense escape mutants shows that genes 132 and 141 are involved in recognition by the Sbash defense system and are proposed to activate a loss in membrane potential mediated by Sbash gp30 and gp31.IMPORTANCE Viral infection is an ongoing challenge to bacterial survival, and there is strong selection for development or acquisition of defense systems that promote survival when bacteria are attacked by bacteriophages. Temperate phages play central roles in these dynamics through lysogenic expression of genes that defend against phage attack, including those unrelated to the prophage. Few prophage-mediated viral defense systems have been characterized, but they are likely widespread both in phage genomes and in the prophages integrated in bacterial chromosomes.},
}

@article {pmid30645710,
year = {2019},
author = {Lee, K and Eggenberger, AL and Banakar, R and McCaw, ME and Zhu, H and Main, M and Kang, M and Gelvin, SB and Wang, K},
title = {CRISPR/Cas9-mediated targeted T-DNA integration in rice.},
journal = {Plant molecular biology},
volume = {99},
number = {4-5},
pages = {317-328},
doi = {10.1007/s11103-018-00819-1},
pmid = {30645710},
issn = {1573-5028},
support = {2016-06247//National Institute of Food and Agriculture/ ; IOS 1725122//National Science Foundation/ ; },
mesh = {Agrobacterium/genetics ; Base Sequence ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems/*genetics ; DNA, Bacterial/*genetics ; Exons ; Gene Editing ; Gene Expression Regulation, Plant/genetics ; Gene Frequency ; Gene Targeting ; Genes, Plant/genetics ; Genetic Vectors/genetics ; Genome, Plant/*genetics ; INDEL Mutation ; Luminescent Proteins/genetics ; Mutagenesis, Insertional/*methods ; Oryza/*genetics/*metabolism ; Plant Proteins/genetics ; Plants, Genetically Modified/*genetics ; Promoter Regions, Genetic ; RNA, Guide/genetics/metabolism ; Sequence Analysis ; },
abstract = {KEY MESSAGE: Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome. Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50-80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.},
}

Agrobacterium/genetics
Base Sequence
CRISPR-Associated Proteins/metabolism
CRISPR-Cas Systems/*genetics
DNA, Bacterial/*genetics
Exons
Gene Editing
Gene Expression Regulation, Plant/genetics
Gene Frequency
Gene Targeting
Genes, Plant/genetics
Genetic Vectors/genetics
Genome, Plant/*genetics
INDEL Mutation
Luminescent Proteins/genetics
Mutagenesis, Insertional/*methods
Oryza/*genetics/*metabolism
Plant Proteins/genetics
Plants, Genetically Modified/*genetics
Promoter Regions, Genetic
RNA, Guide/genetics/metabolism
Sequence Analysis

@article {pmid30622266,
year = {2019},
author = {Kandul, NP and Liu, J and Sanchez C, HM and Wu, SL and Marshall, JM and Akbari, OS},
title = {Transforming insect population control with precision guided sterile males with demonstration in flies.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {84},
doi = {10.1038/s41467-018-07964-7},
pmid = {30622266},
issn = {2041-1723},
support = {5K22AI113060/NH/NIH HHS/United States ; 1R21AI123937/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Drosophila/*genetics/physiology ; Female ; Gene Editing/*methods ; Genome, Insect/genetics ; Insect Vectors/*genetics/physiology ; Male ; Models, Biological ; Pest Control, Biological/*methods ; Population Control/methods ; RNA, Guide/genetics ; *Sexual Behavior, Animal ; },
abstract = {The sterile insect technique (SIT) is an environmentally safe and proven technology to suppress wild populations. To further advance its utility, a novel CRISPR-based technology termed precision guided SIT (pgSIT) is described. PgSIT mechanistically relies on a dominant genetic technology that enables simultaneous sexing and sterilization, facilitating the release of eggs into the environment ensuring only sterile adult males emerge. Importantly, for field applications, the release of eggs will eliminate burdens of manually sexing and sterilizing males, thereby reducing overall effort and increasing scalability. Here, to demonstrate efficacy, we systematically engineer multiple pgSIT systems in Drosophila which consistently give rise to 100% sterile males. Importantly, we demonstrate that pgSIT-generated sterile males are fit and competitive. Using mathematical models, we predict pgSIT will induce substantially greater population suppression than can be achieved by currently-available self-limiting suppression technologies. Taken together, pgSIT offers to potentially transform our ability to control insect agricultural pests and disease vectors.},
}

Animals
CRISPR-Cas Systems/genetics
Drosophila/*genetics/physiology
Female
Gene Editing/*methods
Genome, Insect/genetics
Insect Vectors/*genetics/physiology
Male
Models, Biological
Pest Control, Biological/*methods
Population Control/methods
RNA, Guide/genetics
*Sexual Behavior, Animal

@article {pmid30622252,
year = {2019},
author = {Balmus, G and Pilger, D and Coates, J and Demir, M and Sczaniecka-Clift, M and Barros, AC and Woods, M and Fu, B and Yang, F and Chen, E and Ostermaier, M and Stankovic, T and Ponstingl, H and Herzog, M and Yusa, K and Martinez, FM and Durant, ST and Galanty, Y and Beli, P and Adams, DJ and Bradley, A and Metzakopian, E and Forment, JV and Jackson, SP},
title = {ATM orchestrates the DNA-damage response to counter toxic non-homologous end-joining at broken replication forks.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {87},
doi = {10.1038/s41467-018-07729-2},
pmid = {30622252},
issn = {2041-1723},
mesh = {Animals ; Antineoplastic Agents/*pharmacology/therapeutic use ; Ataxia Telangiectasia Mutated Proteins/*genetics/metabolism ; BRCA1 Protein/metabolism ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Cell Survival/genetics ; DNA Breaks, Double-Stranded/drug effects ; DNA End-Joining Repair/*genetics ; DNA Ligase ATP/metabolism ; DNA Replication/drug effects/genetics ; Drug Resistance, Neoplasm/*genetics ; Female ; Humans ; Mice ; Mice, Inbred NOD ; Mice, Knockout ; Mouse Embryonic Stem Cells ; Mutation ; Neoplasms, Experimental/drug therapy/genetics/pathology ; Phthalazines/pharmacology/therapeutic use ; Piperazines/pharmacology/therapeutic use ; Topotecan/pharmacology/therapeutic use ; },
abstract = {Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM-mutant backgrounds are different to those in BRCA1-mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation.},
}

Animals
Antineoplastic Agents/*pharmacology/therapeutic use
Ataxia Telangiectasia Mutated Proteins/*genetics/metabolism
BRCA1 Protein/metabolism
CRISPR-Cas Systems/genetics
Cell Line, Tumor
Cell Survival/genetics
DNA Breaks, Double-Stranded/drug effects
DNA End-Joining Repair/*genetics
DNA Ligase ATP/metabolism
DNA Replication/drug effects/genetics
Drug Resistance, Neoplasm/*genetics
Female
Humans
Mice
Mice, Inbred NOD
Mice, Knockout
Mouse Embryonic Stem Cells
Mutation
Neoplasms, Experimental/drug therapy/genetics/pathology
Phthalazines/pharmacology/therapeutic use
Piperazines/pharmacology/therapeutic use
Topotecan/pharmacology/therapeutic use

@article {pmid30604771,
year = {2019},
author = {Sun, J and Carlson-Stevermer, J and Das, U and Shen, M and Delenclos, M and Snead, AM and Koo, SY and Wang, L and Qiao, D and Loi, J and Petersen, AJ and Stockton, M and Bhattacharyya, A and Jones, MV and Zhao, X and McLean, PJ and Sproul, AA and Saha, K and Roy, S},
title = {CRISPR/Cas9 editing of APP C-terminus attenuates β-cleavage and promotes α-cleavage.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {53},
doi = {10.1038/s41467-018-07971-8},
pmid = {30604771},
issn = {2041-1723},
support = {R01 AG048218/AG/NIA NIH HHS/United States ; R21 AG052404/AG/NIA NIH HHS/United States ; },
mesh = {Amyloid Precursor Protein Secretases/*metabolism ; Amyloid beta-Peptides/*metabolism ; Amyloid beta-Protein Precursor/*genetics/metabolism ; Animals ; Brain/cytology/pathology ; CRISPR-Cas Systems/genetics ; Dependovirus/genetics ; Disease Models, Animal ; Female ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors/administration & dosage/genetics ; HEK293 Cells ; Humans ; Induced Pluripotent Stem Cells ; Injections, Intraventricular ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Neurodegenerative Diseases/genetics/pathology/*therapy ; Neurons ; Stereotaxic Techniques ; Transfection ; Treatment Outcome ; },
abstract = {CRISPR/Cas9 guided gene-editing is a potential therapeutic tool, however application to neurodegenerative disease models has been limited. Moreover, conventional mutation correction by gene-editing would only be relevant for the small fraction of neurodegenerative cases that are inherited. Here we introduce a CRISPR/Cas9-based strategy in cell and animal models to edit endogenous amyloid precursor protein (APP) at the extreme C-terminus and reciprocally manipulate the amyloid pathway, attenuating APP-β-cleavage and Aβ production, while up-regulating neuroprotective APP-α-cleavage. APP N-terminus and compensatory APP-homologues remain intact, with no apparent effects on neurophysiology in vitro. Robust APP-editing is seen in human iPSC-derived neurons and mouse brains with no detectable off-target effects. Our strategy likely works by limiting APP and BACE-1 approximation, and we also delineate mechanistic events that abrogates APP/BACE-1 convergence in this setting. Our work offers conceptual proof for a selective APP silencing strategy.},
}

Amyloid Precursor Protein Secretases/*metabolism
Amyloid beta-Peptides/*metabolism
Amyloid beta-Protein Precursor/*genetics/metabolism
Animals
Brain/cytology/pathology
CRISPR-Cas Systems/genetics
Dependovirus/genetics
Disease Models, Animal
Female
Gene Editing/*methods
Genetic Therapy/*methods
Genetic Vectors/administration & dosage/genetics
HEK293 Cells
Humans
Induced Pluripotent Stem Cells
Injections, Intraventricular
Male
Mice
Mice, Inbred C57BL
Mice, Transgenic
Neurodegenerative Diseases/genetics/pathology/*therapy
Neurons
Stereotaxic Techniques
Transfection
Treatment Outcome

@article {pmid30604745,
year = {2019},
author = {Gdula, MR and Nesterova, TB and Pintacuda, G and Godwin, J and Zhan, Y and Ozadam, H and McClellan, M and Moralli, D and Krueger, F and Green, CM and Reik, W and Kriaucionis, S and Heard, E and Dekker, J and Brockdorff, N},
title = {The non-canonical SMC protein SmcHD1 antagonises TAD formation and compartmentalisation on the inactive X chromosome.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {30},
doi = {10.1038/s41467-018-07907-2},
pmid = {30604745},
issn = {2041-1723},
support = {//Wellcome Trust/United Kingdom ; 340081//European Research Council/International ; BB/M001873/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; R01 HG003143/HG/NHGRI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Cell Line ; Chromosomal Proteins, Non-Histone/*genetics/metabolism ; CpG Islands ; DNA Methylation/*genetics ; Exons/genetics ; Female ; Fibroblasts ; Gene Knockout Techniques ; Histones/genetics/metabolism ; Male ; Mice ; Point Mutation ; Polycomb-Group Proteins/metabolism ; Transcriptional Activation/*genetics ; *X Chromosome Inactivation ; },
abstract = {The inactive X chromosome (Xi) in female mammals adopts an atypical higher-order chromatin structure, manifested as a global loss of local topologically associated domains (TADs), A/B compartments and formation of two mega-domains. Here we demonstrate that the non-canonical SMC family protein, SmcHD1, which is important for gene silencing on Xi, contributes to this unique chromosome architecture. Specifically, allelic mapping of the transcriptome and epigenome in SmcHD1 mutant cells reveals the appearance of sub-megabase domains defined by gene activation, CpG hypermethylation and depletion of Polycomb-mediated H3K27me3. These domains, which correlate with sites of SmcHD1 enrichment on Xi in wild-type cells, additionally adopt features of active X chromosome higher-order chromosome architecture, including A/B compartments and partial restoration of TAD boundaries. Xi chromosome architecture changes also occurred following SmcHD1 knockout in a somatic cell model, but in this case, independent of Xi gene derepression. We conclude that SmcHD1 is a key factor in defining the unique chromosome architecture of Xi.},
}

Alleles
Animals
CRISPR-Cas Systems
Cell Line
Chromosomal Proteins, Non-Histone/*genetics/metabolism
CpG Islands
DNA Methylation/*genetics
Exons/genetics
Female
Fibroblasts
Gene Knockout Techniques
Histones/genetics/metabolism
Male
Mice
Point Mutation
Polycomb-Group Proteins/metabolism
Transcriptional Activation/*genetics
*X Chromosome Inactivation

@article {pmid30307989,
year = {2018},
author = {Ng, KP and Manjeri, A and Lee, LM and Chan, ZE and Tan, CY and Tan, QD and Majeed, A and Lee, KL and Chuah, C and Suda, T and Ong, ST},
title = {The arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) induces apoptosis in leukemic cells specifically under hypoxic conditions but CRISPR/Cas9 excludes arginase 2 (ARG2) as the functional target.},
journal = {PloS one},
volume = {13},
number = {10},
pages = {e0205254},
pmid = {30307989},
issn = {1932-6203},
mesh = {Antineoplastic Agents/*pharmacology/therapeutic use ; Apoptosis/*drug effects ; Arginase/*antagonists & inhibitors/genetics/metabolism ; Arginine/*analogs & derivatives/pharmacology/therapeutic use ; CRISPR-Cas Systems/genetics ; Cell Cycle/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Drug Screening Assays, Antitumor ; Enzyme Assays ; Feasibility Studies ; Gene Knockout Techniques ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy/pathology ; Primary Cell Culture ; RNA, Small Interfering/metabolism ; },
abstract = {Cancer cells, including in chronic myeloid leukemia (CML), depend on the hypoxic response to persist in hosts and evade therapy. Accordingly, there is significant interest in drugging cancer-specific hypoxic responses. However, a major challenge in leukemia is identifying differential and druggable hypoxic responses between leukemic and normal cells. Previously, we found that arginase 2 (ARG2), an enzyme of the urea cycle, is overexpressed in CML but not normal progenitors. ARG2 is a target of the hypoxia inducible factors (HIF1-α and HIF2-α), and is required for the generation of polyamines which are required for cell growth. We therefore explored if the clinically-tested arginase inhibitor Nω-hydroxy-nor-arginine (nor-NOHA) would be effective against leukemic cells under hypoxic conditions. Remarkably, nor-NOHA effectively induced apoptosis in ARG2-expressing cells under hypoxia but not normoxia. Co-treatment with nor-NOHA overcame hypoxia-mediated resistance towards BCR-ABL1 kinase inhibitors. While nor-NOHA itself is promising in targeting the leukemia hypoxic response, we unexpectedly found that its anti-leukemic activity was independent of ARG2 inhibition. Genetic ablation of ARG2 using CRISPR/Cas9 had no effect on the viability of leukemic cells and their sensitivity towards nor-NOHA. This discrepancy was further evidenced by the distinct effects of ARG2 knockouts and nor-NOHA on cellular respiration. In conclusion, we show that nor-NOHA has significant but off-target anti-leukemic activity among ARG2-expressing hypoxic cells. Since nor-NOHA has been employed in clinical trials, and is widely used in studies on endothelial dysfunction, immunosuppression and metabolism, the diverse biological effects of nor-NOHA must be cautiously evaluated before attributing its activity to ARG inhibition.},
}

Antineoplastic Agents/*pharmacology/therapeutic use
Apoptosis/*drug effects
Arginase/*antagonists & inhibitors/genetics/metabolism
Arginine/*analogs & derivatives/pharmacology/therapeutic use
CRISPR-Cas Systems/genetics
Cell Cycle/drug effects
Cell Line, Tumor
Cell Proliferation/drug effects
Drug Screening Assays, Antitumor
Enzyme Assays
Feasibility Studies
Gene Knockout Techniques
Humans
Leukemia, Myelogenous, Chronic, BCR-ABL Positive/*drug therapy/pathology
Primary Cell Culture
RNA, Small Interfering/metabolism

@article {pmid29797485,
year = {2018},
author = {Xu, X and Zhang, Z and Yang, Y and Huang, S and Li, K and He, L and Zhou, X},
title = {Genome editing reveals the function of Yorkie during the embryonic and early larval development in silkworm, Bombyx mori.},
journal = {Insect molecular biology},
volume = {27},
number = {6},
pages = {675-685},
doi = {10.1111/imb.12502},
pmid = {29797485},
issn = {1365-2583},
mesh = {Animals ; Bombyx/*embryology/genetics/growth & development/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression ; Gene Knockout Techniques ; Insect Proteins/genetics/*metabolism ; Larva/growth & development ; Molting ; Phenotype ; Signal Transduction ; },
abstract = {As a transcriptional coactivator, Yorkie (Yki) is a major downstream target of the Hippo signalling pathway to regulate the organ size during animal development and regeneration. Previous microarray analysis in the silkworm, Bombyx mori, has shown that genes associated with the Hippo pathway were primarily expressed in gonads and imaginal discs. The RNA-interference-mediated silencing of Yki at the early wandering stage delayed B. mori development and ovary maturation, whereas baculovirus-mediated overexpression at the late larval instar facilitated organ growth and accelerated metamorphosis. Here, we employed CRISPR/Cas9-mediated mutagenesis to investigate the function of Yki in B. mori (BmYki) at the embryonic and early larval stages. Knocking out of BmYki led to reduced body size, moulting defects and, eventually, larval lethality. Sequence analysis of CRISPR/Cas9 mutants exhibited an array of deletions in BmYki. As a critical downstream effector of the Hippo kinase cassette, silencing of BmYki at the embryonic stage is indispensable and the consequence is lethal. Given that the Hippo signalling pathway is evolutionarily conserved, Yki has the potential to be a novel molecular target for genetic-based pest management practices.},
}

Animals
Bombyx/*embryology/genetics/growth & development/metabolism
CRISPR-Cas Systems
Gene Editing
Gene Expression
Gene Knockout Techniques
Insect Proteins/genetics/*metabolism
Larva/growth & development
Molting
Phenotype
Signal Transduction

@article {pmid29763675,
year = {2018},
author = {Wang, Q and Cobine, PA and Coleman, JJ},
title = {Efficient genome editing in Fusarium oxysporum based on CRISPR/Cas9 ribonucleoprotein complexes.},
journal = {Fungal genetics and biology : FG & B},
volume = {117},
number = {},
pages = {21-29},
doi = {10.1016/j.fgb.2018.05.003},
pmid = {29763675},
issn = {1096-0937},
support = {K22 AI100983/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Fusarium/*genetics ; Gene Editing ; Gene Targeting ; Genome, Fungal/*genetics ; Ribonucleoproteins/*genetics ; },
abstract = {The Fusarium oxysporum species complex (FOSC) is an economically important group of pathogenic filamentous fungi that are able to infect both animals and plants. Reverse genetic techniques, including gene disruption/deletion methods, to study these fungi are available although limitations exist resulting in decreased efficiency. Herein we describe a gene editing system developed using a F. oxysporum-optimized Cas9 ribonucleoprotein (RNP) and protoplast transformation method. The Cas9 protein and sgRNA were assembled to form a stable RNP in vitro and this complex was transferred into fungal protoplasts for gene editing with PEG-mediated transformation. In order to determine if the Cas9 RNP system is functional in the FOSC protoplasts and assess the efficacy of the system, two genes, URA5 and URA3, were selected for targeted disruption generating uracil auxotroph mutants that are resistant to 5-fluoroorotic acid, 5-FOA. In addition, a gene in a secondary metabolite biosynthetic cluster, the ortholog of BIK1, was mutated using this system and the maximum efficiency of this gene disruption was about 50%. Further analysis of the bik1 mutant confirmed that this polyketide synthase was involved in the synthesis of the red pigment, bikaverin. The mutants generated in this study displayed the strong expected phenotypes, demonstrating this F. oxysporum-optimized CRISPR/Cas9 system is stable and can efficiently disrupt the genes of interest.},
}

CRISPR-Cas Systems/*genetics
Fusarium/*genetics
Gene Editing
Gene Targeting
Genome, Fungal/*genetics
Ribonucleoproteins/*genetics

@article {pmid29606351,
year = {2018},
author = {Yan, S and Tu, Z and Liu, Z and Fan, N and Yang, H and Yang, S and Yang, W and Zhao, Y and Ouyang, Z and Lai, C and Yang, H and Li, L and Liu, Q and Shi, H and Xu, G and Zhao, H and Wei, H and Pei, Z and Li, S and Lai, L and Li, XJ},
title = {A Huntingtin Knockin Pig Model Recapitulates Features of Selective Neurodegeneration in Huntington's Disease.},
journal = {Cell},
volume = {173},
number = {4},
pages = {989-1002.e13},
pmid = {29606351},
issn = {1097-4172},
support = {R01 NS041669/NS/NINDS NIH HHS/United States ; R01 AG019206/AG/NIA NIH HHS/United States ; R01 NS095181/NS/NINDS NIH HHS/United States ; R01 NS095279/NS/NINDS NIH HHS/United States ; R01 NS045016/NS/NINDS NIH HHS/United States ; R01 AG031153/AG/NIA NIH HHS/United States ; R01 NS101701/NS/NINDS NIH HHS/United States ; R01 NS036232/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Body Weight ; Brain/diagnostic imaging/metabolism/pathology ; CRISPR-Cas Systems/genetics ; Cerebral Cortex/pathology/ultrastructure ; Corpus Striatum/pathology/ultrastructure ; Disease Models, Animal ; Huntingtin Protein/*genetics/metabolism ; Huntington Disease/mortality/*pathology ; Magnetic Resonance Imaging ; Neurons/metabolism/pathology ; Nuclear Transfer Techniques ; Survival Rate ; Swine ; Trinucleotide Repeats ; },
abstract = {Huntington's disease (HD) is characterized by preferential loss of the medium spiny neurons in the striatum. Using CRISPR/Cas9 and somatic nuclear transfer technology, we established a knockin (KI) pig model of HD that endogenously expresses full-length mutant huntingtin (HTT). By breeding this HD pig model, we have successfully obtained F1 and F2 generation KI pigs. Characterization of founder and F1 KI pigs shows consistent movement, behavioral abnormalities, and early death, which are germline transmittable. More importantly, brains of HD KI pig display striking and selective degeneration of striatal medium spiny neurons. Thus, using a large animal model of HD, we demonstrate for the first time that overt and selective neurodegeneration seen in HD patients can be recapitulated by endogenously expressed mutant proteins in large mammals, a finding that also underscores the importance of using large mammals to investigate the pathogenesis of neurodegenerative diseases and their therapeutics.},
}

Animals
Body Weight
Brain/diagnostic imaging/metabolism/pathology
CRISPR-Cas Systems/genetics
Cerebral Cortex/pathology/ultrastructure
Corpus Striatum/pathology/ultrastructure
Disease Models, Animal
Huntingtin Protein/*genetics/metabolism
Huntington Disease/mortality/*pathology
Magnetic Resonance Imaging
Neurons/metabolism/pathology
Nuclear Transfer Techniques
Survival Rate
Swine
Trinucleotide Repeats

@article {pmid29500242,
year = {2018},
author = {Caprara, G and Prosperini, E and Piccolo, V and Sigismondo, G and Melacarne, A and Cuomo, A and Boothby, M and Rescigno, M and Bonaldi, T and Natoli, G},
title = {PARP14 Controls the Nuclear Accumulation of a Subset of Type I IFN-Inducible Proteins.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {200},
number = {7},
pages = {2439-2454},
doi = {10.4049/jimmunol.1701117},
pmid = {29500242},
issn = {1550-6606},
mesh = {Active Transport, Cell Nucleus/physiology ; Animals ; CRISPR-Cas Systems ; Cell Line ; Endotoxins/immunology ; Gene Editing ; Interferon-beta/*immunology ; Macrophage Activation/genetics/*immunology ; Macrophages/*immunology/microbiology ; Mice ; Poly(ADP-ribose) Polymerases/*genetics ; RAW 264.7 Cells ; RNA Interference ; RNA, Small Interfering/genetics ; Salmonella typhimurium/growth & development/*immunology ; },
abstract = {The enzymes of the poly-ADP-ribose polymerase (PARP) superfamily control many relevant cellular processes, but a precise understanding of their activities in different physiological or disease contexts is largely incomplete. We found that transcription of several Parp genes was dynamically regulated upon murine macrophage activation by endotoxin. PARP14 was strongly induced by several inflammatory stimuli and translocated into the nucleus of stimulated cells. Quantitative mass spectrometry analysis showed that PARP14 bound to a group of IFN-stimulated gene (ISG)-encoded proteins, most with an unknown function, and it was required for their nuclear accumulation. Moreover, PARP14 depletion attenuated transcription of primary antiviral response genes regulated by the IFN regulatory transcription factor 3, including Ifnb1, thus reducing IFN-β production and activation of ISGs involved in the secondary antiviral response. In agreement with the above-mentioned data, PARP14 hindered Salmonella typhimurium proliferation in murine macrophages. Overall, these data hint at a role of PARP14 in the control of antimicrobial responses and specifically in nuclear activities of a subgroup of ISG-encoded proteins.},
}

Active Transport, Cell Nucleus/physiology
Animals
CRISPR-Cas Systems
Cell Line
Endotoxins/immunology
Gene Editing
Interferon-beta/*immunology
Macrophage Activation/genetics/*immunology
Macrophages/*immunology/microbiology
Mice
Poly(ADP-ribose) Polymerases/*genetics
RAW 264.7 Cells
RNA Interference
RNA, Small Interfering/genetics
Salmonella typhimurium/growth & development/*immunology

@article {pmid29463318,
year = {2018},
author = {Di Cristina, M and Carruthers, VB},
title = {New and emerging uses of CRISPR/Cas9 to genetically manipulate apicomplexan parasites.},
journal = {Parasitology},
volume = {145},
number = {9},
pages = {1119-1126},
pmid = {29463318},
issn = {1469-8161},
support = {R01 AI046675/AI/NIAID NIH HHS/United States ; R01 AI120607/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Apicomplexa/*genetics ; CRISPR-Associated Protein 9/*genetics ; *CRISPR-Cas Systems ; Cryptosporidium/genetics ; Gene Knockout Techniques ; Genetic Engineering/*methods ; *Genome, Protozoan ; Humans ; Mice ; Plasmodium/genetics ; Recombination, Genetic ; Toxoplasma/genetics ; },
abstract = {Although the application of CRISPR/Cas9 genome engineering approaches was first reported in apicomplexan parasites only 3 years ago, this technology has rapidly become an essential component of research on apicomplexan parasites. This review briefly describes the history of CRISPR/Cas9 and the principles behind its use along with documenting its implementation in apicomplexan parasites, especially Plasmodium spp. and Toxoplasma gondii. We also discuss the recent use of CRISPR/Cas9 for whole genome screening of gene knockout mutants in T. gondii and highlight its use for seminal genetic manipulations of Cryptosporidium spp. Finally, we consider new variations of CRISPR/Cas9 that have yet to be implemented in apicomplexans. Whereas CRISPR/Cas9 has already accelerated rapid interrogation of gene function in apicomplexans, the full potential of this technology is yet to be realized as new variations and innovations are integrated into the field.},
}

Animals
Apicomplexa/*genetics
CRISPR-Associated Protein 9/*genetics
*CRISPR-Cas Systems
Cryptosporidium/genetics
Gene Knockout Techniques
Genetic Engineering/*methods
*Genome, Protozoan
Humans
Mice
Plasmodium/genetics
Recombination, Genetic
Toxoplasma/genetics

@article {pmid29445007,
year = {2018},
author = {Kornete, M and Marone, R and Jeker, LT},
title = {Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {200},
number = {7},
pages = {2489-2501},
pmid = {29445007},
issn = {1550-6606},
support = {144860//Swiss National Science Foundation/Switzerland ; R01 AI106923/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; CD4-Positive T-Lymphocytes/*immunology/transplantation ; CRISPR-Cas Systems/genetics ; Cell Engineering/*methods ; Cell Line, Tumor ; Forkhead Transcription Factors/*genetics ; Gene Deletion ; Gene Editing/*methods ; Immunotherapy, Adoptive/methods ; Leukocyte Common Antigens/immunology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Plasmids/*genetics ; Polymorphism, Single Nucleotide/genetics ; },
abstract = {Adoptive cell transfer is an important approach for basic research and emerges as an effective treatment for various diseases, including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. In this article, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology-directed repair, systematically optimize experimental parameters, and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells, with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Repairing the cause of the scurfy syndrome, a 2-bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells.},
}

Animals
CD4-Positive T-Lymphocytes/*immunology/transplantation
CRISPR-Cas Systems/genetics
Cell Engineering/*methods
Cell Line, Tumor
Forkhead Transcription Factors/*genetics
Gene Deletion
Gene Editing/*methods
Immunotherapy, Adoptive/methods
Leukocyte Common Antigens/immunology
Mice
Mice, Inbred C57BL
Mice, Transgenic
Plasmids/*genetics
Polymorphism, Single Nucleotide/genetics

@article {pmid29327444,
year = {2018},
author = {Che, P and Anand, A and Wu, E and Sander, JD and Simon, MK and Zhu, W and Sigmund, AL and Zastrow-Hayes, G and Miller, M and Liu, D and Lawit, SJ and Zhao, ZY and Albertsen, MC and Jones, TJ},
title = {Developing a flexible, high-efficiency Agrobacterium-mediated sorghum transformation system with broad application.},
journal = {Plant biotechnology journal},
volume = {16},
number = {7},
pages = {1388-1395},
pmid = {29327444},
issn = {1467-7652},
mesh = {Agrobacterium/*genetics ; CRISPR-Cas Systems ; Gene Transfer Techniques ; Genetic Engineering/*methods ; Genetic Markers/genetics ; Genetic Vectors/genetics ; Plants, Genetically Modified/genetics ; Sorghum/*genetics ; Transformation, Genetic/genetics ; },
abstract = {Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi-arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here, we report a ternary vector (also known as cohabitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium-mediated transformation of sorghum. We report regeneration frequencies ranging from 6% to 29% in Tx430 using different selectable markers and single copy, backbone free 'quality events' ranging from 45% to 66% of the total events produced. Furthermore, we successfully applied this ternary system to develop transformation protocols for popular but recalcitrant African varieties including Macia, Malisor 84-7 and Tegemeo. In addition, we report the use of this technology to develop the first stable CRISPR/Cas9-mediated gene knockouts in Tx430.},
}

Agrobacterium/*genetics
CRISPR-Cas Systems
Gene Transfer Techniques
Genetic Engineering/*methods
Genetic Markers/genetics
Genetic Vectors/genetics
Plants, Genetically Modified/genetics
Sorghum/*genetics
Transformation, Genetic/genetics

@article {pmid29230929,
year = {2018},
author = {Lin, CS and Hsu, CT and Yang, LH and Lee, LY and Fu, JY and Cheng, QW and Wu, FH and Hsiao, HC and Zhang, Y and Zhang, R and Chang, WJ and Yu, CT and Wang, W and Liao, LJ and Gelvin, SB and Shih, MC},
title = {Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration.},
journal = {Plant biotechnology journal},
volume = {16},
number = {7},
pages = {1295-1310},
pmid = {29230929},
issn = {1467-7652},
mesh = {Arabidopsis/genetics ; Brassica/genetics ; *CRISPR-Cas Systems/genetics ; Genes, Plant/genetics ; Millets/genetics ; Mutagenesis, Site-Directed/*methods ; Mutation/genetics ; Oryza/genetics ; Oxidoreductases/genetics ; *Protoplasts ; Sasa/genetics ; Tobacco/genetics ; Zea mays/genetics ; },
abstract = {Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.},
}

Arabidopsis/genetics
Brassica/genetics
*CRISPR-Cas Systems/genetics
Genes, Plant/genetics
Millets/genetics
Mutagenesis, Site-Directed/*methods
Mutation/genetics
Oryza/genetics
Oxidoreductases/genetics
*Protoplasts
Sasa/genetics
Tobacco/genetics
Zea mays/genetics

@article {pmid29209043,
year = {2018},
author = {Tirado-Gonzalez, I and Czlonka, E and Nevmerzhitskaya, A and Soetopo, D and Bergonzani, E and Mahmoud, A and Contreras, A and Jeremias, I and Platzbecker, U and Bourquin, JP and Kloz, U and Van der Hoeven, F and Medyouf, H},
title = {CRISPR/Cas9-edited NSG mice as PDX models of human leukemia to address the role of niche-derived SPARC.},
journal = {Leukemia},
volume = {32},
number = {4},
pages = {1049-1052},
pmid = {29209043},
issn = {1476-5551},
support = {639795//European Research Council/International ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Disease Progression ; Gene Editing/methods ; Hematologic Neoplasms/genetics/pathology ; Heterografts ; Humans ; Leukemia/*genetics/pathology ; Mice ; Mice, Inbred C57BL ; Mice, Inbred NOD ; Osteonectin/*genetics ; Tumor Microenvironment/genetics ; },
}

Animals
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Disease Progression
Gene Editing/methods
Hematologic Neoplasms/genetics/pathology
Heterografts
Humans
Leukemia/*genetics/pathology
Mice
Mice, Inbred C57BL
Mice, Inbred NOD
Osteonectin/*genetics
Tumor Microenvironment/genetics

@article {pmid29204946,
year = {2018},
author = {DiEuliis, D and Giordano, J},
title = {Gene editing using CRISPR/Cas9: implications for dual-use and biosecurity.},
journal = {Protein & cell},
volume = {9},
number = {3},
pages = {239-240},
pmid = {29204946},
issn = {1674-8018},
support = {UL1 TR001409/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Containment of Biohazards ; Gene Editing/ethics/*methods ; Humans ; },
}

Animals
*CRISPR-Cas Systems
Containment of Biohazards
Gene Editing/ethics/*methods
Humans

@article {pmid28864683,
year = {2018},
author = {Du, J and Shang, J and Chen, F and Zhang, Y and Yin, N and Xie, T and Zhang, H and Yu, J and Liu, F},
title = {A CRISPR/Cas9-Based Screening for Non-Homologous End Joining Inhibitors Reveals Ouabain and Penfluridol as Radiosensitizers.},
journal = {Molecular cancer therapeutics},
volume = {17},
number = {2},
pages = {419-431},
doi = {10.1158/1535-7163.MCT-17-0090},
pmid = {28864683},
issn = {1538-8514},
mesh = {CRISPR-Cas Systems/*genetics ; Humans ; Ouabain/pharmacology/*therapeutic use ; Penfluridol/pharmacology/*therapeutic use ; Radiation-Sensitizing Agents ; Transfection ; },
abstract = {Non-homologous end joining (NHEJ) is the major pathway responsible for the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSB), and correspondingly regulates the cellular response to IR. Identification of NHEJ inhibitors could substantially enhance the tumor radiosensitivity and improve the therapeutic efficiency of radiotherapy. In this study, we demonstrated a screening for NHEJ inhibitors using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system and high-resolution melting (HRM) analysis. Because NHEJ is regarded as an error-prone mechanism, the NHEJ-mediated ligation of the site-specific DSB induced by Cas9 nuclease would eventually cause the mutation of the targeted sequence. Then, HRM analysis, a reliable and rapid assay for detecting sequence variation, was performed to evaluate the mutation efficiency of the targeted site. Validating analysis confirmed the NHEJ activities were positively correlated with the mutation frequencies. Next, an approved drug library containing 1,540 compounds was interrogated by using this screening strategy. Our results identified ouabain, a cardiotonic agent, and penfluridol, an antipsychotic agent, have the capacity to restrain NHEJ activity. Further experiments in vitro revealed the radiosensitizing effects of these compounds. Overall, we presented a cell-based screening for NHEJ inhibitors, which could promote the discovery of novel radiosensitizers. Mol Cancer Ther; 17(2); 419-31. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."},
}

CRISPR-Cas Systems/*genetics
Humans
Ouabain/pharmacology/*therapeutic use
Penfluridol/pharmacology/*therapeutic use
Radiation-Sensitizing Agents
Transfection

@article {pmid28785007,
year = {2017},
author = {Ong, SH and Li, Y and Koike-Yusa, H and Yusa, K},
title = {Optimised metrics for CRISPR-KO screens with second-generation gRNA libraries.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {7384},
pmid = {28785007},
issn = {2045-2322},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Algorithms ; CRISPR-Cas Systems ; Gene Knockout Techniques/*methods ; Gene Library ; Humans ; RNA, Guide/*genetics ; },
abstract = {Genome-wide CRISPR-based knockout (CRISPR-KO) screening is an emerging technique which enables systematic genetic analysis of a cellular or molecular phenotype in question. Continuous improvements, such as modifications to the guide RNA (gRNA) scaffold and the development of gRNA on-target prediction algorithms, have since been made to increase their screening performance. We compared the performance of three available second-generation human genome-wide CRISPR-KO libraries that included at least one of the improvements, and examined the effect of gRNA scaffold, number of gRNAs per gene and number of replicates on screen performance. We identified duplicated screens using a library with 6 gRNAs per gene as providing the best trade-off. Despite the improvements, we found that each improved library still has library-specific false negatives and, for the first time, estimated the false negative rates of CRISPR-KO screens, which are between 10% and 20%. Our newly-defined optimal screening parameters would be helpful in designing screens and constructing bespoke gRNA libraries.},
}

Algorithms
CRISPR-Cas Systems
Gene Knockout Techniques/*methods
Gene Library
Humans
RNA, Guide/*genetics

@article {pmid28761125,
year = {2017},
author = {Feehan, JM and Chiu, CN and Stanar, P and Tam, BM and Ahmed, SN and Moritz, OL},
title = {Modeling Dominant and Recessive Forms of Retinitis Pigmentosa by Editing Three Rhodopsin-Encoding Genes in Xenopus Laevis Using Crispr/Cas9.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {6920},
pmid = {28761125},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; *Disease Models, Animal ; Female ; Gene Editing/*methods ; Genes, Dominant ; Genes, Recessive ; Green Fluorescent Proteins/genetics ; Humans ; Male ; Phenotype ; Point Mutation ; RNA, Guide/genetics ; Retinitis Pigmentosa/*genetics/pathology ; Rhodopsin/*genetics ; Sequence Deletion ; Xenopus Proteins/genetics ; Xenopus laevis/embryology ; },
abstract = {The utility of Xenopus laevis, a common research subject for developmental biology, retinal physiology, cell biology, and other investigations, has been limited by lack of a robust gene knockout or knock-down technology. Here we describe manipulation of the X. laevis genome using CRISPR/Cas9 to model the human disorder retinitis pigmentosa, and to introduce point mutations or exogenous DNA sequences. We introduced and characterized in-frame and out-of-frame insertions and deletions in three genes encoding rhodopsin by co-injection of Cas9 mRNA, eGFP mRNA, and single guide RNAs into fertilized eggs. Deletions were characterized by direct sequencing and cloning; phenotypes were assessed by assays of rod opsin in retinal extracts, and confocal microscopy of cryosectioned and immunolabeled contralateral eyes. We obtained germline transmission of editing to F1 offspring. In-frame deletions frequently caused dominant retinal degeneration associated with rhodopsin biosynthesis defects, while frameshift phenotypes were consistent with knockout. We inserted eGFP or point mutations into rhodopsin genes by co-injection of repair fragments with homology to the Cas9 target sites. Our techniques can produce high frequency gene editing in X. laevis, permitting analysis in the F0 generation, and advancing the utility of X. laevis as a subject for biological research and disease modeling.},
}

Animals
CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems
*Disease Models, Animal
Female
Gene Editing/*methods
Genes, Dominant
Genes, Recessive
Green Fluorescent Proteins/genetics
Humans
Male
Phenotype
Point Mutation
RNA, Guide/genetics
Retinitis Pigmentosa/*genetics/pathology
Rhodopsin/*genetics
Sequence Deletion
Xenopus Proteins/genetics
Xenopus laevis/embryology

@article {pmid28383804,
year = {2017},
author = {Keeler, AM and ElMallah, MK and Flotte, TR},
title = {Gene Therapy 2017: Progress and Future Directions.},
journal = {Clinical and translational science},
volume = {10},
number = {4},
pages = {242-248},
pmid = {28383804},
issn = {1752-8062},
support = {K08 HD077040/HD/NICHD NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; R01 DK098252/DK/NIDDK NIH HHS/United States ; R21 NS098131/NS/NINDS NIH HHS/United States ; },
mesh = {Adenoviridae/genetics ; CRISPR-Cas Systems/genetics ; Drug Approval ; Gammaretrovirus/genetics ; Gene Editing/legislation & jurisprudence/*methods ; Genetic Therapy/legislation & jurisprudence/*methods/trends ; Genetic Vectors/genetics/*therapeutic use ; Humans ; Lentivirus/genetics ; Oncolytic Viruses/genetics ; *RNA Interference ; },
}

Adenoviridae/genetics
CRISPR-Cas Systems/genetics
Drug Approval
Gammaretrovirus/genetics
Gene Editing/legislation & jurisprudence/*methods
Genetic Therapy/legislation & jurisprudence/*methods/trends
Genetic Vectors/genetics/*therapeutic use
Humans
Lentivirus/genetics
Oncolytic Viruses/genetics
*RNA Interference

@article {pmid28257845,
year = {2017},
author = {Zheng, X and Li, SY and Zhao, GP and Wang, J},
title = {An efficient system for deletion of large DNA fragments in Escherichia coli via introduction of both Cas9 and the non-homologous end joining system from Mycobacterium smegmatis.},
journal = {Biochemical and biophysical research communications},
volume = {485},
number = {4},
pages = {768-774},
doi = {10.1016/j.bbrc.2017.02.129},
pmid = {28257845},
issn = {1090-2104},
mesh = {Bacillus subtilis/genetics ; Base Sequence ; *CRISPR-Cas Systems ; *DNA End-Joining Repair ; DNA, Bacterial/*genetics ; Escherichia coli/*genetics ; Genetic Engineering/methods ; Lac Operon/genetics ; Models, Genetic ; Mutagenesis ; Mycobacterium smegmatis/*genetics ; Mycobacterium tuberculosis/genetics ; Operon/genetics ; Reproducibility of Results ; *Sequence Deletion ; Sequence Homology, Nucleic Acid ; },
abstract = {Accompanied with the internal non-homologous end joining (NHEJ) system, Cas9 can be used to easily inactivate a gene or delete a fragment through introduction of DNA double-stranded breaks (DSBs) in eukaryotic cells. While in most prokaryotes (e.g. Escherichia coli), due to the lack of NHEJ, homologous recombination (HR) is required for repair of DSBs, which is less convenient. Here, a markerless system was developed for rapid gene inactivation or fragment deletion in E. coli via introduction of both Cas9 and a bacterial NHEJ system. Three bacterial NHEJ systems, i.e. Mycobacterium smegmatis (Msm), Mycobacterium tuberculosis (Mtb) and Bacillus subtilis (Bs), were tested in E. coli, and the MsmNHEJ system showed the best efficiency. With the employment of Cas9 and MsmNHEJ, we efficiently mutated lacZ gene, deleted glnALG operon and two large DNA fragments (67 kb and 123 kb) in E. coli, respectively. Moreover, the system was further designed to allow for continuous inactivation of genes or deletion of DNA fragments in E. coli. We envision this system can be extended to other bacteria, especially those with low HR efficiency.},
}

Bacillus subtilis/genetics
Base Sequence
*CRISPR-Cas Systems
*DNA End-Joining Repair
DNA, Bacterial/*genetics
Escherichia coli/*genetics
Genetic Engineering/methods
Lac Operon/genetics
Models, Genetic
Mutagenesis
Mycobacterium smegmatis/*genetics
Mycobacterium tuberculosis/genetics
Operon/genetics
Reproducibility of Results
*Sequence Deletion
Sequence Homology, Nucleic Acid

@article {pmid28251316,
year = {2017},
author = {Hohmann, S},
title = {Editor's comment on "CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein".},
journal = {Molecular genetics and genomics : MGG},
volume = {292},
number = {3},
pages = {535-536},
pmid = {28251316},
issn = {1617-4623},
mesh = {CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems/*genetics ; Favism/genetics/prevention & control ; Gene Editing/*ethics/*methods ; Genetic Therapy/*ethics/*methods ; Glucosephosphate Dehydrogenase/genetics ; Humans ; beta-Globins/genetics ; beta-Thalassemia/genetics/prevention & control ; },
}

CRISPR-Associated Protein 9/genetics
CRISPR-Cas Systems/*genetics
Favism/genetics/prevention & control
Gene Editing/*ethics/*methods
Genetic Therapy/*ethics/*methods
Glucosephosphate Dehydrogenase/genetics
Humans
beta-Globins/genetics
beta-Thalassemia/genetics/prevention & control

@article {pmid28062498,
year = {2017},
author = {Zhu, QM and Ko, KA and Ture, S and Mastrangelo, MA and Chen, MH and Johnson, AD and O'Donnell, CJ and Morrell, CN and Miano, JM and Lowenstein, CJ},
title = {Novel Thrombotic Function of a Human SNP in STXBP5 Revealed by CRISPR/Cas9 Gene Editing in Mice.},
journal = {Arteriosclerosis, thrombosis, and vascular biology},
volume = {37},
number = {2},
pages = {264-270},
pmid = {28062498},
issn = {1524-4636},
support = {R01 HL124018/HL/NHLBI NIH HHS/United States ; R01 HL117907/HL/NHLBI NIH HHS/United States ; R01 HL078635/HL/NHLBI NIH HHS/United States ; R01 HL112793/HL/NHLBI NIH HHS/United States ; R56 HL124042/HL/NHLBI NIH HHS/United States ; 13PRE17050105//American Heart Association-American Stroke Association/United States ; R21 HL108372/HL/NHLBI NIH HHS/United States ; T32 HL007937/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Blood Coagulation/*genetics ; Blood Platelets/metabolism ; CRISPR-Associated Proteins/*genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Disease Models, Animal ; Down-Regulation ; Exocytosis ; Gene Editing/*methods ; Genome-Wide Association Study ; Genotype ; Humans ; Mice, Transgenic ; Nerve Tissue Proteins/*genetics ; Phenotype ; Platelet Activation ; *Polymorphism, Single Nucleotide ; R-SNARE Proteins/*genetics ; Thrombosis/blood/genetics/*prevention & control ; von Willebrand Factor/metabolism ; },
abstract = {OBJECTIVE: To identify and characterize the effect of a SNP (single-nucleotide polymorphism) in the STXBP5 locus that is associated with altered thrombosis in humans. GWAS (genome-wide association studies) have identified numerous SNPs associated with human thrombotic phenotypes, but determining the functional significance of an individual candidate SNP can be challenging, particularly when in vivo modeling is required. Recent GWAS led to the discovery of STXBP5 as a regulator of platelet secretion in humans. Further clinical studies have identified genetic variants of STXBP5 that are linked to altered plasma von Willebrand factor levels and thrombosis in humans, but the functional significance of these variants in STXBP5 is not understood.

APPROACH AND RESULTS: We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) techniques to produce a precise mouse model carrying a human coding SNP rs1039084 (encoding human p. N436S) in the STXBP5 locus associated with decreased thrombosis. Mice carrying the orthologous human mutation (encoding p. N437S in mouse STXBP5) have lower plasma von Willebrand factor levels, decreased thrombosis, and decreased platelet secretion compared with wild-type mice. This thrombosis phenotype recapitulates the phenotype of humans carrying the minor allele of rs1039084. Decreased plasma von Willebrand factor and platelet activation may partially explain the decreased thrombotic phenotype in mutant mice.

CONCLUSIONS: Using precise mammalian genome editing, we have identified a human nonsynonymous SNP rs1039084 in the STXBP5 locus as a causal variant for a decreased thrombotic phenotype. CRISPR/Cas9 genetic editing facilitates the rapid and efficient generation of animals to study the function of human genetic variation in vascular diseases.},
}

Animals
Blood Coagulation/*genetics
Blood Platelets/metabolism
CRISPR-Associated Proteins/*genetics/metabolism
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Disease Models, Animal
Down-Regulation
Exocytosis
Gene Editing/*methods
Genome-Wide Association Study
Genotype
Humans
Mice, Transgenic
Nerve Tissue Proteins/*genetics
Phenotype
Platelet Activation
*Polymorphism, Single Nucleotide
R-SNARE Proteins/*genetics
Thrombosis/blood/genetics/*prevention & control
von Willebrand Factor/metabolism

@article {pmid30886355,
year = {2019},
author = {Varble, A and Meaden, S and Barrangou, R and Westra, ER and Marraffini, LA},
title = {Recombination between phages and CRISPR-cas loci facilitates horizontal gene transfer in staphylococci.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41564-019-0400-2},
pmid = {30886355},
issn = {2058-5276},
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats) loci and their associated (cas) genes encode an adaptive immune system that protects prokaryotes from viral1 and plasmid2 invaders. Following viral (phage) infection, a small fraction of the prokaryotic cells are able to integrate a small sequence of the invader's genome into the CRISPR array1. These sequences, known as spacers, are transcribed and processed into small CRISPR RNA guides3-5 that associate with Cas nucleases to specify a viral target for destruction6-9. Although CRISPR-cas loci are widely distributed throughout microbial genomes and often display hallmarks of horizontal gene transfer10-12, the drivers of CRISPR dissemination remain unclear. Here, we show that spacers can recombine with phage target sequences to mediate a form of specialized transduction of CRISPR elements. Phage targets in phage 85, ΦNM1, ΦNM4 and Φ12 can recombine with spacers in either chromosomal or plasmid-borne CRISPR loci in Staphylococcus, leading to either the transfer of CRISPR-adjacent genes or the propagation of acquired immunity to other bacteria in the population, respectively. Our data demonstrate that spacer sequences not only specify the targets of Cas nucleases but also can promote horizontal gene transfer.},
}

@article {pmid30885348,
year = {2019},
author = {Perumal, E and So Youn, K and Sun, S and Seung-Hyun, J and Suji, M and Jieying, L and Yeun-Jun, C},
title = {PTEN inactivation induces epithelial-mesenchymal transition and metastasis by intranuclear translocation of β-catenin and snail/slug in non-small cell lung carcinoma cells.},
journal = {Lung cancer (Amsterdam, Netherlands)},
volume = {130},
number = {},
pages = {25-34},
doi = {10.1016/j.lungcan.2019.01.013},
pmid = {30885348},
issn = {1872-8332},
abstract = {OBJECTIVE: Epithelial-mesenchymal transition (EMT) is the key event in distant metastasis of diverse tumors including lung cancer. Recent evidence suggests the involvement of phosphatase and tensin homolog (PTEN) in EMT phenotypes. However, the molecular mechanism of EMT induced by PTEN inactivation is not clear in lung cancer. We aimed to investigate the role of PTEN inactivation in acquisition of EMT in lung cancer cells.

METHODS: We knocked out the PTEN in PTEN proficient lung cancer cells lines (A549 and NCI-H460) using CRISPR/Cas-9 system and observed the growth, EMT phenotypes, and EMT related molecules. We also explored the in vivo effect of PTEN inactivation on tumor cell growth and distant metastasis using nude mouse injection.

RESULTS: PTEN knockout (KO) cells showed faster growth, migration and invasion than PTEN wild-type (WT) cells. When we injected the cells into nude mice, PTEN-KO cells showed faster growth and higher metastatic potential. In PTEN-KO cells, the levels of phosphorylated AKT (Ser-473 and Thr-308) were profoundly elevated and the expressions of phosphorylated GSK-3β (Ser9, inactive form) increased, while that of β-catenin decreased. Regarding the EMT markers, the expression of E-cadherin decreased but those of N-cadherin, vimentin and MMP-2 increased in the PTEN-KO cells. Especially, PTEN-KO cells showed the almost complete intra-nuclear shift of β-catenin and no β-catenin signal was observed in the cell membrane. Accordingly, PTEN-KO cells exhibited morphological changes such as loss of cell-to-cell contact, pseudopodia and the round shape, which are the typical phenotypes of EMT. Snail and Slug were also dominantly accumulated in the nucleus after PTEN inactivation.

CONCLUSION: All these data consistently support that PTEN inactivation contributes to EMT by nuclear translocation of β-catenin and Snail/Slug in lung cancer cells.},
}

@article {pmid30237171,
year = {2018},
author = {Benoit, CR and Stanton, AE and Tartanian, AC and Motzer, AR and McGaughey, DM and Bond, SR and Brody, LC},
title = {Functional and phylogenetic characterization of noncanonical vitamin B12-binding proteins in zebrafish suggests involvement in cobalamin transport.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {45},
pages = {17606-17621},
pmid = {30237171},
issn = {1083-351X},
mesh = {Animals ; CRISPR-Cas Systems ; Protein Domains ; *Transcobalamins/chemistry/genetics/metabolism ; Vitamin B 12/chemistry/genetics/metabolism ; *Zebrafish/genetics/metabolism ; Zebrafish Proteins/chemistry/genetics/metabolism ; },
abstract = {In humans, transport of food-derived cobalamin (vitamin B12) from the digestive system into the bloodstream involves three paralogous proteins: transcobalamin (TC), haptocorrin (HC), and intrinsic factor (IF). Each of these proteins contains two domains, an α-domain and a β-domain, which together form a cleft in which cobalamin binds. Zebrafish (Danio rerio) are thought to possess only a single cobalamin transport protein, referred to as Tcn2, which is a transcobalamin homolog. Here, we used CRISPR/Cas9 mutagenesis to create null alleles of tcn2 in zebrafish. Fish homozygous for tcn2-null alleles were viable and exhibited no obvious developmentally or behaviorally abnormal phenotypes. For this reason, we hypothesized that previously unidentified cobalamin-carrier proteins encoded in the zebrafish genome may provide an additional pathway for cobalamin transport. We identified genes predicted to code for two such proteins, Tcn-beta-a (Tcnba) and Tcn-beta-b (Tcnbb), which differ from all previously characterized cobalamin transport proteins as they lack the α-domain. These β-domain-only proteins are representative of an undescribed class of cobalamin-carrier proteins that are highly conserved throughout the ray-finned fishes. We observed that the genes encoding the three cobalamin transport homologs, tcn2, tcnba, and tcnbb, are expressed in unique spatial and temporal patterns in the developing zebrafish. Moreover, exogenously expressed recombinant Tcnba and Tcnbb bound cobalamin with high affinity, comparable with binding by full-length Tcn2. Taken together, our results suggest that this noncanonical protein structure has evolved to fully function as a cobalamin-carrier protein, thereby allowing for a compensatory cobalamin transport mechanism in the tcn2-/- zebrafish.},
}

Animals
CRISPR-Cas Systems
Protein Domains
*Transcobalamins/chemistry/genetics/metabolism
Vitamin B 12/chemistry/genetics/metabolism
*Zebrafish/genetics/metabolism
Zebrafish Proteins/chemistry/genetics/metabolism

@article {pmid30118678,
year = {2018},
author = {Tan, Y and Jin, C and Ma, W and Hu, Y and Tanasa, B and Oh, S and Gamliel, A and Ma, Q and Yao, L and Zhang, J and Ohgi, K and Liu, W and Aggarwal, AK and Rosenfeld, MG},
title = {Dismissal of RNA Polymerase II Underlies a Large Ligand-Induced Enhancer Decommissioning Program.},
journal = {Molecular cell},
volume = {71},
number = {4},
pages = {526-539.e8},
pmid = {30118678},
issn = {1097-4164},
support = {R01 NS034934/NS/NINDS NIH HHS/United States ; R37 DK039949/DK/NIDDK NIH HHS/United States ; R01 CA213371/CA/NCI NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; R01 DK018477/DK/NIDDK NIH HHS/United States ; },
mesh = {Base Sequence ; Binding Sites ; CRISPR-Cas Systems ; *Enhancer Elements, Genetic ; Estradiol/pharmacology ; Estrogen Receptor alpha/*genetics/metabolism ; F-Box Proteins/*genetics/metabolism ; Gene Editing/methods ; Gene Expression Regulation/drug effects ; HEK293 Cells ; Hepatocyte Nuclear Factor 3-alpha/*genetics/metabolism ; Histones/genetics/metabolism ; Humans ; Jumonji Domain-Containing Histone Demethylases/*genetics/metabolism ; MCF-7 Cells ; Nedd4 Ubiquitin Protein Ligases/*genetics/metabolism ; Protein Binding ; RNA/genetics/metabolism ; RNA Polymerase II/*genetics/metabolism ; Signal Transduction ; Transcription, Genetic/drug effects ; Ubiquitination/drug effects ; },
abstract = {Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.},
}

Base Sequence
Binding Sites
CRISPR-Cas Systems
*Enhancer Elements, Genetic
Estradiol/pharmacology
Estrogen Receptor alpha/*genetics/metabolism
F-Box Proteins/*genetics/metabolism
Gene Editing/methods
Gene Expression Regulation/drug effects
HEK293 Cells
Hepatocyte Nuclear Factor 3-alpha/*genetics/metabolism
Histones/genetics/metabolism
Humans
Jumonji Domain-Containing Histone Demethylases/*genetics/metabolism
MCF-7 Cells
Nedd4 Ubiquitin Protein Ligases/*genetics/metabolism
Protein Binding
RNA/genetics/metabolism
RNA Polymerase II/*genetics/metabolism
Signal Transduction
Transcription, Genetic/drug effects
Ubiquitination/drug effects

@article {pmid30078338,
year = {2018},
author = {Blokhin, I and Khorkova, O and Hsiao, J and Wahlestedt, C},
title = {Developments in lncRNA drug discovery: where are we heading?.},
journal = {Expert opinion on drug discovery},
volume = {13},
number = {9},
pages = {837-849},
doi = {10.1080/17460441.2018.1501024},
pmid = {30078338},
issn = {1746-045X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Drug Discovery/*methods/trends ; Humans ; *Molecular Biology ; Oligonucleotides/administration & dosage ; RNA, Long Noncoding/*genetics ; },
abstract = {INTRODUCTION: The central dogma of molecular biology, which states that the only role of long RNA transcripts is to convey information from gene to protein, was brought into question in recent years due to discovery of the extensive presence and complex roles of long noncoding RNAs (lncRNAs). Furthermore, lncRNAs were found to be involved in pathogenesis of multiple diseases and thus represent a new class of therapeutic targets. Translational efforts in the lncRNA field have been augmented by progress in optimizing the chemistry and delivery platforms of lncRNA-targeting modalities, including oligonucleotide-based drugs and CRISPR-Cas9. Areas covered: This review covers the current advances in characterizing diversity and biological functions of lncRNA focusing on their therapeutic potential in selected therapeutic areas. Expert opinion: Due to accelerating parallel progress in lncRNA biology and lncRNA-compatible therapeutic modalities, it is likely that lncRNA-dependent mechanisms of pathogenesis will soon be targeted in various disorders, including neurological, psychiatric, cardiovascular, infectious diseases, and cancer. Significant efforts, however, are still required to better understand the biology of both lncRNAs and lncRNA-targeting drugs. Further work is needed in the areas of lncRNA nomenclature, database representation, intra/interfield communication, and education of the community at large.},
}

Animals
CRISPR-Cas Systems/genetics
Drug Discovery/*methods/trends
Humans
*Molecular Biology
Oligonucleotides/administration & dosage
RNA, Long Noncoding/*genetics

@article {pmid30033371,
year = {2018},
author = {Shou, J and Li, J and Liu, Y and Wu, Q},
title = {Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion.},
journal = {Molecular cell},
volume = {71},
number = {4},
pages = {498-509.e4},
doi = {10.1016/j.molcel.2018.06.021},
pmid = {30033371},
issn = {1097-4164},
mesh = {Base Sequence ; CRISPR-Associated Protein 9/*genetics/metabolism ; *CRISPR-Cas Systems ; Carrier Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/metabolism ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; Fanconi Anemia Complementation Group D2 Protein/genetics/metabolism ; Gene Duplication ; Gene Editing/*methods ; Genome, Human ; HEK293 Cells ; Humans ; Mutagenesis, Insertional ; Nuclear Proteins/genetics/metabolism ; RNA, Guide/*genetics/metabolism ; Sequence Deletion ; Sequence Inversion ; },
abstract = {Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site in vivo and in vitro, generating double-strand break ends with 5' overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.},
}

Base Sequence
CRISPR-Associated Protein 9/*genetics/metabolism
*CRISPR-Cas Systems
Carrier Proteins/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA/genetics/metabolism
DNA Breaks, Double-Stranded
*DNA End-Joining Repair
Fanconi Anemia Complementation Group D2 Protein/genetics/metabolism
Gene Duplication
Gene Editing/*methods
Genome, Human
HEK293 Cells
Humans
Mutagenesis, Insertional
Nuclear Proteins/genetics/metabolism
RNA, Guide/*genetics/metabolism
Sequence Deletion
Sequence Inversion

@article {pmid30017060,
year = {2018},
author = {Contreras, LM},
title = {Methods and advances in RNA characterization and design.},
journal = {Methods (San Diego, Calif.)},
volume = {143},
number = {},
pages = {1-3},
doi = {10.1016/j.ymeth.2018.06.003},
pmid = {30017060},
issn = {1095-9130},
mesh = {CRISPR-Cas Systems/genetics ; Computational Biology/instrumentation/*methods ; Gene Editing/instrumentation/*methods ; *Gene Expression Regulation ; High-Throughput Nucleotide Sequencing/instrumentation/methods ; Nanotechnology/instrumentation/*methods ; Nucleic Acid Conformation ; RNA/chemistry/*genetics/isolation & purification/metabolism ; RNA Processing, Post-Transcriptional ; Sequence Analysis, RNA/instrumentation/methods ; Structure-Activity Relationship ; },
}

CRISPR-Cas Systems/genetics
Computational Biology/instrumentation/*methods
Gene Editing/instrumentation/*methods
*Gene Expression Regulation
High-Throughput Nucleotide Sequencing/instrumentation/methods
Nanotechnology/instrumentation/*methods
Nucleic Acid Conformation
RNA/chemistry/*genetics/isolation & purification/metabolism
RNA Processing, Post-Transcriptional
Sequence Analysis, RNA/instrumentation/methods
Structure-Activity Relationship

@article {pmid29458767,
year = {2018},
author = {Paul, MW and Zelensky, AN and Wyman, C and Kanaar, R},
title = {Single-Molecule Dynamics and Localization of DNA Repair Proteins in Cells.},
journal = {Methods in enzymology},
volume = {600},
number = {},
pages = {375-406},
doi = {10.1016/bs.mie.2017.11.015},
pmid = {29458767},
issn = {1557-7988},
mesh = {Animals ; BRCA2 Protein/*analysis/chemistry/metabolism ; CRISPR-Cas Systems/genetics ; Cell Culture Techniques/instrumentation/*methods ; Cells, Cultured ; DNA Breaks, Double-Stranded ; Fluorescence Recovery After Photobleaching/instrumentation/methods ; Gene Editing/methods ; Green Fluorescent Proteins/chemistry ; Intravital Microscopy/instrumentation/*methods ; Luminescent Agents/chemistry ; Mice ; Microscopy, Fluorescence/instrumentation/methods ; Mouse Embryonic Stem Cells ; Protein Binding ; Rad51 Recombinase/*analysis/chemistry/metabolism ; *Recombinational DNA Repair ; Single Molecule Imaging/instrumentation/*methods ; },
abstract = {Direct observation of individual protein molecules in their native environment, at nanometer resolution, in a living cell, in motion is not only fascinating but also uniquely informative. Several recent major technological advances in genomic engineering, protein and synthetic fluorophore development, and light microscopy have dramatically increased the accessibility of this approach. This chapter describes the procedures for modifying endogenous genomic loci to producing fluorescently tagged proteins, their high-resolution visualization, and analysis of their dynamics in mammalian cells, using DNA repair proteins BRCA2 and RAD51 as an example.},
}

Animals
BRCA2 Protein/*analysis/chemistry/metabolism
CRISPR-Cas Systems/genetics
Cell Culture Techniques/instrumentation/*methods
Cells, Cultured
DNA Breaks, Double-Stranded
Fluorescence Recovery After Photobleaching/instrumentation/methods
Gene Editing/methods
Green Fluorescent Proteins/chemistry
Intravital Microscopy/instrumentation/*methods
Luminescent Agents/chemistry
Mice
Microscopy, Fluorescence/instrumentation/methods
Mouse Embryonic Stem Cells
Protein Binding
Rad51 Recombinase/*analysis/chemistry/metabolism
*Recombinational DNA Repair
Single Molecule Imaging/instrumentation/*methods

@article {pmid29309838,
year = {2018},
author = {Lee, YJ and Moon, TS},
title = {Design rules of synthetic non-coding RNAs in bacteria.},
journal = {Methods (San Diego, Calif.)},
volume = {143},
number = {},
pages = {58-69},
doi = {10.1016/j.ymeth.2018.01.001},
pmid = {29309838},
issn = {1095-9130},
mesh = {Aptamers, Nucleotide/chemical synthesis/genetics ; Bacteria/*genetics ; CRISPR-Cas Systems/genetics ; Computational Biology/instrumentation/methods ; *Gene Expression Regulation, Bacterial ; RNA Interference ; RNA, Antisense/*chemical synthesis/genetics ; RNA, Untranslated/*chemical synthesis/genetics ; Synthetic Biology/instrumentation/methods ; },
abstract = {One of the long-term goals of synthetic biology is to develop designable genetic parts with predictable behaviors that can be utilized to implement diverse cellular functions. The discovery of non-coding RNAs and their importance in cellular processing have rapidly attracted researchers' attention towards designing functional non-coding RNA molecules. These synthetic non-coding RNAs have simple design principles governed by Watson-Crick base pairing, but exhibit increasingly complex functions. Importantly, due to their specific and modular behaviors, synthetic non-coding RNAs have been widely adopted to modulate transcription and translation of target genes. In this review, we summarize various design rules and strategies employed to engineer synthetic non-coding RNAs. Specifically, we discuss how RNA molecules can be transformed into powerful regulators and utilized to control target gene expression. With the establishment of generalizable non-coding RNA design rules, the research community will shift its focus to RNA regulators from protein regulators.},
}

Aptamers, Nucleotide/chemical synthesis/genetics
Bacteria/*genetics
CRISPR-Cas Systems/genetics
Computational Biology/instrumentation/methods
*Gene Expression Regulation, Bacterial
RNA Interference
RNA, Antisense/*chemical synthesis/genetics
RNA, Untranslated/*chemical synthesis/genetics
Synthetic Biology/instrumentation/methods

@article {pmid30883991,
year = {2019},
author = {Moses, C and Kaur, P},
title = {Applications of CRISPR systems in respiratory health: Entering a new 'red pen' era in genome editing.},
journal = {Respirology (Carlton, Vic.)},
volume = {},
number = {},
pages = {},
doi = {10.1111/resp.13527},
pmid = {30883991},
issn = {1440-1843},
abstract = {Respiratory diseases, such as influenza infection, acute tracheal bronchitis, pneumonia, tuberculosis, chronic obstructive pulmonary disease, asthma, lung cancer and nasopharyngeal carcinoma, continue to significantly impact human health. Diseases of the lung and respiratory tract are influenced by environmental conditions and socio-economic factors; however, many of these serious respiratory disorders are also rooted in genetic or epigenetic causes. Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, isolated from the immune system of prokaryotes, provide a tool to manipulate gene sequences and gene expression with significant implications for respiratory research. CRISPR/Cas systems allow preclinical modelling of causal factors involved in many respiratory diseases, providing new insights into their underlying mechanisms. CRISPR can also be used to screen for genes involved in respiratory processes, development and pathology, identifying novel disease drivers or drug targets. Finally, CRISPR/Cas systems can potentially correct genetic mutations and edit epigenetic marks that contribute to respiratory disorders, providing a form of personalized medicine that could be used in conjunction with other technologies such as stem cell reprogramming and transplantation. CRISPR gene editing is a young field of research, and concerns regarding its specificity, as well as the need for efficient and safe delivery methods, need to be addressed further. However, CRISPR/Cas systems represent a significant step forward for research and therapy in respiratory health, and it is likely we will see the breakthroughs generated from this technology continue.},
}

@article {pmid30881132,
year = {2019},
author = {Lu, ZJ and Yu, Q and Zhou, SH and Fan, J and Shen, LF and Bao, YY and Wu, TT and Zhou, ML and Huang, YP},
title = {Construction of a GLUT-1 and HIF-1α gene knockout cell model in HEp-2 cells using the CRISPR/Cas9 technique.},
journal = {Cancer management and research},
volume = {11},
number = {},
pages = {2087-2096},
doi = {10.2147/CMAR.S183859},
pmid = {30881132},
issn = {1179-1322},
abstract = {Background: Glucose transporter (GLUT)-mediated glucose uptake is an important process in the development of laryngeal carcinoma, one of the most common malignancies of the head and neck. GLUT-1, together with HIF-1α, is also an indicator of hypoxia. Both proteins play a critical role in glucose uptake and glycolysis in laryngeal carcinoma cells under hypoxic stress. A double gene knockout model in which HIF-1α and GLUT-1 are no longer expressed can provide important information about carcinogenesis in laryngeal carcinoma.

Purpose: In this study we used the CRISPR/Cas 9 system to induce HIF-1α and GLUT-1 double gene knockout in HEp-2 cells and then used the knocked-out cells to study the role of these markers in laryngeal carcinoma, including in chemoradioresistance.

Methods: High-grade small-guide RNAs (sgRNAs) of HIF-1α and GLUT-1 were designed using an online tool and inserted into the pUC57-T7-gRNA vector. The recombinant plasmids were transfected into HEp-2 cells and positive cells were screened using the dilution method. Gene mutation and expression were determined by sequence analysis and immunoblotting.

Results: In HIF-1α and GLUT-1 double gene knockout HEp-2 cells, a 171-bp deletion in the HIF-1α genomic sequence was detected, whereas multiple base insertions resulted in frameshift mutations in the GLUT-1 gene. Neither HIF-1α nor GLUT-1 protein was expressed in positive cells. The proliferation, migration, and invasion of HEp-2 cells were significantly decreased afterward. The possible mechanism may be that the inhibition PI3K/AKT/mTOR pathway by HIF-1α and GLUT-1 double gene knockout using CRISPR/Cas9 technique lead to reduction of glucose uptake and lactic acid generation.

Conclusion: Our HIF-1α and GLUT-1 double gene knockout HEp-2 cell model, obtained using a CRISPR/Cas9-based system, may facilitate studies of the pathogenesis of laryngeal carcinoma.},
}

@article {pmid30584102,
year = {2019},
author = {Xiao, B and Yin, S and Hu, Y and Sun, M and Wei, J and Huang, Z and Wen, Y and Dai, X and Chen, H and Mu, J and Cui, L and Jiang, L},
title = {Epigenetic editing by CRISPR/dCas9 in Plasmodium falciparum.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {116},
number = {1},
pages = {255-260},
doi = {10.1073/pnas.1813542116},
pmid = {30584102},
issn = {1091-6490},
support = {R01 AI116466/AI/NIAID NIH HHS/United States ; U19 AI089672/AI/NIAID NIH HHS/United States ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; Erythrocytes/parasitology ; Gene Editing/*methods ; Gene Knockdown Techniques ; Genes, Protozoan/genetics ; Histone Acetyltransferases/genetics ; Histone Deacetylases/genetics ; Humans ; Malaria, Falciparum/parasitology ; Plasmodium falciparum/*genetics/physiology ; Recombinant Proteins ; },
abstract = {Genetic manipulation remains a major obstacle for understanding the functional genomics of the deadliest malaria parasite Plasmodium falciparum Although the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system has been successfully applied to introduce permanent changes in the parasite genome, its use is still limited. Here we show that fusing different epigenetic effector domains to a Cas9 null mutant efficiently and specifically reprograms the expression of target genes in P. falciparum By precisely writing and erasing histone acetylation at the transcription start site regions of the invasion-related genes reticulocyte binding protein homolog 4 (rh4) and erythrocyte binding protein 175 (eba-175), respectively, we achieved significant activation of rh4 and repression of eba-175, leading to the switch of the parasite invasion pathways into human erythrocytes. By using the epigenetic knockdown system, we have also characterized the effects of PfSET1, previously identified as an essential gene, on expression of mainly trophozoite- and schizont-specific genes, and therefore regulation of the growth of the mature forms of P. falciparum This epigenetic CRISPR/dCas9 system provides a powerful approach for regulating gene expression at the transcriptional level in P. falciparum.},
}

CRISPR-Associated Protein 9/genetics
*CRISPR-Cas Systems
*Epigenesis, Genetic
Erythrocytes/parasitology
Gene Editing/*methods
Gene Knockdown Techniques
Genes, Protozoan/genetics
Histone Acetyltransferases/genetics
Histone Deacetylases/genetics
Humans
Malaria, Falciparum/parasitology
Plasmodium falciparum/*genetics/physiology
Recombinant Proteins

@article {pmid30529264,
year = {2019},
author = {Shi, X and He, W and Guo, S and Zhang, B and Ren, S and Liu, K and Sun, T and Cui, J},
title = {RNA-seq Analysis of the SCN1A-KO Model based on CRISPR/Cas9 Genome Editing Technology.},
journal = {Neuroscience},
volume = {398},
number = {},
pages = {1-11},
doi = {10.1016/j.neuroscience.2018.11.052},
pmid = {30529264},
issn = {1873-7544},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Line ; Epilepsies, Myoclonic/genetics/metabolism ; *Gene Editing ; Gene Expression ; *Gene Knockout Techniques/methods ; Mice ; NAV1.1 Voltage-Gated Sodium Channel/*deficiency/genetics ; RNA, Messenger/metabolism ; Sequence Analysis, RNA ; Signal Transduction ; },
abstract = {Dravet syndrome (DS) is a disease that is primarily caused by the inactivation of the SCN1A-encoded voltage-gated sodium channel alpha subunit (Nav1.1). In this study, we constructed an SCN1A gene knockout model using CRISPR/Cas9 genome editing technology to deprive the Nav1.1 function in vitro. With mRNA-seq analysis we found abundant gene changes after SCN1A knockout, which associated with various signaling pathways, such as cancer pathways, the PI3K-AKT signaling pathway, the MAPK signaling pathway, and pathways involved in HTLV-I infection. We also noticed changes in the spliceosome, decreased glycolytic capacity, disturbances in calcium signaling pathways, and changes in the potassium, sodium, chloride, and calcium plasma channels after SCN1A knockout. In this study, we have been the first time to discover these changes and summarize them here and hope it would provide some clue for the study of Nav1.1 in the nervous system.},
}

Animals
*CRISPR-Cas Systems
Cell Line
Epilepsies, Myoclonic/genetics/metabolism
*Gene Editing
Gene Expression
*Gene Knockout Techniques/methods
Mice
NAV1.1 Voltage-Gated Sodium Channel/*deficiency/genetics
RNA, Messenger/metabolism
Sequence Analysis, RNA
Signal Transduction

@article {pmid30476497,
year = {2019},
author = {Fujii, M and Clevers, H and Sato, T},
title = {Modeling Human Digestive Diseases With CRISPR-Cas9-Modified Organoids.},
journal = {Gastroenterology},
volume = {156},
number = {3},
pages = {562-576},
doi = {10.1053/j.gastro.2018.11.048},
pmid = {30476497},
issn = {1528-0012},
mesh = {CRISPR-Cas Systems/*genetics ; Colorectal Neoplasms/genetics/pathology ; Esophageal Neoplasms/genetics/pathology ; Female ; Gastrointestinal Diseases/genetics/pathology ; Gastrointestinal Neoplasms/*genetics/*pathology ; Gastrointestinal Tract/pathology ; *Genetic Predisposition to Disease ; Humans ; Male ; *Organoids ; Pancreatic Neoplasms/genetics/pathology ; Sensitivity and Specificity ; },
abstract = {Insights into the stem cell niche have allowed researchers to cultivate adult tissue stem cells as organoids that display structural and phenotypic features of healthy and diseased epithelial tissues. Organoids derived from patients' tissues are used as models of disease and to test drugs. CRISPR-Cas9 technology can be used to genetically engineer organoids for studies of monogenic diseases and cancer. We review the derivation of organoids from human gastrointestinal tissues and how CRISPR-Cas9 technology can be used to study these organoids. We discuss burgeoning technologies that are broadening our understanding of diseases of the digestive system.},
}

CRISPR-Cas Systems/*genetics
Colorectal Neoplasms/genetics/pathology
Esophageal Neoplasms/genetics/pathology
Female
Gastrointestinal Diseases/genetics/pathology
Gastrointestinal Neoplasms/*genetics/*pathology
Gastrointestinal Tract/pathology
*Genetic Predisposition to Disease
Humans
Male
*Organoids
Pancreatic Neoplasms/genetics/pathology
Sensitivity and Specificity

@article {pmid30258161,
year = {2018},
author = {DeWeerdt, S},
title = {The genomics of brain cancer.},
journal = {Nature},
volume = {561},
number = {7724},
pages = {S54-S55},
doi = {10.1038/d41586-018-06711-8},
pmid = {30258161},
issn = {1476-4687},
mesh = {Adult ; Animals ; Brain Neoplasms/diagnosis/*genetics/*therapy ; CRISPR-Cas Systems/genetics ; Chromosome Deletion ; Chromosomes, Human, Pair 19/genetics ; Gene Editing ; *Genetic Predisposition to Disease ; Genome, Human/*genetics ; *Genomics ; Glioblastoma/diagnosis/genetics/therapy ; Glioma/diagnosis/genetics/therapy ; Humans ; Isocitrate Dehydrogenase/genetics ; Mice ; Mutation ; Oligodendroglioma/diagnosis/pathology/therapy ; *Pharmacogenomic Testing ; Precision Medicine ; Prognosis ; Survival Analysis ; Telomere Homeostasis/genetics ; Treatment Outcome ; World Health Organization ; },
}

Adult
Animals
Brain Neoplasms/diagnosis/*genetics/*therapy
CRISPR-Cas Systems/genetics
Chromosome Deletion
Chromosomes, Human, Pair 19/genetics
Gene Editing
*Genetic Predisposition to Disease
Genome, Human/*genetics
*Genomics
Glioblastoma/diagnosis/genetics/therapy
Glioma/diagnosis/genetics/therapy
Humans
Isocitrate Dehydrogenase/genetics
Mice
Mutation
Oligodendroglioma/diagnosis/pathology/therapy
*Pharmacogenomic Testing
Precision Medicine
Prognosis
Survival Analysis
Telomere Homeostasis/genetics
Treatment Outcome
World Health Organization

@article {pmid30224336,
year = {2018},
author = {Wang, H and Park, H and Liu, J and Sternberg, PW},
title = {An Efficient Genome Editing Strategy To Generate Putative Null Mutants in Caenorhabditis elegans Using CRISPR/Cas9.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {11},
pages = {3607-3616},
pmid = {30224336},
issn = {2160-1836},
support = {K99 GM126137/GM/NIGMS NIH HHS/United States ; P40 OD010440/OD/NIH HHS/United States ; R24 OD023041/OD/NIH HHS/United States ; T32 GM007616/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/genetics ; *Gene Editing ; Mutation ; },
abstract = {Null mutants are essential for analyzing gene function. Here, we describe a simple and efficient method to generate Caenorhabditis elegans null mutants using CRISPR/Cas9 and short single stranded DNA oligo repair templates to insert a universal 43-nucleotide-long knock-in cassette (STOP-IN) into the early exons of target genes. This STOP-IN cassette has stop codons in all three reading frames and leads to frameshifts, which will generate putative null mutations regardless of the reading frame of the insertion position in exons. The STOP-IN cassette also contains an exogenous Cas9 target site that allows further genome editing and provides a unique sequence that simplifies the identification of successful insertion events via PCR. As a proof of concept, we inserted the STOP-IN cassette at a Cas9 target site in aex-2 to generate new putative null alleles by injecting preassembled Cas9 ribonucleoprotein and a short synthetic single stranded DNA repair template containing the STOP-IN cassette and two ∼35-nucleotide-long homology arms identical to the sequences flanking the Cas9 cut site. We showed that these new aex-2 alleles phenocopied an existing loss-of-function allele of aex-2 We further showed that the new aex-2 null alleles could be reverted back to the wild-type sequence by targeting the exogenous Cas9 cut site included in the STOP-IN cassette and providing a single stranded wild-type DNA repair oligo. We applied our STOP-IN method to generate new putative null mutants for 20 additional genes, including three pharyngeal muscle-specific genes (clik-1, clik-2, and clik-3), and reported a high insertion rate (46%) based on the animals we screened. We showed that null mutations of clik-2 cause recessive lethality with a severe pumping defect and clik-3 null mutants have a mild pumping defect, while clik-1 is dispensable for pumping. We expect that the knock-in method using the STOP-IN cassette will facilitate the generation of new null mutants to understand gene function in C. elegans and other genetic model organisms.},
}

Animals
*CRISPR-Cas Systems
Caenorhabditis elegans/*genetics
Caenorhabditis elegans Proteins/genetics
*Gene Editing
Mutation

@article {pmid30213867,
year = {2018},
author = {Huynh, N and Zeng, J and Liu, W and King-Jones, K},
title = {A Drosophila CRISPR/Cas9 Toolkit for Conditionally Manipulating Gene Expression in the Prothoracic Gland as a Test Case for Polytene Tissues.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {11},
pages = {3593-3605},
pmid = {30213867},
issn = {2160-1836},
mesh = {Animals ; CRISPR-Cas Systems ; Cytochrome P-450 Enzyme System/genetics ; Drosophila/*genetics ; Drosophila Proteins/genetics ; Exocrine Glands/*metabolism ; Gene Expression Regulation ; Mixed Function Oxygenases/genetics ; },
abstract = {Targeting gene function with spatial or temporal specificity is a key goal in molecular genetics. CRISPR-Cas9 has greatly facilitated this strategy, but some standard approaches are problematic. For instance, simple tissue-specific or global overexpression of Cas9 can cause significant lethality or developmental delays even in the absence of gRNAs. In particular, we found that Gal4-mediated expression of UAS-Cas9 in the Drosophila prothoracic gland (PG) was not a suitable strategy to disrupt gene expression, since Cas9 alone caused widespread lethality. The PG is widely used for studying endocrine gland function during animal development, but tools validating PG-specific RNAi phenotypes are lacking. Here, we present a collection of modular gateway-compatible CRISPR-Cas9 tools that allow precise modulation of target gene activity with temporal and spatial specificity. We also demonstrate that Cas9 fused to the progesterone ligand-binding domain can be used to activate gene expression via RU486. Using these approaches, we were able to avoid the lethality associated with simple GAL4-mediated overexpression of Cas9 in the PG. Given that the PG is a polytene tissue, we conclude that these tools work effectively in endoreplicating cells where Cas9 has to target multiple copies of the same locus. Our toolkit can be easily adapted for other tissues and can be used both for gain- and loss-of-function studies.},
}

Animals
CRISPR-Cas Systems
Cytochrome P-450 Enzyme System/genetics
Drosophila/*genetics
Drosophila Proteins/genetics
Exocrine Glands/*metabolism
Gene Expression Regulation
Mixed Function Oxygenases/genetics

@article {pmid30169977,
year = {2018},
author = {Luther, DC and Lee, YW and Nagaraj, H and Scaletti, F and Rotello, VM},
title = {Delivery approaches for CRISPR/Cas9 therapeutics in vivo: advances and challenges.},
journal = {Expert opinion on drug delivery},
volume = {15},
number = {9},
pages = {905-913},
doi = {10.1080/17425247.2018.1517746},
pmid = {30169977},
issn = {1744-7593},
support = {R01 EB022641/EB/NIBIB NIH HHS/United States ; R01 GM077173/GM/NIGMS NIH HHS/United States ; T32 GM008515/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/*methods ; Gene Transfer Techniques ; Genetic Therapy/*methods ; Humans ; RNA, Messenger/genetics ; },
abstract = {INTRODUCTION: Therapeutic gene editing is becoming a viable biomedical tool with the emergence of the CRISPR/Cas9 system. CRISPR-based technologies have promise as a therapeutic platform for many human genetic diseases previously considered untreatable, providing a flexible approach to high-fidelity gene editing. For many diseases, such as sickle-cell disease and beta thalassemia, curative therapy may already be on the horizon, with CRISPR-based clinical trials slated for the next few years. Translation of CRISPR-based therapy to in vivo application however, is no small feat, and major hurdles remain for efficacious use of the CRISPR/Cas9 system in clinical contexts. Areas covered: In this topical review, we highlight recent advances to in vivo delivery of the CRISPR/Cas9 system using various packaging formats, including viral, mRNA, plasmid, and protein-based approaches. We also discuss some of the barriers which have yet to be overcome for successful translation of this technology. Expert opinion: This review focuses on the challenges to efficacy for various delivery formats, with specific emphasis on overcoming these challenges through the development of carrier vehicles for transient approaches to CRISPR/Cas9 delivery in vivo.},
}

Animals
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/*methods
Gene Transfer Techniques
Genetic Therapy/*methods
Humans
RNA, Messenger/genetics

@article {pmid30061297,
year = {2018},
author = {Callahan, SJ and Tepan, S and Zhang, YM and Lindsay, H and Burger, A and Campbell, NR and Kim, IS and Hollmann, TJ and Studer, L and Mosimann, C and White, RM},
title = {Cancer modeling by Transgene Electroporation in Adult Zebrafish (TEAZ).},
journal = {Disease models & mechanisms},
volume = {11},
number = {9},
pages = {},
pmid = {30061297},
issn = {1754-8411},
support = {DP2 CA186572/CA/NCI NIH HHS/United States ; F30 CA220954/CA/NCI NIH HHS/United States ; K08 AR055368/AR/NIAMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; F99 CA212436/CA/NCI NIH HHS/United States ; T32 GM007739/GM/NIGMS NIH HHS/United States ; F31 CA196305/CA/NCI NIH HHS/United States ; },
mesh = {Aging/*genetics ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems/genetics ; Carcinogenesis/genetics/pathology ; Disease Models, Animal ; Disease Progression ; *Electroporation ; Embryo, Nonmammalian/metabolism ; Gene Transfer Techniques ; Melanoma/pathology ; Plasmids/genetics ; Promoter Regions, Genetic ; *Transgenes ; Zebrafish/embryology/*genetics ; },
abstract = {Transgenic animals are invaluable for modeling cancer genomics, but often require complex crosses of multiple germline alleles to obtain the desired combinations. Zebrafish models have advantages in that transgenes can be rapidly tested by mosaic expression, but typically lack spatial and temporal control of tumor onset, which limits their utility for the study of tumor progression and metastasis. To overcome these limitations, we have developed a method referred to as Transgene Electroporation in Adult Zebrafish (TEAZ). TEAZ can deliver DNA constructs with promoter elements of interest to drive fluorophores, oncogenes or CRISPR-Cas9-based mutagenic cassettes in specific cell types. Using TEAZ, we created a highly aggressive melanoma model via Cas9-mediated inactivation of Rb1 in the context of BRAFV600E in spatially constrained melanocytes. Unlike prior models that take ∼4 months to develop, we found that TEAZ leads to tumor onset in ∼7 weeks, and these tumors develop in fully immunocompetent animals. As the resulting tumors initiated at highly defined locations, we could track their progression via fluorescence, and documented deep invasion into tissues and metastatic deposits. TEAZ can be deployed to other tissues and cell types, such as the heart, with the use of suitable transgenic promoters. The versatility of TEAZ makes it widely accessible for rapid modeling of somatic gene alterations and cancer progression at a scale not achievable in other in vivo systems.},
}

Aging/*genetics
Animals
Animals, Genetically Modified
CRISPR-Cas Systems/genetics
Carcinogenesis/genetics/pathology
Disease Models, Animal
Disease Progression
*Electroporation
Embryo, Nonmammalian/metabolism
Gene Transfer Techniques
Melanoma/pathology
Plasmids/genetics
Promoter Regions, Genetic
*Transgenes
Zebrafish/embryology/*genetics

@article {pmid29981117,
year = {2018},
author = {Loyola-Vargas, VM and Avilez-Montalvo, RN},
title = {Plant Tissue Culture: A Battle Horse in the Genome Editing Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1815},
number = {},
pages = {131-148},
doi = {10.1007/978-1-4939-8594-4_7},
pmid = {29981117},
issn = {1940-6029},
mesh = {CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Genome, Plant ; Plants/*genetics ; Tissue Culture Techniques/*methods ; },
abstract = {Plant tissue culture (PTC) is a set of techniques for culturing cells, tissues, or organs in an aseptic medium with a defined chemical composition, in a controlled environment. Tissue culture, when combined with molecular biology techniques, becomes a powerful tool for the study of metabolic pathways, elucidation of cellular processes, genetic improvement and, through genetic engineering, the generation of cell lines resistant to biotic and abiotic stress, obtaining improved plants of agronomic interest, or studying the complex cellular genome. In this chapter, we analyze in general the use of plant tissue culture, in particular protoplasts and calli, in the implementation of CRISPR/Cas9 technology.},
}

CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
*Gene Editing
*Genome, Plant
Plants/*genetics
Tissue Culture Techniques/*methods

@article {pmid29963983,
year = {2018},
author = {Rouillon, C and Athukoralage, JS and Graham, S and Grüschow, S and White, MF},
title = {Control of cyclic oligoadenylate synthesis in a type III CRISPR system.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {29963983},
issn = {2050-084X},
support = {Project grant BB/M000400/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adenine Nucleotides/*biosynthesis ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/genetics/metabolism ; Endodeoxyribonucleases/*genetics/metabolism ; Endoribonucleases/genetics/metabolism ; Escherichia coli Proteins/genetics/metabolism ; Kinetics ; Oligoribonucleotides/*biosynthesis ; Phosphorothioate Oligonucleotides/pharmacology ; RNA Cleavage ; RNA Viruses/*genetics/metabolism ; RNA, Viral/*genetics/metabolism ; RNA-Binding Proteins/genetics/metabolism ; Sulfolobus solfataricus/drug effects/*genetics/immunology/metabolism ; Time Factors ; },
abstract = {The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3' end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The 'RNA shredding' activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence.},
}

Adenine Nucleotides/*biosynthesis
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA-Binding Proteins/genetics/metabolism
Endodeoxyribonucleases/*genetics/metabolism
Endoribonucleases/genetics/metabolism
Escherichia coli Proteins/genetics/metabolism
Kinetics
Oligoribonucleotides/*biosynthesis
Phosphorothioate Oligonucleotides/pharmacology
RNA Cleavage
RNA Viruses/*genetics/metabolism
RNA, Viral/*genetics/metabolism
RNA-Binding Proteins/genetics/metabolism
Sulfolobus solfataricus/drug effects/*genetics/immunology/metabolism
Time Factors

@article {pmid29174564,
year = {2018},
author = {Rauch, F and Geng, Y and Lamplugh, L and Hekmatnejad, B and Gaumond, MH and Penney, J and Yamanaka, Y and Moffatt, P},
title = {Crispr-Cas9 engineered osteogenesis imperfecta type V leads to severe skeletal deformities and perinatal lethality in mice.},
journal = {Bone},
volume = {107},
number = {},
pages = {131-142},
doi = {10.1016/j.bone.2017.11.013},
pmid = {29174564},
issn = {1873-2763},
support = {MOP-137102//CIHR/Canada ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; *Disease Models, Animal ; Gene Editing/methods ; Gene Knock-In Techniques ; Inflammation/genetics/pathology ; Membrane Proteins/*genetics ; Mesenchymal Stem Cells/metabolism/pathology ; Mice ; Mutation ; Osteoblasts/metabolism/*pathology ; Osteogenesis/genetics ; Osteogenesis Imperfecta/*genetics/*pathology ; },
abstract = {Osteogenesis imperfecta (OI) type V is caused by an autosomal dominant mutation in the IFITM5 gene, also known as BRIL. The c.-14C>T mutation in the 5'UTR of BRIL creates a novel translational start site adding 5 residues (MALEP) in frame with the natural coding of BRIL. A neomorphic function has been proposed for the MALEP-BRIL but the mechanisms at play are still unknown. In order to further understand the effects of MALEP-BRIL in vivo, we generated a knockin (KI) mouse model having the exact genetic -14C>T replica of patients with OI type V. Live KI descendants were never obtained from 2 male mosaic founders. Skeletal staining with alizarin red/alcian blue and μCT imaging of KI embryos revealed striking skeletal anomalies such as hypomineralized skull, short and bent long bones, and frail and wavy ribs. Histology and histochemical labeling revealed that midshaft of long bones was filled with hypertrophic chondrocytes, lacked a defined primary ossification center with the absence of defined cortices. Gene expression monitoring at E15.5 and E17.5 showed no change in Osx but decreased Bril itself as well as other differentiated osteoblast markers (Ibsp, Bglap, Sost). However, upregulation of Ptgs2 and Nr4a3 suggested that a pro-inflammatory reaction was activated. Primary osteoblasts from KI calvaria showed delayed differentiation and mineralization, with decreased abundance of BRIL. However, the upregulation AdipoQ and Fabp4 in young cultures indicated a possible switch in fate towards adipogenesis. Altogether our data suggest that the low level expression of MALEP-BRIL in Osx+ mesenchymal progenitors blunted their further differentiation into mature osteoblasts, which may have resulted in part from an inflammatory response.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems
Cell Differentiation/genetics
*Disease Models, Animal
Gene Editing/methods
Gene Knock-In Techniques
Inflammation/genetics/pathology
Membrane Proteins/*genetics
Mesenchymal Stem Cells/metabolism/pathology
Mice
Mutation
Osteoblasts/metabolism/*pathology
Osteogenesis/genetics
Osteogenesis Imperfecta/*genetics/*pathology

@article {pmid30878030,
year = {2018},
author = {Makarova, SS and Khromov, AV and Spechenkova, NA and Taliansky, ME and Kalinina, NO},
title = {Application of the CRISPR/Cas System for Generation of Pathogen-Resistant Plants.},
journal = {Biochemistry. Biokhimiia},
volume = {83},
number = {12},
pages = {1552-1562},
doi = {10.1134/S0006297918120131},
pmid = {30878030},
issn = {1608-3040},
abstract = {The use of the CRISPR/Cas9 prokaryotic adaptive immune system has led to a breakthrough in targeted genome editing in eukaryotes. The CRISPR/Cas technology allows to generate organisms with desirable characteristics by introducing deletions/insertions into selected genome loci resulting in the knockout or modification of target genes. This review focuses on the current state of the CRISPR/Cas use for the generation of plants resistant to viruses, bacteria, and parasitic fungi. Resistance to DNA- and RNA-containing viruses is usually provided by expression in transgenic plants of the Cas endonuclease gene and short guide RNAs (sgRNAs) targeting certain sites in the viral or the host plant genomes to ensure either direct cleavage of the viral genome or modification of the plant host genome in order to decrease the efficiency of virus replication. Editing of plant genes involved in the defense response to pathogens increases plants resistance to bacteria and pathogenic fungi. The review explores strategies and prospects of the development of pathogen-resistant plants with a focus on the generation of non-transgenic (non-genetically modified) organisms, in particular, by using plasmid (DNA)-free systems for delivery of the Cas/sgRNA editing complex into plant cells.},
}

@article {pmid30877356,
year = {2019},
author = {Zhang, YT and Jiang, JY and Shi, TQ and Sun, XM and Zhao, QY and Huang, H and Ren, LJ},
title = {Application of the CRISPR/Cas system for genome editing in microalgae.},
journal = {Applied microbiology and biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00253-019-09726-x},
pmid = {30877356},
issn = {1432-0614},
support = {21878151//National Natural Science Foundation of China/ ; BK20160092//Outstanding Youth Foundation of Jiangsu Province of China/ ; 2015//Program for Innovative Research Teams in Universities of Jiangsu Province/ ; XTE1829//Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture/ ; 18KJB530007//General Program on Natural Science Research Projects of Higher Education of Jiangsu/ ; },
abstract = {Microalgae are arguably the most abundant single-celled eukaryotes and are widely distributed in oceans and freshwater lakes. Moreover, microalgae are widely used in biotechnology to produce bioenergy and high-value products such as polyunsaturated fatty acids (PUFAs), bioactive peptides, proteins, antioxidants and so on. In general, genetic editing techniques were adapted to increase the production of microalgal metabolites. The main genome editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease system. Due to its high genome editing efficiency, the CRISPR/Cas system is emerging as the most important genome editing method. In this review, we summarized the available literature on the application of CRISPR/Cas in microalgal genetic engineering, including transformation methods, strategies for the expression of Cas9 and sgRNA, the CRISPR/Cas9-mediated gene knock-in/knock-out strategies, and CRISPR interference expression modification strategies.},
}

@article {pmid30875129,
year = {2019},
author = {Musharova, O and Sitnik, V and Vlot, M and Savitskaya, E and Datsenko, KA and Krivoy, A and Fedorov, I and Semenova, E and Brouns, SJJ and Severinov, K},
title = {Systematic analysis of Type I-E Escherichia coli CRISPR-Cas PAM sequences ability to promote interference and primed adaptation.},
journal = {Molecular microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/mmi.14237},
pmid = {30875129},
issn = {1365-2958},
abstract = {CRISPR interference occurs when a protospacer recognized by the CRISPR RNA is destroyed by Cas effectors. In Type I CRISPR-Cas systems, protospacer recognition can lead to «primed adaptation» - acquisition of new spacers from in cis located sequences. Type I CRISPR-Cas systems require the presence of a trinucleotide protospacer adjacent motif (PAM) for efficient interference. Here, we investigated the ability of each of 64 possible trinucleotides located at the PAM position to induce CRISPR interference and primed adaptation by the Escherichia coli Type I-E CRISPR-Cas system. We observed clear separation of PAM variants into three groups: those unable to cause interference, those that support rapid interference, and those that lead to reduced interference that occurs over extended periods of time. PAM variants unable to support interference also did not support primed adaptation; those that supported rapid interference led to no or low levels of adaptation, while those that caused attenuated levels of interference consistently led to highest levels of adaptation. The results suggest that primed adaptation is fueled by the products of CRISPR interference. Extended over time interference with targets containing «attenuated» PAM variants provides a continuous source of new spacers leading to high overall level of spacer acquisition. This article is protected by copyright. All rights reserved.},
}

@article {pmid30874768,
year = {2019},
author = {Atmadjaja, AN and Holby, V and Harding, AJ and Krabben, P and Smith, HK and Jenkinson, ER},
title = {CRISPR-Cas, a highly effective tool for genome editing in Clostridium saccharoperbutylacetonicum N1-4(HMT).},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnz059},
pmid = {30874768},
issn = {1574-6968},
abstract = {The solventogenic clostridia have long been known for their ability to convert sugars from complex feedstocks into commercially important solvents. Although the acetone-butanol-ethanol (ABE) process fell out of favour decades ago, renewed interest in sustainability and 'green' chemistry has re-established our appetite for reviving technologies such as these, albeit with 21st century improvements. As CRISPR-Cas genome editing tools are being developed and applied to the solventogenic clostridia, their industrial potential is growing. Through integration of new pathways, the beneficial traits and historical track record of clostridial fermentation can be exploited to generate a much wider range of industrially relevant products. Here we show the application of genome editing using the endogenous CRISPR-Cas mechanism of Clostridium saccharoperbutylacetonicum N1-4(HMT), to generate a deletion, SNP and to integrate new DNA into the genome. These technological advancements pave the way for application of clostridial species to the production of an array of products.},
}

@article {pmid30871003,
year = {2019},
author = {Ashley, CL and Abendroth, A and McSharry, BP and Slobedman, B},
title = {Interferon-Independent Upregulation of Interferon-Stimulated Genes during Human Cytomegalovirus Infection is Dependent on IRF3 Expression.},
journal = {Viruses},
volume = {11},
number = {3},
pages = {},
doi = {10.3390/v11030246},
pmid = {30871003},
issn = {1999-4915},
support = {1063738//National Health and Medical Research Council/ ; },
abstract = {The antiviral activity of type I interferons (IFNs) is primarily mediated by interferon-stimulated genes (ISGs). Induction of ISG transcription is achieved when type I IFNs bind to their cognate receptor and activate the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathways. Recently it has become clear that a number of viruses are capable of directly upregulating a subset of ISGs in the absence of type I IFN production. Using cells engineered to block either the response to, or production of type I IFN, the regulation of IFN-independent ISGs was examined in the context of human cytomegalovirus (HCMV) infection. Several ISGs, including IFIT1, IFIT2, IFIT3, Mx1, Mx2, CXCL10 and ISG15 were found to be upregulated transcriptionally following HCMV infection independently of type I IFN-initiated JAK-STAT signaling, but dependent on intact IRF3 signaling. ISG15 protein regulation mirrored that of its transcript with IFNβ neutralization failing to completely inhibit ISG15 expression post HCMV infection. In addition, no detectable ISG15 protein expression was observed following HCMV infection in IRF3 knockdown CRISPR/Cas-9 clones indicating that IFN-independent control of ISG expression during HCMV infection of human fibroblasts is absolutely dependent on IRF3 expression.},
}

@article {pmid30260998,
year = {2018},
author = {Veach, RA and Wilson, MH},
title = {CRISPR/Cas9 engineering of a KIM-1 reporter human proximal tubule cell line.},
journal = {PloS one},
volume = {13},
number = {9},
pages = {e0204487},
pmid = {30260998},
issn = {1932-6203},
support = {I01 BX002190/BX/BLRD VA/United States ; I01 BX004258/BX/BLRD VA/United States ; R01 DK093660/DK/NIDDK NIH HHS/United States ; },
mesh = {Acute Kidney Injury/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line ; Cisplatin/pharmacology ; Gene Knock-In Techniques ; Gene Targeting ; Genes, Reporter ; Genetic Engineering ; Glucose/pharmacology ; Green Fluorescent Proteins/genetics ; Hepatitis A Virus Cellular Receptor 1/*genetics/*metabolism ; Homologous Recombination ; Humans ; Kidney Tubules, Proximal/cytology/drug effects/*metabolism ; Luciferases/genetics ; Up-Regulation/drug effects ; },
abstract = {We used the CRISPR/Cas9 system to knock-in reporter transgenes at the kidney injury molecule-1 (KIM-1) locus and isolated human proximal tubule cell (HK-2) clones. PCR verified targeted knock-in of the luciferase and eGFP reporter at the KIM-1 locus. HK-2-KIM-1 reporter cells responded to various stimuli including hypoxia, cisplatin, and high glucose, indicative of upregulation of KIM-1 expression. We attempted using CRISPR/Cas9 to also engineer the KIM-1 reporter in telomerase-immortalized human RPTEC cells. However, these cells demonstrated an inability to undergo homologous recombination at the target locus. KIM-1-reporter human proximal tubular cells could be valuable tools in drug discovery for molecules inhibiting kidney injury. Additionally, our gene targeting strategy could be used in other cell lines to evaluate the biology of KIM-1 in vitro or in vivo.},
}

Acute Kidney Injury/genetics/metabolism
CRISPR-Cas Systems
Cell Line
Cisplatin/pharmacology
Gene Knock-In Techniques
Gene Targeting
Genes, Reporter
Genetic Engineering
Glucose/pharmacology
Green Fluorescent Proteins/genetics
Hepatitis A Virus Cellular Receptor 1/*genetics/*metabolism
Homologous Recombination
Humans
Kidney Tubules, Proximal/cytology/drug effects/*metabolism
Luciferases/genetics
Up-Regulation/drug effects

@article {pmid30043304,
year = {2018},
author = {Curtin, SJ},
title = {Editing the Medicago truncatula Genome: Targeted Mutagenesis Using the CRISPR-Cas9 Reagent.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1822},
number = {},
pages = {161-174},
doi = {10.1007/978-1-4939-8633-0_12},
pmid = {30043304},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Chromosome Deletion ; Chromosomes, Plant ; *Gene Editing ; Gene Knockout Techniques ; Genes, Plant ; Genetic Engineering ; *Genome, Plant ; *Genomics/methods ; Mutagenesis ; },
abstract = {Medicago truncatula is an annual plant used for studying legume biology, in particular symbioses with nitrogen-fixing rhizobia and arbuscular mycorrhizal fungi. Efforts to decipher the genetic basis of these ecologically and economically important traits are a major goal of plant and crop biology. M. truncatula is an excellent model system for this purpose, as it has several publicly available sequenced genomes, has a rapid seed-to-seed generation time, and is highly transformable. Various mutagenesis platforms such as Tnt1 retrotransposons and RNAi knockdown have been used successfully in forward and reverse genetic studies to identify and functionally characterize candidate genes. The CRISPR/Cas9 reagent is the most recent mutagenesis platform and is highly effective at generating site-directed double-stranded breaks (DSB) in M. truncatula. This protocol will demonstrate the construction of reagents using two genome engineering platforms that have successfully generated mutant plants in M. truncatula, M. sativa, and soybean systems. The reagents are easy to assemble, can be quickly retrofitted to test novel regulatory sequences for improved efficiency, and can be used for more advanced genome engineering strategies such as gene insertion or gene replacement.},
}

*CRISPR-Cas Systems
Chromosome Deletion
Chromosomes, Plant
*Gene Editing
Gene Knockout Techniques
Genes, Plant
Genetic Engineering
*Genome, Plant
*Genomics/methods
Mutagenesis

@article {pmid29475732,
year = {2018},
author = {Fehse, B and Abramowski-Mock, U},
title = {The Time Is Ripe for Somatic Genome Editing: NIH Program to Strengthen Translation.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {26},
number = {3},
pages = {671-674},
pmid = {29475732},
issn = {1525-0024},
mesh = {Animals ; CRISPR-Cas Systems ; Capital Financing ; *Gene Editing/methods ; *Genome ; *Genomics/methods ; Humans ; National Institutes of Health (U.S.) ; Transcription Activator-Like Effector Nucleases ; Translational Medical Research/methods/organization & administration ; United States ; },
}

Animals
CRISPR-Cas Systems
Capital Financing
*Gene Editing/methods
*Genome
*Genomics/methods
Humans
National Institutes of Health (U.S.)
Transcription Activator-Like Effector Nucleases
Translational Medical Research/methods/organization & administration
United States

@article {pmid29324392,
year = {2018},
author = {Banaszak, LG and Giudice, V and Zhao, X and Wu, Z and Gao, S and Hosokawa, K and Keyvanfar, K and Townsley, DM and Gutierrez-Rodrigues, F and Fernandez Ibanez, MDP and Kajigaya, S and Young, NS},
title = {Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing.},
journal = {Blood cells, molecules & diseases},
volume = {69},
number = {},
pages = {10-22},
doi = {10.1016/j.bcmd.2017.12.002},
pmid = {29324392},
issn = {1096-0961},
support = {/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Apoptosis/genetics ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cell Line, Tumor ; Cell Proliferation ; DNA (Cytosine-5-)-Methyltransferases/*genetics/metabolism ; DNA Damage ; *Gene Editing ; *Genomic Instability ; Humans ; K562 Cells ; Mutation ; *RNA Splicing ; Sequence Analysis, DNA ; Spliceosomes/metabolism ; },
abstract = {DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Here, we successfully created four DNMT3A-mutated K562 cell lines with frameshift mutations resulting in truncated DNMT3A proteins. DNMT3A-mutated cell lines exhibited significantly impaired growth and increased apoptotic activity compared to wild-type (WT) cells. Consistent with previous studies, DNMT3A-mutated cells displayed impaired differentiation capacity. RNA-seq was used to compare transcriptomes of DNMT3A-mutated and WT cells; DNMT3A ablation resulted in downregulation of genes involved in spliceosome function, causing dysfunction of RNA splicing. Unexpectedly, we observed DNMT3A-mutated cells to exhibit marked genomic instability and an impaired DNA damage response compared to WT. CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy.},
}

Apoptosis/genetics
*CRISPR-Cas Systems
Cell Differentiation/genetics
Cell Line, Tumor
Cell Proliferation
DNA (Cytosine-5-)-Methyltransferases/*genetics/metabolism
DNA Damage
*Gene Editing
*Genomic Instability
Humans
K562 Cells
Mutation
*RNA Splicing
Sequence Analysis, DNA
Spliceosomes/metabolism

@article {pmid28811591,
year = {2017},
author = {Zhang, X and Li, W and Liu, C and Peng, X and Lin, J and He, S and Li, X and Han, B and Zhang, N and Wu, Y and Chen, L and Wang, L and MaYila, and Huang, J and Liu, M},
title = {Alteration of sheep coat color pattern by disruption of ASIP gene via CRISPR Cas9.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {8149},
pmid = {28811591},
issn = {2045-2322},
mesh = {Agouti Signaling Protein/*genetics ; Amino Acid Sequence ; Animals ; Base Sequence ; *CRISPR-Cas Systems ; Caspase 9/metabolism ; Gene Editing ; Gene Targeting ; Genetic Loci ; Genotype ; Hair Color/*genetics ; Phenotype ; Polymorphism, Single Nucleotide ; RNA, Guide ; RNA, Messenger/genetics ; Sequence Analysis, DNA ; Sheep/*genetics ; Zygote/metabolism ; },
abstract = {Coat color is an important characteristic and economic trait in domestic sheep. Aiming at alteration of Chinese merino sheep coat color by genome manipulation, we disrupted sheep agouti signaling protein gene by CRISPR/Cas9. A total of seven indels were identified in 5 of 6 born lambs. Each targeted lamb happened at least two kinds of modifications, and targeted lambs with multiple modifications displayed variety of coat color patterns. Three lambs with 4 bp deletion showed badgerface with black body coat color in two lambs, and brown coat color with light ventral pigmentation in another one. The black-white spotted color was observed in two lambs with 2 bp deletion. Further analysis unraveled that modifications happened in one or more than two copies of ASIP gene, and moreover, the additional spontaneous mutations of D9 and/or D5 preceding the targeting modification could also involve the formation of coat color patterns. Taken together, the entanglement of ASIP modifications by CRISPR/Cas9, spontaneous D9/D5 mutations, and ASIP gene duplications contributed to the variety of coat color patterns in targeted lambs.},
}

Agouti Signaling Protein/*genetics
Amino Acid Sequence
Animals
Base Sequence
*CRISPR-Cas Systems
Caspase 9/metabolism
Gene Editing
Gene Targeting
Genetic Loci
Genotype
Hair Color/*genetics
Phenotype
Polymorphism, Single Nucleotide
RNA, Guide
RNA, Messenger/genetics
Sequence Analysis, DNA
Sheep/*genetics
Zygote/metabolism

@article {pmid30870431,
year = {2019},
author = {Thomas, M and Burgio, G and Adams, DJ and Iyer, V},
title = {Collateral damage and CRISPR genome editing.},
journal = {PLoS genetics},
volume = {15},
number = {3},
pages = {e1007994},
doi = {10.1371/journal.pgen.1007994},
pmid = {30870431},
issn = {1553-7404},
abstract = {The simplicity and the versatility of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR-Cas) systems have enabled the genetic modification of virtually every organism and offer immense therapeutic potential for the treatment of human disease. Although these systems may function efficiently within eukaryotic cells, there remain concerns about the accuracy of Cas endonuclease effectors and their use for precise gene editing. Recently, two independent reports investigating the editing accuracy of the CRISPR-Cas9 system were published by separate groups at the Wellcome Sanger Institute; our study-Iyer and colleagues [1]-defined the landscape of off-target mutations, whereas the other by Kosicki and colleagues [2] detailed the existence of on-target, potentially deleterious deletions. Although both studies found evidence of large on-target CRISPR-induced deletions, they reached seemingly very different conclusions.},
}

@article {pmid30867524,
year = {2019},
author = {Odamaki, T and Bottacini, F and Mitsuyama, E and Yoshida, K and Kato, K and Xiao, JZ and van Sinderen, D},
title = {Impact of a bathing tradition on shared gut microbe among Japanese families.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {4380},
doi = {10.1038/s41598-019-40938-3},
pmid = {30867524},
issn = {2045-2322},
support = {FEMS-RG-2016-0103//Federation of European Microbiological Societies (FEMS)/ ; 17/IFB/5429//Science Foundation Ireland (SFI)/ ; SFI/12/RC/2273//Science Foundation Ireland (SFI)/ ; },
abstract = {Sharing of Bifidobacterium longum strains had recently been shown to occur among Japanese family members, a phenomenon that is not confined to mother-infant pairs. In the current study, we investigated if bathtub water is a possible vehicle for the exchange of strains as a consequence of a Japanese custom to share bathtub water by family members during bathing practices. A total of twenty-one subjects from five Japanese families, each consisting of parents with either 2 or 3 children, were enrolled in this study and the fecal microbiota of all participants was determined. Viable bifidobacterial strains were isolated from all bathtub water samples. A subsequent comparative genome analysis using ninety-eight strains indicated that certain strain-sets, which were isolated from feces and bathtub water, share near identical genome sequences, including CRISPR/Cas protospacers. By means of unweighted UniFrac distance analysis based on 16S rRNA gene analysis of 59 subjects from sixteen Japanese families, we showed that the fecal microbiota composition among family members that share bathtub water is significantly closer than that between family members that do not engage in this practice. Our results indicate that bathtub water represents a vehicle for the transmission of gut bacteria, and that the Japanese custom of sharing bathtub water contributes to the exchange of gut microbes, in particular bifidobacteria, among family members.},
}

@article {pmid30867223,
year = {2019},
author = {Thormann, V and Glaser, LV and Rothkegel, MC and Borschiwer, M and Bothe, M and Fuchs, A and Meijsing, SH},
title = {Expanding the repertoire of glucocorticoid receptor target genes by engineering genomic response elements.},
journal = {Life science alliance},
volume = {2},
number = {2},
pages = {},
doi = {10.26508/lsa.201800283},
pmid = {30867223},
issn = {2575-1077},
abstract = {The glucocorticoid receptor (GR), a hormone-activated transcription factor, binds to a myriad of genomic binding sites yet seems to regulate a much smaller number of genes. Genome-wide analysis of GR binding and gene regulation has shown that the likelihood of GR-dependent regulation increases with decreased distance of its binding to the transcriptional start site of a gene. To test if we can adopt this knowledge to expand the repertoire of GR target genes, we used CRISPR/Cas-mediated homology-directed repair to add a single GR-binding site directly upstream of the transcriptional start site of each of four genes. To our surprise, we found that the addition of a single GR-binding site can be enough to convert a gene into a GR target. The gain of GR-dependent regulation was observed for two of four genes analyzed and coincided with acquired GR binding at the introduced binding site. However, the gene-specific gain of GR-dependent regulation could not be explained by obvious differences in chromatin accessibility between converted genes and their non-converted counterparts. Furthermore, by introducing GR-binding sequences with different nucleotide compositions, we show that activation can be facilitated by distinct sequences without obvious differences in activity between the GR-binding sequence variants we tested. The approach to use genome engineering to build genomic response elements facilitates the generation of cell lines with tailored repertoires of GR-responsive genes and a framework to test and refine our understanding of the cis-regulatory logic of gene regulation by testing if engineered response elements behave as predicted.},
}

@article {pmid30520725,
year = {2018},
author = {OhAinle, M and Helms, L and Vermeire, J and Roesch, F and Humes, D and Basom, R and Delrow, JJ and Overbaugh, J and Emerman, M},
title = {A virus-packageable CRISPR screen identifies host factors mediating interferon inhibition of HIV.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
doi = {10.7554/eLife.39823},
pmid = {30520725},
issn = {2050-084X},
support = {P30 CA015704/CA/NCI NIH HHS/United States ; CFAR New Investigator Award, P30 AI027757/AI/NIAID NIH HHS/United States ; DP1 DA039543/DA/NIDA NIH HHS/United States ; P30 CA015704-43/CA/NCI NIH HHS/United States ; P30 DK056465/DK/NIDDK NIH HHS/United States ; R01 AI030927/AI/NIAID NIH HHS/United States ; CCEH Pilot Grant, DK56465/DK/NIDDK NIH HHS/United States ; P30 AI027757/AI/NIAID NIH HHS/United States ; R01 AI30927/AI/NIAID NIH HHS/United States ; R37 AI030927/AI/NIAID NIH HHS/United States ; },
mesh = {Antigens, CD/genetics/immunology ; Antigens, Differentiation/genetics/immunology ; CRISPR-Cas Systems ; Carrier Proteins/genetics/immunology ; Cell Line, Tumor ; Epithelial Cells/drug effects/*immunology/virology ; GPI-Linked Proteins/genetics/immunology ; Gene Editing/*methods ; Gene Expression Regulation ; Genetic Vectors/chemistry/immunology ; HEK293 Cells ; HIV-1/drug effects/*genetics/growth & development/immunology ; *Host-Pathogen Interactions ; Humans ; Interferon-alpha/pharmacology ; Lentivirus/genetics/metabolism ; Myxovirus Resistance Proteins/genetics/immunology ; Nuclear Proteins/deficiency/*genetics/immunology ; Phosphotransferases (Alcohol Group Acceptor)/deficiency/*genetics/immunology ; Receptors, CCR5/genetics/immunology ; Receptors, CXCR4/genetics/immunology ; Signal Transduction ; THP-1 Cells ; Viral Tropism/genetics ; Virus Assembly/drug effects ; Virus Replication/drug effects ; },
abstract = {Interferon (IFN) inhibits HIV replication by inducing antiviral effectors. To comprehensively identify IFN-induced HIV restriction factors, we assembled a CRISPR sgRNA library of Interferon Stimulated Genes (ISGs) into a modified lentiviral vector that allows for packaging of sgRNA-encoding genomes in trans into budding HIV-1 particles. We observed that knockout of Zinc Antiviral Protein (ZAP) improved the performance of the screen due to ZAP-mediated inhibition of the vector. A small panel of IFN-induced HIV restriction factors, including MxB, IFITM1, Tetherin/BST2 and TRIM5alpha together explain the inhibitory effects of IFN on the CXCR4-tropic HIV-1 strain, HIV-1LAI, in THP-1 cells. A second screen with a CCR5-tropic primary strain, HIV-1Q23.BG505, described an overlapping, but non-identical, panel of restriction factors. Further, this screen also identifies HIV dependency factors. The ability of IFN-induced restriction factors to inhibit HIV strains to replicate in human cells suggests that these human restriction factors are incompletely antagonized.

Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).},
}

Antigens, CD/genetics/immunology
Antigens, Differentiation/genetics/immunology
CRISPR-Cas Systems
Carrier Proteins/genetics/immunology
Cell Line, Tumor
Epithelial Cells/drug effects/*immunology/virology
GPI-Linked Proteins/genetics/immunology
Gene Editing/*methods
Gene Expression Regulation
Genetic Vectors/chemistry/immunology
HEK293 Cells
HIV-1/drug effects/*genetics/growth & development/immunology
*Host-Pathogen Interactions
Humans
Interferon-alpha/pharmacology
Lentivirus/genetics/metabolism
Myxovirus Resistance Proteins/genetics/immunology
Nuclear Proteins/deficiency/*genetics/immunology
Phosphotransferases (Alcohol Group Acceptor)/deficiency/*genetics/immunology
Receptors, CCR5/genetics/immunology
Receptors, CXCR4/genetics/immunology
Signal Transduction
THP-1 Cells
Viral Tropism/genetics
Virus Assembly/drug effects
Virus Replication/drug effects

@article {pmid30332437,
year = {2018},
author = {Morozova, KN and Suldina, LA and Malankhanova, TB and Grigor'eva, EV and Zakian, SM and Kiseleva, E and Malakhova, AA},
title = {Introducing an expanded CAG tract into the huntingtin gene causes a wide spectrum of ultrastructural defects in cultured human cells.},
journal = {PloS one},
volume = {13},
number = {10},
pages = {e0204735},
pmid = {30332437},
issn = {1932-6203},
mesh = {CRISPR-Cas Systems ; Clone Cells/metabolism/ultrastructure ; Endoplasmic Reticulum/ultrastructure ; Gene Knockout Techniques ; HEK293 Cells ; Humans ; Huntingtin Protein/antagonists & inhibitors/*genetics ; Huntington Disease/*genetics/*pathology ; Lysosomes/ultrastructure ; Microscopy, Electron, Transmission ; Mitochondria/ultrastructure ; Mutant Proteins/*genetics ; Mutation ; *Trinucleotide Repeat Expansion ; },
abstract = {Modeling of neurodegenerative diseases in vitro holds great promise for biomedical research. Human cell lines harboring a mutations in disease-causing genes are thought to recapitulate early stages of the development an inherited disease. Modern genome-editing tools allow researchers to create isogenic cell clones with an identical genetic background providing an adequate "healthy" control for biomedical and pharmacological experiments. Here, we generated isogenic mutant cell clones with 150 CAG repeats in the first exon of the huntingtin (HTT) gene using the CRISPR/Cas9 system and performed ultrastructural and morphometric analyses of the internal organization of the mutant cells. Electron microscopy showed that deletion of three CAG triplets or an HTT gene knockout had no significant influence on the cell structure. The insertion of 150 CAG repeats led to substantial changes in quantitative and morphological parameters of mitochondria and increased the association of mitochondria with the smooth and rough endoplasmic reticulum while causing accumulation of small autolysosomes in the cytoplasm. Our data indicate for the first time that expansion of the CAG repeat tract in HTT introduced via the CRISPR/Cas9 technology into a human cell line initiates numerous ultrastructural defects that are typical for Huntington's disease.},
}

CRISPR-Cas Systems
Clone Cells/metabolism/ultrastructure
Endoplasmic Reticulum/ultrastructure
Gene Knockout Techniques
HEK293 Cells
Humans
Huntingtin Protein/antagonists & inhibitors/*genetics
Huntington Disease/*genetics/*pathology
Lysosomes/ultrastructure
Microscopy, Electron, Transmission
Mitochondria/ultrastructure
Mutant Proteins/*genetics
Mutation
*Trinucleotide Repeat Expansion

@article {pmid30864562,
year = {2019},
author = {Veigl, SJ},
title = {A use/disuse paradigm for CRISPR-Cas systems.},
journal = {Biology & philosophy},
volume = {34},
number = {1},
pages = {13},
doi = {10.1007/s10539-018-9661-z},
pmid = {30864562},
issn = {0169-3867},
}

@article {pmid30353190,
year = {2018},
author = {Neff, EP},
title = {CRISPR-barcoding the mouse.},
journal = {Lab animal},
volume = {47},
number = {11},
pages = {309},
doi = {10.1038/s41684-018-0190-9},
pmid = {30353190},
issn = {1548-4475},
mesh = {Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Mice ; Mutation ; },
}

Animals
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Mice
Mutation

@article {pmid30353159,
year = {2018},
author = {Parthasarathy, S},
title = {Use the patent system to regulate gene editing.},
journal = {Nature},
volume = {562},
number = {7728},
pages = {486-488},
doi = {10.1038/d41586-018-07108-3},
pmid = {30353159},
issn = {1476-4687},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clinical Trials as Topic/ethics/legislation & jurisprudence ; Congresses as Topic ; European Union ; Gene Editing/*ethics/*legislation & jurisprudence/statistics & numerical data ; *Government Regulation ; History, 19th Century ; History, 20th Century ; History, 21st Century ; Human Embryonic Stem Cells ; Humans ; Licensure ; Patents as Topic/history/*legislation & jurisprudence/statistics & numerical data ; Swine/genetics ; United States ; },
}

Animals
CRISPR-Cas Systems/*genetics
Clinical Trials as Topic/ethics/legislation & jurisprudence
Congresses as Topic
European Union
Gene Editing/*ethics/*legislation & jurisprudence/statistics & numerical data
*Government Regulation
History, 19th Century
History, 20th Century
History, 21st Century
Human Embryonic Stem Cells
Humans
Licensure
Patents as Topic/history/*legislation & jurisprudence/statistics & numerical data
Swine/genetics
United States

@article {pmid30087231,
year = {2018},
author = {Ibba, G and Piu, C and Uleri, E and Serra, C and Dolei, A},
title = {Disruption by SaCas9 Endonuclease of HERV-Kenv, a Retroviral Gene with Oncogenic and Neuropathogenic Potential, Inhibits Molecules Involved in Cancer and Amyotrophic Lateral Sclerosis.},
journal = {Viruses},
volume = {10},
number = {8},
pages = {},
pmid = {30087231},
issn = {1999-4915},
mesh = {Amyotrophic Lateral Sclerosis/*pathology/therapy ; CRISPR-Cas Systems ; Carcinogenesis/genetics ; Cell Line, Tumor ; DNA-Binding Proteins/genetics ; Endogenous Retroviruses/*genetics/pathogenicity ; Gene Editing ; Gene Expression Regulation ; Humans ; Male ; NF-kappa B/genetics ; Neoplasms/*virology ; Oncogenes/*genetics ; Prostatic Neoplasms ; RNA, Viral ; Serine-Arginine Splicing Factors/genetics ; Staphylococcus aureus/enzymology ; Viral Envelope Proteins/*genetics ; },
abstract = {The human endogenous retrovirus (HERV)-K, human mouse mammary tumor virus like-2 (HML-2) subgroup of HERVs is activated in several tumors and has been related to prostate cancer progression and motor neuron diseases. The cellular splicing factor 2/alternative splicing factor (SF2/ASF) is a positive regulator of gene expression, coded by a potent proto-oncogene, amplified, and abnormally expressed in tumors. TAR DNA-binding protein-43 (TDP-43) is a DNA/RNA-binding protein, negative regulator of alternative splicing, known for causing neurodegeneration, and with complex roles in oncogenesis. We used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, with the Cas9 system from Staphylococcus aureus (SaCas9), to disrupt the HERV-K(HML-2)env gene, and evaluated the effects on cultured cells. The tool was tested on human prostate cancer LNCaP cells, whose HERV-Kenv transcription profile is known. It caused HERV-K(HML-2)env disruption (the first reported of a HERV gene), as evaluated by DNA sequencing, and inhibition of env transcripts and proteins. The HERV-K(HML-2)env disruption was found to interfere with important regulators of cell expression and proliferation, involved in manaling, RNA-binding, and alternative splicing, such as epidermal growth factor receptor (EGF-R), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), SF2/ASF, and TDP-43. These novel findings suggest that HERV-K is not an innocent bystander, they reinforce its links to oncogenesis and motor neuron diseases, and they open potential innovative therapeutic options.},
}

Amyotrophic Lateral Sclerosis/*pathology/therapy
CRISPR-Cas Systems
Carcinogenesis/genetics
Cell Line, Tumor
DNA-Binding Proteins/genetics
Endogenous Retroviruses/*genetics/pathogenicity
Gene Editing
Gene Expression Regulation
Humans
Male
NF-kappa B/genetics
Neoplasms/*virology
Oncogenes/*genetics
Prostatic Neoplasms
RNA, Viral
Serine-Arginine Splicing Factors/genetics
Staphylococcus aureus/enzymology
Viral Envelope Proteins/*genetics

@article {pmid29961307,
year = {2018},
author = {Kim, YK and Nam, SA and Yang, CW},
title = {Applications of kidney organoids derived from human pluripotent stem cells.},
journal = {The Korean journal of internal medicine},
volume = {33},
number = {4},
pages = {649-659},
pmid = {29961307},
issn = {2005-6648},
mesh = {CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Humans ; Kidney ; *Kidney Diseases/therapy ; *Organoids ; *Pluripotent Stem Cells ; },
abstract = {The establishment of protocols to differentiate kidney organoids from human pluripotent stem cells provides potential applications of kidney organoids in regenerative medicine. Modeling of renal diseases, drug screening, nephrotoxicity testing of compounds, and regenerative therapy are attractive applications. Although much progress still remains to be made in the development of kidney organoids, recent advances in clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated system 9 (Cas9) genome editing and three-dimensional bioprinting technologies have contributed to the application of kidney organoids in clinical fields. In this section, we review recent advances in the applications of kidney organoids to kidney disease modelling, drug screening, nephrotoxicity testing, and regenerative therapy.},
}

CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Editing
Humans
Kidney
*Kidney Diseases/therapy
*Organoids
*Pluripotent Stem Cells

@article {pmid29906440,
year = {2018},
author = {},
title = {CRISPR Inspirations.},
journal = {Cell},
volume = {173},
number = {7},
pages = {1560-1561},
doi = {10.1016/j.cell.2018.05.055},
pmid = {29906440},
issn = {1097-4172},
mesh = {Bacteriophages/genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Escherichia coli/genetics/metabolism ; Gene Editing ; Haloferax volcanii/genetics/metabolism ; Immunity/genetics ; Streptococcus thermophilus/genetics ; },
}

Bacteriophages/genetics/metabolism
CRISPR-Cas Systems/*genetics
Escherichia coli/genetics/metabolism
Gene Editing
Haloferax volcanii/genetics/metabolism
Immunity/genetics
Streptococcus thermophilus/genetics

@article {pmid29906439,
year = {2018},
author = {},
title = {The Ongoing Shakeup in Organelle Biology.},
journal = {Cell},
volume = {173},
number = {7},
pages = {1557-1559},
doi = {10.1016/j.cell.2018.05.054},
pmid = {29906439},
issn = {1097-4172},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Organelles/*metabolism ; Protein Interaction Maps ; Proteins/genetics/metabolism ; },
abstract = {With the complexities of organelle communication and their dynamics under intense investigation, what are the new principles that are emerging, and where is the field headed? Cell's Robert Kruger recently discussed these questions with Erika Holzbaur, Jennifer Lippincott-Schwartz, and Ivan Dikic. Annotated excerpts from this conversation are presented below, and the full conversation is available with the article online.},
}

Animals
CRISPR-Cas Systems/genetics
Organelles/*metabolism
Protein Interaction Maps
Proteins/genetics/metabolism

@article {pmid29812974,
year = {2018},
author = {Luo, JJ and Bian, WP and Liu, Y and Huang, HY and Yin, Q and Yang, XJ and Pei, DS},
title = {CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {32},
number = {9},
pages = {5132-5142},
doi = {10.1096/fj.201800077RR},
pmid = {29812974},
issn = {1530-6860},
mesh = {Animals ; Animals, Genetically Modified/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Gene Transfer Techniques ; Genetic Engineering/methods ; Genetic Therapy/methods ; Genome/*genetics ; Promoter Regions, Genetic/genetics ; Zebrafish/*genetics ; },
abstract = {Numerous feasible methods for inserting large fragments of exogenous DNA sequences into the zebrafish genome have been developed, as has genome editing technology using programmable nucleases. However, the coding sequences of targeted endogenous genes are disrupted, and the expression patterns of inserted exogenous genes cannot completely recapitulate those of endogenous genes. Here we describe the establishment of a novel strategy for endogenous promoter-driven and microhomology-mediated end-joining-dependent integration of a donor vector using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9. We successfully integrated mCherry into the final coding sequence of targeted genes to generate seamless transgenic zebrafish lines with high efficiency. This novel seamless transgenesis technique not only maintained the integrity of the endogenous gene but also did not disrupt the function of targeted gene. Therefore, our microhomology-mediated end-joining-mediated transgenesis strategy may have broader applications in gene therapy. Moreover, this novel seamless gene-editing strategy in zebrafish provides a valuable new transgenesis technique, which was driven by endogenous promoters and in vivo animal reporter modes for translational medicine. It is expected to be a standard gene-editing technique in the field of zebrafish, leading to some important breakthroughs for studies in early embryogenesis.-Luo, J.-J., Bian, W.-P., Liu, Y., Huang, H.-Y., Yin, Q., Yang, X.-J., Pei, D.-S. CRISPR/Cas9-based genome engineering of zebrafish using a seamless integration strategy.},
}

Animals
Animals, Genetically Modified/genetics
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/methods
Gene Transfer Techniques
Genetic Engineering/methods
Genetic Therapy/methods
Genome/*genetics
Promoter Regions, Genetic/genetics
Zebrafish/*genetics

@article {pmid29794019,
year = {2018},
author = {Atanasov, KE and Liu, C and Erban, A and Kopka, J and Parker, JE and Alcázar, R},
title = {NLR Mutations Suppressing Immune Hybrid Incompatibility and Their Effects on Disease Resistance.},
journal = {Plant physiology},
volume = {177},
number = {3},
pages = {1152-1169},
pmid = {29794019},
issn = {1532-2548},
mesh = {3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/genetics ; Arabidopsis/genetics/*immunology/*microbiology ; Arabidopsis Proteins/*genetics/immunology ; CRISPR-Cas Systems ; Chimera ; Disease Resistance/*genetics/immunology ; Epistasis, Genetic ; Gene Expression Regulation, Plant ; *Mutation ; NLR Proteins/genetics ; Oomycetes/pathogenicity ; Plant Diseases/*genetics ; Plants, Genetically Modified ; Poland ; Proto-Oncogene Proteins c-myb/genetics ; Quantitative Trait Loci ; Self-Incompatibility in Flowering Plants/genetics/immunology ; Tobacco ; },
abstract = {Genetic divergence between populations can lead to reproductive isolation. Hybrid incompatibilities (HI) represent intermediate points along a continuum toward speciation. In plants, genetic variation in disease resistance (R) genes underlies several cases of HI. The progeny of a cross between Arabidopsis (Arabidopsis thaliana) accessions Landsberg erecta (Ler, Poland) and Kashmir2 (Kas2, central Asia) exhibits immune-related HI. This incompatibility is due to a genetic interaction between a cluster of eight TNL (TOLL/INTERLEUKIN1 RECEPTOR-NUCLEOTIDE BINDING-LEU RICH REPEAT) RPP1 (RECOGNITION OF PERONOSPORA PARASITICA1)-like genes (R1-R8) from Ler and central Asian alleles of a Strubbelig-family receptor-like kinase (SRF3) from Kas2. In characterizing mutants altered in Ler/Kas2 HI, we mapped multiple mutations to the RPP1-like Ler locus. Analysis of these suppressor of Ler/Kas2 incompatibility (sulki) mutants reveals complex, additive and epistatic interactions underlying RPP1-like Ler locus activity. The effects of these mutations were measured on basal defense, global gene expression, primary metabolism, and disease resistance to a local Hyaloperonospora arabidopsidis isolate (Hpa Gw) collected from Gorzów (Gw), where the Landsberg accession originated. Gene expression sectors and metabolic hallmarks identified for HI are both dependent and independent of RPP1-like Ler members. We establish that mutations suppressing immune-related Ler/Kas2 HI do not compromise resistance to Hpa Gw. QTL mapping analysis of Hpa Gw resistance point to RPP7 as the causal locus. This work provides insight into the complex genetic architecture of the RPP1-like Ler locus and immune-related HI in Arabidopsis and into the contributions of RPP1-like genes to HI and defense.},
}

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase/genetics
Arabidopsis/genetics/*immunology/*microbiology
Arabidopsis Proteins/*genetics/immunology
CRISPR-Cas Systems
Chimera
Disease Resistance/*genetics/immunology
Epistasis, Genetic
Gene Expression Regulation, Plant
*Mutation
NLR Proteins/genetics
Oomycetes/pathogenicity
Plant Diseases/*genetics
Plants, Genetically Modified
Poland
Proto-Oncogene Proteins c-myb/genetics
Quantitative Trait Loci
Self-Incompatibility in Flowering Plants/genetics/immunology
Tobacco

@article {pmid29779948,
year = {2018},
author = {Koren, I and Timms, RT and Kula, T and Xu, Q and Li, MZ and Elledge, SJ},
title = {The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons.},
journal = {Cell},
volume = {173},
number = {7},
pages = {1622-1635.e14},
pmid = {29779948},
issn = {1097-4172},
support = {//Wellcome Trust/United Kingdom ; /HHMI/Howard Hughes Medical Institute/United States ; R01 AG011085/AG/NIA NIH HHS/United States ; 201387/Z/16/Z//Wellcome Trust/United Kingdom ; },
mesh = {Amino Acid Motifs ; Animals ; Antigens, Neoplasm/metabolism ; CRISPR-Cas Systems/genetics ; Computational Biology/methods ; Genetic Vectors/genetics/metabolism ; HEK293 Cells ; Humans ; Lentivirus/genetics ; Leupeptins/pharmacology ; Open Reading Frames/genetics ; Peptides/metabolism ; Proteasome Endopeptidase Complex/chemistry/metabolism ; Protein Stability/drug effects ; Protein Subunits/metabolism ; Proteolysis ; Proteome/genetics/*metabolism ; Receptors, Cytokine/genetics/metabolism ; Ubiquitin-Protein Ligases/*metabolism ; },
abstract = {Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.},
}

Amino Acid Motifs
Animals
Antigens, Neoplasm/metabolism
CRISPR-Cas Systems/genetics
Computational Biology/methods
Genetic Vectors/genetics/metabolism
HEK293 Cells
Humans
Lentivirus/genetics
Leupeptins/pharmacology
Open Reading Frames/genetics
Peptides/metabolism
Proteasome Endopeptidase Complex/chemistry/metabolism
Protein Stability/drug effects
Protein Subunits/metabolism
Proteolysis
Proteome/genetics/*metabolism
Receptors, Cytokine/genetics/metabolism
Ubiquitin-Protein Ligases/*metabolism

@article {pmid29512112,
year = {2018},
author = {Howe, DK and Yeargan, M and Simpson, L and Dangoudoubiyam, S},
title = {Molecular Genetic Manipulation of Sarcocystis neurona.},
journal = {Current protocols in microbiology},
volume = {48},
number = {},
pages = {20D.2.1-20D.2.14},
doi = {10.1002/cpmc.48},
pmid = {29512112},
issn = {1934-8533},
mesh = {Animals ; CRISPR-Cas Systems ; Encephalomyelitis/parasitology/*veterinary ; *Genetic Techniques ; Horse Diseases/parasitology ; Horses ; Sarcocystis/*genetics/physiology ; Transfection/*methods ; },
abstract = {Sarcocystis neurona is a member of the important phylum Apicomplexa and the primary cause of equine protozoal myeloencephalitis (EPM). Moreover, S. neurona is the best-studied species in the genus Sarcocystis, one of the most successful parasite taxa, as virtually all vertebrate animals may be infected by at least one species. Consequently, scientific investigation of S. neurona will aid in the control of EPM and neurologic disease in sea mammals, while also improving our understanding of a prominent branch on the apicomplexan phylogenetic tree. These protocols describe methods that expand the capabilities to study this prominent member of the Apicomplexa. © 2018 by John Wiley & Sons, Inc.},
}

Animals
CRISPR-Cas Systems
Encephalomyelitis/parasitology/*veterinary
*Genetic Techniques
Horse Diseases/parasitology
Horses
Sarcocystis/*genetics/physiology
Transfection/*methods

@article {pmid30176395,
year = {2018},
author = {Wu, Y and Chen, T and Liu, Y and Lv, X and Li, J and Du, G and Ledesma-Amaro, R and Liu, L},
title = {CRISPRi allows optimal temporal control of N-acetylglucosamine bioproduction by a dynamic coordination of glucose and xylose metabolism in Bacillus subtilis.},
journal = {Metabolic engineering},
volume = {49},
number = {},
pages = {232-241},
doi = {10.1016/j.ymben.2018.08.012},
pmid = {30176395},
issn = {1096-7184},
mesh = {Acetylglucosamine/*biosynthesis/genetics ; *Bacillus subtilis/genetics/metabolism ; Bacterial Proteins/biosynthesis/genetics ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Bacterial ; Glucose/genetics/*metabolism ; Xylose/genetics/*metabolism ; },
abstract = {Glucose and xylose are the two most abundant sugars in renewable lignocellulose sources; however, typically they cannot be simultaneously utilized due to carbon catabolite repression. N-acetylglucosamine (GlcNAc) is a typical nutraceutical and has many applications in the field of healthcare. Here, we have developed a gene repressor system based on xylose-induced CRISPR interference (CRISPRi) in Bacillus subtilis, aimed at downregulating the expression of three genes (zwf, pfkA, glmM) that control the major competing reactions of GlcNAc synthesis (pentose phosphate pathway (HMP), glycolysis, and peptidoglycan synthesis pathway (PSP)), with the potential to relieve glucose repression and allow the co-utilization of both glucose and xylose. Simultaneous repression of these three genes by CRISPRi improved GlcNAc titer by 13.2% to 17.4 ± 0.47 g/L, with the GlcNAc yield on glucose and xylose showing an 84.1% improvement, reaching 0.42 ± 0.036 g/g. In order to further engineer the synergetic utilization of glucose and xylose, a combinatorial approach was developed based on 27 arrays containing sgRNAs with different repression capacities targeting the three genes. We further optimized the temporal control of the system and found that when 15 g/L xylose was added 6 h after inoculation, the most efficient strain, BNX122, synthesized 20.5 ± 0.85 g/L GlcNAc with a yield of 0.46 ± 0.010 g/g glucose and xylose in shake flask culture. Finally, the GlcNAc titer and productivity in a 3-L fed-batch bioreactor reached 103.1 ± 2.11 g/L and 1.17 ± 0.024 g/L/h, which were 5.0-fold and 2.7-fold of that in shake flask culture, respectively. Taken together, these findings suggest that a CRISPRi-enabled regulation method provides a simple, efficient, and universal way to promote the synergetic utilization of multiple carbon sources by microbial cell factories.},
}

Acetylglucosamine/*biosynthesis/genetics
*Bacillus subtilis/genetics/metabolism
Bacterial Proteins/biosynthesis/genetics
*CRISPR-Cas Systems
*Gene Expression Regulation, Bacterial
Glucose/genetics/*metabolism
Xylose/genetics/*metabolism

@article {pmid30126863,
year = {2018},
author = {Karlgren, M and Simoff, I and Keiser, M and Oswald, S and Artursson, P},
title = {CRISPR-Cas9: A New Addition to the Drug Metabolism and Disposition Tool Box.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {46},
number = {11},
pages = {1776-1786},
doi = {10.1124/dmd.118.082842},
pmid = {30126863},
issn = {1521-009X},
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Clustered Regularly Interspaced Short Palindromic Repeats/*physiology ; Cytochrome P-450 Enzyme System/metabolism ; Gene Editing/methods ; Humans ; Inactivation, Metabolic/*physiology ; Pharmaceutical Preparations/*metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9), i.e., CRISPR-Cas9, has been extensively used as a gene-editing technology during recent years. Unlike earlier technologies for gene editing or gene knockdown, such as zinc finger nucleases and RNA interference, CRISPR-Cas9 is comparably easy to use, affordable, and versatile. Recently, CRISPR-Cas9 has been applied in studies of drug absorption, distribution, metabolism, and excretion (ADME) and for ADME model generation. To date, about 50 papers have been published describing in vitro or in vivo CRISPR-Cas9 gene editing of ADME and ADME-related genes. Twenty of these papers describe gene editing of clinically relevant genes, such as ATP-binding cassette drug transporters and cytochrome P450 drug-metabolizing enzymes. With CRISPR-Cas9, the ADME tool box has been substantially expanded. This new technology allows us to develop better and more predictive in vitro and in vivo ADME models and map previously underexplored ADME genes and gene families. In this mini-review, we give an overview of the CRISPR-Cas9 technology and summarize recent applications of CRISPR-Cas9 within the ADME field. We also speculate about future applications of CRISPR-Cas9 in ADME research.},
}

Animals
CRISPR-Cas Systems/*physiology
Clustered Regularly Interspaced Short Palindromic Repeats/*physiology
Cytochrome P-450 Enzyme System/metabolism
Gene Editing/methods
Humans
Inactivation, Metabolic/*physiology
Pharmaceutical Preparations/*metabolism

@article {pmid29695624,
year = {2018},
author = {Vyas, VK and Bushkin, GG and Bernstein, DA and Getz, MA and Sewastianik, M and Barrasa, MI and Bartel, DP and Fink, GR},
title = {New CRISPR Mutagenesis Strategies Reveal Variation in Repair Mechanisms among Fungi.},
journal = {mSphere},
volume = {3},
number = {2},
pages = {},
pmid = {29695624},
issn = {2379-5042},
support = {T32 GM007287/GM/NIGMS NIH HHS/United States ; R15 AI130950/AI/NIAID NIH HHS/United States ; R35 GM118135/GM/NIGMS NIH HHS/United States ; R01 GM035010/GM/NIGMS NIH HHS/United States ; F32 GM108201/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Candida albicans/genetics ; Candida glabrata/genetics ; DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; Fungi/*genetics ; Gene Editing/*methods ; Genetic Vectors ; *Mutagenesis ; RNA, Guide/genetics ; Saccharomyces cerevisiae/genetics ; },
abstract = {We have created new vectors for clustered regularly interspaced short palindromic repeat (CRISPR) mutagenesis in Candida albicans, Saccharomyces cerevisiae, Candida glabrata, and Naumovozyma castellii These new vectors permit a comparison of the requirements for CRISPR mutagenesis in each of these species and reveal different dependencies for repair of the Cas9 double-stranded break. Both C. albicans and S. cerevisiae rely heavily on homology-directed repair, whereas C. glabrata and N. castellii use both homology-directed and nonhomologous end-joining pathways. The high efficiency of these vectors permits the creation of unmarked deletions in each of these species and the recycling of the dominant selection marker for serial mutagenesis in prototrophs. A further refinement, represented by the "Unified" Solo vectors, incorporates Cas9, guide RNA, and repair template into a single vector, thus enabling the creation of vector libraries for pooled screens. To facilitate the design of such libraries, we have identified guide sequences for each of these species with updated guide selection algorithms.IMPORTANCE CRISPR-mediated genome engineering technologies have revolutionized genetic studies in a wide range of organisms. Here we describe new vectors and guide sequences for CRISPR mutagenesis in the important human fungal pathogens C. albicans and C. glabrata, as well as in the related yeasts S. cerevisiae and N. castellii The design of these vectors enables efficient serial mutagenesis in each of these species by leaving few, if any, exogenous sequences in the genome. In addition, we describe strategies for the creation of unmarked deletions in each of these species and vector designs that permit the creation of vector libraries for pooled screens. These tools and strategies promise to advance genetic engineering of these medically and industrially important species.},
}

*CRISPR-Cas Systems
Candida albicans/genetics
Candida glabrata/genetics
DNA Breaks, Double-Stranded
*DNA End-Joining Repair
Fungi/*genetics
Gene Editing/*methods
Genetic Vectors
*Mutagenesis
RNA, Guide/genetics
Saccharomyces cerevisiae/genetics

@article {pmid29587177,
year = {2018},
author = {O'Donnell, KA},
title = {Advances in functional genetic screening with transposons and CRISPR/Cas9 to illuminate cancer biology.},
journal = {Current opinion in genetics & development},
volume = {49},
number = {},
pages = {85-94},
doi = {10.1016/j.gde.2018.03.006},
pmid = {29587177},
issn = {1879-0380},
support = {R01 CA207763/CA/NCI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/genetics ; DNA Transposable Elements/*genetics ; Early Detection of Cancer ; *Genetic Testing ; Humans ; Mutagenesis, Insertional/*genetics ; Mutation/genetics ; Neoplasms/diagnosis/*genetics/pathology ; },
abstract = {Large-scale genome sequencing studies have identified a wealth of mutations in human tumors and have dramatically advanced the field of cancer genetics. However, the functional consequences of an altered gene in tumor progression cannot always be inferred from mutation status alone. This underscores the critical need for complementary methods to assign functional significance to mutated genes in cancer. Transposons are mobile genetic elements that serve as powerful tools for insertional mutagenesis. Over the last decade, investigators have employed mouse models with on-demand transposon-mediated mutagenesis to perform unbiased genetic screens to identify clinically relevant genes that participate in the pathogenesis of human cancer. Two distinct DNA transposon mutagenesis systems, Sleeping Beauty (SB) and PiggyBac (PB), have been applied extensively in vivo and more recently, in ex vivo settings. These studies have informed our understanding of the genes and pathways that drive cancer initiation, progression, and metastasis. This review highlights the latest progress on cancer gene identification for specific cancer subtypes, as well as new technological advances and incorporation of the CRISPR/Cas9 toolbox into transposon-mediated functional genetic studies.},
}

CRISPR-Cas Systems/genetics
DNA Transposable Elements/*genetics
Early Detection of Cancer
*Genetic Testing
Humans
Mutagenesis, Insertional/*genetics
Mutation/genetics
Neoplasms/diagnosis/*genetics/pathology

@article {pmid30856357,
year = {2019},
author = {Le Rhun, A and Escalera-Maurer, A and Bratovič, M and Charpentier, E},
title = {CRISPR-Cas in Streptococcus pyogenes.},
journal = {RNA biology},
volume = {},
number = {},
pages = {1-10},
doi = {10.1080/15476286.2019.1582974},
pmid = {30856357},
issn = {1555-8584},
abstract = {The discovery and characterization of the prokaryotic CRISPR-Cas immune system has led to a revolution in genome editing and engineering technologies. Despite the fact that most applications emerged after the discovery of the type II-A CRISPR-Cas9 system of Streptococcus pyogenes, its biological importance in this organism has received little attention. Here, we provide a comprehensive overview of the current knowledge about CRISPR-Cas systems from S. pyogenes. We discuss how the interplay between CRISPR-mediated immunity and horizontal gene transfer might have modeled the evolution of this pathogen. We review the current literature about the CRISPR-Cas systems present in S. pyogenes (types I-C and II-A), and describe their distinctive biochemical and functional features. Finally, we summarize the main biotechnological applications that have arisen from the discovery of the CRISPR-Cas9 system in S. pyogenes.},
}

@article {pmid30853970,
year = {2019},
author = {Parmeciano Di Noto, G and Molina, MC and Quiroga, C},
title = {Insights Into Non-coding RNAs as Novel Antimicrobial Drugs.},
journal = {Frontiers in genetics},
volume = {10},
number = {},
pages = {57},
doi = {10.3389/fgene.2019.00057},
pmid = {30853970},
issn = {1664-8021},
abstract = {Multidrug resistant bacteria are a serious worldwide problem, especially carbapenem-resistant Enterobacteriaceae (such as Klebsiella pneumoniae and Escherichia coli), Acinetobacter baumannii and Pseudomonas aeruginosa. Since the emergence of extensive and pan-drug resistant bacteria there are few antibiotics left to treat patients, thus novel RNA-based strategies are being considered. Here, we examine the current situation of different non-coding RNAs found in bacteria as well as their function and potential application as antimicrobial agents. Furthermore, we discuss the factors that may contribute in the efficient development of RNA-based drugs, the limitations for their implementation and the use of nanocarriers for delivery.},
}

@article {pmid30853940,
year = {2019},
author = {Jaiswal, S and Singh, DK and Shukla, P},
title = {Gene Editing and Systems Biology Tools for Pesticide Bioremediation: A Review.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {87},
doi = {10.3389/fmicb.2019.00087},
pmid = {30853940},
issn = {1664-302X},
abstract = {Bioremediation is the degradation potential of microorganisms to dissimilate the complex chemical compounds from the surrounding environment. The genetics and biochemistry of biodegradation processes in datasets opened the way of systems biology. Systemic biology aid the study of interacting parts involved in the system. The significant keys of system biology are biodegradation network, computational biology, and omics approaches. Biodegradation network consists of all the databases and datasets which aid in assisting the degradation and deterioration potential of microorganisms for bioremediation processes. This review deciphers the bio-degradation network, i.e., the databases and datasets (UM-BBD, PAN, PTID, etc.) aiding in assisting the degradation and deterioration potential of microorganisms for bioremediation processes, computational biology and multi omics approaches like metagenomics, genomics, transcriptomics, proteomics, and metabolomics for the efficient functional gene mining and their validation for bioremediation experiments. Besides, the present review also describes the gene editing tools like CRISPR Cas, TALEN, and ZFNs which can possibly make design microbe with functional gene of interest for degradation of particular recalcitrant for improved bioremediation.},
}

@article {pmid30809052,
year = {2019},
author = {Maxmen, A},
title = {Faster, better, cheaper: the rise of CRISPR in disease detection.},
journal = {Nature},
volume = {566},
number = {7745},
pages = {437},
doi = {10.1038/d41586-019-00601-3},
pmid = {30809052},
issn = {1476-4687},
mesh = {CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Nucleic Acids ; },
}

CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Nucleic Acids