@article {pmid30785668,
year = {2019},
author = {Zhang, T and Zhao, Y and Ye, J and Cao, X and Xu, C and Chen, B and An, H and Jiao, Y and Zhang, F and Yang, X and Zhou, G},
title = {Establishing CRISPR/Cas13a immune system conferring RNA virus resistance in both dicot and monocot plants.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.13095},
pmid = {30785668},
issn = {1467-7652},
abstract = {Besides its powerful capability for genome editing, the clustered, regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) (CRISPR/Cas) systems, has been exploited to combat virus infection in eukaryotic organisms (Zaidi et al., 2016). By harnessing CRISPR/Cas system, its compelling inhibiting activities against DNA viruses (Ali et al., 2015; Baltes et al., 2015; Ji et al., 2015) or RNA viruses (Aman et al., 2018; Zhang et al., 2018) were reported in many cases. Moreover, due to the facts that the eukaryotic viruses themselves do not equip the ability to counter this prokaryotic immune defense, by utilization of such strategy, we could establish effective control and eradiation strategy against the eukaryotic virus. This article is protected by copyright. All rights reserved.},
}

@article {pmid30779874,
year = {2019},
author = {Rouet, R and Christ, D},
title = {Efficient intracellular delivery of CRISPR-Cas ribonucleoproteins through receptor mediated endocytosis.},
journal = {ACS chemical biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acschembio.9b00116},
pmid = {30779874},
issn = {1554-8937},
abstract = {We recently reported a new delivery system harnessing surface receptors for targeted uptake of CRISPR-Cas9 ribonucleoprotein (RNP) into mammalian cells (Rouet et al., JACS 2018). For this purpose, Cas9 protein was labeled with the small molecule ligand ASGRL, specific for the asialoglycoprotein receptor, enabling endosomal uptake of the RNP into human cells expressing the receptor. However, detailed mechanistic insights had remained unknown and editing efficiency low. Here we investigate the mechanism of endosomal escape as mediated by the ppTG21 peptide and outline the development of novel Cas9 or Cas12a RNP complexes with increased editing efficiency.},
}

@article {pmid30775868,
year = {2016},
author = {Liang, Z and Zong, Y and Gao, C},
title = {An Efficient Targeted Mutagenesis System Using CRISPR/Cas in Monocotyledons.},
journal = {Current protocols in plant biology},
volume = {1},
number = {2},
pages = {329-344},
doi = {10.1002/cppb.20021},
pmid = {30775868},
issn = {2379-8068},
abstract = {Precise genome modification using artificial nucleases is a powerful tool for in-depth understanding of gene functions and for creating new varieties. The CRISPR/Cas system, derived from an adaptive immunity system in bacteria and archaea, can introduce DNA double-strand breaks (DSBs) into pre-selected genomic loci and lead to loss of gene function due to error-prone non-homologous end joining (NHEJ). RNA-guided nucleases have been widely used in several eukaryotic organisms. In this article, we provide a detailed protocol for designing and constructing gRNA targets, detecting nuclease activity in transient protoplast assays, and identifying mutations in transgenic plants (including rice, wheat and maize). Targeted mutations in T0 plants can be generated in 14 to 18 weeks. © 2016 by John Wiley & Sons, Inc.},
}

@article {pmid29916068,
year = {2018},
author = {Kapusi, E and Stöger, E},
title = {Detection of CRISPR/Cas9-Induced Genomic Fragment Deletions in Barley and Generation of Homozygous Edited Lines via Embryogenic Pollen Culture.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1789},
number = {},
pages = {9-20},
doi = {10.1007/978-1-4939-7856-4_2},
pmid = {29916068},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; DNA, Plant/genetics ; GC Rich Sequence ; Gene Deletion ; Gene Editing/*methods ; Genome, Plant ; Haploidy ; Hordeum/embryology/*genetics ; Mutation Rate ; Pollen/embryology/*genetics ; Tissue Culture Techniques/methods ; Transformation, Genetic ; },
abstract = {The CRISPR/Cas9 system from Streptococcus pyogenes is an increasingly popular tool for genome editing due to its ease of application. Here we demonstrate genomic DNA fragment removal using RNA directed Cas9 nuclease in barley. The high mutation frequency confirms the exceptional efficiency of the system and its suitability for generating loss-of-function mutant lines that may be used in functional genetics approaches to study endomembrane trafficking pathways and posttranslational protein modifications. The generation of doubled haploids from genome edited plants allows the recovery of true breeding lines that are instantly homozygous for the edited alleles.},
}

*CRISPR-Cas Systems
DNA, Plant/genetics
GC Rich Sequence
Gene Deletion
Gene Editing/*methods
Genome, Plant
Haploidy
Hordeum/embryology/*genetics
Mutation Rate
Pollen/embryology/*genetics
Tissue Culture Techniques/methods
Transformation, Genetic

@article {pmid29856031,
year = {2018},
author = {Vachey, G and Déglon, N},
title = {CRISPR/Cas9-Mediated Genome Editing for Huntington's Disease.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1780},
number = {},
pages = {463-481},
doi = {10.1007/978-1-4939-7825-0_21},
pmid = {29856031},
issn = {1940-6029},
mesh = {Animals ; Brain/pathology ; CRISPR-Cas Systems/*genetics ; Cell Culture Techniques/instrumentation/methods ; Cloning, Molecular/methods ; Disease Models, Animal ; Gene Editing/instrumentation/*methods ; Genetic Therapy/instrumentation/*methods ; Genetic Vectors/administration & dosage/genetics ; HEK293 Cells ; Humans ; Huntingtin Protein/*genetics ; Huntington Disease/genetics/pathology/*therapy ; Lentivirus/genetics ; Mice ; Mice, Transgenic ; Mutation ; RNA, Guide/genetics ; Stereotaxic Techniques/instrumentation ; Transfection/instrumentation/methods ; },
abstract = {This chapter describes the potential use of viral-mediated gene transfer in the central nervous system for genome editing in the context of Huntington's disease. Here, we provide protocols that cover the design of various genome editing strategies, the cloning of CRISPR/Cas9 elements into lentiviral vectors, and the assessment of cleavage efficiency, as well as potential unwanted effects.},
}

Animals
Brain/pathology
CRISPR-Cas Systems/*genetics
Cell Culture Techniques/instrumentation/methods
Cloning, Molecular/methods
Disease Models, Animal
Gene Editing/instrumentation/*methods
Genetic Therapy/instrumentation/*methods
Genetic Vectors/administration & dosage/genetics
HEK293 Cells
Humans
Huntingtin Protein/*genetics
Huntington Disease/genetics/pathology/*therapy
Lentivirus/genetics
Mice
Mice, Transgenic
Mutation
RNA, Guide/genetics
Stereotaxic Techniques/instrumentation
Transfection/instrumentation/methods

@article {pmid29734775,
year = {2018},
author = {Miao, J and Ying, B and Li, R and Tollefson, AE and Spencer, JF and Wold, WSM and Song, SH and Kong, IK and Toth, K and Wang, Y and Wang, Z},
title = {Characterization of an N-Terminal Non-Core Domain of RAG1 Gene Disrupted Syrian Hamster Model Generated by CRISPR Cas9.},
journal = {Viruses},
volume = {10},
number = {5},
pages = {},
pmid = {29734775},
issn = {1999-4915},
support = {R41 OD021979/OD/NIH HHS/United States ; HHSN272201000021I/AI/NIAID NIH HHS/United States ; },
mesh = {Adenoviridae Infections/*immunology ; Adenoviruses, Human ; Animals ; B-Lymphocytes/cytology ; CRISPR-Cas Systems ; Disease Models, Animal ; Frameshift Mutation ; Genes, RAG-1/*genetics/*immunology ; Immunologic Deficiency Syndromes/*genetics ; Killer Cells, Natural/cytology ; Mesocricetus ; Spleen/immunology/pathology ; T-Lymphocytes/cytology ; },
abstract = {The accumulating evidence demonstrates that Syrian hamsters have advantages as models for various diseases. To develop a Syrian hamster (Mesocricetus auratus) model of human immunodeficiency caused by RAG1 gene mutations, we employed the CRISPR/Cas9 system and introduced an 86-nucleotide frameshift deletion in the hamster RAG1 gene encoding part of the N-terminal non-core domain of RAG1. Histological and immunohistochemical analyses demonstrated that these hamsters (referred herein as RAG1-86nt hamsters) had atrophic spleen and thymus, and developed significantly less white pulp and were almost completely devoid of splenic lymphoid follicles. The RAG1-nt86 hamsters had barely detectable CD3⁺ and CD4⁺ T cells. The expression of B and T lymphocyte-specific genes (CD3γ and CD4 for T cell-specific) and (CD22 and FCMR for B cell-specific) was dramatically reduced, whereas the expression of macrophage-specific (CD68) and natural killer (NK) cell-specific (CD94 and KLRG1) marker genes was increased in the spleen of RAG1-nt86 hamsters compared to wildtype hamsters. Interestingly, despite the impaired development of B and T lymphocytes, the RAG1-86nt hamsters still developed neutralizing antibodies against human adenovirus type C6 (HAdV-C6) upon intranasal infection and were capable of clearing the infectious viruses, albeit with slower kinetics. Therefore, the RAG1-86nt hamster reported herein (similar to the hypomorphic RAG1 mutations in humans that cause Omenn syndrome), may provide a useful model for studying the pathogenesis of the specific RAG1-mutation-induced human immunodeficiency, the host immune response to adenovirus infection and other pathogens as well as for evaluation of cell and gene therapies for treatment of this subset of RAG1 mutation patients.},
}

Adenoviridae Infections/*immunology
Adenoviruses, Human
Animals
B-Lymphocytes/cytology
CRISPR-Cas Systems
Disease Models, Animal
Frameshift Mutation
Genes, RAG-1/*genetics/*immunology
Immunologic Deficiency Syndromes/*genetics
Killer Cells, Natural/cytology
Mesocricetus
Spleen/immunology/pathology
T-Lymphocytes/cytology

@article {pmid29724057,
year = {2018},
author = {Fan, S and Xu, X and Liao, Y and Wang, Y and Wang, J and Feng, M and Wang, L and Zhang, Y and He, Z and Yang, F and Fraser, NW and Li, Q},
title = {Attenuated Phenotype and Immunogenic Characteristics of a Mutated Herpes Simplex Virus 1 Strain in the Rhesus Macaque.},
journal = {Viruses},
volume = {10},
number = {5},
pages = {},
pmid = {29724057},
issn = {1999-4915},
mesh = {Animals ; Antigens, Viral/genetics/*immunology ; CRISPR-Cas Systems ; Herpes Simplex/immunology/*prevention & control ; Herpesvirus 1, Human/*genetics ; Herpesvirus Vaccines/genetics/*immunology ; Immunogenicity, Vaccine ; Macaca mulatta/virology ; Mutation ; Neutralization Tests ; Phenotype ; Trigeminal Ganglion/virology ; Vaccines, Attenuated/genetics/immunology ; Viral Matrix Proteins/genetics/immunology ; Viral Proteins/genetics/immunology ; Virus Replication ; },
abstract = {Herpes simplex virus type 1(HSV-1) presents a conundrum to public health worldwide because of its specific pathogenicity and clinical features. Some experimental vaccines, such as the recombinant viral glycoproteins, exhibit the viral immunogenicity of a host-specific immune response, but none of these has achieved a valid epidemiological protective efficacy in the human population. In the present study, we constructed an attenuated HSV-1 strain M3 through the partial deletion of UL7, UL41, and the latency-associated transcript (LAT) using the CRISPR/Cas9 system. The mutant strain exhibited lowered infectivity and virulence in macaques. Neutralization testing and ELISpot detection of the specific T-cell responses confirmed the specific immunity induced by M3 immunization and this immunity defended against the challenges of the wild-type strain and restricted the entry of the wild-type strain into the trigeminal ganglion. These results in rhesus macaques demonstrated the potential of the attenuated vaccine for the prevention of HSV-1 in humans.},
}

Animals
Antigens, Viral/genetics/*immunology
CRISPR-Cas Systems
Herpes Simplex/immunology/*prevention & control
Herpesvirus 1, Human/*genetics
Herpesvirus Vaccines/genetics/*immunology
Immunogenicity, Vaccine
Macaca mulatta/virology
Mutation
Neutralization Tests
Phenotype
Trigeminal Ganglion/virology
Vaccines, Attenuated/genetics/immunology
Viral Matrix Proteins/genetics/immunology
Viral Proteins/genetics/immunology
Virus Replication

@article {pmid29534717,
year = {2018},
author = {Sun, J and Wang, Q and Jiang, Y and Wen, Z and Yang, L and Wu, J and Yang, S},
title = {Genome editing and transcriptional repression in Pseudomonas putida KT2440 via the type II CRISPR system.},
journal = {Microbial cell factories},
volume = {17},
number = {1},
pages = {41},
pmid = {29534717},
issn = {1475-2859},
support = {No. 21676240//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; Endonucleases/metabolism ; Gene Deletion ; *Gene Editing ; Gene Expression ; Green Fluorescent Proteins ; Mutagenesis, Insertional ; Pseudomonas putida/*genetics/metabolism ; },
abstract = {BACKGROUND: The soil bacterium Pseudomonas putida KT2440 is a "generally recognized as safe"-certified strain with robust property and versatile metabolism. Thus, it is an ideal candidate for synthetic biology, biodegradation, and other biotechnology applications. The known genome editing approaches of Pseudomonas are suboptimal; thus, it is necessary to develop a high efficiency genome editing tool.

RESULTS: In this study, we established a fast and convenient CRISPR-Cas9 method in P. putida KT2440. Gene deletion, gene insertion and gene replacement could be achieved within 5 days, and the mutation efficiency reached > 70%. Single nucleotide replacement could be realized, overcoming the limitations of protospacer adjacent motif sequences. We also applied nuclease-deficient Cas9 binding at three locations upstream of enhanced green fluorescent protein (eGFP) for transcriptional inhibition, and the expression intensity of eGFP reduced to 28.5, 29.4, and 72.1% of the control level, respectively. Furthermore, based on this CRISPR-Cas9 system, we also constructed a CRISPR-Cpf1 system, which we validated for genome editing in P. putida KT2440.

CONCLUSIONS: In this research, we established CRISPR based genome editing and regulation control systems in P. putida KT2440. These fast and efficient approaches will greatly facilitate the application of P. putida KT2440.},
}

*CRISPR-Cas Systems
Endonucleases/metabolism
Gene Deletion
*Gene Editing
Gene Expression
Green Fluorescent Proteins
Mutagenesis, Insertional
Pseudomonas putida/*genetics/metabolism

@article {pmid30773816,
year = {2019},
author = {Krupovic, M and Makarova, KS and Wolf, YI and Medvedeva, S and Prangishvili, D and Forterre, P and Koonin, EV},
title = {Integrated Mobile Genetic Elements in Thaumarchaeota.},
journal = {Environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1462-2920.14564},
pmid = {30773816},
issn = {1462-2920},
abstract = {To explore the diversity of mobile genetic elements (MGE) associated with archaea of the phylum Thaumarchaeota, we exploited the property of most MGE to integrate into the genomes of their hosts. Integrated MGE (iMGE) were identified in 20 thaumarchaeal genomes amounting to 2 Mbp of mobile thaumarchaeal DNA. These iMGE group into five major classes: (i) proviruses, (ii) casposons, (iii) insertion sequence-like transposons, (iv) integrative-conjugative elements, and (v) cryptic integrated elements. The majority of the iMGE belong to the latter category and might represent novel families of viruses or plasmids. The identified proviruses are related to tailed viruses of the order Caudovirales and to tailless icosahedral viruses with the double jelly-roll capsid proteins. The thaumarchaeal iMGE are all connected within a gene sharing network, highlighting pervasive gene exchange between MGE occupying the same ecological niche. The thaumarchaeal mobilome carries multiple auxiliary metabolic genes, including multicopper oxidases and ammonia monooxygenase subunit C, and stress response genes, such as those for universal stress response proteins (UspA). Thus, iMGE might make important contributions to the fitness and adaptation of their hosts. We identified several iMGE carrying type I-B CRISPR-Cas systems and spacers matching other thaumarchaeal iMGE, suggesting antagonistic interactions between coexisting MGE and symbiotic relationships with the hosts. This article is protected by copyright. All rights reserved.},
}

@article {pmid30770599,
year = {2019},
author = {Liu, CY and Wang, HC},
title = {The fibroblast of radicular cyst facilitate osteoclastogenesis via the autocrine of Fibronectin containing extra domain A.},
journal = {Oral diseases},
volume = {},
number = {},
pages = {},
doi = {10.1111/odi.13064},
pmid = {30770599},
issn = {1601-0825},
abstract = {OBJECTIVE: To investigate the alternative spliced isoforms of Fibronectin (FN) in the stroma of radicular cysts; and analyze the associations between these isoforms and the osteoclastogenic effects of fibroblasts.

METHODS AND MATERIALS: The specimens of radicular cysts were stained with immunohistochemistry, and the associations between each FN isoform and clinical parameters were assessed. The fibroblasts isolated from cysts or jaw bone were cultured to induce the Trap+MNCs. In the conditioned medium, the Fibronectin containing extra domain A (EDA+FN) were neutralized by antibody IST-9, and the EDA exon of fibroblasts were knockout by CRISPR/Cas system, for assessing the osteolastogenic effects. The mRNA level of FN isoforms and the osteoclastogenesis related genes were analyzed by quantitive PCR.

RESULTS: EDA+FN staining was positively associate with the size of the lesions (P<0.05). In contrast with the controls, the ratio of EDA+FN/total FN in the fibroblasts from radicular cysts was significantly higher (P<0.05), and positively associate with Trap+MNCs counting, it was consistent with increased expression of COX-2, IL-6, IL-17 and the RANKL/OPG (P<0.05). The Trap+MNCs counting and osteoclastogenesis related genes were decreased by IST-9 blocking and EDA exon knockout in fibroblasts, but the blockage of the interaction between EDA+FN and pre-osteoclasts exhibited little effects on Trap+MNCs formation.

CONCLUSION: The microenvironment of the fibrous capsule of radicular cysts facilitate the splicing of EDA exon, it endues EDA+FN with autocrine effects on fibroblast itself, and increases the expression of osteoclastogenesis related genes, by which the osteoclastogenesis in radicular cysts could be initiated. This article is protected by copyright. All rights reserved.},
}

@article {pmid30766726,
year = {2019},
author = {Goeckel, ME and Basgall, EM and Lewis, IC and Goetting, SC and Yan, Y and Halloran, M and Finnigan, GC},
title = {Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae.},
journal = {Fungal biology and biotechnology},
volume = {6},
number = {},
pages = {2},
doi = {10.1186/s40694-019-0065-x},
pmid = {30766726},
issn = {2054-3085},
abstract = {Background: The bacterial CRISPR/Cas genome editing system has provided a major breakthrough in molecular biology. One use of this technology is within a nuclease-based gene drive. This type of system can install a genetic element within a population at unnatural rates. Combatting of vector-borne diseases carried by metazoans could benefit from a delivery system that bypasses traditional Mendelian laws of segregation. Recently, laboratory studies in fungi, insects, and even mice, have demonstrated successful propagation of CRISPR gene drives and the potential utility of this type of mechanism. However, current gene drives still face challenges including evolved resistance, containment, and the consequences of application in wild populations. Additional research into molecular mechanisms that would allow for control, titration, and inhibition of drive systems is needed.

Results: In this study, we use artificial gene drives in budding yeast to explore mechanisms to modulate nuclease activity of Cas9 through its nucleocytoplasmic localization. We examine non-native nuclear localization sequences (both NLS and NES) on Cas9 fusion proteins in vivo through fluorescence microscopy and genomic editing. Our results demonstrate that mutational substitutions to nuclear signals and combinatorial fusions can both modulate the level of gene drive activity within a population of cells.

Conclusions: These findings have implications for control of traditional nuclease-dependent editing and use of gene drive systems within other organisms. For instance, initiation of a nuclear export mechanism to Cas9 could serve as a molecular safeguard within an active gene drive to reduce or eliminate editing.},
}

@article {pmid30377126,
year = {2018},
author = {Zhang, H and Chen, M and Fang, T and Zhang, T and Ni, W},
title = {[Establishment and verification of a mouse model of Gata4 gene H435Y mutation].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {38},
number = {10},
pages = {1245-1249},
doi = {10.3969/j.issn.1673-4254.2018.10.15},
pmid = {30377126},
issn = {1673-4254},
mesh = {Animals ; *CRISPR-Cas Systems ; GATA4 Transcription Factor/*genetics ; Gene Targeting/*methods ; Mice ; Mice, Inbred C57BL ; Models, Animal ; Point Mutation/*genetics ; Polymerase Chain Reaction ; },
abstract = {OBJECTIVE: To establish a mouse model of H435Y mutation of Gata4 gene using CRISPR/Cas9- mediated gene targeting.

METHODS: The single-stranded guide RNA (sgRNA) specific to the H435Y loci of Gata4 gene was designed based on the sequence of Gata4 gene. After activity assessment, the active sgRNA and Cas9 were in vitro transcribed into RNA and microinjected along with the donor DNA fragment with point mutations into fertilized mouse eggs. The microinjected eggs were transferred into pseudopregnant mice to obtain the F0 generation mice with the target Gata4 gene mutation confirmed by PCR and gene sequencing. Gata4 gene mutations in the offsprings of the F0 generation mice were analyzed.

RESULTS: Gene sequencing confirmed the successful establishment of mouse models carrying H435Y mutation of Gata4 gene in 4 of the F0 generation mice. The positive F0 generation mice were crossed with wild-type C57BL/6J mice to obtain the F1 generation mice, and PCR confirmed the presence of H435Y mutations of Gata4 gene in 6 of the F1 mice. Then F2 generation mice were obtained by F1 generation matting with each other. PCR showed that H435Y mutation of Gata4 gene in F2 mice was found, indicating the mousemodel of Gata4 gene mutation in H435Y was established and propagated successfully.

CONCLUSIONS: We successfully established Gata4 gene H435Y mutant mouse models using CRISPR/Cas9 technique.},
}

Animals
*CRISPR-Cas Systems
GATA4 Transcription Factor/*genetics
Gene Targeting/*methods
Mice
Mice, Inbred C57BL
Models, Animal
Point Mutation/*genetics
Polymerase Chain Reaction

@article {pmid30296255,
year = {2018},
author = {Baskoylu, SN and Yersak, J and O'Hern, P and Grosser, S and Simon, J and Kim, S and Schuch, K and Dimitriadi, M and Yanagi, KS and Lins, J and Hart, AC},
title = {Single copy/knock-in models of ALS SOD1 in C. elegans suggest loss and gain of function have different contributions to cholinergic and glutamatergic neurodegeneration.},
journal = {PLoS genetics},
volume = {14},
number = {10},
pages = {e1007682},
pmid = {30296255},
issn = {1553-7404},
mesh = {Amino Acid Sequence ; Amyotrophic Lateral Sclerosis/*genetics/pathology ; Animals ; Animals, Genetically Modified ; Base Sequence ; CRISPR-Cas Systems ; Caenorhabditis elegans/*genetics ; Caenorhabditis elegans Proteins/*genetics ; Cholinergic Neurons/metabolism/*pathology ; Disease Models, Animal ; Gain of Function Mutation ; Gene Frequency ; Gene Knock-In Techniques ; Glutamic Acid/metabolism ; Humans ; Loss of Function Mutation ; Motor Neurons/metabolism/*pathology ; Superoxide Dismutase/*genetics ; Superoxide Dismutase-1/*genetics ; },
abstract = {Mutations in Cu/Zn superoxide dismutase 1 (SOD1) lead to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that disproportionately affects glutamatergic and cholinergic motor neurons. Previous work with SOD1 overexpression models supports a role for SOD1 toxic gain of function in ALS pathogenesis. However, the impact of SOD1 loss of function in ALS cannot be directly examined in overexpression models. In addition, overexpression may obscure the contribution of SOD1 loss of function in the degeneration of different neuronal populations. Here, we report the first single-copy, ALS knock-in models in C. elegans generated by transposon- or CRISPR/Cas9- mediated genome editing of the endogenous sod-1 gene. Introduction of ALS patient amino acid changes A4V, H71Y, L84V, G85R or G93A into the C. elegans sod-1 gene yielded single-copy/knock-in ALS SOD1 models. These differ from previously reported overexpression models in multiple assays. In single-copy/knock-in models, we observed differential impact of sod-1 ALS alleles on glutamatergic and cholinergic neurodegeneration. A4V, H71Y, G85R, and G93A animals showed increased SOD1 protein accumulation and oxidative stress induced degeneration, consistent with a toxic gain of function in cholinergic motor neurons. By contrast, H71Y, L84V, and G85R lead to glutamatergic neuron degeneration due to sod-1 loss of function after oxidative stress. However, dopaminergic and serotonergic neuronal populations were spared in single-copy ALS models, suggesting a neuronal-subtype specificity previously not reported in invertebrate ALS SOD1 models. Combined, these results suggest that knock-in models may reproduce the neurotransmitter-type specificity of ALS and that both SOD1 loss and gain of toxic function differentially contribute to ALS pathogenesis in different neuronal populations.},
}

Amino Acid Sequence
Amyotrophic Lateral Sclerosis/*genetics/pathology
Animals
Animals, Genetically Modified
Base Sequence
CRISPR-Cas Systems
Caenorhabditis elegans/*genetics
Caenorhabditis elegans Proteins/*genetics
Cholinergic Neurons/metabolism/*pathology
Disease Models, Animal
Gain of Function Mutation
Gene Frequency
Gene Knock-In Techniques
Glutamic Acid/metabolism
Humans
Loss of Function Mutation
Motor Neurons/metabolism/*pathology
Superoxide Dismutase/*genetics
Superoxide Dismutase-1/*genetics

@article {pmid30284145,
year = {2018},
author = {Lau, CH and Suh, Y},
title = {In vivo epigenome editing and transcriptional modulation using CRISPR technology.},
journal = {Transgenic research},
volume = {27},
number = {6},
pages = {489-509},
pmid = {30284145},
issn = {1573-9368},
support = {R01 CA180126/CA/NCI NIH HHS/United States ; R01 GM104459/GM/NIGMS NIH HHS/United States ; U19 AG056278/AG/NIA NIH HHS/United States ; P01 AG017242/AG/NIA NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; DNA Methylation ; *Epigenesis, Genetic ; *Epigenomics ; *Gene Editing ; Humans ; Promoter Regions, Genetic ; *Transcription, Genetic ; },
abstract = {The rapid advancement of CRISPR technology has enabled targeted epigenome editing and transcriptional modulation in the native chromatin context. However, only a few studies have reported the successful editing of the epigenome in adult animals in contrast to the rapidly growing number of in vivo genome editing over the past few years. In this review, we discuss the challenges facing in vivo epigenome editing and new strategies to overcome the huddles. The biggest challenge has been the difficulty in packaging dCas9 fusion proteins required for manipulation of epigenome into the adeno-associated virus (AAV) delivery vehicle. We review the strategies to address the AAV packaging issue, including small dCas9 orthologues, truncated dCas9 mutants, a split-dCas9 system, and potent truncated effector domains. We discuss the dCas9 conjugation strategies to recruit endogenous chromatin modifiers and remodelers to specific genomic loci, and recently developed methods to recruit multiple copies of the dCas9 fusion protein, or to simultaneous express multiple gRNAs for robust epigenome editing or synergistic transcriptional modulation. The use of Cre-inducible dCas9-expressing mice or a genetic cross between dCas9- and sgRNA-expressing flies has also helped overcome the transgene delivery issue. We provide perspective on how a combination use of these strategies can facilitate in vivo epigenome editing and transcriptional modulation.},
}

*CRISPR-Cas Systems
DNA Methylation
*Epigenesis, Genetic
*Epigenomics
*Gene Editing
Humans
Promoter Regions, Genetic
*Transcription, Genetic

@article {pmid30284144,
year = {2018},
author = {Vilarino, M and Suchy, FP and Rashid, ST and Lindsay, H and Reyes, J and McNabb, BR and van der Meulen, T and Huising, MO and Nakauchi, H and Ross, PJ},
title = {Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep.},
journal = {Transgenic research},
volume = {27},
number = {6},
pages = {525-537},
pmid = {30284144},
issn = {1573-9368},
mesh = {Animals ; *Animals, Genetically Modified ; Biopsy ; *Blastocyst ; *CRISPR-Cas Systems ; Embryo Transfer ; *Embryo, Mammalian ; Embryonic Development ; Female ; Gene Editing/methods/*veterinary ; Homeodomain Proteins/*genetics ; Male ; Mosaicism ; *Mutation ; Sheep ; Trans-Activators/*genetics ; },
abstract = {The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in ~ 50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.},
}

Animals
*Animals, Genetically Modified
Biopsy
*Blastocyst
*CRISPR-Cas Systems
Embryo Transfer
*Embryo, Mammalian
Embryonic Development
Female
Gene Editing/methods/*veterinary
Homeodomain Proteins/*genetics
Male
Mosaicism
*Mutation
Sheep
Trans-Activators/*genetics

@article {pmid29381698,
year = {2018},
author = {Hirst, J and Itzhak, DN and Antrobus, R and Borner, GHH and Robinson, MS},
title = {Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval.},
journal = {PLoS biology},
volume = {16},
number = {1},
pages = {e2004411},
pmid = {29381698},
issn = {1545-7885},
support = {086598//Wellcome Trust/United Kingdom ; 100140//Wellcome Trust/United Kingdom ; },
mesh = {Adaptor Proteins, Vesicular Transport/*genetics/metabolism/*physiology ; CRISPR-Cas Systems ; Endosomes/physiology ; Golgi Apparatus/physiology ; HeLa Cells ; Humans ; Lysosomes/genetics/physiology ; Mass Spectrometry ; Membrane Proteins/metabolism ; Phenotype ; Protein Transport ; Spastic Paraplegia, Hereditary/genetics ; Vesicular Transport Proteins/metabolism ; },
abstract = {The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR), GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5-associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.},
}

Adaptor Proteins, Vesicular Transport/*genetics/metabolism/*physiology
CRISPR-Cas Systems
Endosomes/physiology
Golgi Apparatus/physiology
HeLa Cells
Humans
Lysosomes/genetics/physiology
Mass Spectrometry
Membrane Proteins/metabolism
Phenotype
Protein Transport
Spastic Paraplegia, Hereditary/genetics
Vesicular Transport Proteins/metabolism

@article {pmid29352001,
year = {2018},
author = {Noel, S and Lee, SA and Sadasivam, M and Hamad, ARA and Rabb, H},
title = {KEAP1 Editing Using CRISPR/Cas9 for Therapeutic NRF2 Activation in Primary Human T Lymphocytes.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {200},
number = {5},
pages = {1929-1936},
pmid = {29352001},
issn = {1550-6606},
support = {R01 DK111209/DK/NIDDK NIH HHS/United States ; },
mesh = {Antigens, CD/genetics ; Antigens, Differentiation, T-Lymphocyte/genetics ; Antioxidants/metabolism ; CRISPR-Cas Systems/*genetics ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Expression/genetics ; Heme Oxygenase-1/genetics ; Humans ; Interleukin-10/genetics ; Jurkat Cells ; Kelch-Like ECH-Associated Protein 1/*genetics ; Lectins, C-Type/genetics ; NAD(P)H Dehydrogenase (Quinone)/genetics ; NF-E2-Related Factor 2/*genetics ; RNA, Messenger/genetics ; T-Lymphocytes/metabolism/*physiology ; Up-Regulation/genetics ; },
abstract = {Oxidant stress modifies T lymphocyte activation and function. Previous work demonstrated that murine T cell-specific kelch like-ECH-associated protein 1 (Keap1) deletion enhances antioxidant capacity and protects from experimental acute kidney injury. In this study, we used CRISPR technology to develop clinically translatable human T cell-specific KEAP1 deletion. Delivery of KEAP1 exon 2 specific Cas9:guide RNA in Jurkat T cells led to significant (∼70%) editing and upregulation of NRF2-regulated antioxidant genes NADPH dehydrogenase quinone 1 (NQO1) (up to 11-fold), heme oxygenase 1 (HO1) (up to 11-fold), and GCLM (up to 2-fold). In primary human T cells, delivery of KEAP1 exon 2 target site 2-specific ATTO 550-labeled Cas9:guide RNA edited KEAP1 in ∼40% cells and significantly (p ≤ 0.04) increased NQO1 (16-fold), HO1 (9-fold), and GCLM (2-fold) expression. To further enrich KEAP1-edited cells, ATTO 550-positive cells were sorted 24 h after electroporation. Assessment of ATTO 550-positive cells showed KEAP1 editing in ∼55% cells. There was no detectable off-target cleavage in the top three predicted genes in the ATTO 550-positive cells. Gene expression analysis found significantly (p ≤ 0.01) higher expression of NQO1 mRNA in ATTO 550-positive cells compared with control cells. Flow cytometric assessment showed increased (p ≤ 0.01) frequency of CD4-, CD25-, and CD69-expressing KEAP1 edited cells whereas frequency of CD8- (p ≤ 0.01) and IL-17- (p ≤ 0.05) expressing cells was reduced compared with control cells. Similar experimental conditions resulted in significant KEAP1 editing, increased antioxidant gene expression, and frequency of CD69 and IL-10 positive cells in highly enriched KEAP1-edited regulatory T cells. KEAP1-edited T cells could potentially be used for treating multiple human diseases.},
}

Antigens, CD/genetics
Antigens, Differentiation, T-Lymphocyte/genetics
Antioxidants/metabolism
CRISPR-Cas Systems/*genetics
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Expression/genetics
Heme Oxygenase-1/genetics
Humans
Interleukin-10/genetics
Jurkat Cells
Kelch-Like ECH-Associated Protein 1/*genetics
Lectins, C-Type/genetics
NAD(P)H Dehydrogenase (Quinone)/genetics
NF-E2-Related Factor 2/*genetics
RNA, Messenger/genetics
T-Lymphocytes/metabolism/*physiology
Up-Regulation/genetics

@article {pmid28958488,
year = {2017},
author = {He, Y and Zhang, T and Yang, N and Xu, M and Yan, L and Wang, L and Wang, R and Zhao, Y},
title = {Self-cleaving ribozymes enable the production of guide RNAs from unlimited choices of promoters for CRISPR/Cas9 mediated genome editing.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {44},
number = {9},
pages = {469-472},
pmid = {28958488},
issn = {1673-8527},
support = {R01 GM114660/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Editing ; *Genomics ; Promoter Regions, Genetic/*genetics ; RNA, Catalytic/*metabolism ; RNA, Guide/*genetics ; },
}

CRISPR-Cas Systems/*genetics
*Gene Editing
*Genomics
Promoter Regions, Genetic/*genetics
RNA, Catalytic/*metabolism
RNA, Guide/*genetics

@article {pmid28943147,
year = {2017},
author = {Wei, L and Wang, X and Yang, S and Yuan, W and Li, J},
title = {Efficient generation of the mouse model with a defined point mutation through haploid cell-mediated gene editing.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {44},
number = {9},
pages = {461-463},
doi = {10.1016/j.jgg.2017.07.004},
pmid = {28943147},
issn = {1673-8527},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Gene Editing/*methods ; *Haploidy ; Mice ; Models, Animal ; Point Mutation/*genetics ; },
}

Animals
Base Sequence
CRISPR-Cas Systems/genetics
Gene Editing/*methods
*Haploidy
Mice
Models, Animal
Point Mutation/*genetics

@article {pmid30763533,
year = {2019},
author = {Rostøl, JT and Marraffini, L},
title = {(Ph)ighting Phages: How Bacteria Resist Their Parasites.},
journal = {Cell host & microbe},
volume = {25},
number = {2},
pages = {184-194},
doi = {10.1016/j.chom.2019.01.009},
pmid = {30763533},
issn = {1934-6069},
abstract = {Bacteria are under constant attack from bacteriophages (phages), bacterial parasites that are the most abundant biological entity on earth. To resist phage infection, bacteria have evolved an impressive arsenal of anti-phage systems. Recent advances have significantly broadened and deepened our understanding of how bacteria battle phages, spearheaded by new systems like CRISPR-Cas. This review aims to summarize bacterial anti-phage mechanisms, with an emphasis on the most recent developments in the field.},
}

@article {pmid30762876,
year = {2019},
author = {Scaltriti, E and Carminati, D and Cortimiglia, C and Ramoni, R and Sørensen, KI and Giraffa, G and Zago, M},
title = {Survey on the CRISPR arrays in Lactobacillus helveticus genomes.},
journal = {Letters in applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/lam.13128},
pmid = {30762876},
issn = {1472-765X},
abstract = {Lactobacillus helveticus is a homofermentative thermophilic LAB that is mainly used in the manufacture of Swiss type and long-ripened Italian hard cheeses. In this study, the presence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) were analysed in 25 L. helveticus genomes and identified in 23 of these genomes. A total of 40 CRISPR loci were identified and classified into five main families based on CRISPR repeats: Ldbu1, Lsal1, Lhel1, Lhel2 and a new repeat family named Lhel3. Spacers had a size between 30 and 40 bp whereas repeats have an average size of 30 bp, with three longer repeats. The analysis displayed the presence of conserved spacers in 23 out of the 40 CRISPR loci. A geographical distribution of L. helveticus isolates with similar CRISPR spacer array profiles were not observed. Based on the presence of the signature protein Cas3, all CRISPR loci belonged to Type I. This analysis demonstrated a great CRISPR array variability within L. helveticus, which could be a useful tool for genotypic strain differentiation. A next step will be to understand the possible role of CRISPR/Cas system for the resistance of L. helveticus to phage infection. This article is protected by copyright. All rights reserved.},
}

@article {pmid30761103,
year = {2019},
author = {Broecker, F and Moelling, K},
title = {Evolution of Immune Systems From Viruses and Transposable Elements.},
journal = {Frontiers in microbiology},
volume = {10},
number = {},
pages = {51},
doi = {10.3389/fmicb.2019.00051},
pmid = {30761103},
issn = {1664-302X},
abstract = {Virus-derived sequences and transposable elements constitute a substantial portion of many cellular genomes. Recent insights reveal the intimate evolutionary relationship between these sequences and various cellular immune pathways. At the most basic level, superinfection exclusion may be considered a prototypical virus-mediated immune system that has been described in both prokaryotes and eukaryotes. More complex immune mechanisms fully or partially derived from mobile genetic elements include CRISPR-Cas of prokaryotes and the RAG1/2 system of vertebrates, which provide immunological memory of foreign genetic elements and generate antibody and T cell receptor diversity, respectively. In this review, we summarize the current knowledge on the contribution of mobile genetic elements to the evolution of cellular immune pathways. A picture is emerging in which the various cellular immune systems originate from and are spread by viruses and transposable elements. Immune systems likely evolved from simple superinfection exclusion to highly complex defense strategies.},
}

@article {pmid30244086,
year = {2018},
author = {Kim, MS and Shin, MJ and Kim, KH},
title = {Increase of viral hemorrhagic septicemia virus growth by knockout of IRF9 gene in Epithelioma papulosum cyprini cells.},
journal = {Fish & shellfish immunology},
volume = {83},
number = {},
pages = {443-448},
doi = {10.1016/j.fsi.2018.09.025},
pmid = {30244086},
issn = {1095-9947},
mesh = {Animals ; CRISPR-Cas Systems ; Cell Line ; Fishes ; Gene Editing ; Gene Knockout Techniques ; Interferon Type I/immunology ; Interferon-Stimulated Gene Factor 3, gamma Subunit/*genetics ; Novirhabdovirus/*physiology ; Poly I-C/pharmacology ; Virus Replication ; },
abstract = {Viral hemorrhagic septicemia virus (VHSV) has been a notorious pathogen in freshwater and marine fish. Due to the lack of effective treatment measures against VHSV disease, the development of prophylactic vaccines has been required, and methods that can produce high-titered viruses would be advantageous in producing cost-effective vaccines. Type I interferon (IFN) responses are the key elements of vertebrates' antiviral activities, and IFN-stimulated gene factor 3 (ISGF3) complex formed through type I IFNs up-regulates the expression of IFN-stimulated genes (ISGs). IFN regulatory factor 9 (IRF9) is a key component of ISGF3, so the inhibition of IRF9 would compromise host's type I IFN responses, which would weaken host antiviral activity. In this study, to increase the replication of VHSV, we generated IRF9 knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector that contains an EPC cell's U6 promoter-driven guide RNA cassette (targeting IRF9 gene) and a Cas9 expressing cassette. In the clones of IRF9 knockout EPC cells, there were no increase in ISG15 gene by poly I:C, and in Mx1 gene by both poly I:C and VHSV. Interestingly, although the increased folds were conspicuously lower than control EPC cells, the expression of ISG 15 gene in all the IRF9 knockout clones was significantly increased by VHSV infection. Control EPC cells pre-treated with poly I:C did not show any CPE when infected with VHSV, however, IRF9 knockout EPC cells showed CPE by VHSV infection in spite of being pretreated with poly I:C. The replication of VHSV in IRF9 knockout EPC cells was significantly faster and higher than that in control EPC cells indicating that the IRF9 knockout-mediated decrease of type I IFN responses allowed VHSV to replicate efficiently. Considering an economical aspect for the production of fish vaccines, the present IRF9 knockout EPC cells can be used to get higher-titered VHSV.},
}

Animals
CRISPR-Cas Systems
Cell Line
Fishes
Gene Editing
Gene Knockout Techniques
Interferon Type I/immunology
Interferon-Stimulated Gene Factor 3, gamma Subunit/*genetics
Novirhabdovirus/*physiology
Poly I-C/pharmacology
Virus Replication

@article {pmid29979095,
year = {2018},
author = {Miao, J and Chi, Y and Lin, D and Tyler, BM and Liu, X},
title = {Mutations in ORP1 Conferring Oxathiapiprolin Resistance Confirmed by Genome Editing using CRISPR/Cas9 in Phytophthora capsici and P. sojae.},
journal = {Phytopathology},
volume = {108},
number = {12},
pages = {1412-1419},
doi = {10.1094/PHYTO-01-18-0010-R},
pmid = {29979095},
issn = {0031-949X},
mesh = {CRISPR-Cas Systems ; Capsicum/*microbiology ; Drug Resistance/*genetics ; Fungicides, Industrial/*pharmacology ; Gene Editing ; Hydrocarbons, Fluorinated/*pharmacology ; Phenotype ; Phytophthora/drug effects/*genetics ; Plant Diseases/*microbiology ; Point Mutation ; Pyrazoles/*pharmacology ; Receptors, Steroid/*genetics ; Sporangia/drug effects/genetics ; },
abstract = {Oxathiapiprolin is a novel fungicide that was recently registered in a number of countries to control plant-pathogenic oomycetes such as Phytophthora capsici. In our previous study, point mutations G770V and G839W in oxysterol binding protein-related protein 1 (ORP1) were detected in oxathiapiprolin-resistant P. capsici isolates (PcORP1). Here, we used the CRISPR/Cas9 system to verify the effects of these two point mutations on P. capsici phenotypes. Transformants containing heterozygous G770V and G839W mutations in PcORP1 showed high levels of oxathiapiprolin resistance. The G770V transformants showed otherwise similar phenotypes compared with the wild-type isolate BYA5, including sporangia and zoospore production, cyst germination, and pathogenicity. However, two independent transformants with heterozygous G839W mutations in PcORP1 could not produce sporangia. Three transformants with an unexpected point mutation in PcORP1 (ΔN837) showed high oxathiapiprolin resistance, and either similar or significantly reduced fitness compared with BYA5. The same deletion (ΔN837) was confirmed to confer oxathiapiprolin resistance in P. sojae by using CRISPR/Cas9. These homozygous P. sojae mutants also showed either similar or strongly reduced fitness compared with the wild-type parent isolate P6497. These results improve our understanding of oxathiapiprolin resistance in Phytophthora spp., and will be useful for the development of novel oxysterol-binding protein homolog inhibitor fungicides.},
}

CRISPR-Cas Systems
Capsicum/*microbiology
Drug Resistance/*genetics
Fungicides, Industrial/*pharmacology
Gene Editing
Hydrocarbons, Fluorinated/*pharmacology
Phenotype
Phytophthora/drug effects/*genetics
Plant Diseases/*microbiology
Point Mutation
Pyrazoles/*pharmacology
Receptors, Steroid/*genetics
Sporangia/drug effects/genetics

@article {pmid29579237,
year = {2018},
author = {Kim, MJ and Jeon, S and Burbulla, LF and Krainc, D},
title = {Acid ceramidase inhibition ameliorates α-synuclein accumulation upon loss of GBA1 function.},
journal = {Human molecular genetics},
volume = {27},
number = {11},
pages = {1972-1988},
pmid = {29579237},
issn = {1460-2083},
support = {P30 CA138313/CA/NCI NIH HHS/United States ; P30 GM103339/GM/NIGMS NIH HHS/United States ; R01 NS076054/NS/NINDS NIH HHS/United States ; },
mesh = {Autophagy/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Ceramides/genetics/metabolism ; Dopaminergic Neurons/metabolism/pathology ; Gene Editing ; Gene Expression/genetics ; Glucosylceramidase/*genetics/pharmacokinetics ; Humans ; Lewy Body Disease/genetics/pathology ; Lysosomes/genetics/metabolism ; Parkinson Disease/*genetics/metabolism/pathology ; Protein Isoforms/genetics ; alpha-Synuclein/*genetics ; rab GTP-Binding Proteins/*genetics ; },
abstract = {GBA1 encodes the lysosomal enzyme β-glucocerebrosidase (GCase) which converts glucosylceramide into ceramide and glucose. Mutations in GBA1 lead to Gaucher's disease and are a major risk factor for Parkinson's disease (PD) and Dementia with Lewy bodies (DLB), synucleinopathies characterized by accumulation of intracellular α-synuclein. In this study, we examined whether decreased ceramide that is observed in GCase-deficient cells contributes to α-synuclein accumulation. We demonstrated that deficiency of GCase leads to a reduction of C18-ceramide species and altered intracellular localization of Rab8a, a small GTPase implicated in secretory autophagy, that contributed to impaired secretion of α-synuclein and accumulation of intracellular α-synuclein. This secretory defect was rescued by exogenous C18-ceramide or chemical inhibition of lysosomal enzyme acid ceramidase that converts lysosomal ceramide into sphingosine. Inhibition of acid ceramidase by carmofur resulted in increased ceramide levels and decreased glucosylsphingosine levels in GCase-deficient cells, and also reduced oxidized α-synuclein and levels of ubiquitinated proteins in GBA1-PD patient-derived dopaminergic neurons. Together, these results suggest that decreased ceramide generation via the catabolic lysosomal salvage pathway in GCase mutant cells contributes to α-synuclein accumulation, potentially due to impaired secretory autophagy. We thus propose that acid ceramidase inhibition which restores ceramide levels may be a potential therapeutic strategy to target synucleinopathies linked to GBA1 mutations including PD and DLB.},
}

Autophagy/genetics
CRISPR-Cas Systems/genetics
Cell Line
Ceramides/genetics/metabolism
Dopaminergic Neurons/metabolism/pathology
Gene Editing
Gene Expression/genetics
Glucosylceramidase/*genetics/pharmacokinetics
Humans
Lewy Body Disease/genetics/pathology
Lysosomes/genetics/metabolism
Parkinson Disease/*genetics/metabolism/pathology
Protein Isoforms/genetics
alpha-Synuclein/*genetics
rab GTP-Binding Proteins/*genetics

@article {pmid30759237,
year = {2019},
author = {Dorsey, BW and Huang, L and Mondragón, A},
title = {Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkz079},
pmid = {30759237},
issn = {1362-4962},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types. Class 1 CRISPR-Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, and provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.},
}

@article {pmid30755626,
year = {2019},
author = {Fernandes, LGV and Guaman, LP and Vasconcellos, SA and Heinemann, MB and Picardeau, M and Nascimento, ALTO},
title = {Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1839},
doi = {10.1038/s41598-018-37949-x},
pmid = {30755626},
issn = {2045-2322},
abstract = {Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the β-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.},
}

@article {pmid30659180,
year = {2019},
author = {Profumo, V and Forte, B and Percio, S and Rotundo, F and Doldi, V and Ferrari, E and Fenderico, N and Dugo, M and Romagnoli, D and Benelli, M and Valdagni, R and Dolfini, D and Zaffaroni, N and Gandellini, P},
title = {LEADeR role of miR-205 host gene as long noncoding RNA in prostate basal cell differentiation.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {307},
doi = {10.1038/s41467-018-08153-2},
pmid = {30659180},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Cell Differentiation ; Cell Line, Tumor ; Cells, Cultured ; Epithelial Cells/metabolism ; Gene Editing ; Humans ; Male ; MicroRNAs/*genetics ; Promoter Regions, Genetic ; Prostate/*cytology/metabolism ; RNA, Long Noncoding/*genetics ; Ribonuclease III/metabolism ; Transcriptome ; },
abstract = {Though miR-205 function has been largely characterized, the nature of its host gene, MIR205HG, is still completely unknown. Here, we show that only lowly expressed alternatively spliced MIR205HG transcripts act as de facto pri-miRNAs, through a process that involves Drosha to prevent unfavorable splicing and directly mediate miR-205 excision. Notably, MIR205HG-specific processed transcripts revealed to be functional per se as nuclear long noncoding RNA capable of regulating differentiation of human prostate basal cells through control of the interferon pathway. At molecular level, MIR205HG directly binds the promoters of its target genes, which have an Alu element in proximity of the Interferon-Regulatory Factor (IRF) binding site, and represses their transcription likely buffering IRF1 activity, with the ultimate effect of preventing luminal differentiation. As MIR205HG functions autonomously from (albeit complementing) miR-205 in preserving the basal identity of prostate epithelial cells, it warrants reannotation as LEADeR (Long Epithelial Alu-interacting Differentiation-related RNA).},
}

CRISPR-Cas Systems
Cell Differentiation
Cell Line, Tumor
Cells, Cultured
Epithelial Cells/metabolism
Gene Editing
Humans
Male
MicroRNAs/*genetics
Promoter Regions, Genetic
Prostate/*cytology/metabolism
RNA, Long Noncoding/*genetics
Ribonuclease III/metabolism
Transcriptome

@article {pmid29941452,
year = {2018},
author = {Wang, S and Ren, S and Bai, R and Xiao, P and Zhou, Q and Zhou, Y and Zhou, Z and Niu, Y and Ji, W and Chen, Y},
title = {No off-target mutations in functional genome regions of a CRISPR/Cas9-generated monkey model of muscular dystrophy.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {30},
pages = {11654-11658},
pmid = {29941452},
issn = {1083-351X},
mesh = {Animals ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Mutational Analysis/methods ; Disease Models, Animal ; *Gene Editing/methods ; Gene Knockout Techniques/methods ; Gene Library ; Macaca mulatta ; Muscular Dystrophy, Duchenne/*genetics ; *Mutation ; *Whole Genome Sequencing/methods ; },
abstract = {CRISPR/Cas9 is now widely used in biomedical research and has great potential for clinical applications. However, the safety and efficacy of this gene-editing technique are significant issues. Recent reports on mouse models and human cells have raised concerns that off-target mutations could hamper applying the CRISPR technology in patients. The high similarities of nonhuman primates to humans in genome content and organization, genetic diversity, physiology, and cognitive abilities have made these animals ideal experimental models for understanding human diseases and developing therapeutics. Off-target mutations of CRISPR/Cas9 have been analyzed in previous studies of nonhuman primates, but no report has investigated genome-wide off-target effects in living monkeys. Here, we used rhesus monkeys in which a genetic disorder mimicking Duchenne muscular dystrophy had previously been produced with CRISPR/Cas9. Using whole-genome sequencing to comprehensively assess on- and off-target mutations in these animals, we found that CRISPR/Cas9-based gene editing is active on the expected genomic sites without producing off-target modifications in other functional regions of the genome. These findings suggest that the CRISPR/Cas9 technique could be relatively safe and effective in modeling genetic disease in nonhuman primates and in future therapeutic research of human diseases.},
}

Animals
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
DNA Mutational Analysis/methods
Disease Models, Animal
*Gene Editing/methods
Gene Knockout Techniques/methods
Gene Library
Macaca mulatta
Muscular Dystrophy, Duchenne/*genetics
*Mutation
*Whole Genome Sequencing/methods

@article {pmid29087011,
year = {2018},
author = {Curtin, SJ and Xiong, Y and Michno, JM and Campbell, BW and Stec, AO and Čermák, T and Starker, C and Voytas, DF and Eamens, AL and Stupar, RM},
title = {CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula.},
journal = {Plant biotechnology journal},
volume = {16},
number = {6},
pages = {1125-1137},
pmid = {29087011},
issn = {1467-7652},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant/*genetics ; Medicago truncatula/*genetics/metabolism ; Mutagenesis, Site-Directed ; RNA/*metabolism ; RNA-Binding Proteins/*genetics ; Soybeans/*genetics/metabolism ; Transcription Activator-Like Effector Nucleases ; },
abstract = {Processing of double-stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL-effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi-allelic double mutant for the two soya bean paralogous Double-stranded RNA-binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9-generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ-line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer-like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer-like3 gene and the GmHen1a gene was observed in the T0 generation, but these mutations failed to transmit to the T1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole-genome sequencing to reveal a spectrum of non-germ-line-targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops.},
}

Base Sequence
CRISPR-Cas Systems
Gene Expression Regulation, Plant/*genetics
Medicago truncatula/*genetics/metabolism
Mutagenesis, Site-Directed
RNA/*metabolism
RNA-Binding Proteins/*genetics
Soybeans/*genetics/metabolism
Transcription Activator-Like Effector Nucleases

@article {pmid30745359,
year = {2019},
author = {Malfacini, D and Patt, J and Annala, S and Harpsøe, K and Eryilmaz, F and Reher, R and Crüsemann, M and Hanke, W and Zhang, H and Tietze, D and Gloriam, DE and Bräuner-Osborne, H and Strømgaard, K and König, GM and Inoue, A and Gomeza, J and Kostenis, E},
title = {Rational design of a heterotrimeric G protein α subunit with artificial inhibitor sensitivity.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA118.007250},
pmid = {30745359},
issn = {1083-351X},
abstract = {Transmembrane signals initiated by a range of extracellular stimuli converge on members of the Gq family of heterotrimeric G proteins, which relay these signals in target cells. Gq family G proteins comprise Gq, G11, G14, and G16, which upon activation mediate their cellular effects via inositol lipid-dependent and -independent signaling to control fundamental processes in mammalian physiology. To date, highly specific inhibition of Gq/11/14 signaling can be achieved only with FR900359 (FR) and YM-254890 (YM), two naturally occurring cyclic depsipeptides. To further development of FR or YM mimics for other Gα subunits, we here set out to rationally design Gα16 proteins with artificial FR/YM sensitivity by introducing an engineered depsipeptide binding site. Thereby we permit control of G16 function through ligands that are inactive on the wild type protein. Using CRISPR/Cas9-generated Gαq/Gα11-null cells and loss- and gain-of-function mutagenesis along with label-free whole-cell biosensing, we determined the molecular coordinates for FR/YM inhibition of Gq and transplanted these to FR/YM-insensitive G16. Intriguingly, despite having close structural similarity, FR and YM yielded biologically distinct activities: it was more difficult to perturb Gq inhibition by FR and easier to install FR inhibition onto G16 than perturb or install inhibition with YM. A unique hydrophobic network utilized by FR accounted for these unexpected discrepancies. Our results suggest that non-Gq/11/14 proteins should be amenable to inhibition by FR scaffold-based inhibitors, provided that these inhibitors mimic the interaction of FR with Gα proteins harboring engineered FR-binding sites.},
}

@article {pmid30700876,
year = {2019},
author = {Braun, M and Meacham, D},
title = {UN could lead debate on gene editing.},
journal = {Nature},
volume = {565},
number = {7741},
pages = {567},
doi = {10.1038/d41586-019-00324-5},
pmid = {30700876},
issn = {1476-4687},
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; Genome, Human ; Humans ; United Nations ; },
}

*CRISPR-Cas Systems
*Gene Editing
Genome, Human
Humans
United Nations

@article {pmid30390160,
year = {2019},
author = {Smirnikhina, SA and Anuchina, AA and Lavrov, AV},
title = {Ways of improving precise knock-in by genome-editing technologies.},
journal = {Human genetics},
volume = {138},
number = {1},
pages = {1-19},
doi = {10.1007/s00439-018-1953-5},
pmid = {30390160},
issn = {1432-1203},
support = {17-75-20095//Russian Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; Gene Knock-In Techniques/*methods ; Genetic Engineering/*methods ; Humans ; },
abstract = {Despite the recent discover of genome-editing methods, today we can say these approaches have firmly entered our life. Two approaches-knocking out malfunctioning gene allele or correcting the mutation with precise knock-in-can be used in hereditary monogenic diseases treatment. The latter approach is relatively ineffective. Modern data about the ways of repair of double-strand DNA breaks formed by nucleases are presented in this review. The main part of the review is devoted to the ways of increasing precise and effective knock-in: inhibition of non-homologous end joining and stimulation of homology-directed repair key factors, use of small molecules with unknown mechanism of action, cell-cycle synchronization and cell-cycle-dependent activity of Cas9, donor molecule design, selection, alternative methods for insertion and other approaches.},
}

*CRISPR-Cas Systems
Gene Editing/*methods
Gene Knock-In Techniques/*methods
Genetic Engineering/*methods
Humans

@article {pmid30320547,
year = {2018},
author = {Vuolo, L and Stevenson, NL and Heesom, KJ and Stephens, DJ},
title = {Dynein-2 intermediate chains play crucial but distinct roles in primary cilia formation and function.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30320547},
issn = {2050-084X},
support = {MR/P000177/1//Medical Research Council/United Kingdom ; BB/N000420/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; MR/K018019/1//Medical Research Council/United Kingdom ; BB/L014181/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {Adaptor Proteins, Signal Transducing/chemistry/*metabolism ; Amino Acid Sequence ; Animals ; CRISPR-Cas Systems/genetics ; Carrier Proteins/chemistry/*metabolism ; Cell Line ; Cell Membrane/metabolism ; Cilia/*metabolism/ultrastructure ; Dyneins/*metabolism ; Gene Knockout Techniques ; Humans ; Mice ; Mutation/genetics ; Phenotype ; Tumor Suppressor Proteins/metabolism ; },
abstract = {The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport.},
}

Adaptor Proteins, Signal Transducing/chemistry/*metabolism
Amino Acid Sequence
Animals
CRISPR-Cas Systems/genetics
Carrier Proteins/chemistry/*metabolism
Cell Line
Cell Membrane/metabolism
Cilia/*metabolism/ultrastructure
Dyneins/*metabolism
Gene Knockout Techniques
Humans
Mice
Mutation/genetics
Phenotype
Tumor Suppressor Proteins/metabolism

@article {pmid30275281,
year = {2018},
author = {Zhang, H and Pan, H and Zhou, C and Wei, Y and Ying, W and Li, S and Wang, G and Li, C and Ren, Y and Li, G and Ding, X and Sun, Y and Li, GL and Song, L and Li, Y and Yang, H and Liu, Z},
title = {Simultaneous zygotic inactivation of multiple genes in mouse through CRISPR/Cas9-mediated base editing.},
journal = {Development (Cambridge, England)},
volume = {145},
number = {20},
pages = {},
doi = {10.1242/dev.168906},
pmid = {30275281},
issn = {1477-9129},
mesh = {Amino Acid Transport Systems, Acidic/metabolism ; Animals ; Base Sequence ; CRISPR-Cas Systems/*genetics ; Cochlea/metabolism ; Deafness/genetics/physiopathology ; Disease Models, Animal ; Electrophysiological Phenomena ; Gene Editing/*methods ; Membrane Proteins/metabolism ; Mice ; Molecular Motor Proteins/metabolism ; Mutation/genetics ; Zygote/*metabolism ; },
abstract = {In vivo genetic mutation has become a powerful tool for dissecting gene function; however, multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations, at best difficult to interpret, and, at worst, misleading. Hence, it is necessary to establish an efficient way to disrupt multiple genes simultaneously. CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon; this is referred to as CRISPR-stop. Its application in generating zygotic mutations has not been well explored yet. Here, we first performed a proof-of-principle test by disrupting Atoh1, a gene crucial for auditory hair cell generation. Next, we individually mutated vGlut3 (Slc17a8), otoferlin (Otof) and prestin (Slc26a5), three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otof and prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for high-throughput screening of mouse developmental and functional genes, matching the efficiency of methods available for model organisms such as Drosophila.},
}

Amino Acid Transport Systems, Acidic/metabolism
Animals
Base Sequence
CRISPR-Cas Systems/*genetics
Cochlea/metabolism
Deafness/genetics/physiopathology
Disease Models, Animal
Electrophysiological Phenomena
Gene Editing/*methods
Membrane Proteins/metabolism
Mice
Molecular Motor Proteins/metabolism
Mutation/genetics
Zygote/*metabolism

@article {pmid30266827,
year = {2018},
author = {Vaid, S and Camp, JG and Hersemann, L and Eugster Oegema, C and Heninger, AK and Winkler, S and Brandl, H and Sarov, M and Treutlein, B and Huttner, WB and Namba, T},
title = {A novel population of Hopx-dependent basal radial glial cells in the developing mouse neocortex.},
journal = {Development (Cambridge, England)},
volume = {145},
number = {20},
pages = {},
doi = {10.1242/dev.169276},
pmid = {30266827},
issn = {1477-9129},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Proliferation ; Embryo, Mammalian/metabolism ; Ependymoglial Cells/*metabolism ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Homeodomain Proteins/*metabolism ; Humans ; Lateral Ventricles/embryology ; Mice, Inbred C57BL ; Neocortex/*cytology/*embryology/metabolism ; PAX6 Transcription Factor/metabolism ; Stem Cells/cytology ; },
abstract = {A specific subpopulation of neural progenitor cells, the basal radial glial cells (bRGCs) of the outer subventricular zone (OSVZ), are thought to have a key role in the evolutionary expansion of the mammalian neocortex. In the developing lissencephalic mouse neocortex, bRGCs exist at low abundance and show significant molecular differences from bRGCs in developing gyrencephalic species. Here, we demonstrate that the developing mouse medial neocortex (medNcx), in contrast to the canonically studied lateral neocortex (latNcx), exhibits an OSVZ and an abundance of bRGCs similar to that in developing gyrencephalic neocortex. Unlike bRGCs in developing mouse latNcx, the bRGCs in medNcx exhibit human bRGC-like gene expression, including expression of Hopx, a human bRGC marker. Disruption of Hopx expression in mouse embryonic medNcx and forced Hopx expression in mouse embryonic latNcx demonstrate that Hopx is required and sufficient, respectively, for bRGC abundance as found in the developing gyrencephalic neocortex. Taken together, our data identify a novel bRGC subpopulation in developing mouse medNcx that is highly related to bRGCs of developing gyrencephalic neocortex.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Proliferation
Embryo, Mammalian/metabolism
Ependymoglial Cells/*metabolism
Gene Expression Profiling
Gene Expression Regulation, Developmental
Homeodomain Proteins/*metabolism
Humans
Lateral Ventricles/embryology
Mice, Inbred C57BL
Neocortex/*cytology/*embryology/metabolism
PAX6 Transcription Factor/metabolism
Stem Cells/cytology

@article {pmid29764955,
year = {2018},
author = {Logsdon, GA and Black, BE},
title = {The new normal of structure/function studies in the era of CRISPR/Cas9.},
journal = {The Biochemical journal},
volume = {475},
number = {9},
pages = {1635-1642},
doi = {10.1042/BCJ20170025},
pmid = {29764955},
issn = {1470-8728},
support = {R01 GM082989/GM/NIGMS NIH HHS/United States ; R01 GM105654/GM/NIGMS NIH HHS/United States ; T32 GM007229/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing ; *Genome, Human ; Humans ; Proteins/*chemistry/genetics/*metabolism ; Structure-Activity Relationship ; },
abstract = {Major advances in gene-editing technologies have enabled the rapid dissection of proteins in complex biological systems, facilitating biological experiments to complement biochemical studies with purified components. In this editorial, we highlight CRISPR/Cas9-based strategies to rapidly manipulate endogenous genes - strategies that have already transformed functional studies of proteins in metazoan systems. We further describe emerging tools using a catalytically dead version of Cas9 (dCas9) that do not cleave DNA, but can alter gene expression and/or local chromatin states, edit single nucleotide bases, and permit the visualization of specific genomic loci. Looking to the not-too-distant future, CRISPR/Cas9-based methodologies promise to lead to discoveries of new biology, opening the door for bold new synthetic biology platforms.},
}

*CRISPR-Cas Systems
*Gene Editing
*Genome, Human
Humans
Proteins/*chemistry/genetics/*metabolism
Structure-Activity Relationship

@article {pmid29603460,
year = {2018},
author = {Harrison, P and Hart, S},
title = {Gene editing and gene regulation with CRISPR.},
journal = {Experimental physiology},
volume = {103},
number = {4},
pages = {437-438},
doi = {10.1113/EP086864},
pmid = {29603460},
issn = {1469-445X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Editing/methods ; Gene Expression Regulation/*genetics ; Humans ; Mutation/genetics ; RNA, Guide/genetics ; },
}

Animals
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Editing/methods
Gene Expression Regulation/*genetics
Humans
Mutation/genetics
RNA, Guide/genetics

@article {pmid29544468,
year = {2018},
author = {Maurer, JM and Sagerström, CG},
title = {A parental requirement for dual-specificity phosphatase 6 in zebrafish.},
journal = {BMC developmental biology},
volume = {18},
number = {1},
pages = {6},
pmid = {29544468},
issn = {1471-213X},
support = {R01 NS038183/NS/NINDS NIH HHS/United States ; R56 NS038183/NS/NINDS NIH HHS/United States ; NS038183//Foundation for the National Institutes of Health/International ; },
mesh = {Alleles ; Animals ; Base Sequence ; CRISPR-Cas Systems/genetics ; Cell Division/drug effects ; Embryo, Nonmammalian/cytology/drug effects/metabolism ; Embryonic Development/drug effects ; Female ; Gastrulation/drug effects ; Gene Editing ; Gene Expression Regulation, Developmental/drug effects ; Gene Knockdown Techniques ; Germ Cells/drug effects/metabolism ; Homozygote ; Male ; Morpholinos/pharmacology ; Mutation/genetics ; Ovary/metabolism ; Phenotype ; Rhombencephalon/drug effects/metabolism ; Testis/metabolism ; Zebrafish/embryology/*metabolism ; Zebrafish Proteins/genetics/*metabolism ; },
abstract = {BACKGROUND: Signaling cascades, such as the extracellular signal-regulated kinase (ERK) pathway, play vital roles in early vertebrate development. Signals through these pathways are initiated by a growth factor or hormone, are transduced through a kinase cascade, and result in the expression of specific downstream genes that promote cellular proliferation, growth, or differentiation. Tight regulation of these signals is provided by positive or negative modulators at varying levels in the pathway, and is required for proper development and function. Two members of the dual-specificity phosphatase (Dusp) family, dusp6 and dusp2, are believed to be negative regulators of the ERK pathway and are expressed in both embryonic and adult zebrafish, but their specific roles in embryogenesis remain to be fully understood.

RESULTS: Using CRISPR/Cas9 genome editing technology, we generated zebrafish lines harboring germ line deletions in dusp6 and dusp2. We do not detect any overt defects in dusp2 mutants, but we find that approximately 50% of offspring from homozygous dusp6 mutants do not proceed through embryonic development. These embryos are fertilized, but are unable to proceed past the first zygotic mitosis and stall at the 1-cell stage for several hours before dying by 10 h post fertilization. We demonstrate that dusp6 is expressed in gonads of both male and female zebrafish, suggesting that loss of dusp6 causes defects in germ cell production. Notably, the 50% of homozygous dusp6 mutants that complete the first cell division appear to progress through embryogenesis normally and give rise to fertile adults.

CONCLUSIONS: The fact that offspring of homozygous dusp6 mutants stall prior to activation of the zygotic genome, suggests that loss of dusp6 affects gametogenesis and/or parentally-directed early development. Further, since only approximately 50% of homozygous dusp6 mutants are affected, we postulate that ERK signaling is tightly regulated and that dusp6 is required to keep ERK signaling within a range that is permissive for proper embryogenesis. Lastly, since dusp6 is expressed throughout zebrafish embryogenesis, but dusp6 mutants do not exhibit defects after the first cell division, it is possible that other regulators of the ERK pathway compensate for loss of dusp6 at later stages.},
}

Alleles
Animals
Base Sequence
CRISPR-Cas Systems/genetics
Cell Division/drug effects
Embryo, Nonmammalian/cytology/drug effects/metabolism
Embryonic Development/drug effects
Female
Gastrulation/drug effects
Gene Editing
Gene Expression Regulation, Developmental/drug effects
Gene Knockdown Techniques
Germ Cells/drug effects/metabolism
Homozygote
Male
Morpholinos/pharmacology
Mutation/genetics
Ovary/metabolism
Phenotype
Rhombencephalon/drug effects/metabolism
Testis/metabolism
Zebrafish/embryology/*metabolism
Zebrafish Proteins/genetics/*metabolism

@article {pmid29282799,
year = {2018},
author = {Harrison, PT and Hart, S},
title = {A beginner's guide to gene editing.},
journal = {Experimental physiology},
volume = {103},
number = {4},
pages = {439-448},
doi = {10.1113/EP086047},
pmid = {29282799},
issn = {1469-445X},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/genetics ; Gene Editing/methods ; Genome/*genetics ; Humans ; Zinc Finger Nucleases/genetics ; },
abstract = {NEW FINDINGS: What is the topic of this review? This review summarizes the development of gene editing from early proof-of-concept studies in the 1980s to contemporary programmable and RNA-guided nucleases, which enable rapid and precise alteration of DNA sequences of almost any living cell. What advances does it highlight? With an average of one clustered regularly interspaced short palindromic repeat (CRISPR) Cas9 paper published every 4 h in 2017, this review cannot highlight all new developments, but a number of key improvements, including increases in efficiency, a range of new options to reduce off-target effects and plans for CRISPR to enter clinical trials in 2018, are discussed.

ABSTRACT: Genome editing enables precise changes to be made in the genome of living cells. The technique was originally developed in the 1980s but largely limited to use in mice. The discovery that a targeted double-stranded break at a unique site in the genome, close to the site to be changed, could substantially increase the efficiency of editing raised the possibility of using the technique in a broader range of animal models and, potentially, human cells. But the challenge was to identify reagents that could create targeted breaks at a unique genomic location with minimal off-target effects. In 2005, the demonstration that programmable zinc finger nucleases (ZFNs) could perform this task led to a number of proof-of-concept studies, but a limitation was the ease with which effective ZFNs could be produced. In 2009, the development of TAL effector nucleases (TALENs) increased the specificity of gene editing and the ease of design and production. However, it was not until 2013 and the development of the clustered regularly interspaced short palindromic repeat (CRISPR) Cas9/guide RNA that gene editing became a research tool that any laboratory could use.},
}

Animals
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Endonucleases/genetics
Gene Editing/methods
Genome/*genetics
Humans
Zinc Finger Nucleases/genetics

@article {pmid28706248,
year = {2017},
author = {Yoon, S and Wang, P and Peng, Q and Wang, Y and Shung, KK},
title = {Acoustic-transfection for genomic manipulation of single-cells using high frequency ultrasound.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {5275},
pmid = {28706248},
issn = {2045-2322},
support = {P41 EB002182/EB/NIBIB NIH HHS/United States ; R01 GM125379/GM/NIGMS NIH HHS/United States ; K02 HL109142/HL/NHLBI NIH HHS/United States ; R33 CA204704/CA/NCI NIH HHS/United States ; R01 HL121365/HL/NHLBI NIH HHS/United States ; K99 GM120493/GM/NIGMS NIH HHS/United States ; R21 CA209629/CA/NCI NIH HHS/United States ; },
mesh = {*Acoustics ; *CRISPR-Cas Systems ; *Gene Editing ; Genome, Human ; Genomics/*methods ; Green Fluorescent Proteins/*genetics ; HeLa Cells ; Humans ; Plasmids/administration & dosage ; Single-Cell Analysis ; Transfection/*methods ; Ultrasonography/*instrumentation/methods ; },
abstract = {Efficient intracellular delivery of biologically active macromolecules has been a challenging but important process for manipulating live cells for research and therapeutic purposes. There have been limited transfection techniques that can deliver multiple types of active molecules simultaneously into single-cells as well as different types of molecules into physically connected individual neighboring cells separately with high precision and low cytotoxicity. Here, a high frequency ultrasound-based remote intracellular delivery technique capable of delivery of multiple DNA plasmids, messenger RNAs, and recombinant proteins is developed to allow high spatiotemporal visualization and analysis of gene and protein expressions as well as single-cell gene editing using clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9), a method called acoustic-transfection. Acoustic-transfection has advantages over typical sonoporation because acoustic-transfection utilizing ultra-high frequency ultrasound over 150 MHz can directly deliver gene and proteins into cytoplasm without microbubbles, which enables controlled and local intracellular delivery to acoustic-transfection technique. Acoustic-transfection was further demonstrated to deliver CRISPR-Cas9 systems to successfully modify and reprogram the genome of single live cells, providing the evidence of the acoustic-transfection technique for precise genome editing using CRISPR-Cas9.},
}

*Acoustics
*CRISPR-Cas Systems
*Gene Editing
Genome, Human
Genomics/*methods
Green Fluorescent Proteins/*genetics
HeLa Cells
Humans
Plasmids/administration & dosage
Single-Cell Analysis
Transfection/*methods
Ultrasonography/*instrumentation/methods

@article {pmid28303677,
year = {2018},
author = {Lee, CM and Davis, TH and Bao, G},
title = {Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.},
journal = {Experimental physiology},
volume = {103},
number = {4},
pages = {456-460},
doi = {10.1113/EP086043},
pmid = {28303677},
issn = {1469-445X},
mesh = {Animals ; CRISPR-Associated Protein 9/*metabolism ; CRISPR-Cas Systems/*genetics ; DNA/genetics ; Gene Editing/methods ; Genome/genetics ; Humans ; RNA, Guide/genetics ; },
abstract = {NEW FINDINGS: What is the topic of this review? In this review, we analyse the performance of recently described tools for CRISPR/Cas9 guide RNA design, in particular, design tools that predict CRISPR/Cas9 activity. What advances does it highlight? Recently, many tools designed to predict CRISPR/Cas9 activity have been reported. However, the majority of these tools lack experimental validation. Our analyses indicate that these tools have poor predictive power. Our preliminary results suggest that target site accessibility should be considered in order to develop better guide RNA design tools with improved predictive power. The recent adaptation of the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system for targeted genome engineering has led to its widespread application in many fields worldwide. In order to gain a better understanding of the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design, these studies have spawned a plethora of guide RNA (gRNA) design tools with algorithms based solely on direct or indirect sequence features. Here, we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore, we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimization, we hypothesize that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics.},
}

Animals
CRISPR-Associated Protein 9/*metabolism
CRISPR-Cas Systems/*genetics
DNA/genetics
Gene Editing/methods
Genome/genetics
Humans
RNA, Guide/genetics

@article {pmid28271571,
year = {2018},
author = {March, OP and Reichelt, J and Koller, U},
title = {Gene editing for skin diseases: designer nucleases as tools for gene therapy of skin fragility disorders.},
journal = {Experimental physiology},
volume = {103},
number = {4},
pages = {449-455},
doi = {10.1113/EP086044},
pmid = {28271571},
issn = {1469-445X},
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genetic Therapy/methods ; Humans ; Skin Diseases/*genetics/*therapy ; Transcription Activator-Like Effector Nucleases/genetics ; },
abstract = {NEW FINDINGS: What is the topic of this review? This review concerns current gene editing strategies for blistering skin diseases with respect to individual genetic constellations and distinct conditions. What advances does it highlight? Specificity and safety dominate the discussion of gene editing applications for gene therapy, where a number of tools are implemented. Recent developments in this rapidly progressing field pose further questions regarding which tool is best suited for each particular use. The current treatment of inherited blistering skin diseases, such as epidermolysis bullosa (EB), is largely restricted to wound care and pain management. More effective therapeutic strategies are urgently required, and targeting the genetic basis of these severe diseases is now within reach. Here, we describe current gene editing tools and their potential to correct gene function in monogenetic blistering skin diseases. We present the features of the most frequently used gene editing techniques, transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), determining their preferential application for specific genetic conditions, including the type of mutational inheritance, the targeting site within the gene or the possibility to target the mutation specifically. Both tools have traits beneficial in specific situations. Promising developments in the field engender gene editing as a potentially powerful therapeutic option for future clinical applications.},
}

Animals
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Editing/methods
Genetic Therapy/methods
Humans
Skin Diseases/*genetics/*therapy
Transcription Activator-Like Effector Nucleases/genetics

@article {pmid30742049,
year = {2019},
author = {McKenzie, RE and Almendros, C and Vink, JNA and Brouns, SJJ},
title = {Using CAPTURE to detect spacer acquisition in native CRISPR arrays.},
journal = {Nature protocols},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41596-018-0123-5},
pmid = {30742049},
issn = {1750-2799},
abstract = {CRISPR-Cas systems are able to acquire immunological memories (spacers) from bacteriophages and plasmids in order to survive infection; however, this often occurs at low frequency within a population, which can make it difficult to detect. Here we describe CAPTURE (CRISPR adaptation PCR technique using reamplification and electrophoresis), a versatile and adaptable protocol to detect spacer-acquisition events by electrophoresis imaging with high-enough sensitivity to identify spacer acquisition in 1 in 105 cells. Our method harnesses two simple PCR steps, separated by automated electrophoresis and extraction of size-selected DNA amplicons, thus allowing the removal of unexpanded arrays from the sample pool and enabling 1,000-times more sensitive detection of new spacers than alternative PCR protocols. CAPTURE is a straightforward method that requires only 1 d to enable the detection of spacer acquisition in all native CRISPR systems and facilitate studies aimed both at unraveling the mechanism of spacer integration and more sensitive tracing of integration events in natural ecosystems.},
}

@article {pmid30368976,
year = {2019},
author = {Tanihara, F and Hirata, M and Nguyen, NT and Le, QA and Hirano, T and Takemoto, T and Nakai, M and Fuchimoto, DI and Otoi, T},
title = {Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation.},
journal = {Animal science journal = Nihon chikusan Gakkaiho},
volume = {90},
number = {1},
pages = {55-61},
doi = {10.1111/asj.13129},
pmid = {30368976},
issn = {1740-0929},
support = {//Funds for the Development of Human Resources in Science and Technology/ ; //Home for Innovative Researchers and Academic Knowledge Users (HIRAKU)/ ; JP17H03938//JSPS KAKENHI/ ; JP17K19325//JSPS KAKENHI/ ; //SATREPS/ ; },
mesh = {Animals ; *Blastocyst ; CRISPR-Associated Protein 9/*genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; *Electroporation ; Female ; Fertilization in Vitro ; Gene Editing/*methods ; Homeodomain Proteins/*genetics ; *Mutation ; RNA, Guide/genetics ; Swine/*genetics ; Trans-Activators/*genetics ; *Zygote ; },
abstract = {Recently, we established the GEEP ("gene editing by electroporation of Cas9 protein") method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome-editing efficiency. To produce an animal model for diabetes, we targeted PDX-1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX-1 exon 1 was introduced into in vitro-fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX-1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX-1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.},
}

Animals
*Blastocyst
CRISPR-Associated Protein 9/*genetics
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
*Electroporation
Female
Fertilization in Vitro
Gene Editing/*methods
Homeodomain Proteins/*genetics
*Mutation
RNA, Guide/genetics
Swine/*genetics
Trans-Activators/*genetics
*Zygote

@article {pmid30143771,
year = {2018},
author = {Nelson, C},
title = {Change the ORF, please.},
journal = {Lab animal},
volume = {47},
number = {9},
pages = {232},
doi = {10.1038/s41684-018-0140-6},
pmid = {30143771},
issn = {1548-4475},
mesh = {Animals ; *Arthropods ; CRISPR-Cas Systems ; Female ; *Gene Editing ; Ovary ; Ribonucleoproteins ; },
}

Animals
*Arthropods
CRISPR-Cas Systems
Female
*Gene Editing
Ovary
Ribonucleoproteins

@article {pmid29997084,
year = {2018},
author = {Liu, YC and Chen, Q and Yang, XL and Tang, QS and Yao, KT and Xu, Y},
title = {[Generation of a new strain of NOD/SCID/IL2Rγ-/- mice with targeted disruption of Prkdc and IL2Rγ genes using CRISPR/Cas9 system].},
journal = {Nan fang yi ke da xue xue bao = Journal of Southern Medical University},
volume = {38},
number = {6},
pages = {639-646},
pmid = {29997084},
issn = {1673-4254},
mesh = {Animals ; B-Lymphocytes ; *CRISPR-Cas Systems ; DNA-Activated Protein Kinase/*genetics ; DNA-Binding Proteins/*genetics ; Interleukin Receptor Common gamma Subunit/*genetics ; Killer Cells, Natural ; Mice ; Mice, Inbred NOD/*genetics ; Mice, Knockout ; Mice, Nude ; Mice, SCID/*genetics ; Models, Animal ; Nuclear Proteins/*genetics ; Selective Breeding/genetics ; Species Specificity ; T-Lymphocytes ; *Transplantation, Heterologous ; },
abstract = {OBJECTIVE: The NOD/SCID/IL2Rγ- /- (NSG) mouse strain is the most widely used immunodeficient strain for xenograft transplantation. However, the existing SCID mutation is a spontaneous mutation of the Prkdc gene, which leads to leaky T cell developmental block and difficulty in genotyping. It is therefore important to develop a new strain of NSG mice with targeted disruption of Prkdc and IL2Rγ genes.

METHODS: Targeted disruption of Prkdc and IL2Rγ genes was achieved using the CRISPR/Cas9 system. By intercrossing the knockout and NOD mice, we obtained a novel strain of NOD/SCID/IL2Rγ- /-(NSG) mice, denoted as cNSG (Chinese NSG) mice.

RESULTS: In addition to the NOD mutation, cNSG mice exhibited a complete absence of T cells, B cells and NK cells. cNSG mice allowed more efficient engraftment of human cancer cells than the commonly used immunodeficient nude mice.

CONCLUSION: cNSG mice will provide an important xenotransplantation model for biomedical research.},
}

Animals
B-Lymphocytes
*CRISPR-Cas Systems
DNA-Activated Protein Kinase/*genetics
DNA-Binding Proteins/*genetics
Interleukin Receptor Common gamma Subunit/*genetics
Killer Cells, Natural
Mice
Mice, Inbred NOD/*genetics
Mice, Knockout
Mice, Nude
Mice, SCID/*genetics
Models, Animal
Nuclear Proteins/*genetics
Selective Breeding/genetics
Species Specificity
T-Lymphocytes
*Transplantation, Heterologous

@article {pmid29677511,
year = {2018},
author = {Bester, AC and Lee, JD and Chavez, A and Lee, YR and Nachmani, D and Vora, S and Victor, J and Sauvageau, M and Monteleone, E and Rinn, JL and Provero, P and Church, GM and Clohessy, JG and Pandolfi, PP},
title = {An Integrated Genome-wide CRISPRa Approach to Functionalize lncRNAs in Drug Resistance.},
journal = {Cell},
volume = {173},
number = {3},
pages = {649-664.e20},
pmid = {29677511},
issn = {1097-4172},
support = {P50 HG005550/HG/NHGRI NIH HHS/United States ; R35 CA197529/CA/NCI NIH HHS/United States ; RM1 HG008525/HG/NHGRI NIH HHS/United States ; T32 CA009216/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Cytarabine/pharmacology ; *Drug Resistance, Neoplasm ; Female ; Gene Expression Profiling ; Gene Regulatory Networks ; *Genome, Human ; HEK293 Cells ; HL-60 Cells ; Humans ; K562 Cells ; Leukemia, Myeloid, Acute/drug therapy/genetics ; Male ; Mice ; Pharmacogenetics ; Proteins/genetics ; RNA/analysis ; RNA, Long Noncoding/*genetics ; RNA, Messenger/genetics ; Signal Transduction ; },
abstract = {Resistance to chemotherapy plays a significant role in cancer mortality. To identify genetic units affecting sensitivity to cytarabine, the mainstay of treatment for acute myeloid leukemia (AML), we developed a comprehensive and integrated genome-wide platform based on a dual protein-coding and non-coding integrated CRISPRa screening (DICaS). Putative resistance genes were initially identified using pharmacogenetic data from 760 human pan-cancer cell lines. Subsequently, genome scale functional characterization of both coding and long non-coding RNA (lncRNA) genes by CRISPR activation was performed. For lncRNA functional assessment, we developed a CRISPR activation of lncRNA (CaLR) strategy, targeting 14,701 lncRNA genes. Computational and functional analysis identified novel cell-cycle, survival/apoptosis, and cancer signaling genes. Furthermore, transcriptional activation of the GAS6-AS2 lncRNA, identified in our analysis, leads to hyperactivation of the GAS6/TAM pathway, a resistance mechanism in multiple cancers including AML. Thus, DICaS represents a novel and powerful approach to identify integrated coding and non-coding pathways of therapeutic relevance.},
}

Animals
*CRISPR-Cas Systems
Cytarabine/pharmacology
*Drug Resistance, Neoplasm
Female
Gene Expression Profiling
Gene Regulatory Networks
*Genome, Human
HEK293 Cells
HL-60 Cells
Humans
K562 Cells
Leukemia, Myeloid, Acute/drug therapy/genetics
Male
Mice
Pharmacogenetics
Proteins/genetics
RNA/analysis
RNA, Long Noncoding/*genetics
RNA, Messenger/genetics
Signal Transduction

@article {pmid29622782,
year = {2018},
author = {Jang, DE and Lee, JY and Lee, JH and Koo, OJ and Bae, HS and Jung, MH and Bae, JH and Hwang, WS and Chang, YJ and Lee, YH and Lee, HW and Yeom, SC},
title = {Multiple sgRNAs with overlapping sequences enhance CRISPR/Cas9-mediated knock-in efficiency.},
journal = {Experimental & molecular medicine},
volume = {50},
number = {4},
pages = {16},
pmid = {29622782},
issn = {2092-6413},
mesh = {Animals ; Base Sequence ; *CRISPR-Cas Systems ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats ; Fibroblasts ; *Gene Knock-In Techniques ; Genetic Loci ; High-Throughput Nucleotide Sequencing ; Homologous Recombination ; Mice ; Polymorphism, Single Nucleotide ; RNA, Guide/*genetics ; Zygote ; },
abstract = {The CRISPR/Cas9 system is widely applied in genome engineering due to its simplicity and versatility. Although this has revolutionized genome-editing technology, knockin animal generation via homology directed repair (HDR) is not as efficient as nonhomologous end-joining DNA-repair-dependent knockout. Although its double-strand break activity may vary, Cas9 derived from Streptococcus pyogenens allows robust design of single-guide RNAs (sgRNAs) within the target sequence; However, prescreening for different sgRNA activities delays the process of transgenic animal generation. To overcome this limitation, multiple sets of different sgRNAs were examined for their knockin efficiency. We discovered profound advantages associated with single-stranded oligo-donor-mediated HDR processes using overlapping sgRNAs (sharing at least five base pairs of the target sites) as compared with using non-overlapping sgRNAs for knock-in mouse generation. Studies utilizing cell lines revealed shorter sequence deletions near target mutations using overlapping sgRNAs as compared with those observed using non-overlapping sgRNAs, which may favor the HDR process. Using this simple method, we successfully generated several transgenic mouse lines harboring loxP insertions or single-nucleotide substitutions with a highly efficiency of 18-38%. Our results demonstrate a simple and efficient method for generating transgenic animals harboring foreign-sequence knockins or short-nucleotide substitutions by the use of overlapping sgRNAs.},
}

Animals
Base Sequence
*CRISPR-Cas Systems
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats
Fibroblasts
*Gene Knock-In Techniques
Genetic Loci
High-Throughput Nucleotide Sequencing
Homologous Recombination
Mice
Polymorphism, Single Nucleotide
RNA, Guide/*genetics
Zygote

@article {pmid29578069,
year = {2018},
author = {Burnight, ER and Giacalone, JC and Cooke, JA and Thompson, JR and Bohrer, LR and Chirco, KR and Drack, AV and Fingert, JH and Worthington, KS and Wiley, LA and Mullins, RF and Stone, EM and Tucker, BA},
title = {CRISPR-Cas9 genome engineering: Treating inherited retinal degeneration.},
journal = {Progress in retinal and eye research},
volume = {65},
number = {},
pages = {28-49},
doi = {10.1016/j.preteyeres.2018.03.003},
pmid = {29578069},
issn = {1873-1635},
support = {T32 GM007337/GM/NIGMS NIH HHS/United States ; R01 EY024588/EY/NEI NIH HHS/United States ; R01 EY026008/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Genetic Therapy/*methods ; Humans ; Induced Pluripotent Stem Cells ; Retinal Degeneration/genetics/*therapy ; *Stem Cell Transplantation ; },
abstract = {Gene correction is a valuable strategy for treating inherited retinal degenerative diseases, a major cause of irreversible blindness worldwide. Single gene defects cause the majority of these retinal dystrophies. Gene augmentation holds great promise if delivered early in the course of the disease, however, many patients carry mutations in genes too large to be packaged into adeno-associated viral vectors and some, when overexpressed via heterologous promoters, induce retinal toxicity. In addition to the aforementioned challenges, some patients have sustained significant photoreceptor cell loss at the time of diagnosis, rendering gene replacement therapy insufficient to treat the disease. These patients will require cell replacement to restore useful vision. Fortunately, the advent of induced pluripotent stem cell and CRISPR-Cas9 gene editing technologies affords researchers and clinicians a powerful means by which to develop strategies to treat patients with inherited retinal dystrophies. In this review we will discuss the current developments in CRISPR-Cas9 gene editing in vivo in animal models and in vitro in patient-derived cells to study and treat inherited retinal degenerative diseases.},
}

Animals
CRISPR-Cas Systems/*genetics
Gene Editing/*methods
Genetic Therapy/*methods
Humans
Induced Pluripotent Stem Cells
Retinal Degeneration/genetics/*therapy
*Stem Cell Transplantation

@article {pmid29509513,
year = {2018},
author = {Tang, YD and Guo, JC and Wang, TY and Zhao, K and Liu, JT and Gao, JC and Tian, ZJ and An, TQ and Cai, XH},
title = {CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {32},
number = {8},
pages = {4293-4301},
doi = {10.1096/fj.201701129R},
pmid = {29509513},
issn = {1530-6860},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Cell Line ; Cercopithecus aethiops ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; DNA Viruses/*genetics ; Gene Editing/methods ; Gene Knockout Techniques/methods ; Genome, Viral/genetics ; Herpesvirus 1, Suid/genetics ; RNA, Guide/*genetics ; Transfection/methods ; Vero Cells ; },
abstract = {Several groups have used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) for DNA virus editing. In most cases, one single-guide RNA (sgRNA) is used, which produces inconsistencies in gene editing. In this study, we used a swine herpesvirus, pseudorabies virus, as a model to systematically explore the application of CRISPR/Cas9 in DNA virus editing. In our current report, we demonstrated that cotransfection of 2 sgRNAs and a viral genome resulted in significantly better knockout efficiency than the transfection-infection-based approach. This method could result in 100% knockout of ≤3500 bp of viral nonessential large fragments. Furthermore, knockin efficiency was significantly improved by using 2 sgRNAs and was also correlated with the number of background viruses. We also demonstrated that the background viruses were all 2-sgRNA-mediated knockout mutants. Finally, this study demonstrated that the efficacy of gene knockin is determined by the replicative kinetics of background viruses. We propose that CRISPR/Cas9 coupled with 2 sgRNAs creates a powerful tool for DNA virus editing and offers great potential for future applications.-Tang, Y.-D., Guo, J.-C., Wang, T.-Y., Zhao, K., Liu, J.-T., Gao, J.-C., Tian, Z.-J., An, T.-Q., Cai, X.-H. CRISPR/Cas9-mediated 2-sgRNA cleavage facilitates pseudorabies virus editing.},
}

Animals
CRISPR-Cas Systems/*genetics
Cell Line
Cercopithecus aethiops
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
DNA Viruses/*genetics
Gene Editing/methods
Gene Knockout Techniques/methods
Genome, Viral/genetics
Herpesvirus 1, Suid/genetics
RNA, Guide/*genetics
Transfection/methods
Vero Cells

@article {pmid29330178,
year = {2018},
author = {Li, K and Cai, D and Wang, Z and He, Z and Chen, S},
title = {Development of an Efficient Genome Editing Tool in Bacillus licheniformis Using CRISPR-Cas9 Nickase.},
journal = {Applied and environmental microbiology},
volume = {84},
number = {6},
pages = {},
pmid = {29330178},
issn = {1098-5336},
mesh = {Bacillus licheniformis/enzymology/*genetics ; Bacterial Proteins/*genetics/metabolism ; CRISPR-Cas Systems/*genetics ; Deoxyribonuclease I/*genetics/metabolism ; Gene Editing/*methods ; Subtilisins/*genetics/metabolism ; },
abstract = {Bacillus strains are important industrial bacteria that can produce various biochemical products. However, low transformation efficiencies and a lack of effective genome editing tools have hindered its widespread application. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 techniques have been utilized in many organisms as genome editing tools because of their high efficiency and easy manipulation. In this study, an efficient genome editing method was developed for Bacillus licheniformis using a CRISPR-Cas9 nickase integrated into the genome of B. licheniformis DW2 with overexpression driven by the P43 promoter. The yvmC gene was deleted using the CRISPR-Cas9n technique with homology arms of 1.0 kb as a representative example, and an efficiency of 100% was achieved. In addition, two genes were simultaneously disrupted with an efficiency of 11.6%, and the large DNA fragment bacABC (42.7 kb) was deleted with an efficiency of 79.0%. Furthermore, the heterologous reporter gene aprN, which codes for nattokinase in Bacillus subtilis, was inserted into the chromosome of B. licheniformis with an efficiency of 76.5%. The activity of nattokinase in the DWc9nΔ7/pP43SNT-SsacC strain reached 59.7 fibrinolytic units (FU)/ml, which was 25.7% higher than that of DWc9n/pP43SNT-SsacC Finally, the engineered strain DWc9nΔ7 (Δepr ΔwprA Δmpr ΔaprE Δvpr ΔbprA ΔbacABC), with multiple disrupted genes, was constructed using the CRISPR-Cas9n technique. Taken together, we have developed an efficient genome editing tool based on CRISPR-Cas9n in B. licheniformis This tool could be applied to strain improvement for future research.IMPORTANCE As important industrial bacteria, Bacillus strains have attracted significant attention due to their production of biological products. However, genetic manipulation of these bacteria is difficult. The CRISPR-Cas9 system has been applied to genome editing in some bacteria, and CRISPR-Cas9n was proven to be an efficient and precise tool in previous reports. The significance of our research is the development of an efficient, more precise, and systematic genome editing method for single-gene deletion, multiple-gene disruption, large DNA fragment deletion, and single-gene integration in Bacillus licheniformis via Cas9 nickase. We also applied this method to the genetic engineering of the host strain for protein expression.},
}

Bacillus licheniformis/enzymology/*genetics
Bacterial Proteins/*genetics/metabolism
CRISPR-Cas Systems/*genetics
Deoxyribonuclease I/*genetics/metabolism
Gene Editing/*methods
Subtilisins/*genetics/metabolism

@article {pmid30738385,
year = {2019},
author = {Emmert, AS and Vuong, SM and Shula, C and Lindquist, D and Yuan, W and Hu, YC and Mangano, FT and Goto, J},
title = {Characterization of a novel rat model of X-linked hydrocephalus by CRISPR-mediated mutation in L1cam.},
journal = {Journal of neurosurgery},
volume = {},
number = {},
pages = {1-14},
doi = {10.3171/2018.10.JNS181015},
pmid = {30738385},
issn = {1933-0693},
abstract = {OBJECTIVEEmergence of CRISPR/Cas9 genome editing provides a robust method for gene targeting in a variety of cell types, including fertilized rat embryos. The authors used this method to generate a transgenic rat L1cam knockout model of X-linked hydrocephalus (XLH) with human genetic etiology. The object of this study was to use diffusion tensor imaging (DTI) in studying perivascular white matter tract injury in the rat model and to characterize its pathological definition in histology.METHODSTwo guide RNAs designed to disrupt exon 4 of the L1cam gene on the X chromosome were injected into Sprague-Dawley rat embryos. Following embryo transfer into pseudopregnant females, rats were born and their DNA was sequenced for evidence of L1cam mutation. The mutant and control wild-type rats were monitored for growth and hydrocephalus phenotypes. Their macro- and microbrain structures were studied with T2-weighted MRI, DTI, immunohistochemistry, and transmission electron microscopy (TEM).RESULTSThe authors successfully obtained 2 independent L1cam knockout alleles and 1 missense mutant allele. Hemizygous male mutants from all 3 alleles developed hydrocephalus and delayed development. Significant reductions in fractional anisotropy and axial diffusivity were observed in the corpus callosum, external capsule, and internal capsule at 3 months of age. The mutant rats did not show reactive gliosis by then but exhibited hypomyelination and increased extracellular fluid in the corpus callosum.CONCLUSIONSThe CRISPR/Cas9-mediated genome editing system can be harnessed to efficiently disrupt the L1cam gene in rats for creation of a larger XLH animal model than previously available. This study provides evidence that the early pathology of the periventricular white matter tracts in hydrocephalus can be detected in DTI. Furthermore, TEM-based morphometric analysis of the corpus callosum elucidates the underlying cytopathological changes accompanying hydrocephalus-derived variations in DTI. The CRISPR/Cas9 system offers opportunities to explore novel surgical and imaging techniques on larger mammalian models.},
}

@article {pmid30737174,
year = {2019},
author = {Uribe, RV and van der Helm, E and Misiakou, MA and Lee, SW and Kol, S and Sommer, MOA},
title = {Discovery and Characterization of Cas9 Inhibitors Disseminated across Seven Bacterial Phyla.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2019.01.003},
pmid = {30737174},
issn = {1934-6069},
abstract = {CRISPR-Cas systems in bacteria and archaea provide immunity against bacteriophages and plasmids. To overcome CRISPR immunity, phages have acquired anti-CRISPR genes that reduce CRISPR-Cas activity. Using a synthetic genetic circuit, we developed a high-throughput approach to discover anti-CRISPR genes from metagenomic libraries based on their functional activity rather than sequence homology or genetic context. We identified 11 DNA fragments from soil, animal, and human metagenomes that circumvent Streptococcus pyogenes Cas9 activity in our selection strain. Further in vivo and in vitro characterization of a subset of these hits validated the activity of four anti-CRISPRs. Notably, homologs of some of these anti-CRISPRs were detected in seven different phyla, namely Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, Spirochaetes, and Balneolaeota, and have high sequence identity suggesting recent horizontal gene transfer. Thus, anti-CRISPRs against type II-A CRISPR-Cas systems are widely distributed across bacterial phyla, suggesting a more complex ecological role than previously appreciated.},
}

@article {pmid30735951,
year = {2019},
author = {Lee, HS and Lee, SH and Park, Y},
title = {Enhancement of androgen transcriptional activation assay based on genome edited glucocorticoid knock out human prostate cancer cell line.},
journal = {Environmental research},
volume = {171},
number = {},
pages = {437-443},
doi = {10.1016/j.envres.2019.01.027},
pmid = {30735951},
issn = {1096-0953},
abstract = {Endocrine-disrupting chemicals (EDCs) interfere with the biological activity of hormones. Among EDC's, (anti-)androgenic compounds potentially cause several androgen-related diseases. To improve the accuracy of an in vitro transactivation assay (TA) for detection of (anti-)androgenic compounds, We established the glucocorticoid receptor (GR) knockout 22Rv1/MMTV cell line by using an RNA-guided engineered nuclease (RGEN)-derived CRISPR/Cas system. The 22Rv1/MMTV GRKO cell line was characterized and validated by androgen receptor (AR)-mediated TA assay compared with the AR-TA assay using 22Rv1/MMTV. In conclusion, the AR-TA assay with the 22Rv1/MMTV GRKO cell line was more accurate, excluding the misleading signals derived from glucocorticoids or equivalent chemicals, and might be an effective method for screening potential (anti-)androgenic compounds.},
}

@article {pmid30733471,
year = {2019},
author = {Wang, J and Goh, KM and Salem, DR and Sani, RK},
title = {Genome analysis of a thermophilic exopolysaccharide-producing bacterium - Geobacillus sp. WSUCF1.},
journal = {Scientific reports},
volume = {9},
number = {1},
pages = {1608},
doi = {10.1038/s41598-018-36983-z},
pmid = {30733471},
issn = {2045-2322},
support = {1736255//National Science Foundation (NSF)/ ; 1736255//National Science Foundation (NSF)/ ; },
abstract = {Geobacillus sp. WSUCF1 is a Gram-positive, spore-forming, aerobic and thermophilic bacterium, isolated from a soil sample obtained from a compost facility. Strain WSUCF1 demonstrated EPS producing capability using different sugars as the carbon source. The whole-genome analysis of WSUCF1 was performed to disclose the essential genes correlated with nucleotide sugar precursor biosynthesis, assembly of monosaccharide units, export of the polysaccharide chain, and regulation of EPS production. Both the biosynthesis pathway and export mechanism of EPS were proposed based on functional annotation. Additionally, the genome description of strain WSUCF1 suggests sophisticated systems for its adaptation under thermophilic conditions. The presence of genes associated with CRISPR-Cas system, quorum quenching lactonase, polyketide synthesis and arsenic resistance makes this strain a potential candidate for various applications in biotechnology and biomedicine. The present study indicates that strain WSUCF1 has promise as a thermophilic EPS producer for a broad range of industrial applications. To the best of our knowledge, this is the first report on genome analysis of a thermophilic Geobacillus species focusing on its EPS biosynthesis and transportation, which will likely pave the way for both enhanced yield and tailor-made EPS production by thermophilic bacteria.},
}

@article {pmid30731369,
year = {2019},
author = {Tang, B and Gong, T and Zhou, X and Lu, M and Zeng, J and Peng, X and Wang, S and Li, Y},
title = {Deletion of cas3 gene in Streptococcus mutans affects biofilm formation and increases fluoride sensitivity.},
journal = {Archives of oral biology},
volume = {99},
number = {},
pages = {190-197},
doi = {10.1016/j.archoralbio.2019.01.016},
pmid = {30731369},
issn = {1879-1506},
abstract = {OBJECTIVE: The goal of this study was to analyze the impact of cas3 gene on the biofilm formation and virulence gene expression in S. mutans, since our previous studies have found a connection between CRISPR/Cas systems and biofilm formation in S. mutans.

METHODS: The cas3 gene in-frame deletion strains of S. mutans UA159 was constructed by a two-step transformation procedure and the cas3 mutant strain was complemented in trans. The biofilm biomass was measured by crystal violet staining, and the synthesis of exopolysaccharides (EPS) was measured by the anthrone-sulfuric method. Biofilm analysis and structural imaging was using confocal laser scanning microscope (CLSM) and scanning electron microscope (SEM) assays. The fluorescence in situ hybridization (FISH) was used to analyze the spatiotemporal interactions between S. mutans and Streptococcus sanguinis. Fluoride sensitivity was determined using fluoride tolerance assays. The expression of biofilm formation related genes was evaluated by qRT-PCR.

RESULTS: Our results showed that S. mutans cas3 deletion strain formed less biofilm and became less competitive when it was co-cultured with S. sanguinis under fluoride treatment. The expression levels of virulence genes including vicR, gtfC, smu0630 and comDE were significantly downregulated.

CONCLUSIONS: The cas3 gene in S. mutans could regulate biofilm formation and fluoride resistance, consequently affecting S. mutans competitiveness in a dual-species biofilm model under fluoride treatment. These results also provide a potential strategy for enhancing fluoride specificity, with cas3 gene as a potential genetic target in the modulation of oral microecology and the treatment of dental caries.},
}

@article {pmid30730208,
year = {2019},
author = {Finkler, M and Ott, A},
title = {Bead-based assay for spatiotemporal gene expression control in cell-free transcription-translation systems.},
journal = {BioTechniques},
volume = {66},
number = {1},
pages = {29-33},
doi = {10.2144/btn-2018-0097},
pmid = {30730208},
issn = {1940-9818},
abstract = {Cell-free gene expression has applications in synthetic biology, biotechnology and biomedicine. In this technique gene expression regulation plays an important role. Transcription factors do not completely suppress expression while other methods for expression control, for example CRISPR/Cas, often require important biochemical modifications. Here we use an all Escherichia coli-based cell-free expression system and present a bead-based method to instantly start and, at a later stage, completely stop gene expression. Magnetic beads coated with DNA of the gene of interest trigger gene expression. The expression stops if we remove the bead-bound DNA as well as transcribed mRNA by hybridization to bead-bound ssDNA. Our method is a simple way to control expression duration very accurately in time and space.},
}

@article {pmid30710124,
year = {2019},
author = {Conklin, BR},
title = {On the road to a gene drive in mammals.},
journal = {Nature},
volume = {566},
number = {7742},
pages = {43-45},
doi = {10.1038/d41586-019-00185-y},
pmid = {30710124},
issn = {1476-4687},
mesh = {Animals ; Automobile Driving ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; *Gene Drive Technology ; Mammals ; Mice ; },
}

Animals
Automobile Driving
*CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Female
*Gene Drive Technology
Mammals
Mice

@article {pmid30674877,
year = {2019},
author = {Spence, EF and Dube, S and Uezu, A and Locke, M and Soderblom, EJ and Soderling, SH},
title = {In vivo proximity proteomics of nascent synapses reveals a novel regulator of cytoskeleton-mediated synaptic maturation.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {386},
doi = {10.1038/s41467-019-08288-w},
pmid = {30674877},
issn = {2041-1723},
support = {MH103374/NH/NIH HHS/United States ; NS059957/NH/NIH HHS/United States ; F31NS092402/NH/NIH HHS/United States ; },
mesh = {Actin Capping Proteins/genetics/metabolism ; Animals ; Biotin ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cytoskeletal Proteins/metabolism ; Cytoskeleton/*metabolism ; Dendritic Spines/metabolism ; Excitatory Postsynaptic Potentials/*physiology ; GTPase-Activating Proteins ; Gene Expression Regulation ; HEK293 Cells ; Humans ; Mice, Inbred C57BL ; Microfilament Proteins/genetics/metabolism ; Microtubules/metabolism ; Neurogenesis/genetics/physiology ; Neurons/metabolism ; Proteome/genetics/*metabolism ; *Proteomics ; Synapses/genetics/*metabolism ; },
abstract = {Excitatory synapse formation during development involves the complex orchestration of both structural and functional alterations at the postsynapse. However, the molecular mechanisms that underlie excitatory synaptogenesis are only partially resolved, in part because the internal machinery of developing synapses is largely unknown. To address this, we apply a chemicogenetic approach, in vivo biotin identification (iBioID), to discover aspects of the proteome of nascent synapses. This approach uncovered sixty proteins, including a previously uncharacterized protein, CARMIL3, which interacts in vivo with the synaptic cytoskeletal regulator proteins SrGAP3 (or WRP) and actin capping protein. Using new CRISPR-based approaches, we validate that endogenous CARMIL3 is localized to developing synapses where it facilitates the recruitment of capping protein and is required for spine structural maturation and AMPAR recruitment associated with synapse unsilencing. Together these proteomic and functional studies reveal a previously unknown mechanism important for excitatory synapse development in the developing perinatal brain.},
}

Actin Capping Proteins/genetics/metabolism
Animals
Biotin
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Cytoskeletal Proteins/metabolism
Cytoskeleton/*metabolism
Dendritic Spines/metabolism
Excitatory Postsynaptic Potentials/*physiology
GTPase-Activating Proteins
Gene Expression Regulation
HEK293 Cells
Humans
Mice, Inbred C57BL
Microfilament Proteins/genetics/metabolism
Microtubules/metabolism
Neurogenesis/genetics/physiology
Neurons/metabolism
Proteome/genetics/*metabolism
*Proteomics
Synapses/genetics/*metabolism

@article {pmid30642460,
year = {2019},
author = {Javed, MR and Noman, M and Shahid, M and Ahmed, T and Khurshid, M and Rashid, MH and Ismail, M and Sadaf, M and Khan, F},
title = {Current situation of biofuel production and its enhancement by CRISPR/Cas9-mediated genome engineering of microbial cells.},
journal = {Microbiological research},
volume = {219},
number = {},
pages = {1-11},
doi = {10.1016/j.micres.2018.10.010},
pmid = {30642460},
issn = {1618-0623},
mesh = {Bacteria/*genetics/metabolism ; *Biofuels ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Fungi/*genetics/metabolism ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome, Bacterial/genetics ; Genome, Fungal/genetics ; Renewable Energy ; },
abstract = {Geopolitical and economic factors have motivated the scientific community to utilize renewable energy resources. In addition to the modifications in major steps and processes of biofuel production, manipulation of microbial genome engineering tools is essential in order to find sustainable solution of continuous depletion of fossil-fuels. Recently, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated nuclease 9), a prokaryotic molecular immunity system, has emerged as a novel technology for targeted genomic engineering. This genetic machinery seems to be a groundbreaking discovery to engineer the microbial genomes for desired traits such as enhancing the biofuel tolerance, inhibitor tolerance and thermotolerance as well as modifying the cellulases and hemicelluloses enzymes. In this review, a summary of different generations of biofuels, integrated processes of bioconversion of raw materials into biofuels and role of microbes in biofuel production has been presented. However, the ultimate focus of the review is on major discoveries of CRISPR/Cas9-mediated genome editing in microorganisms and exploitation of these discoveries for enhanced biofuel production.},
}

Bacteria/*genetics/metabolism
*Biofuels
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Fungi/*genetics/metabolism
Gene Editing/*methods
Genetic Engineering/*methods
Genome, Bacterial/genetics
Genome, Fungal/genetics
Renewable Energy

@article {pmid30555080,
year = {2018},
author = {Turner, AN and Andersen, RS and Bookout, IE and Brashear, LN and Davis, JC and Gahan, DM and Davis, JC and Gotham, JP and Hijaz, BA and Kaushik, AS and Mcgill, JB and Miller, VL and Moseley, ZP and Nowell, CL and Patel, RK and Rodgers, MC and Patel, RK and Shihab, YA and Walker, AP and Glover, SR and Foster, SD and Challa, AK},
title = {Analysis of novel domain-specific mutations in the zebrafish ndr2/cyclops gene generated using CRISPR-Cas9 RNPs.},
journal = {Journal of genetics},
volume = {97},
number = {5},
pages = {1315-1325},
pmid = {30555080},
issn = {0973-7731},
mesh = {Amino Acid Sequence ; Animals ; Base Sequence ; Binding Sites/genetics ; *CRISPR-Cas Systems ; Embryo, Nonmammalian/embryology/metabolism ; Holoprosencephaly/genetics ; Intracellular Signaling Peptides and Proteins/chemistry/*genetics ; Models, Molecular ; *Mutation ; Phenotype ; Protein Domains ; Ribonucleoproteins/*genetics/metabolism ; Zebrafish/embryology/genetics/metabolism ; Zebrafish Proteins/chemistry/*genetics ; },
abstract = {Nodal-related protein (ndr2) is amember of the transforming growth factor type β superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced and the remaining alleles were obtained via random insertion, gene targeting (TALEN) or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos.We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted polymerase chain reaction amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in the loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into the structure-function of the ndr2 gene.},
}

Amino Acid Sequence
Animals
Base Sequence
Binding Sites/genetics
*CRISPR-Cas Systems
Embryo, Nonmammalian/embryology/metabolism
Holoprosencephaly/genetics
Intracellular Signaling Peptides and Proteins/chemistry/*genetics
Models, Molecular
*Mutation
Phenotype
Protein Domains
Ribonucleoproteins/*genetics/metabolism
Zebrafish/embryology/genetics/metabolism
Zebrafish Proteins/chemistry/*genetics

@article {pmid30403660,
year = {2018},
author = {Rousset, F and Cui, L and Siouve, E and Becavin, C and Depardieu, F and Bikard, D},
title = {Genome-wide CRISPR-dCas9 screens in E. coli identify essential genes and phage host factors.},
journal = {PLoS genetics},
volume = {14},
number = {11},
pages = {e1007749},
pmid = {30403660},
issn = {1553-7404},
mesh = {*CRISPR-Cas Systems ; Escherichia coli/*genetics/virology ; *Genes, Essential ; *Genetic Association Studies ; *Genome, Bacterial ; *Genome-Wide Association Study ; Host-Pathogen Interactions ; },
abstract = {High-throughput genetic screens are powerful methods to identify genes linked to a given phenotype. The catalytic null mutant of the Cas9 RNA-guided nuclease (dCas9) can be conveniently used to silence genes of interest in a method also known as CRISPRi. Here, we report a genome-wide CRISPR-dCas9 screen using a starting pool of ~ 92,000 sgRNAs which target random positions in the chromosome of E. coli. To benchmark our method, we first investigate its utility to predict gene essentiality in the genome of E. coli during growth in rich medium. We could identify 79% of the genes previously reported as essential and demonstrate the non-essentiality of some genes annotated as essential. In addition, we took advantage of the intermediate repression levels obtained when targeting the template strand of genes to show that cells are very sensitive to the expression level of a limited set of essential genes. Our data can be visualized on CRISPRbrowser, a custom web interface available at crispr.pasteur.fr. We then apply the screen to discover E. coli genes required by phages λ, T4 and 186 to kill their host, highlighting the involvement of diverse host pathways in the infection process of the three tested phages. We also identify colanic acid capsule synthesis as a shared resistance mechanism to all three phages. Finally, using a plasmid packaging system and a transduction assay, we identify genes required for the formation of functional λ capsids, thus covering the entire phage cycle. This study demonstrates the usefulness and convenience of pooled genome-wide CRISPR-dCas9 screens in bacteria and paves the way for their broader use as a powerful tool in bacterial genomics.},
}

*CRISPR-Cas Systems
Escherichia coli/*genetics/virology
*Genes, Essential
*Genetic Association Studies
*Genome, Bacterial
*Genome-Wide Association Study
Host-Pathogen Interactions

@article {pmid30383747,
year = {2018},
author = {Combs, PA and Fraser, HB},
title = {Spatially varying cis-regulatory divergence in Drosophila embryos elucidates cis-regulatory logic.},
journal = {PLoS genetics},
volume = {14},
number = {11},
pages = {e1007631},
pmid = {30383747},
issn = {1553-7404},
mesh = {Alleles ; Animals ; CRISPR-Cas Systems ; Drosophila/*genetics ; Drosophila Proteins/genetics ; *Embryo, Nonmammalian ; Gene Editing ; Gene Expression Profiling ; Gene Expression Regulation ; Gene Targeting ; Genome-Wide Association Study ; Polymorphism, Single Nucleotide ; *Regulatory Sequences, Nucleic Acid ; },
abstract = {Spatial patterning of gene expression is a key process in development, yet how it evolves is still poorly understood. Both cis- and trans-acting changes could participate in complex interactions, so to isolate the cis-regulatory component of patterning evolution, we measured allele-specific spatial gene expression patterns in D. melanogaster × simulans hybrid embryos. RNA-seq of cryo-sectioned slices revealed 66 genes with strong spatially varying allele-specific expression. We found that hunchback, a major regulator of developmental patterning, had reduced expression of the D. simulans allele specifically in the anterior tip of hybrid embryos. Mathematical modeling of hunchback cis-regulation suggested a candidate transcription factor binding site variant, which we verified as causal using CRISPR-Cas9 genome editing. In sum, even comparing morphologically near-identical species we identified surprisingly extensive spatial variation in gene expression, suggesting not only that development is robust to many such changes, but also that natural selection may have ample raw material for evolving new body plans via changes in spatial patterning.},
}

Alleles
Animals
CRISPR-Cas Systems
Drosophila/*genetics
Drosophila Proteins/genetics
*Embryo, Nonmammalian
Gene Editing
Gene Expression Profiling
Gene Expression Regulation
Gene Targeting
Genome-Wide Association Study
Polymorphism, Single Nucleotide
*Regulatory Sequences, Nucleic Acid

@article {pmid30265349,
year = {2018},
author = {Muhr, M and Paulat, M and Awwanah, M and Brinkkötter, M and Teichmann, T},
title = {CRISPR/Cas9-mediated knockout of Populus BRANCHED1 and BRANCHED2 orthologs reveals a major function in bud outgrowth control.},
journal = {Tree physiology},
volume = {38},
number = {10},
pages = {1588-1597},
doi = {10.1093/treephys/tpy088},
pmid = {30265349},
issn = {1758-4469},
mesh = {*CRISPR-Cas Systems ; Gene Knockout Techniques ; Phenotype ; Plant Leaves/genetics/growth & development ; Plant Proteins/*genetics/metabolism ; Plant Stems/genetics/*growth & development ; Populus/*genetics/*growth & development/metabolism ; Transcription Factors/*genetics/metabolism ; },
abstract = {The TCP-type transcription factors BRANCHED1 and BRANCHED2 shape plant architecture by suppressing bud outgrowth, with BRANCHED2 only playing a minor role in Arabidopsis. Here, we investigated the function of orthologs of these genes in the model tree Populus. We used CRISPR/Cas9 to generate loss-of-function mutants of previously identified Populus BRANCHED1-1 and BRANCHED2-1 candidate genes. BRANCHED1-1 mutants exhibited strongly enhanced bud outgrowth. BRANCHED2-1 mutants had an extreme bud outgrowth phenotype and possessed two ectopic leaves at each node. While BRANCHED1 function is conserved in poplar, BRANCHED2, in contrast to its Arabidopsis counterpart, plays an even more critical role in bud outgrowth regulation. In addition, we identified a new, not yet reported association of this gene to leaf development.},
}

*CRISPR-Cas Systems
Gene Knockout Techniques
Phenotype
Plant Leaves/genetics/growth & development
Plant Proteins/*genetics/metabolism
Plant Stems/genetics/*growth & development
Populus/*genetics/*growth & development/metabolism
Transcription Factors/*genetics/metabolism

@article {pmid30142205,
year = {2018},
author = {Song, L and Ouedraogo, JP and Kolbusz, M and Nguyen, TTM and Tsang, A},
title = {Efficient genome editing using tRNA promoter-driven CRISPR/Cas9 gRNA in Aspergillus niger.},
journal = {PloS one},
volume = {13},
number = {8},
pages = {e0202868},
pmid = {30142205},
issn = {1932-6203},
mesh = {Aspergillus niger/*genetics ; CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; *Genomics ; Mutagenesis ; Promoter Regions, Genetic/*genetics ; RNA, Guide/*genetics ; RNA, Transfer/*genetics ; Transcription, Genetic ; },
abstract = {As a powerful tool for fast and precise genome editing, the CRISPR/Cas9 system has been applied in filamentous fungi to improve the efficiency of genome alteration. However, the method of delivering guide RNA (gRNA) remains a bottleneck in performing CRISPR mutagenesis in Aspergillus species. Here we report a gRNA transcription driven by endogenous tRNA promoters which include a tRNA gene plus 100 base pairs of upstream sequence. Co-transformation of a cas9-expressing plasmid with a linear DNA coding for gRNA demonstrated that 36 of the 37 tRNA promoters tested were able to generate the intended mutation in A. niger. When gRNA and cas9 were expressed in a single extra-chromosomal plasmid, the efficiency of gene mutation was as high as 97%. Co-transformation with DNA template for homologous recombination, the CRISPR/Cas9 system resulted ~42% efficiency of gene replacement in a strain with a functioning non-homologous end joining machinery (kusA+), and an efficiency of >90% gene replacement in a kusA- background. Our results demonstrate that tRNA promoter-mediated gRNA expressions are reliable and efficient in genome editing in A. niger.},
}

Aspergillus niger/*genetics
CRISPR-Cas Systems/*genetics
Gene Editing/*methods
*Genomics
Mutagenesis
Promoter Regions, Genetic/*genetics
RNA, Guide/*genetics
RNA, Transfer/*genetics
Transcription, Genetic

@article {pmid29995861,
year = {2018},
author = {Roth, TL and Puig-Saus, C and Yu, R and Shifrut, E and Carnevale, J and Li, PJ and Hiatt, J and Saco, J and Krystofinski, P and Li, H and Tobin, V and Nguyen, DN and Lee, MR and Putnam, AL and Ferris, AL and Chen, JW and Schickel, JN and Pellerin, L and Carmody, D and Alkorta-Aranburu, G and Del Gaudio, D and Matsumoto, H and Morell, M and Mao, Y and Cho, M and Quadros, RM and Gurumurthy, CB and Smith, B and Haugwitz, M and Hughes, SH and Weissman, JS and Schumann, K and Esensten, JH and May, AP and Ashworth, A and Kupfer, GM and Greeley, SAW and Bacchetta, R and Meffre, E and Roncarolo, MG and Romberg, N and Herold, KC and Ribas, A and Leonetti, MD and Marson, A},
title = {Reprogramming human T cell function and specificity with non-viral genome targeting.},
journal = {Nature},
volume = {559},
number = {7714},
pages = {405-409},
pmid = {29995861},
issn = {1476-4687},
support = {DP3 DK111914/DK/NIDDK NIH HHS/United States ; T32 DK007418/DK/NIDDK NIH HHS/United States ; T32 GM007618/GM/NIGMS NIH HHS/United States ; R35 CA197633/CA/NCI NIH HHS/United States ; P50 GM082250/GM/NIGMS NIH HHS/United States ; K23 DK094866/DK/NIDDK NIH HHS/United States ; K23 AI115001/AI/NIAID NIH HHS/United States ; P30 DK020595/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Autoimmunity/genetics ; CRISPR-Cas Systems/genetics ; Cells, Cultured ; Cellular Reprogramming/*genetics ; *Gene Editing ; Genome, Human/*genetics ; Humans ; Interleukin-2 Receptor alpha Subunit/genetics ; Male ; Mice ; Neoplasm Transplantation ; Protein Engineering ; Receptors, Antigen, T-Cell/genetics ; T-Lymphocytes/cytology/*immunology/*metabolism ; },
abstract = {Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.},
}

Animals
Autoimmunity/genetics
CRISPR-Cas Systems/genetics
Cells, Cultured
Cellular Reprogramming/*genetics
*Gene Editing
Genome, Human/*genetics
Humans
Interleukin-2 Receptor alpha Subunit/genetics
Male
Mice
Neoplasm Transplantation
Protein Engineering
Receptors, Antigen, T-Cell/genetics
T-Lymphocytes/cytology/*immunology/*metabolism

@article {pmid29777776,
year = {2018},
author = {Gurung, S and Asante, E and Hummel, D and Williams, A and Feldman-Schultz, O and Halloran, MC and Sittaramane, V and Chandrasekhar, A},
title = {Distinct roles for the cell adhesion molecule Contactin2 in the development and function of neural circuits in zebrafish.},
journal = {Mechanisms of development},
volume = {152},
number = {},
pages = {1-12},
doi = {10.1016/j.mod.2018.05.005},
pmid = {29777776},
issn = {1872-6356},
support = {R01 NS086934/NS/NINDS NIH HHS/United States ; R01 NS040449/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Axons/metabolism ; CRISPR-Cas Systems ; Cell Adhesion/*genetics ; Cell Movement/genetics ; Cell Polarity/genetics ; Contactin 2/*genetics ; Gene Expression Regulation, Developmental/genetics ; Humans ; Mice ; Morpholinos/genetics/metabolism ; Motor Neurons/metabolism ; Neurogenesis/*genetics ; Zebrafish/*genetics/growth & development ; Zebrafish Proteins/*genetics ; },
abstract = {Contactin2 (Cntn2)/Transient Axonal Glycoprotein 1 (Tag1), a neural cell adhesion molecule, has established roles in neuronal migration and axon fasciculation in chick and mouse. In zebrafish, antisense morpholino-based studies have indicated roles for cntn2 in the migration of facial branchiomotor (FBM) neurons, the guidance of the axons of the nucleus of the medial longitudinal fascicle (nucMLF), and the outgrowth of Rohon-Beard (RB) central axons. To study functions of Cntn2 in later stages of neuronal development, we generated cntn2 mutant zebrafish using CRISPR-Cas9. Using a null mutant allele, we detected genetic interactions between cntn2 and the planar cell polarity gene vangl2, as shown previously with cntn2 morphants, demonstrating a function for cntn2 during FBM neuron migration in a sensitized background of reduced planar cell polarity signaling. In addition, maternal-zygotic (MZ) cntn2 mutant larvae exhibited aberrant touch responses and swimming, suggestive of defects in sensorimotor circuits, consistent with studies in mice. However, the nucMLF axon convergence, FBM neuron migration, and RB outgrowth defects seen in morphants were not seen in the mutants, and we show here that they are likely off-target effects of morpholinos. However, MLF axons exhibited local defasciculation in MZcntn2 mutants, consistent with a role for Cntn2 in axon fasciculation. These data demonstrate distinct roles for zebrafish cntn2 in neuronal migration and axon fasciculation, and in the function of sensorimotor circuits.},
}

Animals
Axons/metabolism
CRISPR-Cas Systems
Cell Adhesion/*genetics
Cell Movement/genetics
Cell Polarity/genetics
Contactin 2/*genetics
Gene Expression Regulation, Developmental/genetics
Humans
Mice
Morpholinos/genetics/metabolism
Motor Neurons/metabolism
Neurogenesis/*genetics
Zebrafish/*genetics/growth & development
Zebrafish Proteins/*genetics

@article {pmid29669863,
year = {2018},
author = {He, X and Kläsener, K and Iype, JM and Becker, M and Maity, PC and Cavallari, M and Nielsen, PJ and Yang, J and Reth, M},
title = {Continuous signaling of CD79b and CD19 is required for the fitness of Burkitt lymphoma B cells.},
journal = {The EMBO journal},
volume = {37},
number = {11},
pages = {},
pmid = {29669863},
issn = {1460-2075},
mesh = {Antigens, CD19/*genetics ; B-Lymphocytes/pathology ; Burkitt Lymphoma/*genetics/pathology ; CD79 Antigens/*genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Leukemic/genetics ; Genetic Fitness/*genetics ; Humans ; Immunoglobulins/genetics ; Immunoreceptor Tyrosine-Based Activation Motif/genetics ; Phosphatidylinositol 3-Kinases/genetics ; Signal Transduction ; },
abstract = {Expression of the B-cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B-cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine-based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B-cell co-receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co-receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igβ can be expressed on the B-cell surface, where it is found in close proximity to CD19 and signals in an ITAM-dependent manner. These data suggest that Igβ and CD19 are part of an alternative B-cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.},
}

Antigens, CD19/*genetics
B-Lymphocytes/pathology
Burkitt Lymphoma/*genetics/pathology
CD79 Antigens/*genetics
CRISPR-Cas Systems
Gene Expression Regulation, Leukemic/genetics
Genetic Fitness/*genetics
Humans
Immunoglobulins/genetics
Immunoreceptor Tyrosine-Based Activation Motif/genetics
Phosphatidylinositol 3-Kinases/genetics
Signal Transduction

@article {pmid29489018,
year = {2018},
author = {Ibrahim, SH and Robertson, KD},
title = {Use of the CRISPR/Cas9-based epigenetic gene activation system In Vivo: A new potential therapeutic modality.},
journal = {Hepatology (Baltimore, Md.)},
volume = {68},
number = {3},
pages = {1191-1193},
pmid = {29489018},
issn = {1527-3350},
support = {K08 DK111397/DK/NIDDK NIH HHS/United States ; R01 DK110024/DK/NIDDK NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Epigenomics ; Gene Transfer Techniques ; Genetic Therapy ; *Transcriptional Activation ; },
}

*CRISPR-Cas Systems
Epigenomics
Gene Transfer Techniques
Genetic Therapy
*Transcriptional Activation

@article {pmid29480201,
year = {2018},
author = {Tan, DCS and Yao, S and Ittner, A and Bertz, J and Ke, YD and Ittner, LM and Delerue, F},
title = {Generation of a New Tau Knockout (tauΔex1) Line Using CRISPR/Cas9 Genome Editing in Mice.},
journal = {Journal of Alzheimer's disease : JAD},
volume = {62},
number = {2},
pages = {571-578},
doi = {10.3233/JAD-171058},
pmid = {29480201},
issn = {1875-8908},
mesh = {Animals ; *CRISPR-Cas Systems ; Exons ; Gene Editing/*methods ; Mice ; Mice, Inbred C57BL ; *Mice, Knockout ; tau Proteins/*genetics ; },
abstract = {Alzheimer's disease and other dementias present with tau pathology. Several mouse lines with knockout of the tau-encoding Mapt gene have been reported, yet findings often differed between lines and sites. Here, we report a new tau knockout strain (tauΔex1), generated by CRISPR/Cas9-mediated genome editing of intron -1/exon 1 of Mapt in C57Bl/6J mice. TauΔex1 mice had no overt phenotype, but, in line with previous models, they showed a significantly reduced susceptibility to excitotoxic seizures, with normal memory formation in young mice. This new in vivo resource will be made freely available to the research community.},
}

Animals
*CRISPR-Cas Systems
Exons
Gene Editing/*methods
Mice
Mice, Inbred C57BL
*Mice, Knockout
tau Proteins/*genetics

@article {pmid29260561,
year = {2018},
author = {Huang, M and Zhou, X and Wang, H and Xing, D},
title = {Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Triggered Isothermal Amplification for Site-Specific Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {90},
number = {3},
pages = {2193-2200},
doi = {10.1021/acs.analchem.7b04542},
pmid = {29260561},
issn = {1520-6882},
mesh = {CRISPR-Associated Protein 9/*chemistry ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Methylation ; DNA, Bacterial/*analysis/genetics ; Limit of Detection ; Listeria monocytogenes/genetics ; Nucleic Acid Amplification Techniques/*methods ; RNA, Bacterial/*analysis/genetics ; RNA, Guide/genetics ; },
abstract = {A novel CRISPR/Cas9 triggered isothermal exponential amplification reaction (CAS-EXPAR) strategy based on CRISPR/Cas9 cleavage and nicking endonuclease (NEase) mediated nucleic acids amplification was developed for rapid and site-specific nucleic acid detection. CAS-EXPAR was primed by the target DNA fragment produced by cleavage of CRISPR/Cas9, and the amplification reaction performed cyclically to generate a large number of DNA replicates which were detected using a real-time fluorescence monitoring method. This strategy that combines the advantages of CRISPR/Cas9 and exponential amplification showed high specificity as well as rapid amplification kinetics. Unlike conventional nucleic acids amplification reactions, CAS-EXPAR does not require exogenous primers, which often cause target-independent amplification. Instead, primers are first generated by Cas9/sgRNA directed site-specific cleavage of target and accumulated during the reaction. It was demonstrated this strategy gave a detection limit of 0.82 amol and showed excellent specificity in discriminating single-base mismatch. Moreover, the applicability of this method to detect DNA methylation and L. monocytogenes total RNA was also verified. Therefore, CAS-EXPAR may provide a new paradigm for efficient nucleic acid amplification and hold the potential for molecular diagnostic applications.},
}

CRISPR-Associated Protein 9/*chemistry
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
DNA Methylation
DNA, Bacterial/*analysis/genetics
Limit of Detection
Listeria monocytogenes/genetics
Nucleic Acid Amplification Techniques/*methods
RNA, Bacterial/*analysis/genetics
RNA, Guide/genetics

@article {pmid30724156,
year = {2019},
author = {Xue, C and Sashital, DG},
title = {Mechanisms of Type I-E and I-F CRISPR-Cas Systems in Enterobacteriaceae.},
journal = {EcoSal Plus},
volume = {8},
number = {2},
pages = {},
doi = {10.1128/ecosalplus.ESP-0008-2018},
pmid = {30724156},
issn = {2324-6200},
abstract = {CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against invasion by bacteriophages and other mobile genetic elements. Short fragments of invader DNA are stored as immunological memories within CRISPR (clustered regularly interspaced short palindromic repeat) arrays in the host chromosome. These arrays provide a template for RNA molecules that can guide CRISPR-associated (Cas) proteins to specifically neutralize viruses upon subsequent infection. Over the past 10 years, our understanding of CRISPR-Cas systems has benefited greatly from a number of model organisms. In particular, the study of several members of the Gram-negative Enterobacteriaceae family, especially Escherichia coli and Pectobacterium atrosepticum, have provided significant insights into the mechanisms of CRISPR-Cas immunity. In this review, we provide an overview of CRISPR-Cas systems present in members of the Enterobacteriaceae. We also detail the current mechanistic understanding of the type I-E and type I-F CRISPR-Cas systems that are commonly found in enterobacteria. Finally, we discuss how phages can escape or inactivate CRISPR-Cas systems and the measures bacteria can enact to counter these types of events.},
}

@article {pmid30722720,
year = {2019},
author = {Common, J and Westra, ER},
title = {CRISPR evolution and bacteriophage persistence in the context of population bottlenecks.},
journal = {RNA biology},
volume = {},
number = {},
pages = {},
doi = {10.1080/15476286.2019.1578608},
pmid = {30722720},
issn = {1555-8584},
abstract = {Population bottlenecks often cause strong reductions in genetic diversity and alter population structure. In the context of host-parasite interactions, bottlenecks could in theory benefit either the host or the pathogen. We predicted that bottlenecking of bacterial populations that evolve CRISPR immunity against bacteriophages (phage) would benefit the pathogen, because CRISPR spacer diversity can rapidly drive phages extinct. To test this, we bottlenecked populations of bacteria and phage, tracking phage persistence and the evolution of bacterial resistance mechanisms. Contrary to our prediction, bottlenecking worked in the advantage of the host. With some possible exceptions, this effect was not caused by CRISPR immunity. This host benefit is consistent with a dilution effect disproportionately affecting phage. This study provides further insight into how bottlenecking influences bacteria-phage dynamics, the role of dilution in bacteria-phage interactions, and the evolution of host immune systems.},
}

@article {pmid30286211,
year = {2018},
author = {Ye, W and Chew, M and Hou, J and Lai, F and Leopold, SJ and Loo, HL and Ghose, A and Dutta, AK and Chen, Q and Ooi, EE and White, NJ and Dondorp, AM and Preiser, P and Chen, J},
title = {Microvesicles from malaria-infected red blood cells activate natural killer cells via MDA5 pathway.},
journal = {PLoS pathogens},
volume = {14},
number = {10},
pages = {e1007298},
pmid = {30286211},
issn = {1553-7374},
support = {R01 NS099064/NS/NINDS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell-Derived Microparticles/*immunology ; Cells, Cultured ; Cytoplasm/metabolism ; Erythrocytes/*immunology/metabolism/parasitology ; Humans ; Interferon-Induced Helicase, IFIH1/antagonists & inhibitors/genetics/*metabolism ; Killer Cells, Natural/*immunology/metabolism/parasitology ; Lymphocyte Activation ; Malaria, Falciparum/*immunology/metabolism/parasitology ; Plasmodium falciparum/*immunology/isolation & purification ; },
abstract = {Natural killer (NK) cells provide the first line of defense against malaria parasite infection. However, the molecular mechanisms through which NK cells are activated by parasites are largely unknown, so is the molecular basis underlying the variation in NK cell responses to malaria infection in the human population. Here, we compared transcriptional profiles of responding and non-responding NK cells following exposure to Plasmodium-infected red blood cells (iRBCs) and identified MDA5, a RIG-I-like receptor involved in sensing cytosolic RNAs, to be differentially expressed. Knockout of MDA5 in responding human NK cells by CRISPR/cas9 abolished NK cell activation, IFN-γ secretion, lysis of iRBCs. Similarly, inhibition of TBK1/IKKε, an effector molecule downstream of MDA5, also inhibited activation of responding NK cells. Conversely, activation of MDA5 by liposome-packaged poly I:C restored non-responding NK cells to lyse iRBCs. We further show that microvesicles containing large parasite RNAs from iRBCs activated NK cells by fusing with NK cells. These findings suggest that NK cells are activated through the MDA5 pathway by parasite RNAs that are delivered to the cytoplasm of NK cells by microvesicles from iRBCs. The difference in MDA5 expression between responding and non-responding NK cells following exposure to iRBCs likely contributes to the variation in NK cell responses to malaria infection in the human population.},
}

CRISPR-Cas Systems
Cell-Derived Microparticles/*immunology
Cells, Cultured
Cytoplasm/metabolism
Erythrocytes/*immunology/metabolism/parasitology
Humans
Interferon-Induced Helicase, IFIH1/antagonists & inhibitors/genetics/*metabolism
Killer Cells, Natural/*immunology/metabolism/parasitology
Lymphocyte Activation
Malaria, Falciparum/*immunology/metabolism/parasitology
Plasmodium falciparum/*immunology/isolation & purification

@article {pmid29883600,
year = {2018},
author = {Hou, Z and Zhang, Y},
title = {Insights into a Mysterious CRISPR Adaptation Factor, Cas4.},
journal = {Molecular cell},
volume = {70},
number = {5},
pages = {757-758},
doi = {10.1016/j.molcel.2018.05.028},
pmid = {29883600},
issn = {1097-4164},
support = {R00 GM117268/GM/NIGMS NIH HHS/United States ; },
mesh = {Adaptation, Physiological ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA ; Endonucleases ; },
abstract = {Cas4 nucleases are conserved factors in many CRISPR systems, yet their molecular role has remained enigmatic. In this issue of Molecular Cell, Shiimori et al. (2018) report that two Cas4 nucleases together determine the size, orientation, and PAM for foreign DNA snippets acquired by CRISPR loci as immunological memory.},
}

Adaptation, Physiological
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA
Endonucleases

@article {pmid29593249,
year = {2018},
author = {Vanden Oever, M and Twaroski, K and Osborn, MJ and Wagner, JE and Tolar, J},
title = {Inside out: regenerative medicine for recessive dystrophic epidermolysis bullosa.},
journal = {Pediatric research},
volume = {83},
number = {1-2},
pages = {318-324},
pmid = {29593249},
issn = {1530-0447},
support = {R01 AR063070/AR/NIAMS NIH HHS/United States ; T32 GM113846/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Epidermolysis Bullosa Dystrophica/*genetics/*therapy ; Gene Editing ; Genes, Recessive ; Genetic Therapy/*methods ; Humans ; Keratinocytes/cytology ; Mice ; Regenerative Medicine/*methods ; Skin/metabolism ; Stem Cell Transplantation/*methods ; Stem Cells/cytology ; Wound Healing ; },
abstract = {Epidermolysis bullosa is classified as a genodermatosis, an inherited genetic skin disorder that results in severe, chronic skin blistering with painful and life-threatening complications. Although there is currently no cure for epidermolysis bullosa, concurrent advances in gene and stem cell therapies are converging toward combinatorial therapies that hold the promise of clinically meaningful and lifelong improvement. Recent studies using hematopoietic stem cells and mesenchymal stromal/stem cells to treat epidermolysis bullosa have demonstrated the potential for sustained, effective management of the most severe cases. Furthermore, advances in the use of gene therapy and gene-editing techniques, coupled with the development of induced pluripotent stem cells from patients with epidermolysis bullosa, allow for autologous therapies derived from a renewable population of cells that are patient-specific. Here we describe emerging treatments for epidermolysis bullosa and other genodermatoses, along with a discussion of their benefits and limitations as effective therapies.},
}

Animals
CRISPR-Cas Systems
Epidermolysis Bullosa Dystrophica/*genetics/*therapy
Gene Editing
Genes, Recessive
Genetic Therapy/*methods
Humans
Keratinocytes/cytology
Mice
Regenerative Medicine/*methods
Skin/metabolism
Stem Cell Transplantation/*methods
Stem Cells/cytology
Wound Healing

@article {pmid28246396,
year = {2017},
author = {Kato, T and Hara, S and Goto, Y and Ogawa, Y and Okayasu, H and Kubota, S and Tamano, M and Terao, M and Takada, S},
title = {Creation of mutant mice with megabase-sized deletions containing custom-designed breakpoints by means of the CRISPR/Cas9 system.},
journal = {Scientific reports},
volume = {7},
number = {1},
pages = {59},
pmid = {28246396},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; Female ; *Gene Deletion ; Male ; Mice, Mutant Strains/*genetics ; Microinjections ; *Mutation ; Plasmids ; Zygote ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for creation of mutant mice with mutations mirroring those in human patients. Various methods have been developed for this purpose, including deletions, inversions, and translocations. So far, mutant mice with deletions of up to 1.2 megabases (Mb) have been generated by microinjection of the CRISPR/Cas9 system into fertilized eggs; however, a method for generation of mutant mice with a deletion of more than several Mb size is necessary because such deletions have often been identified as possible causes of human diseases. With an aim to enable the generation of disease models carrying large deletions with a breakpoint in custom-designed sequences, we developed a method for induction of an Mb-sized deletion by microinjection of a pair of sgRNAs, Cas9, and a donor plasmid into fertilized eggs. Using this method, we efficiently and rapidly generated mutant mice carrying deletions up to 5 Mb.},
}

Animals
*CRISPR-Cas Systems
Disease Models, Animal
Female
*Gene Deletion
Male
Mice, Mutant Strains/*genetics
Microinjections
*Mutation
Plasmids
Zygote

@article {pmid30717668,
year = {2019},
author = {McDonald, ND and Regmi, A and Morreale, DP and Borowski, JD and Fidelma Boyd, E},
title = {CRISPR-Cas systems are present predominantly on mobile genetic elements in Vibrio species.},
journal = {BMC genomics},
volume = {20},
number = {1},
pages = {105},
doi = {10.1186/s12864-019-5439-1},
pmid = {30717668},
issn = {1471-2164},
support = {IOS-1656688//National Science Foundation/ ; 5T32GM008550//National Institutes of Health/ ; P20GM103446//National Institutes of Health/ ; },
abstract = {BACKGROUND: Bacteria are prey for many viruses that hijack the bacterial cell in order to propagate, which can result in bacterial cell lysis and death. Bacteria have developed diverse strategies to counteract virus predation, one of which is the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR associated (Cas) proteins immune defense system. Species within the bacterial family Vibrionaceae are marine organisms that encounter large numbers of phages. Our goal was to determine the significance of CRISPR-Cas systems as a mechanism of defense in this group by investigating their prevalence, phylogenetic distribution, and genome context.

RESULTS: Herein, we describe all the CRISPR-Cas system types and their distribution within the family Vibrionaceae. In Vibrio cholerae genomes, we identified multiple variant type I-F systems, which were also present in 41 additional species. In a large number of Vibrio species, we identified a mini type I-F system comprised of tniQcas5cas7cas6f, which was always associated with Tn7-like transposons. The Tn7-like elements, in addition to the CRISPR-Cas system, also contained additional cargo genes such as restriction modification systems and type three secretion systems. A putative hybrid CRISPR-Cas system was identified containing type III-B genes followed by a type I-F cas6f and a type I-F CRISPR that was associated with a prophage in V. cholerae and V. metoecus strains. Our analysis identified CRISPR-Cas types I-C, I-E, I-F, II-B, III-A, III-B, III-D, and the rare type IV systems as well as cas loci architectural variants among 70 species. All systems described contained a CRISPR array that ranged in size from 3 to 179 spacers. The systems identified were present predominantly within mobile genetic elements (MGEs) such as genomic islands, plasmids, and transposon-like elements. Phylogenetic analysis of Cas proteins indicated that the CRISPR-Cas systems were acquired by horizontal gene transfer.

CONCLUSIONS: Our data show that CRISPR-Cas systems are phylogenetically widespread but sporadic in occurrence, actively evolving, and present on MGEs within Vibrionaceae.},
}

@article {pmid30714208,
year = {2019},
author = {Nasseri, S and Nikkho, B and Parsa, S and Ebadifar, A and Soleimani, F and Rahimi, K and Vahabzadeh, Z and Khadem-Erfan, MB and Rostamzadeh, J and Baban, B and Banafshi, O and Assadollahi, V and Mirzaie, S and Fathi, F},
title = {Generation of Fam83h knockout mice by CRISPR/Cas9-mediated gene engineering.},
journal = {Journal of cellular biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/jcb.28381},
pmid = {30714208},
issn = {1097-4644},
support = {//Medical University of Kurdistan/ ; //Kurdistan University of Medical Sciences/ ; },
abstract = {Family with sequence similarity 83 member H (FAM83H) protein-coding geneplay an essential role in the structural organization, calcification of developing enamel, and keratin cytoskeleton disassembly by recruiting Casein kinase 1 alpha (CSNK1A1) to keratin filaments. In this study, we have applied CRISPR Cas9 nickase (D10A) to knockout (KO) the Fam83h gene in NMRI outbred mice. We generated homozygous Fam83h KO mice (Fam83h Ko/Ko) through a premature termination codon, which was validated by Sanger sequencing in F0 generation. Next, we also bred the FAM83H KO for two generations. Reverse-transcription polymerase chain reaction and Western blot analysis approved the Fam83h KO mice. The Fam83h KO mice had evidence of normal morphology at the cervical loops, secretory and maturation stages, and mandibular molars. In comparison with the normal wild-type mice (Fam83h W/W), the F2 homozygous KO (Fam83h Ko/Ko) had sparse, scruffy coats with small body size and decreased general activity. Also, they had the natural reproductive ability and natural lifespan. In addition, delay in opening the eyes and dry eyes among infant mice were seen. The F1 heterozygous mice looked comparable to the normal wild-type mice (Fam83h W/W), which showed autosomal recessive inheritance of these phenotypes. The KO of FAM83H had controversial effects on the development of teeth and the formation of enamel. The phenotype defect in dental development and the enamel formation were seen in three mice among four generations. It can be concluded that null FAM83H in outbred mice not only showed the reported phenotypes in null inbred mouse but also showed normal lifespan and reproductive ability; dental deficiency in three homozygous mice; and the symptoms that were similar to the symptoms of dry eye syndrome and curly coat dog syndrome in all four evaluated KO generations.},
}

@article {pmid30664650,
year = {2019},
author = {Campagne, A and Lee, MK and Zielinski, D and Michaud, A and Le Corre, S and Dingli, F and Chen, H and Shahidian, LZ and Vassilev, I and Servant, N and Loew, D and Pasmant, E and Postel-Vinay, S and Wassef, M and Margueron, R},
title = {BAP1 complex promotes transcription by opposing PRC1-mediated H2A ubiquitylation.},
journal = {Nature communications},
volume = {10},
number = {1},
pages = {348},
doi = {10.1038/s41467-018-08255-x},
pmid = {30664650},
issn = {2041-1723},
mesh = {Animals ; B-Lymphocytes/cytology/*metabolism ; CRISPR-Cas Systems ; Cell Cycle Proteins/*genetics/metabolism ; Cell Line, Tumor ; Drosophila melanogaster ; Gene Editing ; Haploidy ; HeLa Cells ; Histones/genetics/*metabolism ; Humans ; Polycomb Repressive Complex 1/genetics/metabolism ; *Protein Processing, Post-Translational ; Repressor Proteins/genetics/metabolism ; Sf9 Cells ; Spodoptera ; Transcription Factors/genetics/metabolism ; Transcriptional Activation ; Tumor Suppressor Proteins/*genetics/metabolism ; Ubiquitin Thiolesterase/*genetics/metabolism ; Ubiquitination ; },
abstract = {In Drosophila, a complex consisting of Calypso and ASX catalyzes H2A deubiquitination and has been reported to act as part of the Polycomb machinery in transcriptional silencing. The mammalian homologs of these proteins (BAP1 and ASXL1/2/3, respectively), are frequently mutated in various cancer types, yet their precise functions remain unclear. Using an integrative approach based on isogenic cell lines generated with CRISPR/Cas9, we uncover an unanticipated role for BAP1 in gene activation. This function requires the assembly of an enzymatically active BAP1-associated core complex (BAP1.com) containing one of the redundant ASXL proteins. We investigate the mechanism underlying BAP1.com-mediated transcriptional regulation and show that it does not participate in Polycomb-mediated silencing. Instead, our results establish that the function of BAP1.com is to safeguard transcriptionally active genes against silencing by the Polycomb Repressive Complex 1.},
}

Animals
B-Lymphocytes/cytology/*metabolism
CRISPR-Cas Systems
Cell Cycle Proteins/*genetics/metabolism
Cell Line, Tumor
Drosophila melanogaster
Gene Editing
Haploidy
HeLa Cells
Histones/genetics/*metabolism
Humans
Polycomb Repressive Complex 1/genetics/metabolism
*Protein Processing, Post-Translational
Repressor Proteins/genetics/metabolism
Sf9 Cells
Spodoptera
Transcription Factors/genetics/metabolism
Transcriptional Activation
Tumor Suppressor Proteins/*genetics/metabolism
Ubiquitin Thiolesterase/*genetics/metabolism
Ubiquitination

@article {pmid30409154,
year = {2018},
author = {Duan, J and Lu, G and Hong, Y and Hu, Q and Mai, X and Guo, J and Si, X and Wang, F and Zhang, Y},
title = {Live imaging and tracking of genome regions in CRISPR/dCas9 knock-in mice.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {192},
pmid = {30409154},
issn = {1474-760X},
mesh = {Animals ; *CRISPR-Cas Systems ; Cells, Cultured ; *Genome ; Green Fluorescent Proteins/genetics/*metabolism ; HEK293 Cells ; Hep G2 Cells ; Hepatocytes/cytology/*metabolism ; Humans ; Image Processing, Computer-Assisted ; Mice ; Mice, Inbred C57BL ; Molecular Imaging/*methods ; Telomere/genetics/*metabolism ; },
abstract = {CRISPR/dCas9 is a versatile tool that can be used to recruit various effectors and fluorescent molecules to defined genome regions where it can modulate genetic and epigenetic markers, or track the chromatin dynamics in live cells. In vivo applications of CRISPR/dCas9 in animals have been challenged by delivery issues. We generate and characterize a mouse strain with dCas9-EGFP ubiquitously expressed in various tissues. Studying telomere dynamics in these animals reveals surprising results different from those observed in cultured cell lines. The CRISPR/dCas9 knock-in mice provide an important and versatile tool to mechanistically study genome functions in live animals.},
}

Animals
*CRISPR-Cas Systems
Cells, Cultured
*Genome
Green Fluorescent Proteins/genetics/*metabolism
HEK293 Cells
Hep G2 Cells
Hepatocytes/cytology/*metabolism
Humans
Image Processing, Computer-Assisted
Mice
Mice, Inbred C57BL
Molecular Imaging/*methods
Telomere/genetics/*metabolism

@article {pmid30400943,
year = {2018},
author = {Guo, X and Dean, A},
title = {CRISPR/Cas9 offers a new tool for studying the role of chromatin architecture in disease pathogenesis.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {185},
pmid = {30400943},
issn = {1474-760X},
support = {DK075033/DK/NIDDK NIH HHS/United States ; },
mesh = {CCCTC-Binding Factor/genetics/*metabolism ; *CRISPR-Cas Systems ; *Chromatin ; *Gene Expression Regulation, Neoplastic ; Humans ; Male ; Prostatic Neoplasms/genetics/*pathology ; Protein Binding ; *Regulatory Elements, Transcriptional ; },
abstract = {A recent study used CRISPR/Cas9 to reveal long-range looping between disease-related genes and their regulatory elements that is mediated by the CCCTC-binding factor (CTCF) in prostate cancer.},
}

CCCTC-Binding Factor/genetics/*metabolism
*CRISPR-Cas Systems
*Chromatin
*Gene Expression Regulation, Neoplastic
Humans
Male
Prostatic Neoplasms/genetics/*pathology
Protein Binding
*Regulatory Elements, Transcriptional

@article {pmid30400928,
year = {2018},
author = {Yao, Y},
title = {Genome editing: from tools to biological insights.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {186},
pmid = {30400928},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; Gene Editing/*methods ; *Genetic Engineering ; *Genome ; },
}

*CRISPR-Cas Systems
Gene Editing/*methods
*Genetic Engineering
*Genome

@article {pmid30400804,
year = {2018},
author = {Smith, JL and Mou, H and Xue, W},
title = {Understanding and repurposing CRISPR-mediated alternative splicing.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {184},
pmid = {30400804},
issn = {1474-760X},
support = {DP2 HL137167/HL/NHLBI NIH HHS/United States ; P01 HL131471/HL/NHLBI NIH HHS/United States ; UG3 HL147367/HL/NHLBI NIH HHS/United States ; },
mesh = {*Alternative Splicing ; *CRISPR-Cas Systems ; *Exons ; Gene Editing/*methods ; Humans ; RNA, Guide/*genetics ; },
abstract = {Two new studies refine our understanding of CRISPR-associated exon skipping and redefine its utility in engineering alternative splicing.},
}

*Alternative Splicing
*CRISPR-Cas Systems
*Exons
Gene Editing/*methods
Humans
RNA, Guide/*genetics

@article {pmid30367669,
year = {2018},
author = {Alkan, F and Wenzel, A and Anthon, C and Havgaard, JH and Gorodkin, J},
title = {CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters.},
journal = {Genome biology},
volume = {19},
number = {1},
pages = {177},
pmid = {30367669},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; Endonucleases/metabolism ; *Gene Editing ; Humans ; Nucleic Acids/*chemistry ; RNA, Guide/*genetics ; Substrate Specificity ; },
abstract = {BACKGROUND: Recent experimental efforts of CRISPR-Cas9 systems have shown that off-target binding and cleavage are a concern for the system and that this is highly dependent on the selected guide RNA (gRNA) design. Computational predictions of off-targets have been proposed as an attractive and more feasible alternative to tedious experimental efforts. However, accurate scoring of the high number of putative off-targets plays a key role for the success of computational off-targeting assessment.

RESULTS: We present an approximate binding energy model for the Cas9-gRNA-DNA complex, which systematically combines the energy parameters obtained for RNA-RNA, DNA-DNA, and RNA-DNA duplexes. Based on this model, two novel off-target assessment methods for gRNA selection in CRISPR-Cas9 applications are introduced: CRISPRoff to assign confidence scores to predicted off-targets and CRISPRspec to measure the specificity of the gRNA. We benchmark the methods against current state-of-the-art methods and show that both are in better agreement with experimental results. Furthermore, we show significant evidence supporting the inverse relationship between the on-target cleavage efficiency and specificity of the system, in which introduced binding energies are key components.

CONCLUSIONS: The impact of the binding energies provides a direction for further studies of off-targeting mechanisms. The performance of CRISPRoff and CRISPRspec enables more accurate off-target evaluation for gRNA selections, prior to any CRISPR-Cas9 genome-editing application. For given gRNA sequences or all potential gRNAs in a given target region, CRISPRoff-based off-target predictions and CRISPRspec-based specificity evaluations can be carried out through our webserver at https://rth.dk/resources/crispr/ .},
}

*CRISPR-Cas Systems
Endonucleases/metabolism
*Gene Editing
Humans
Nucleic Acids/*chemistry
RNA, Guide/*genetics
Substrate Specificity

@article {pmid30328670,
year = {2018},
author = {Yang, Y and Wang, YH and Chen, XE and Tian, D and Xu, X and Li, K and Huang, YP and He, L},
title = {CRISPR/Cas9-mediated Tyrosine hydroxylase knockout resulting in larval lethality in Agrotis ipsilon.},
journal = {Insect science},
volume = {25},
number = {6},
pages = {1017-1024},
doi = {10.1111/1744-7917.12647},
pmid = {30328670},
issn = {1744-7917},
support = {2015CB755703//National Basic Research Program of China/ ; 31772517//National Natural Science Foundation of China/ ; 18140902200//Science and Technology Commission of Shanghai Municipality/ ; //Shanghai Key Lab for Urban Ecological Processes and Eco-Restoration, East China Normal University/ ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Gene Expression Regulation, Developmental ; Larva/*growth & development ; Lepidoptera/enzymology/*genetics/*growth & development ; Mutation ; Pest Control, Biological ; Phenotype ; Tyrosine 3-Monooxygenase/*deficiency/*genetics ; },
abstract = {Tyrosine hydroxylase (TH) is involved in insect melanin and the catecholamine biosynthesis pathway. TH as an enzyme catalyzing the conversion of tyrosine to 3,4-dihydroxyphenylalanine is the first step reaction in the pathway. Although TH has been proven to affect the pigmentation of the epidermis and development in many insects, there is no report about physiological function of the TH gene in Agrotis ipsilon. Here we cloned the TH gene from A. ipsilon. Semi-quantitative real-time polymerase chain reaction (PCR) analysis showed that AiTH was expressed at all development stages. Moreover, its high expression levels in the head and epidermis suggest that it is mainly related to pigment deposition and insect development. Then, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system to target the AiTH gene: deletion events were detected at the target sites. Compared with the control group, a few mutants with the phenomenon of narrowing in the egg shell and embryos can develop but cannot hatch; the other hatched embryos were seriously dehydrated after hatching and died within the first day. Quantitative real-time PCR analysis revealed that TH was down-regulated in AiTH mutants. Here, our work demonstrated that AiTH plays an important role in growth and development of newly hatched larvae; meanwhile, it would be a promising target to explore a control strategy for A. ipsilon.},
}

Animals
CRISPR-Cas Systems/*genetics
Gene Expression Regulation, Developmental
Larva/*growth & development
Lepidoptera/enzymology/*genetics/*growth & development
Mutation
Pest Control, Biological
Phenotype
Tyrosine 3-Monooxygenase/*deficiency/*genetics

@article {pmid30156184,
year = {2018},
author = {Gutierrez-Triana, JA and Tavhelidse, T and Thumberger, T and Thomas, I and Wittbrodt, B and Kellner, T and Anlas, K and Tsingos, E and Wittbrodt, J},
title = {Efficient single-copy HDR by 5' modified long dsDNA donors.},
journal = {eLife},
volume = {7},
number = {},
pages = {},
pmid = {30156184},
issn = {2050-084X},
support = {CRC 873,TP A3//Deutsche Forschungsgemeinschaft/International ; CRC 873 project A3//Deutsche Forschungsgemeinschaft/International ; },
mesh = {Animals ; *CRISPR-Cas Systems ; DNA/*genetics/metabolism ; *DNA End-Joining Repair ; Embryo, Nonmammalian/metabolism ; Fish Proteins/genetics/metabolism ; Gene Editing/*methods ; Green Fluorescent Proteins/genetics/metabolism ; Models, Genetic ; Oryzias ; *Recombinational DNA Repair ; },
abstract = {CRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing, precise, homology-directed repair (HDR) of endogenous DNA stretches is a prerequisite. To favor HDR, many approaches interfere with the repair machinery or manipulate Cas9 itself. Using Medaka we show that the modification of 5' ends of long dsDNA donors strongly enhances HDR, favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging.},
}

Animals
*CRISPR-Cas Systems
DNA/*genetics/metabolism
*DNA End-Joining Repair
Embryo, Nonmammalian/metabolism
Fish Proteins/genetics/metabolism
Gene Editing/*methods
Green Fluorescent Proteins/genetics/metabolism
Models, Genetic
Oryzias
*Recombinational DNA Repair

@article {pmid29876814,
year = {2018},
author = {Hoof, JB and Nødvig, CS and Mortensen, UH},
title = {Genome Editing: CRISPR-Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1775},
number = {},
pages = {119-132},
doi = {10.1007/978-1-4939-7804-5_11},
pmid = {29876814},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Gene Editing/*methods ; Gene Targeting/*methods ; Genetic Engineering/*methods ; Genetic Vectors/genetics ; RNA, Guide/genetics ; },
abstract = {In the present chapter, we present the protocols and guidelines to facilitate implementation of CRISPR-Cas9 technology in fungi where few or no genetic tools are in place. Hence, we firstly explain how to identify dominant markers for genetic transformation. Secondly, we provide a guide for construction of Cas9/sgRNA episomal expression vectors. Thirdly, we present how to mutagenize reporter genes to explore the efficiency of CRISPR-Cas9 in the relevant fungus and to ease subsequent CRISPR-mediated genetic engineering. Lastly, we describe how to make CRISPR-mediated marker-dependent and marker-free gene targeting.},
}

CRISPR-Cas Systems/*genetics
Gene Editing/*methods
Gene Targeting/*methods
Genetic Engineering/*methods
Genetic Vectors/genetics
RNA, Guide/genetics

@article {pmid29858247,
year = {2018},
author = {Fong, LK and Yang, MM and Dos Santos Chaves, R and Reyna, SM and Langness, VF and Woodruff, G and Roberts, EA and Young, JE and Goldstein, LSB},
title = {Full-length amyloid precursor protein regulates lipoprotein metabolism and amyloid-β clearance in human astrocytes.},
journal = {The Journal of biological chemistry},
volume = {293},
number = {29},
pages = {11341-11357},
pmid = {29858247},
issn = {1083-351X},
support = {P50 AG005131/AG/NIA NIH HHS/United States ; RF1 AG048083/AG/NIA NIH HHS/United States ; T32 GM008666/GM/NIGMS NIH HHS/United States ; },
mesh = {Amyloid beta-Peptides/*metabolism ; Amyloid beta-Protein Precursor/genetics/*metabolism ; Astrocytes/cytology/*metabolism ; CRISPR-Cas Systems ; Cell Line ; Cholesterol/*metabolism ; Endocytosis ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism ; Lipoproteins/*metabolism ; Receptors, LDL/metabolism ; Sterol Regulatory Element Binding Proteins/metabolism ; },
abstract = {Mounting evidence suggests that alterations in cholesterol homeostasis are involved in Alzheimer's disease (AD) pathogenesis. Amyloid precursor protein (APP) or multiple fragments generated by proteolytic processing of APP have previously been implicated in the regulation of cholesterol metabolism. However, the physiological function of APP in regulating lipoprotein homeostasis in astrocytes, which are responsible for de novo cholesterol biosynthesis and regulation in the brain, remains unclear. To address this, here we used CRISPR/Cas9 genome editing to generate isogenic APP-knockout (KO) human induced pluripotent stem cells (hiPSCs) and differentiated them into human astrocytes. We found that APP-KO astrocytes have reduced cholesterol and elevated levels of sterol regulatory element-binding protein (SREBP) target gene transcripts and proteins, which were both downstream consequences of reduced lipoprotein endocytosis. To elucidate which APP fragments regulate cholesterol homeostasis and to examine whether familial AD mutations in APP affect lipoprotein metabolism, we analyzed an isogenic allelic series harboring the APP Swedish and APP V717F variants. Only astrocytes homozygous for the APP Swedish (APPSwe/Swe) mutation, which had reduced full-length APP (FL APP) due to increased β-secretase cleavage, recapitulated the APP-KO phenotypes. Astrocytic internalization of β-amyloid (Aβ), another ligand for low-density lipoprotein (LDL) receptors, was also impaired in APP-KO and APPSwe/Swe astrocytes. Finally, impairing cleavage of FL APP through β-secretase inhibition in APPSwe/Swe astrocytes reversed the LDL and Aβ endocytosis defects. In conclusion, FL APP is involved in the endocytosis of LDL receptor ligands and is required for proper cholesterol homeostasis and Aβ clearance in human astrocytes.},
}

Amyloid beta-Peptides/*metabolism
Amyloid beta-Protein Precursor/genetics/*metabolism
Astrocytes/cytology/*metabolism
CRISPR-Cas Systems
Cell Line
Cholesterol/*metabolism
Endocytosis
Humans
Induced Pluripotent Stem Cells/cytology/metabolism
Lipoproteins/*metabolism
Receptors, LDL/metabolism
Sterol Regulatory Element Binding Proteins/metabolism

@article {pmid29851168,
year = {2018},
author = {Lau, RW and Wang, B and Ricardo, SD},
title = {Gene editing of stem cells for kidney disease modelling and therapeutic intervention.},
journal = {Nephrology (Carlton, Vic.)},
volume = {23},
number = {11},
pages = {981-990},
doi = {10.1111/nep.13410},
pmid = {29851168},
issn = {1440-1797},
support = {//Monash University Platform Access Grant/ ; //European Associations for Information and Research on Kidney Disease Genetic/ ; },
mesh = {Animals ; CRISPR-Cas Systems ; *Gene Editing/ethics ; Genetic Therapy ; Humans ; Kidney Diseases/etiology/genetics/*therapy ; Stem Cells/*metabolism ; },
abstract = {Recent developments in targeted gene editing have paved the way for the wide adoption of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nucleases (Cas9) as an RNA-guided molecular tool to modify the genome of eukaryotic cells of animals. Theoretically, the translation of CRISPR-Cas9 can be applied to the treatment of inherited or acquired kidney disease, kidney transplantation and genetic corrections of somatic cells from kidneys with inherited mutations, such as polycystic kidney disease. Human pluripotent stem cells have been used to generate an unlimited source of kidney progenitor cells or, when spontaneously differentiated into three-dimensional kidney organoids, to model kidney organogenesis or the pathogenesis of disease. Gene editing now allows for the tagging and selection of specific kidney cell types or disease-specific gene knock in/out, which enables more precise understanding of kidney organogenesis and genetic diseases. This review discusses the mechanisms of action, in addition to the advantages and disadvantages, of the three major gene editing technologies, namely, CRISPR-Cas9, zinc finger nucleases and transcription activator-like effector nucleases. The implications of using gene editing to better understand kidney disease is reviewed in detail. In addition, the ethical issues of gene editing, which could be easily neglected in the modern, fast-paced research environment, are highlighted.},
}

Animals
CRISPR-Cas Systems
*Gene Editing/ethics
Genetic Therapy
Humans
Kidney Diseases/etiology/genetics/*therapy
Stem Cells/*metabolism

@article {pmid29757419,
year = {2018},
author = {Selvaraj, V and Stocco, DM},
title = {Letter to the Editor: Dubious Conclusions on TSPO Function.},
journal = {Endocrinology},
volume = {159},
number = {7},
pages = {2528-2529},
doi = {10.1210/en.2018-00052},
pmid = {29757419},
issn = {1945-7170},
mesh = {Animals ; *CRISPR-Cas Systems ; Leydig Cells ; Male ; *Membrane Potential, Mitochondrial ; Mice ; Mutation ; Neoplasms ; Receptors, GABA/genetics ; },
}

Animals
*CRISPR-Cas Systems
Leydig Cells
Male
*Membrane Potential, Mitochondrial
Mice
Mutation
Neoplasms
Receptors, GABA/genetics

@article {pmid29754237,
year = {2018},
author = {Schwartz, C and Wheeldon, I},
title = {CRISPR-Cas9-Mediated Genome Editing and Transcriptional Control in Yarrowia lipolytica.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1772},
number = {},
pages = {327-345},
doi = {10.1007/978-1-4939-7795-6_18},
pmid = {29754237},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/genetics ; Gene Editing/methods ; Gene Expression Regulation/genetics ; Genetic Vectors ; Genome, Fungal/*genetics ; Homologous Recombination/genetics ; RNA, Guide/*genetics ; Synthetic Biology/methods ; Transcription, Genetic/*genetics ; Yarrowia/*genetics ; },
abstract = {The discovery and adaptation of RNA-guided nucleases has resulted in the rapid development of efficient, scalable, and easily accessible synthetic biology tools for targeted genome editing and transcriptional control. In these systems, for example CRISPR-Cas9 from Streptococcus pyogenes, a protein with nuclease activity is targeted to a specific nucleotide sequence by a short RNA molecule, whereupon binding it cleaves the targeted nucleotide strand. To extend this genome-editing ability to the industrially important oleaginous yeast Yarrowia lipolytica, we developed a set of easily usable and effective CRISPR-Cas9 episomal vectors. In this protocols chapter, we first present a method by which arbitrary protein-coding genes can be disrupted via indel formation after CRISPR-Cas9 targeting. A second method demonstrates how the same CRISPR-Cas9 system can be used to induce markerless gene cassette integration into the genome by inducing homologous recombination after DNA cleavage by Cas9. Finally, we describe how a catalytically inactive form of Cas9 fused to a transcriptional repressor can be used to control transcription of native genes in Y. lipolytica. The CRISPR-Cas9 tools and strategies described here greatly increase the types of genome editing and transcriptional control that can be achieved in Y. lipolytica, and promise to facilitate more advanced engineering of this important oleaginous host.},
}

CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Endonucleases/genetics
Gene Editing/methods
Gene Expression Regulation/genetics
Genetic Vectors
Genome, Fungal/*genetics
Homologous Recombination/genetics
RNA, Guide/*genetics
Synthetic Biology/methods
Transcription, Genetic/*genetics
Yarrowia/*genetics

@article {pmid29754236,
year = {2018},
author = {Zhang, ZT and Jiménez-Bonilla, P and Seo, SO and Lu, T and Jin, YS and Blaschek, HP and Wang, Y},
title = {Bacterial Genome Editing with CRISPR-Cas9: Taking Clostridium beijerinckii as an Example.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1772},
number = {},
pages = {297-325},
doi = {10.1007/978-1-4939-7795-6_17},
pmid = {29754236},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Chromosome Deletion ; Clostridium beijerinckii/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Genome, Bacterial/*genetics ; Homologous Recombination/genetics ; RNA, Guide/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {CRISPR-Cas9 has been explored as a transformative genome engineering tool for many eukaryotic organisms. However, its utilization in bacteria remains limited and ineffective. This chapter, taking Clostridium beijerinckii as an example, describes the use of Streptococcus pyogenes CRISPR-Cas9 system guided by the single chimeric guide RNA (gRNA) for diverse genome-editing purposes, including chromosomal gene deletion, integration, single nucleotide modification, as well as "clean" mutant selection. The general principle is to use CRISPR-Cas9 as an efficient selection tool for the edited mutant (whose CRISPR-Cas9 target site has been disrupted through a homologous recombination event and thus can survive selection) against? the wild type background cells. This protocol is broadly applicable to other microorganisms for genome-editing purposes.},
}

CRISPR-Cas Systems/*genetics
Chromosome Deletion
Clostridium beijerinckii/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Genome, Bacterial/*genetics
Homologous Recombination/genetics
RNA, Guide/genetics
Streptococcus pyogenes/genetics

@article {pmid29754231,
year = {2018},
author = {Pohl, C and Mózsik, L and Driessen, AJM and Bovenberg, RAL and Nygård, YI},
title = {Genome Editing in Penicillium chrysogenum Using Cas9 Ribonucleoprotein Particles.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1772},
number = {},
pages = {213-232},
doi = {10.1007/978-1-4939-7795-6_12},
pmid = {29754231},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Endonucleases/genetics ; Gene Deletion ; Gene Editing/methods ; Gene Targeting/methods ; Penicillium chrysogenum/*genetics ; Protoplasts/metabolism ; RNA, Guide/*genetics ; Ribonucleoproteins/genetics ; },
abstract = {Several CRISPR/Cas9 tools have been recently established for precise genome editing in a wide range of filamentous fungi. This genome editing platform offers high flexibility in target selection and the possibility of introducing genetic deletions without the introduction of transgenic sequences . This chapter describes an approach for the transformation of Penicillium chrysogenum protoplasts with preassembled ribonucleoprotein particles (RNPs) consisting of purified Cas9 protein and in vitro transcribed single guide RNA (sgRNA) for the deletion of genome sequences or their replacement with alternative sequences. This method is potentially transferable to all fungal strains where protoplasts can be obtained from.},
}

CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Endonucleases/genetics
Gene Deletion
Gene Editing/methods
Gene Targeting/methods
Penicillium chrysogenum/*genetics
Protoplasts/metabolism
RNA, Guide/*genetics
Ribonucleoproteins/genetics

@article {pmid29754229,
year = {2018},
author = {Lebar, T and Jerala, R},
title = {Designed Transcriptional Regulation in Mammalian Cells Based on TALE- and CRISPR/dCas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1772},
number = {},
pages = {191-203},
doi = {10.1007/978-1-4939-7795-6_10},
pmid = {29754229},
issn = {1940-6029},
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems/*genetics ; Cell Line ; Cell Line, Tumor ; Cricetulus ; DNA-Binding Proteins/genetics ; Gene Expression Regulation/*genetics ; HEK293 Cells ; HeLa Cells ; Humans ; Mammals/*genetics ; Mice ; NIH 3T3 Cells ; Transcription Activator-Like Effectors/*genetics ; Transcription, Genetic/*genetics ; },
abstract = {Transcriptional regulation lies at the center of many cellular processes and is the result of cellular response to different external and internal signals. Control of transcription of selected genes enables an unprecedented access to shape the cellular response. While orthogonal transcription factors from bacteria, yeast, plants, or other cells have been used to introduce new cellular logic into mammalian cells, the discovery of designable modular DNA binding domains, such as Transcription Activator-Like Effectors (TALEs) and the CRISPR system, enable targeting of almost any selected DNA sequence. Fusion or conditional association of DNA targeting domain with transcriptional effector domains enables controlled regulation of almost any endogenous or ectopic gene. Moreover, the designed regulators can be linked into genetic circuits to implement complex responses, such as different types of Boolean functions and switches. In this chapter, we describe the protocols for achieving efficient transcriptional regulation with TALE- and CRISPR-based designed transcription factors in mammalian cells.},
}

Animals
CHO Cells
CRISPR-Cas Systems/*genetics
Cell Line
Cell Line, Tumor
Cricetulus
DNA-Binding Proteins/genetics
Gene Expression Regulation/*genetics
HEK293 Cells
HeLa Cells
Humans
Mammals/*genetics
Mice
NIH 3T3 Cells
Transcription Activator-Like Effectors/*genetics
Transcription, Genetic/*genetics

@article {pmid29754227,
year = {2018},
author = {Shin, J and Lee, N and Cho, S and Cho, BK},
title = {Targeted Genome Editing Using DNA-Free RNA-Guided Cas9 Ribonucleoprotein for CHO Cell Engineering.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1772},
number = {},
pages = {151-169},
doi = {10.1007/978-1-4939-7795-6_8},
pmid = {29754227},
issn = {1940-6029},
mesh = {Animals ; CHO Cells ; CRISPR-Cas Systems/*genetics ; Cell Engineering/methods ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Cricetinae ; Cricetulus ; DNA/*genetics ; Gene Deletion ; Gene Editing/methods ; Genome/*genetics ; Mutagenesis, Insertional/methods ; RNA, Guide/*genetics ; Ribonucleoproteins/*genetics ; },
abstract = {Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. This method uses purified Cas9 protein and in vitro transcribed sgRNA to edit the target gene without vector DNA. We have applied the RNP complex to CHO cell engineering to obtain desirable phenotypes and to reduce unintended insertional mutagenesis and off-target effects. Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.},
}

Animals
CHO Cells
CRISPR-Cas Systems/*genetics
Cell Engineering/methods
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Cricetinae
Cricetulus
DNA/*genetics
Gene Deletion
Gene Editing/methods
Genome/*genetics
Mutagenesis, Insertional/methods
RNA, Guide/*genetics
Ribonucleoproteins/*genetics

@article {pmid29524151,
year = {2018},
author = {Klann, TS and Crawford, GE and Reddy, TE and Gersbach, CA},
title = {Screening Regulatory Element Function with CRISPR/Cas9-based Epigenome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1767},
number = {},
pages = {447-480},
doi = {10.1007/978-1-4939-7774-1_25},
pmid = {29524151},
issn = {1940-6029},
support = {S10 OD018164/OD/NIH HHS/United States ; R01 DA036865/DA/NIDA NIH HHS/United States ; U01 HG007900/HG/NHGRI NIH HHS/United States ; DP2 OD008586/OD/NIH HHS/United States ; P30 AR066527/AR/NIAMS NIH HHS/United States ; T32 GM008555/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cloning, Molecular/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Epigenesis, Genetic ; Gene Editing/*methods ; Gene Expression Profiling/methods ; Gene Knock-In Techniques/methods ; Gene Library ; HEK293 Cells ; High-Throughput Screening Assays/methods ; Humans ; K562 Cells ; Luminescent Proteins/genetics ; RNA, Guide/*genetics ; Regulatory Sequences, Nucleic Acid ; },
abstract = {Genomic regulatory elements that control gene expression play an important role in many traits and diseases. Identifying the regulatory elements associated with each gene or phenotype and understanding the function of that element remain a significant challenge. To address this technological need, we developed CRISPR/Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. This protocol includes detailed instructions for design and cloning of gRNA libraries, construction of endogenous reporter cell lines via CRISPR/Cas9-mediated knock-in of fluorescent proteins, overall screen design, and recovery of the gRNA library for enrichment analysis. This protocol will be generally useful for implementing genome engineering technologies for high-throughput functional annotation of putative regulatory elements in their native chromosomal context.},
}

*CRISPR-Cas Systems
Cloning, Molecular/methods
Clustered Regularly Interspaced Short Palindromic Repeats
*Epigenesis, Genetic
Gene Editing/*methods
Gene Expression Profiling/methods
Gene Knock-In Techniques/methods
Gene Library
HEK293 Cells
High-Throughput Screening Assays/methods
Humans
K562 Cells
Luminescent Proteins/genetics
RNA, Guide/*genetics
Regulatory Sequences, Nucleic Acid

@article {pmid29524150,
year = {2018},
author = {Gao, D and Liang, FS},
title = {Chemical Inducible dCas9-Guided Editing of H3K27 Acetylation in Mammalian Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1767},
number = {},
pages = {429-445},
doi = {10.1007/978-1-4939-7774-1_24},
pmid = {29524150},
issn = {1940-6029},
support = {R21 HG008776/HG/NHGRI NIH HHS/United States ; },
mesh = {Abscisic Acid/*metabolism ; Acetylation ; Animals ; *CRISPR-Cas Systems ; Epigenesis, Genetic ; Gene Editing/*methods ; Genetic Loci ; HEK293 Cells ; *Histone Code ; Histones/genetics/metabolism ; Humans ; Interleukin 1 Receptor Antagonist Protein/genetics ; Promoter Regions, Genetic ; Protein Processing, Post-Translational ; Transfection/methods ; p300-CBP Transcription Factors/metabolism ; },
abstract = {The ability to edit specific epigenetic modifications at defined gene loci is pivotal to understand the biological function of these epigenetic marks. Here we describe a new inducible method to integrate the dCas9-based genome targeting with abscisic acid (ABA)-based chemically induced proximity (CIP) technologies to modify histone tail modifications at specific genome loci in living cells. ABA leads to rapid hetero-dimerization of the PYL and ABI proteins, which can be individually fused to dCas9 and a histone-modifying enzyme core domain. In the presence of ABA and locus-specific sgRNAs, this histone-modifying activity can be recruited to a specific genome locus to achieve histone editing with perfect temporal control. Herein, we describe the use of this technique in HEK293T cells to control the recruitment of the p300 acetyltransferase core domain to the human IL1RN locus to ectopically increase the acetylation of H3K27 and induce the expression of IL1RN gene.},
}

Abscisic Acid/*metabolism
Acetylation
Animals
*CRISPR-Cas Systems
Epigenesis, Genetic
Gene Editing/*methods
Genetic Loci
HEK293 Cells
*Histone Code
Histones/genetics/metabolism
Humans
Interleukin 1 Receptor Antagonist Protein/genetics
Promoter Regions, Genetic
Protein Processing, Post-Translational
Transfection/methods
p300-CBP Transcription Factors/metabolism

@article {pmid29524149,
year = {2018},
author = {Morita, S and Horii, T and Hatada, I},
title = {Editing of DNA Methylation Using dCas9-Peptide Repeat and scFv-TET1 Catalytic Domain Fusions.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {1767},
number = {},
pages = {419-428},
doi = {10.1007/978-1-4939-7774-1_23},
pmid = {29524149},
issn = {1940-6029},
mesh = {CRISPR-Cas Systems ; Catalytic Domain ; DNA Demethylation ; *DNA Methylation ; Epigenesis, Genetic ; Gene Editing/*methods ; Humans ; Mixed Function Oxygenases/genetics ; Polymerase Chain Reaction/methods ; RNA, Guide/genetics ; Zinc Fingers ; },
abstract = {DNA methylation, one of the most studied epigenetic modifications, regulates many biological processes. Dysregulation of DNA methylation is implicated in the etiology of several diseases, such as cancer and imprinting diseases. Accordingly, technologies designed to manipulate DNA methylation at specific loci are very important, and many epigenome editing technologies have been developed, based on zinc finger proteins, TALEs, and CRISPR/dCas9 targeting. We describe a protocol to induce and assess DNA demethylation on a target gene. It is based on a modification of the dCas9-SunTag system for efficient, targeted demethylation at specific DNA loci. The original SunTag system consists of ten copies of the GCN4 peptide separated by 5-amino-acid linkers. To achieve efficient recruitment of an anti-GCN4 scFv fused to the ten-eleven (TET) 1 hydroxylase, an enzyme that demethylates DNA, we changed the linker length to 22 amino acids.},
}

CRISPR-Cas Systems
Catalytic Domain
DNA Demethylation
*DNA Methylation
Epigenesis, Genetic
Gene Editing/*methods
Humans
Mixed Function Oxygenases/genetics
Polymerase Chain Reaction/methods
RNA, Guide/genetics
Zinc Fingers