@article {pmid30220031,
year = {2018},
author = {Kim, MS and Kim, KH},
title = {CRISPR/Cas9-mediated knockout of HIF-1α gene in epithelioma papulosum cyprini (EPC) cells inhibited apoptosis and viral hemorrhagic septicemia virus (VHSV) growth.},
journal = {Archives of virology},
volume = {163},
number = {12},
pages = {3395-3402},
doi = {10.1007/s00705-018-4018-0},
pmid = {30220031},
issn = {1432-8798},
mesh = {Animals ; *Apoptosis ; CRISPR-Cas Systems ; Cell Line ; Cyprinidae/virology ; Fish Diseases/*genetics/metabolism/physiopathology/virology ; Gene Knockout Techniques ; Hemorrhagic Septicemia, Viral/*genetics/metabolism/physiopathology/virology ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics/metabolism ; Novirhabdovirus/genetics/*growth & development/physiology ; },
abstract = {Hypoxia-inducible factor-1 (HIF-1) is a heterodimer of HIF-1α and HIF-1β, and its key role in the regulation of cellular responses to hypoxia has been well-demonstrated. The participation of HIF-1α in apoptosis has been reported in mammals, however, a little information is available on the role of HIF-1α in the progression of apoptosis in fish. In this study, to know the role of HIF-1α in the apoptosis of fish cells, we produced HIF-1α knockout Epithelioma papulosum cyprini (EPC) cells using a CRISPR/Cas9 vector, and a single cell clone showing a heterozygous insertion/deletion (indel) mutation (one nucleotide insertion and one nucleotide deletion in HIF-1α gene) was chosen for further experiments. To confirm the knockout of HIF-1α, cells were transfected with a hypoxia reporting vector containing hypoxic response elements (HREs). EPC cells transfected with the reporting plasmids showed significantly increased luminescence by exposure to cobalt chloride, a prolyl hydroxylases inhibitor. On the other hand, HIF-1α knockout EPC cells showed a non-responsiveness to a cobalt chloride exposure, suggesting that functional HIF-1α protein was not produced in the HIF-1α knockout EPC cells. Apoptosis progression induced by camptothecin and viral hemorrhagic septicemia virus (VHSV) infection was severely inhibited by HIF-1α knockout, and the replication of VHSV was significantly retarded in HIF-1α knockout EPC cells. These results suggest that HIF-1α in EPC cells acts as a pro-apoptotic factor in the progression of apoptosis triggered by a DNA damaging agent and rhabdoviral infection.},
}

Animals
*Apoptosis
CRISPR-Cas Systems
Cell Line
Cyprinidae/virology
Fish Diseases/*genetics/metabolism/physiopathology/virology
Gene Knockout Techniques
Hemorrhagic Septicemia, Viral/*genetics/metabolism/physiopathology/virology
Hypoxia-Inducible Factor 1, alpha Subunit/*genetics/metabolism
Novirhabdovirus/genetics/*growth & development/physiology

@article {pmid29956290,
year = {2018},
author = {Cheong, TC and Blasco, RB and Chiarle, R},
title = {The CRISPR/Cas9 System as a Tool to Engineer Chromosomal Translocation In Vivo.},
journal = {Advances in experimental medicine and biology},
volume = {1044},
number = {},
pages = {39-48},
doi = {10.1007/978-981-13-0593-1_4},
pmid = {29956290},
issn = {0065-2598},
support = {R01 CA196703/CA/NCI NIH HHS/United States ; R01 CA222598/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Embryonic Stem Cells/metabolism ; Genetic Engineering/*methods ; Mice ; *Translocation, Genetic ; },
abstract = {The CRISPR/Cas9 system has emerged as a powerful tool to edit the genome. Among many applications, the system generates the exciting possibility of engineering small and large portions of chromosomes to induce a variety of structural alterations such as deletions, inversions, insertions and inter-chromosomal translocations. Furthermore, the availability of viral vectors that express Cas9 has been critical to deliver the CRISPR/Cas9 system directly in vivo to induce chromosomal rearrangements. This review provides an overview of the state-of-the-art CRISPR/Cas9 technology to model a variety of rearrangements in vivo in animal models.},
}

Animals
*CRISPR-Cas Systems
Embryonic Stem Cells/metabolism
Genetic Engineering/*methods
Mice
*Translocation, Genetic

@article {pmid29956288,
year = {2018},
author = {Brunet, E and Jasin, M},
title = {Induction of Chromosomal Translocations with CRISPR-Cas9 and Other Nucleases: Understanding the Repair Mechanisms That Give Rise to Translocations.},
journal = {Advances in experimental medicine and biology},
volume = {1044},
number = {},
pages = {15-25},
doi = {10.1007/978-981-13-0593-1_2},
pmid = {29956288},
issn = {0065-2598},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01 CA185660/CA/NCI NIH HHS/United States ; R35 GM118175/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; *DNA Repair ; *Endonucleases ; Humans ; Mice ; *Translocation, Genetic ; },
abstract = {Chromosomal translocations are associated with several tumor types, including hematopoietic malignancies, sarcomas, and solid tumors of epithelial origin, due to their activation of a proto-oncogene or generation of a novel fusion protein with oncogenic potential. In many cases, the availability of suitable human models has been lacking because of the difficulty in recapitulating precise expression of the fusion protein or other reasons. Further, understanding how translocations form mechanistically has been a goal, as it may suggest ways to prevent their occurrence. Chromosomal translocations arise when DNA ends from double-strand breaks (DSBs) on two heterologous chromosomes are improperly joined. This review provides a summary of DSB repair mechanisms and their contribution to translocation formation, the various programmable nuclease platforms that have been used to generate translocations, and the successes that have been achieved in this area.},
}

Animals
*CRISPR-Cas Systems
DNA Breaks, Double-Stranded
*DNA Repair
*Endonucleases
Humans
Mice
*Translocation, Genetic

@article {pmid29872189,
year = {2018},
author = {Guglielmi, G},
title = {Million-dollar Kavli prize recognizes scientist scooped on CRISPR.},
journal = {Nature},
volume = {558},
number = {7708},
pages = {17-18},
doi = {10.1038/d41586-018-05308-5},
pmid = {29872189},
issn = {1476-4687},
mesh = {Animals ; Astronomy/standards ; *Awards and Prizes ; *CRISPR-Cas Systems ; *Gene Editing ; Hearing ; Humans ; Mice ; Neurosciences/standards ; Patents as Topic/legislation & jurisprudence ; },
}

Animals
Astronomy/standards
*Awards and Prizes
*CRISPR-Cas Systems
*Gene Editing
Hearing
Humans
Mice
Neurosciences/standards
Patents as Topic/legislation & jurisprudence

@article {pmid29514863,
year = {2018},
author = {Tagliabue, G},
title = {Comment on "Why are NGOs sceptical of genome editing?".},
journal = {EMBO reports},
volume = {19},
number = {4},
pages = {},
doi = {10.15252/embr.201745620},
pmid = {29514863},
issn = {1469-3178},
mesh = {CRISPR-Cas Systems ; *Gene Editing ; *Genome ; },
}

CRISPR-Cas Systems
*Gene Editing
*Genome

@article {pmid29330493,
year = {2018},
author = {Sawatsubashi, S and Joko, Y and Fukumoto, S and Matsumoto, T and Sugano, SS},
title = {Development of versatile non-homologous end joining-based knock-in module for genome editing.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {593},
doi = {10.1038/s41598-017-18911-9},
pmid = {29330493},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Cell Line ; *DNA End-Joining Repair ; Gene Editing ; Gene Knock-In Techniques/*methods ; Genetic Engineering ; Humans ; Keratinocytes/cytology/metabolism ; Plasmids/genetics ; Receptors, Calcitriol/*genetics ; },
abstract = {CRISPR/Cas9-based genome editing has dramatically accelerated genome engineering. An important aspect of genome engineering is efficient knock-in technology. For improved knock-in efficiency, the non-homologous end joining (NHEJ) repair pathway has been used over the homology-dependent repair pathway, but there remains a need to reduce the complexity of the preparation of donor vectors. We developed the versatile NHEJ-based knock-in module for genome editing (VIKING). Using the consensus sequence of the time-honored pUC vector to cut donor vectors, any vector with a pUC backbone could be used as the donor vector without customization. Conditions required to minimize random integration rates of the donor vector were also investigated. We attempted to isolate null lines of the VDR gene in human HaCaT keratinocytes using knock-in/knock-out with a selection marker cassette, and found 75% of clones isolated were successfully knocked-in. Although HaCaT cells have hypotetraploid genome composition, the results suggest multiple clones have VDR null phenotypes. VIKING modules enabled highly efficient knock-in of any vectors harboring pUC vectors. Users now can insert various existing vectors into an arbitrary locus in the genome. VIKING will contribute to low-cost genome engineering.},
}

*CRISPR-Cas Systems
Cell Line
*DNA End-Joining Repair
Gene Editing
Gene Knock-In Techniques/*methods
Genetic Engineering
Humans
Keratinocytes/cytology/metabolism
Plasmids/genetics
Receptors, Calcitriol/*genetics

@article {pmid28440293,
year = {2017},
author = {Yeung, ATY and Hale, C and Lee, AH and Gill, EE and Bushell, W and Parry-Smith, D and Goulding, D and Pickard, D and Roumeliotis, T and Choudhary, J and Thomson, N and Skarnes, WC and Dougan, G and Hancock, REW},
title = {Exploiting induced pluripotent stem cell-derived macrophages to unravel host factors influencing Chlamydia trachomatis pathogenesis.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15013},
doi = {10.1038/ncomms15013},
pmid = {28440293},
issn = {2041-1723},
support = {//Wellcome Trust/United Kingdom ; },
mesh = {Adult ; CRISPR-Cas Systems/genetics ; Cell Differentiation ; Chlamydia Infections/*genetics/immunology/microbiology ; Chlamydia trachomatis/immunology/*pathogenicity ; Gene Editing/methods ; Gene Expression Profiling ; Gene Knockout Techniques ; HeLa Cells ; Healthy Volunteers ; Host-Pathogen Interactions/*genetics/immunology ; Humans ; Induced Pluripotent Stem Cells/physiology ; Interferon Regulatory Factors/genetics/immunology ; Interleukin-10 Receptor alpha Subunit/genetics/immunology ; Macrophages/microbiology/*physiology ; Mutation ; Proteomics/methods ; },
abstract = {Chlamydia trachomatis remains a leading cause of bacterial sexually transmitted infections and preventable blindness worldwide. There are, however, limited in vitro models to study the role of host genetics in the response of macrophages to this obligate human pathogen. Here, we describe an approach using macrophages derived from human induced pluripotent stem cells (iPSdMs) to study macrophage-Chlamydia interactions in vitro. We show that iPSdMs support the full infectious life cycle of C. trachomatis in a manner that mimics the infection of human blood-derived macrophages. Transcriptomic and proteomic profiling of the macrophage response to chlamydial infection highlighted the role of the type I interferon and interleukin 10-mediated responses. Using CRISPR/Cas9 technology, we generated biallelic knockout mutations in host genes encoding IRF5 and IL-10RA in iPSCs, and confirmed their roles in limiting chlamydial infection in macrophages. This model can potentially be extended to other pathogens and tissue systems to advance our understanding of host-pathogen interactions and the role of human genetics in influencing the outcome of infections.},
}

Adult
CRISPR-Cas Systems/genetics
Cell Differentiation
Chlamydia Infections/*genetics/immunology/microbiology
Chlamydia trachomatis/immunology/*pathogenicity
Gene Editing/methods
Gene Expression Profiling
Gene Knockout Techniques
HeLa Cells
Healthy Volunteers
Host-Pathogen Interactions/*genetics/immunology
Humans
Induced Pluripotent Stem Cells/physiology
Interferon Regulatory Factors/genetics/immunology
Interleukin-10 Receptor alpha Subunit/genetics/immunology
Macrophages/microbiology/*physiology
Mutation
Proteomics/methods

@article {pmid28134250,
year = {2017},
author = {Qi, S and Li, Z and Schulze-Gahmen, U and Stjepanovic, G and Zhou, Q and Hurley, JH},
title = {Structural basis for ELL2 and AFF4 activation of HIV-1 proviral transcription.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14076},
doi = {10.1038/ncomms14076},
pmid = {28134250},
issn = {2041-1723},
support = {R01 AI095057/AI/NIAID NIH HHS/United States ; R01 AI041757/AI/NIAID NIH HHS/United States ; P50 GM082250/GM/NIGMS NIH HHS/United States ; S10 OD016268/OD/NIH HHS/United States ; P41 GM103393/GM/NIGMS NIH HHS/United States ; },
mesh = {Acquired Immunodeficiency Syndrome/*genetics/virology ; Binding Sites/genetics ; CRISPR-Cas Systems ; Crystallography, X-Ray ; Gene Expression Regulation, Viral ; Gene Knockout Techniques ; HIV-1/pathogenicity/*physiology ; HeLa Cells ; Humans ; Jurkat Cells ; Mutagenesis ; Protein Binding/genetics ; Protein Domains/genetics ; Proviruses/*physiology ; Recombinant Fusion Proteins/chemistry/genetics/metabolism ; Repressor Proteins/*chemistry/genetics/metabolism ; Transcription Elongation, Genetic/*physiology ; Transcriptional Elongation Factors/*chemistry/genetics/metabolism ; Virus Activation/genetics ; Virus Latency/genetics ; tat Gene Products, Human Immunodeficiency Virus/genetics ; },
abstract = {The intrinsically disordered scaffold proteins AFF1/4 and the transcription elongation factors ELL1/2 are core components of the super elongation complex required for HIV-1 proviral transcription. Here we report the 2.0-Å resolution crystal structure of the human ELL2 C-terminal domain bound to its 50-residue binding site on AFF4, the ELLBow. The ELL2 domain has the same arch-shaped fold as the tight junction protein occludin. The ELLBow consists of an N-terminal helix followed by an extended hairpin that we refer to as the elbow joint, and occupies most of the concave surface of ELL2. This surface is important for the ability of ELL2 to promote HIV-1 Tat-mediated proviral transcription. The AFF4-ELL2 interface is imperfectly packed, leaving a cavity suggestive of a potential binding site for transcription-promoting small molecules.},
}

Acquired Immunodeficiency Syndrome/*genetics/virology
Binding Sites/genetics
CRISPR-Cas Systems
Crystallography, X-Ray
Gene Expression Regulation, Viral
Gene Knockout Techniques
HIV-1/pathogenicity/*physiology
HeLa Cells
Humans
Jurkat Cells
Mutagenesis
Protein Binding/genetics
Protein Domains/genetics
Proviruses/*physiology
Recombinant Fusion Proteins/chemistry/genetics/metabolism
Repressor Proteins/*chemistry/genetics/metabolism
Transcription Elongation, Genetic/*physiology
Transcriptional Elongation Factors/*chemistry/genetics/metabolism
Virus Activation/genetics
Virus Latency/genetics
tat Gene Products, Human Immunodeficiency Virus/genetics

@article {pmid30451675,
year = {2018},
author = {Croteau, FR and Rousseau, GM and Moineau, S},
title = {[The CRISPR-Cas system: beyond genome editing].},
journal = {Medecine sciences : M/S},
volume = {34},
number = {10},
pages = {813-819},
doi = {10.1051/medsci/2018215},
pmid = {30451675},
issn = {1958-5381},
abstract = {CRISPR-Cas is an adaptive immune system used by many microbes to defend against nucleic acids invasion such as viral genomes. The microbial system uses its CRISPR locus to store genetic information that will generate short CRISPR RNAs. The latter with endonucleases (Cas) prevent future viral infections. Parts of this system were exploited to develop a powerful genome editing tool that was adapted for a variety of organisms. The ability of the CRISPR-Cas9 technology to effectively and precisely cut a targeted genomic DNA region has the potential to may be one day cure genetic diseases. The malleability of this editing tool also offers a wide range of possibilities from modulations of gene expression to epigenetic modifications. The natural CRISPR loci found in bacteria can be used to differentiate microbial strains or to study the interactions between bacteria and its habitat. Addressing CRISPR-Cas fundamentals in microbes and its popular use in eukaryotes, this review presents an update on a system that has revolutionized biological sciences.},
}

@article {pmid29769725,
year = {2018},
author = {Zhang, R and Kim, AS and Fox, JM and Nair, S and Basore, K and Klimstra, WB and Rimkunas, R and Fong, RH and Lin, H and Poddar, S and Crowe, JE and Doranz, BJ and Fremont, DH and Diamond, MS},
title = {Mxra8 is a receptor for multiple arthritogenic alphaviruses.},
journal = {Nature},
volume = {557},
number = {7706},
pages = {570-574},
doi = {10.1038/s41586-018-0121-3},
pmid = {29769725},
issn = {1476-4687},
support = {HHSN272201400058C/AI/NIAID NIH HHS/United States ; R01 AI095436/AI/NIAID NIH HHS/United States ; R01 AI114816/AI/NIAID NIH HHS/United States ; R01 AI123348/AI/NIAID NIH HHS/United States ; },
mesh = {3T3 Cells ; Animals ; Antibodies, Blocking/immunology ; CRISPR-Cas Systems/genetics ; Chikungunya virus/*metabolism/pathogenicity ; Chondrocytes/metabolism ; Fibroblasts/metabolism ; Humans ; Immunoglobulins/immunology/*metabolism ; Male ; Membrane Proteins/antagonists & inhibitors/immunology/*metabolism ; Mice ; Muscle, Skeletal/cytology/metabolism ; O'nyong-nyong Virus/*metabolism/pathogenicity ; Osteoblasts/metabolism ; Receptors, Fc/metabolism ; Receptors, Virus/deficiency/genetics/*metabolism ; },
abstract = {Arthritogenic alphaviruses comprise a group of enveloped RNA viruses that are transmitted to humans by mosquitoes and cause debilitating acute and chronic musculoskeletal disease 1 . The host factors required for alphavirus entry remain poorly characterized 2 . Here we use a genome-wide CRISPR-Cas9-based screen to identify the cell adhesion molecule Mxra8 as an entry mediator for multiple emerging arthritogenic alphaviruses, including chikungunya, Ross River, Mayaro and O'nyong nyong viruses. Gene editing of mouse Mxra8 or human MXRA8 resulted in reduced levels of viral infection of cells and, reciprocally, ectopic expression of these genes resulted in increased infection. Mxra8 bound directly to chikungunya virus particles and enhanced virus attachment and internalization into cells. Consistent with these findings, Mxra8-Fc fusion protein or anti-Mxra8 monoclonal antibodies blocked chikungunya virus infection in multiple cell types, including primary human synovial fibroblasts, osteoblasts, chondrocytes and skeletal muscle cells. Mutagenesis experiments suggest that Mxra8 binds to a surface-exposed region across the A and B domains of chikungunya virus E2 protein, which are a speculated site of attachment. Finally, administration of the Mxra8-Fc protein or anti-Mxra8 blocking antibodies to mice reduced chikungunya and O'nyong nyong virus infection as well as associated foot swelling. Pharmacological targeting of Mxra8 could form a strategy for mitigating infection and disease by multiple arthritogenic alphaviruses.},
}

3T3 Cells
Animals
Antibodies, Blocking/immunology
CRISPR-Cas Systems/genetics
Chikungunya virus/*metabolism/pathogenicity
Chondrocytes/metabolism
Fibroblasts/metabolism
Humans
Immunoglobulins/immunology/*metabolism
Male
Membrane Proteins/antagonists & inhibitors/immunology/*metabolism
Mice
Muscle, Skeletal/cytology/metabolism
O'nyong-nyong Virus/*metabolism/pathogenicity
Osteoblasts/metabolism
Receptors, Fc/metabolism
Receptors, Virus/deficiency/genetics/*metabolism

@article {pmid29323173,
year = {2018},
author = {Teixeira, M and Py, BF and Bosc, C and Laubreton, D and Moutin, MJ and Marvel, J and Flamant, F and Markossian, S},
title = {Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {474},
doi = {10.1038/s41598-017-18826-5},
pmid = {29323173},
issn = {2045-2322},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; DNA End-Joining Repair ; Electroporation ; Endopeptidases/chemistry/genetics ; Female ; Gene Editing/*methods ; Genetic Loci ; Genotyping Techniques ; INDEL Mutation ; Male ; Mice ; Mice, Inbred C57BL ; Mutation, Missense ; RNA, Guide/*genetics ; Zygote/*metabolism ; },
abstract = {In this report, we present an improved protocol for CRISPR/Cas9 genome editing in mice. The procedure consists in the electroporation of intact mouse zygotes with ribonucleoprotein complexes prepared in vitro from recombinant Cas9 nuclease and synthetic dual guide RNA. This simple cloning-free method proves to be extremely efficient for the generation of indels and small deletions by non-homologous end joining, and for the generation of specific point mutations by homology-directed repair. The procedure, which avoids DNA construction, in vitro transcription and oocyte microinjection, greatly simplifies genome editing in mice.},
}

Animals
CRISPR-Cas Systems/*genetics
DNA End-Joining Repair
Electroporation
Endopeptidases/chemistry/genetics
Female
Gene Editing/*methods
Genetic Loci
Genotyping Techniques
INDEL Mutation
Male
Mice
Mice, Inbred C57BL
Mutation, Missense
RNA, Guide/*genetics
Zygote/*metabolism

@article {pmid29315387,
year = {2018},
author = {Narimatsu, Y and Joshi, HJ and Yang, Z and Gomes, C and Chen, YH and Lorenzetti, FC and Furukawa, S and Schjoldager, KT and Hansen, L and Clausen, H and Bennett, EP and Wandall, HH},
title = {A validated gRNA library for CRISPR/Cas9 targeting of the human glycosyltransferase genome.},
journal = {Glycobiology},
volume = {28},
number = {5},
pages = {295-305},
doi = {10.1093/glycob/cwx101},
pmid = {29315387},
issn = {1460-2423},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Library ; Glycosyltransferases/*genetics/metabolism ; HEK293 Cells ; Humans ; RNA, Guide/*genetics/metabolism ; Reproducibility of Results ; },
abstract = {Over 200 glycosyltransferases are involved in the orchestration of the biosynthesis of the human glycome, which is comprised of all glycan structures found on different glycoconjugates in cells. The glycome is vast, and despite advancements in analytic strategies it continues to be difficult to decipher biological roles of glycans with respect to specific glycan structures, type of glycoconjugate, particular glycoproteins, and distinct glycosites on proteins. In contrast to this, the number of glycosyltransferase genes involved in the biosynthesis of the human glycome is manageable, and the biosynthetic roles of most of these enzymes are defined or can be predicted with reasonable confidence. Thus, with the availability of the facile CRISPR/Cas9 gene editing tool it now seems easier to approach investigation of the functions of the glycome through genetic dissection of biosynthetic pathways, rather than by direct glycan analysis. However, obstacles still remain with design and validation of efficient gene targeting constructs, as well as with the interpretation of results from gene targeting and the translation of gene function to glycan structures. This is especially true for glycosylation steps covered by isoenzyme gene families. Here, we present a library of validated high-efficiency gRNA designs suitable for individual and combinatorial targeting of the human glycosyltransferase genome together with a global view of the predicted functions of human glycosyltransferases to facilitate and guide gene targeting strategies in studies of the human glycome.},
}

CRISPR-Cas Systems/*genetics
*Gene Library
Glycosyltransferases/*genetics/metabolism
HEK293 Cells
Humans
RNA, Guide/*genetics/metabolism
Reproducibility of Results

@article {pmid29197638,
year = {2018},
author = {Lowder, LG and Zhou, J and Zhang, Y and Malzahn, A and Zhong, Z and Hsieh, TF and Voytas, DF and Zhang, Y and Qi, Y},
title = {Robust Transcriptional Activation in Plants Using Multiplexed CRISPR-Act2.0 and mTALE-Act Systems.},
journal = {Molecular plant},
volume = {11},
number = {2},
pages = {245-256},
doi = {10.1016/j.molp.2017.11.010},
pmid = {29197638},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Expression Regulation, Plant/genetics/physiology ; Transcription Factors/genetics/*metabolism ; Transcriptional Activation/genetics/*physiology ; },
abstract = {User-friendly tools for robust transcriptional activation of endogenous genes are highly demanded in plants. We previously showed that a dCas9-VP64 system consisting of the deactivated CRISPR-associated protein 9 (dCas9) fused with four tandem repeats of the transcriptional activator VP16 (VP64) could be used for transcriptional activation of endogenous genes in plants. In this study, we developed a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for dCas9-based transcriptional activation, and found that simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 (designated CRISPR-Act2.0) yielded stronger transcriptional activation than the dCas9-VP64 system. Moreover, we developed a multiplex transcription activator-like effector activation (mTALE-Act) system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0 in most cases tested. In addition, we explored tissue-specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools could be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies, which will facilitate transcriptional activation applications in both dicots and monocots.},
}

CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Expression Regulation, Plant/genetics/physiology
Transcription Factors/genetics/*metabolism
Transcriptional Activation/genetics/*physiology

@article {pmid28565744,
year = {2017},
author = {Cheng, LH and Liu, Y and Niu, T},
title = {[Chromosomal large fragment deletion induced by CRISPR/Cas9 gene editing system].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {38},
number = {5},
pages = {427-431},
doi = {10.3760/cma.j.issn.0253-2727.2017.05.014},
pmid = {28565744},
issn = {0253-2727},
mesh = {Animals ; *CRISPR-Cas Systems ; Chromosome Deletion ; DNA ; Gene Deletion ; *Gene Editing ; HEK293 Cells ; Humans ; Mice ; Multigene Family ; Mutagenesis, Insertional ; NIH 3T3 Cells ; Plasmids ; },
abstract = {Objective: Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. Methods: CRISPR-Cas9 lentiviral plasmid that could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse 11B3 chromosome was constructed via molecular clone. HEK293T cells were transfected to package lentivirus of CRISPR or Cas9 cDNA, then mouse NIH3T3 cells were infected by lentivirus and genomic DNA of these cells was extracted. The deleted fragment was amplified by PCR, TA clone, Sanger sequencing and other techniques were used to confirm the deletion of Aloxe3-Alox12b-Alox8 cluster genes. Results: The CRISPR-Cas9 lentiviral plasmid, which could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes, was successfully constructed. Deletion of target chromosome fragment (Aloxe3-Alox12b-Alox8 cluster genes) was verified by PCR. The deletion of Aloxe3-Alox12b-Alox8 cluster genes was affirmed by TA clone, Sanger sequencing, and the breakpoint junctions of the CRISPR-Cas9 system mediate cutting events were accurately recombined, insertion mutation did not occur between two cleavage sites at all. Conclusion: Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.},
}

Animals
*CRISPR-Cas Systems
Chromosome Deletion
DNA
Gene Deletion
*Gene Editing
HEK293 Cells
Humans
Mice
Multigene Family
Mutagenesis, Insertional
NIH 3T3 Cells
Plasmids

@article {pmid28561021,
year = {2017},
author = {Shin, HY and Wang, C and Lee, HK and Yoo, KH and Zeng, X and Kuhns, T and Yang, CM and Mohr, T and Liu, C and Hennighausen, L},
title = {CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15464},
doi = {10.1038/ncomms15464},
pmid = {28561021},
issn = {2041-1723},
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Female ; Gene Editing/*methods ; Genome/*genetics ; Genotype ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; RNA, Guide/genetics ; Sequence Deletion/*genetics ; },
abstract = {Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.},
}

Animals
CRISPR-Cas Systems/*genetics
Female
Gene Editing/*methods
Genome/*genetics
Genotype
Male
Mice
Mice, Inbred C57BL
Mice, Transgenic
RNA, Guide/genetics
Sequence Deletion/*genetics

@article {pmid28548094,
year = {2017},
author = {Filler Hayut, S and Melamed Bessudo, C and Levy, AA},
title = {Targeted recombination between homologous chromosomes for precise breeding in tomato.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15605},
doi = {10.1038/ncomms15605},
pmid = {28548094},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Gene Targeting/*methods ; Genome, Plant/genetics ; Heterozygote ; High-Throughput Nucleotide Sequencing ; Lycopersicon esculentum/*genetics ; Mutation ; Plant Breeding/*methods ; Plant Proteins/genetics ; Plants, Genetically Modified/*genetics ; Recombinational DNA Repair/*genetics ; Whole Genome Sequencing ; },
abstract = {Homologous recombination (HR) between parental chromosomes occurs stochastically. Here, we report on targeted recombination between homologous chromosomes upon somatic induction of DNA double-strand breaks (DSBs) via CRISPR-Cas9. We demonstrate this via a visual and molecular assay whereby DSB induction between two alleles carrying different mutations in the PHYTOENE SYNTHASE (PSY1) gene results in yellow fruits with wild type red sectors forming via HR-mediated DSB repair. We also show that in heterozygote plants containing one psy1 allele immune and one sensitive to CRISPR, repair of the broken allele using the unbroken allele sequence template is a common outcome. In another assay, we show evidence of a somatically induced DSB in a cross between a psy1 edible tomato mutant and wild type Solanum pimpinellifolium, targeting only the S. pimpinellifolium allele. This enables characterization of germinally transmitted targeted somatic HR events, demonstrating that somatically induced DSBs can be exploited for precise breeding of crops.},
}

CRISPR-Cas Systems/genetics
DNA Breaks, Double-Stranded
Gene Targeting/*methods
Genome, Plant/genetics
Heterozygote
High-Throughput Nucleotide Sequencing
Lycopersicon esculentum/*genetics
Mutation
Plant Breeding/*methods
Plant Proteins/genetics
Plants, Genetically Modified/*genetics
Recombinational DNA Repair/*genetics
Whole Genome Sequencing

@article {pmid28534478,
year = {2017},
author = {Rosenbluh, J and Xu, H and Harrington, W and Gill, S and Wang, X and Vazquez, F and Root, DE and Tsherniak, A and Hahn, WC},
title = {Complementary information derived from CRISPR Cas9 mediated gene deletion and suppression.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15403},
doi = {10.1038/ncomms15403},
pmid = {28534478},
issn = {2041-1723},
support = {U01 CA176058/CA/NCI NIH HHS/United States ; U01 CA199253/CA/NCI NIH HHS/United States ; },
mesh = {A549 Cells ; *CRISPR-Cas Systems ; Catalysis ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; DNA/analysis ; DNA Copy Number Variations ; Endonucleases/metabolism ; False Positive Reactions ; *Gene Deletion ; Gene Editing ; Gene Expression ; Gene Library ; *Genes, Essential ; *Genome, Human ; HT29 Cells ; Humans ; Phenotype ; },
abstract = {CRISPR-Cas9 provides the means to perform genome editing and facilitates loss-of-function screens. However, we and others demonstrated that expression of the Cas9 endonuclease induces a gene-independent response that correlates with the number of target sequences in the genome. An alternative approach to suppressing gene expression is to block transcription using a catalytically inactive Cas9 (dCas9). Here we directly compare genome editing by CRISPR-Cas9 (cutting, CRISPRc) and gene suppression using KRAB-dCas9 (CRISPRi) in loss-of-function screens to identify cell essential genes. CRISPRc identified 98% of previously defined cell essential genes. After optimizing library construction by analysing transcriptional start sites (TSS), CRISRPi identified 92% of core cell essential genes and did not show a bias to regions involved in copy number alterations. However, bidirectional promoters scored as false positives in CRISRPi. We conclude that CRISPRc and CRISPRi have different off-target effects and combining these approaches provides complementary information in loss-of-function genetic screens.},
}

A549 Cells
*CRISPR-Cas Systems
Catalysis
Cell Line
Cell Line, Tumor
Cell Proliferation
DNA/analysis
DNA Copy Number Variations
Endonucleases/metabolism
False Positive Reactions
*Gene Deletion
Gene Editing
Gene Expression
Gene Library
*Genes, Essential
*Genome, Human
HT29 Cells
Humans
Phenotype

@article {pmid28474669,
year = {2017},
author = {Morgens, DW and Wainberg, M and Boyle, EA and Ursu, O and Araya, CL and Tsui, CK and Haney, MS and Hess, GT and Han, K and Jeng, EE and Li, A and Snyder, MP and Greenleaf, WJ and Kundaje, A and Bassik, MC},
title = {Genome-scale measurement of off-target activity using Cas9 toxicity in high-throughput screens.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15178},
doi = {10.1038/ncomms15178},
pmid = {28474669},
issn = {2041-1723},
support = {P50 HG007735/HG/NHGRI NIH HHS/United States ; T32 CA009302/CA/NCI NIH HHS/United States ; T32 GM007790/GM/NIGMS NIH HHS/United States ; T32 HG000044/HG/NHGRI NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Cell Line ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Damage/genetics ; Gene Targeting/*methods ; *Genomic Library ; High-Throughput Screening Assays/*methods ; Humans ; Polysaccharides/biosynthesis ; RNA Interference ; RNA, Guide/*genetics ; Ricin/toxicity ; },
abstract = {CRISPR-Cas9 screens are powerful tools for high-throughput interrogation of genome function, but can be confounded by nuclease-induced toxicity at both on- and off-target sites, likely due to DNA damage. Here, to test potential solutions to this issue, we design and analyse a CRISPR-Cas9 library with 10 variable-length guides per gene and thousands of negative controls targeting non-functional, non-genic regions (termed safe-targeting guides), in addition to non-targeting controls. We find this library has excellent performance in identifying genes affecting growth and sensitivity to the ricin toxin. The safe-targeting guides allow for proper control of toxicity from on-target DNA damage. Using this toxicity as a proxy to measure off-target cutting, we demonstrate with tens of thousands of guides both the nucleotide position-dependent sensitivity to single mismatches and the reduction of off-target cutting using truncated guides. Our results demonstrate a simple strategy for high-throughput evaluation of target specificity and nuclease toxicity in Cas9 screens.},
}

CRISPR-Cas Systems/*genetics
Cell Line
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
DNA Damage/genetics
Gene Targeting/*methods
*Genomic Library
High-Throughput Screening Assays/*methods
Humans
Polysaccharides/biosynthesis
RNA Interference
RNA, Guide/*genetics
Ricin/toxicity

@article {pmid28469274,
year = {2017},
author = {Jiang, Y and Qian, F and Yang, J and Liu, Y and Dong, F and Xu, C and Sun, B and Chen, B and Xu, X and Li, Y and Wang, R and Yang, S},
title = {CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {15179},
doi = {10.1038/ncomms15179},
pmid = {28469274},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Corynebacterium glutamicum/*genetics ; DNA, Single-Stranded/genetics ; Endonucleases/*metabolism ; Francisella/enzymology ; Gene Editing/*methods ; Genetic Engineering/*methods ; Genome, Bacterial/genetics ; },
abstract = {Corynebacterium glutamicum is an important industrial metabolite producer that is difficult to genetically engineer. Although the Streptococcus pyogenes (Sp) CRISPR-Cas9 system has been adapted for genome editing of multiple bacteria, it cannot be introduced into C. glutamicum. Here we report a Francisella novicida (Fn) CRISPR-Cpf1-based genome-editing method for C. glutamicum. CRISPR-Cpf1, combined with single-stranded DNA (ssDNA) recombineering, precisely introduces small changes into the bacterial genome at efficiencies of 86-100%. Large gene deletions and insertions are also obtained using an all-in-one plasmid consisting of FnCpf1, CRISPR RNA, and homologous arms. The two CRISPR-Cpf1-assisted systems enable N iterative rounds of genome editing in 3N+4 or 3N+2 days. A proof-of-concept, codon saturation mutagenesis at G149 of γ-glutamyl kinase relieves L-proline inhibition using Cpf1-assisted ssDNA recombineering. Thus, CRISPR-Cpf1-based genome editing provides a highly efficient tool for genetic engineering of Corynebacterium and other bacteria that cannot utilize the Sp CRISPR-Cas9 system.},
}

CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Corynebacterium glutamicum/*genetics
DNA, Single-Stranded/genetics
Endonucleases/*metabolism
Francisella/enzymology
Gene Editing/*methods
Genetic Engineering/*methods
Genome, Bacterial/genetics

@article {pmid28169275,
year = {2017},
author = {Shin, G and Grimes, SM and Lee, H and Lau, BT and Xia, LC and Ji, HP},
title = {CRISPR-Cas9-targeted fragmentation and selective sequencing enable massively parallel microsatellite analysis.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14291},
doi = {10.1038/ncomms14291},
pmid = {28169275},
issn = {2041-1723},
support = {P01 HG000205/HG/NHGRI NIH HHS/United States ; R01 HG006137/HG/NHGRI NIH HHS/United States ; R33 CA174575/CA/NCI NIH HHS/United States ; },
mesh = {Alleles ; *CRISPR-Cas Systems ; *Genomic Library ; Haplotypes/genetics ; High-Throughput Nucleotide Sequencing/*methods ; *Human Genome Project ; Humans ; Microsatellite Repeats/*genetics ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA/*methods ; },
abstract = {Microsatellites are multi-allelic and composed of short tandem repeats (STRs) with individual motifs composed of mononucleotides, dinucleotides or higher including hexamers. Next-generation sequencing approaches and other STR assays rely on a limited number of PCR amplicons, typically in the tens. Here, we demonstrate STR-Seq, a next-generation sequencing technology that analyses over 2,000 STRs in parallel, and provides the accurate genotyping of microsatellites. STR-Seq employs in vitro CRISPR-Cas9-targeted fragmentation to produce specific DNA molecules covering the complete microsatellite sequence. Amplification-free library preparation provides single molecule sequences without unique molecular barcodes. STR-selective primers enable massively parallel, targeted sequencing of large STR sets. Overall, STR-Seq has higher throughput, improved accuracy and provides a greater number of informative haplotypes compared with other microsatellite analysis approaches. With these new features, STR-Seq can identify a 0.1% minor genome fraction in a DNA mixture composed of different, unrelated samples.},
}

Alleles
*CRISPR-Cas Systems
*Genomic Library
Haplotypes/genetics
High-Throughput Nucleotide Sequencing/*methods
*Human Genome Project
Humans
Microsatellite Repeats/*genetics
Polymorphism, Single Nucleotide
Sequence Analysis, DNA/*methods

@article {pmid30446870,
year = {2018},
author = {Liu, Q and Wang, C and Jiao, X and Zhang, H and Song, L and Li, Y and Gao, C and Wang, K},
title = {Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
doi = {10.1007/s11427-018-9402-9},
pmid = {30446870},
issn = {1869-1889},
abstract = {The CRISPR/Cas system has been extensively applied to make precise genetic modifications in various organisms. Despite its importance and widespread use, large-scale mutation screening remains time-consuming, labour-intensive and costly. Here, we developed Hi-TOM (available at https://doi.org/www.hi-tom.net/hi-tom/), an online tool to track the mutations with precise percentage for multiple samples and multiple target sites. We also described a corresponding next-generation sequencing (NGS) library construction strategy by fixing the bridge sequences and barcoding primers. Analysis of the samples from rice, hexaploid wheat and human cells reveals that the Hi-TOM tool has high reliability and sensitivity in tracking various mutations, especially complex chimeric mutations frequently induced by genome editing. Hi-TOM does not require special design of barcode primers, cumbersome parameter configuration or additional data analysis. Thus, the streamlined NGS library construction and comprehensive result output make Hi-TOM particularly suitable for high-throughput identification of all types of mutations induced by CRISPR/Cas systems.},
}

@article {pmid30444997,
year = {2018},
author = {Mo, CY and Marraffini, LA},
title = {If You'd Like to Stop a Type III CRISPR Ribonuclease, Then You Should Put a Ring (Nuclease) on It.},
journal = {Molecular cell},
volume = {72},
number = {4},
pages = {608-609},
doi = {10.1016/j.molcel.2018.10.048},
pmid = {30444997},
issn = {1097-4164},
abstract = {Athukoralage et al. (2018) identify a new class of nuclease that degrades cyclic oligoadenylate (cOA), a second messenger that activates non-specific RNA degradation by the type III CRISPR-Cas accessory RNase Csm6/Csx1. This discovery provides a mechanism for regulating the degradation of foreign transcripts during the type III CRISPR immune response.},
}

@article {pmid30443021,
year = {2018},
author = {Riad, A and Zeng, C and Weng, CC and Winters, H and Xu, K and Makvandi, M and Metz, T and Carlin, S and Mach, RH},
title = {Sigma-2 Receptor/TMEM97 and PGRMC-1 Increase the Rate of Internalization of LDL by LDL Receptor through the Formation of a Ternary Complex.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {16845},
doi = {10.1038/s41598-018-35430-3},
pmid = {30443021},
issn = {2045-2322},
abstract = {CRISPR/Cas gene studies were conducted in HeLa cells where either PGRMC1, TMEM97 or both proteins were removed via gene editing. A series of radioligand binding studies, confocal microscopy studies, and internalization of radiolabeled or fluorescently tagged LDL particles were then conducted in these cells. The results indicate that PGRMC1 knockout (KO) did not reduce the density of binding sites for the sigma-2 receptor (σ2R) radioligands, [125I]RHM-4 or [3H]DTG, but a reduction in the receptor affinity of both radioligands was observed. TMEM97 KO resulted in a complete loss of binding of [125I]RHM-4 and a significant reduction in binding of [3H]DTG. TMEM97 KO and PGRMC1 KO resulted in an equal reduction in the rate of uptake of fluorescently-tagged or 3H-labeled LDL, and knocking out both proteins did not result in a further rate of reduction of LDL uptake. Confocal microscopy and Proximity Ligation Assay studies indicated a clear co-localization of LDLR, PGRMC1 and TMEM97. These data indicate that the formation of a ternary complex of LDLR-PGRMC1-TMEM97 is necessary for the rapid internalization of LDL by LDLR.},
}

@article {pmid30217040,
year = {2018},
author = {Pan, Q and Wang, J and Gao, Y and Cui, H and Liu, C and Qi, X and Zhang, Y and Wang, Y and Wang, X},
title = {The Natural Large Genomic Deletion Is Unrelated to the Increased Virulence of the Novel Genotype Fowl Adenovirus 4 Recently Emerged in China.},
journal = {Viruses},
volume = {10},
number = {9},
pages = {},
doi = {10.3390/v10090494},
pmid = {30217040},
issn = {1999-4915},
mesh = {Adenoviridae/classification/*genetics ; Adenoviridae Infections/*veterinary ; Animals ; CRISPR-Cas Systems ; Chickens ; China/epidemiology ; Ducks ; Gene Editing ; *Genome, Viral ; Genotype ; Phylogeny ; Poultry Diseases/diagnosis/epidemiology/*virology ; *Sequence Deletion ; Virulence/genetics ; },
abstract = {Since 2015, severe hydropericardium-hepatitis syndrome (HHS), caused by a highly pathogenic fowl adenovirus 4 (FAdV-4), emerged in China. In our previous study, the FAdV-4 has been identified as a novel genotype with a unique 1966-bp nucleotide deletion (1966Del) between open reading frame 42 and 43. In this study, the natural 1966Del was frequently identified among 17 clinical isolates and other reported Chinese clinical strains. To investigate the relationship between 1966Del and the increased virulence of the novel FAdV-4, a CRISPR/Cas9 operating platform for FAdV-4 was developed for the first time in this study. Based on this platform, a Re1966 strain was rescued, inserted the relative 1966Del sequence of a nonpathogenic strain KR5. In the pathogenicity study, the Re1966 strain retained high virulence for specific-pathogen-free chickens, similar to the parental wild-type HLJFAd15, although the survival time of chickens infected with Re1966 was much longer. Therefore, the natural 1966Del was identified as a non-essential site for the increased virulence of the emerged novel FAdV-4. Although further research on the virulence-determining region or point within the genome of the novel FAdV-4 is needed, the CRISPR/Cas9 operating platform for the novel FAdV-4 was developed and successfully applied to edit the genomic DNA for the first time, and it provides a novel powerful tool for both basic virology studies and vaccine vector development of FAdVs.},
}

Adenoviridae/classification/*genetics
Adenoviridae Infections/*veterinary
Animals
CRISPR-Cas Systems
Chickens
China/epidemiology
Ducks
Gene Editing
*Genome, Viral
Genotype
Phylogeny
Poultry Diseases/diagnosis/epidemiology/*virology
*Sequence Deletion
Virulence/genetics

@article {pmid30210230,
year = {2018},
author = {Zelinka, CP and Sotolongo-Lopez, M and Fadool, JM},
title = {Targeted disruption of the endogenous zebrafish rhodopsin locus as models of rapid rod photoreceptor degeneration.},
journal = {Molecular vision},
volume = {24},
number = {},
pages = {587-602},
pmid = {30210230},
issn = {1090-0535},
support = {R21 EY025410/EY/NEI NIH HHS/United States ; },
mesh = {Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Codon, Terminator/genetics ; *Disease Models, Animal ; Frameshift Mutation/genetics ; Gene Targeting ; Immunoblotting ; Polymorphism, Restriction Fragment Length ; RNA, Messenger/genetics ; Retinal Degeneration/*genetics/pathology ; Retinal Rod Photoreceptor Cells/*pathology ; Rhodopsin/*genetics ; Zebrafish/*genetics ; Zebrafish Proteins/*genetics ; },
abstract = {Purpose: Retinitis pigmentosa (RP) is a collection of genetic disorders that results in the degeneration of light-sensitive photoreceptor cells, leading to blindness. RP is associated with more than 70 loci that may display dominant or recessive modes of inheritance, but mutations in the gene encoding the visual pigment rhodopsin (RHO) are the most frequent cause. In an effort to develop precise mutations in zebrafish as novel models of photoreceptor degeneration, we describe the generation and germline transmission of a series of novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-induced insertion and deletion (indel) mutations in the major zebrafish rho locus, rh1-1.

Methods: One- or two-cell staged zebrafish embryos were microinjected with in vitro transcribed mRNA encoding Cas9 and a single guide RNA (gRNA). Mutations were detected by restriction fragment length polymorphism (RFLP) and DNA sequence analyses in injected embryos and offspring. Immunolabeling with rod- and cone-specific antibodies was used to test for histological and cellular changes.

Results: Using gRNAs that targeted highly conserved regions of rh1-1, a series of dominant and recessive alleles were recovered that resulted in the rapid degeneration of rod photoreceptors. No effect on cones was observed. Targeting the 5'-coding sequence of rh1-1 led to the recovery of several indels similar to disease-associated alleles. A frame shift mutation leading to a premature stop codon (T17*) resulted in rod degeneration when brought to homozygosity. Immunoblot and fluorescence labeling with a Rho-specific antibody suggest that this is indeed a null allele, illustrating that the Rho expression is essential for rod survival. Two in-frame mutations were recovered that disrupted the highly conserved N-linked glycosylation consensus sequence at N15. Larvae heterozygous for either of the alleles demonstrated rapid rod degeneration. Targeting of the 3'-coding region of rh1-1 resulted in the recovery of an allele encoding a premature stop codon (S347*) upstream of the conserved VSPA sorting sequence and a second in-frame allele that disrupted the putative phosphorylation site at S339. Both alleles resulted in rod death in a dominant inheritance pattern. Following the loss of the targeting sequence, immunolabeling for Rho was no longer restricted to the rod outer segment, but it was also localized to the plasma membrane.

Conclusions: The efficiency of CRISPR/Cas9 for gene targeting, coupled with the large number of mutations associated with RP, provided a backdrop for the rapid isolation of novel alleles in zebrafish that phenocopy disease. These novel lines will provide much needed in-vivo models for high throughput screens of compounds or genes that protect from photoreceptor degeneration.},
}

Animals
Animals, Genetically Modified
CRISPR-Cas Systems
Codon, Terminator/genetics
*Disease Models, Animal
Frameshift Mutation/genetics
Gene Targeting
Immunoblotting
Polymorphism, Restriction Fragment Length
RNA, Messenger/genetics
Retinal Degeneration/*genetics/pathology
Retinal Rod Photoreceptor Cells/*pathology
Rhodopsin/*genetics
Zebrafish/*genetics
Zebrafish Proteins/*genetics

@article {pmid30076486,
year = {2018},
author = {Kuruvilla, J and Sasmita, AO and Ling, APK},
title = {Therapeutic potential of combined viral transduction and CRISPR/Cas9 gene editing in treating neurodegenerative diseases.},
journal = {Neurological sciences : official journal of the Italian Neurological Society and of the Italian Society of Clinical Neurophysiology},
volume = {39},
number = {11},
pages = {1827-1835},
doi = {10.1007/s10072-018-3521-0},
pmid = {30076486},
issn = {1590-3478},
mesh = {Animals ; CRISPR-Cas Systems/*physiology ; Gene Editing/*methods ; Genetic Therapy/*methods ; Genetic Vectors ; Humans ; Neurodegenerative Diseases/*genetics/*therapy ; },
abstract = {BACKGROUND AND PURPOSE: The central nervous system (CNS) faces unique difficulties in attaining permanent therapy for neurodegenerative disorder (ND). Genomic level forms of therapy have garnered interest in the recent decade, with the novel CRISPR/Cas9 gene editing tool continuing to be explored due to its efficiency, safety, and adaptability to varying conditions. With the aid of viral vectors as transport vectors, the gene editing tool has produced promising in vitro and in vivo findings in study models. Thus, this review focuses on the recent advancements and update of CRISPR/Cas9 to combat neurodegenerative diseases.

METHODS: Articles detailing potential applications of CRISPR/Cas9 in neurodegenerative settings were retrieved from PubMed and Google Scholar with the keywords "CRISPR," "gene editing," and "neurodegenerative diseases." Relevant information was collected and critically reviewed.

RESULTS: The utility of CRISPR/Cas9 coupled with viral transduction ranges from the disruption of amyloid precursor protein (APP) production at a genomic level in Alzheimer's disease (AD) to the deletion of varying exon portions of the Dmd gene in Duchenne muscular dystrophy (DMD) which would increase dystrophin expression. This usage of CRISPR/Cas9 also extends to experimentally ameliorate the neurodegenerative effects caused by viral infections.

CONCLUSION: The CRISPR/Cas9 gene editing tool is a powerful arsenal in the field of gene therapy and molecular medicine; hence, more research should be called to focus on the ample potential this tool has to offer in the field of neurodegenerative diseases.},
}

Animals
CRISPR-Cas Systems/*physiology
Gene Editing/*methods
Genetic Therapy/*methods
Genetic Vectors
Humans
Neurodegenerative Diseases/*genetics/*therapy

@article {pmid29321601,
year = {2018},
author = {Janssen, BD and Chen, YP and Molgora, BM and Wang, SE and Simoes-Barbosa, A and Johnson, PJ},
title = {CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {270},
doi = {10.1038/s41598-017-18442-3},
pmid = {29321601},
issn = {2045-2322},
support = {T32 AI007323/AI/NIAID NIH HHS/United States ; R33 AI119721/AI/NIAID NIH HHS/United States ; R01 AI103182/AI/NIAID NIH HHS/United States ; T32 GM007185/GM/NIGMS NIH HHS/United States ; R21 AI119721/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Female ; *Gene Editing ; Gene Expression ; *Gene Knockout Techniques ; Gene Targeting ; Genes, Protozoan ; Genes, Reporter ; Genome, Protozoan ; Humans ; Trichomonas Vaginitis/parasitology ; Trichomonas vaginalis/*genetics ; },
abstract = {The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T. vaginalis cell. We then created DNA constructs required to express several components essential to drive CRISPR/Cas9-mediated DNA modification: guide RNA (gRNA), the Cas9 endonuclease, short oligonucleotides and large, linearized DNA templates. Using these technical advances, we have established CRISPR/Cas9-mediated repair of mutations in genes contained on circular DNA plasmids harbored by the parasite. We also engineered CRISPR/Cas9 directed homologous recombination to delete (i.e. knock out) two non-essential genes within the T. vaginalis genome. This first report of the use of the CRISPR/Cas9 system in T. vaginalis greatly expands the ability to manipulate the genome of this pathogen and sets the stage for testing of the role of specific genes in many biological processes.},
}

*CRISPR-Cas Systems
Female
*Gene Editing
Gene Expression
*Gene Knockout Techniques
Gene Targeting
Genes, Protozoan
Genes, Reporter
Genome, Protozoan
Humans
Trichomonas Vaginitis/parasitology
Trichomonas vaginalis/*genetics

@article {pmid29311693,
year = {2018},
author = {Peretz, L and Besser, E and Hajbi, R and Casden, N and Ziv, D and Kronenberg, N and Gigi, LB and Sweetat, S and Khawaled, S and Aqeilan, R and Behar, O},
title = {Combined shRNA over CRISPR/cas9 as a methodology to detect off-target effects and a potential compensatory mechanism.},
journal = {Scientific reports},
volume = {8},
number = {1},
pages = {93},
doi = {10.1038/s41598-017-18551-z},
pmid = {29311693},
issn = {2045-2322},
mesh = {Animals ; *CRISPR-Cas Systems ; Cell Death/genetics ; Cell Line, Tumor ; Disease Models, Animal ; Gene Knockdown Techniques ; Glioma/genetics/metabolism/pathology ; Heterografts ; Humans ; Mice ; RNA Interference ; RNA, Small Interfering/*genetics ; Semaphorins/genetics/metabolism ; },
abstract = {Inhibition of genes is a powerful approach to study their function. While RNA interference is a widely used method to achieve this goal, mounting evidence indicates that such an approach is prone to off-target effects. An alternative approach to gene function inhibition is genetic mutation, such as the CRISPR/cas9 method. A recent report, however, demonstrated that genetic mutation and inhibition of gene expression do not always give corresponding results. This can be explained by off-target effects, but it was recently shown, at least in one case, that these differences are the result of a compensatory mechanism induced only by genetic mutation. We present here a combination of RNA inhibition and CRISPR/cas9 methods to identify possible off targets as well as potential compensatory effects. This approach is demonstrated by testing a possible role for Sema4B in glioma biology, in which our results implicate Sema4B as having a critical function. In stark contrast, by using shRNA over CRISPR/cas9 combined methodology, we clearly demonstrate that the Sema4B targeted shRNA effects on cell proliferation is the result of off-target effects. Nevertheless, it also revealed that certain splice variants of Sema4B are important for the ability of glioma cells to grow as individual clones.},
}

Animals
*CRISPR-Cas Systems
Cell Death/genetics
Cell Line, Tumor
Disease Models, Animal
Gene Knockdown Techniques
Glioma/genetics/metabolism/pathology
Heterografts
Humans
Mice
RNA Interference
RNA, Small Interfering/*genetics
Semaphorins/genetics/metabolism

@article {pmid29196861,
year = {2018},
author = {Lau, CH and Suh, Y},
title = {CRISPR-based strategies for studying regulatory elements and chromatin structure in mammalian gene control.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {29},
number = {3-4},
pages = {205-228},
doi = {10.1007/s00335-017-9727-2},
pmid = {29196861},
issn = {1432-1777},
support = {AG017242/NH/NIH HHS/United States ; GM104459/NH/NIH HHS/United States ; CA180126/NH/NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/*genetics ; Chromatin/*chemistry ; DNA, Intergenic/genetics ; *Gene Expression Regulation ; Humans ; Mammals/*genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; },
abstract = {The development of high-throughput methods has enabled the genome-wide identification of putative regulatory elements in a wide variety of mammalian cells at an unprecedented resolution. Extensive genomic studies have revealed the important role of regulatory elements and genetic variation therein in disease formation and risk. In most cases, there is only correlative evidence for the roles of these elements and non-coding changes within these elements in pathogenesis. With the advent of genome- and epigenome-editing tools based on the CRISPR technology, it is now possible to test the functional relevance of the regulatory elements and alterations on a genomic scale. Here, we review the various CRISPR-based strategies that have been developed to functionally validate the candidate regulatory elements in mammals as well as the non-coding genetic variants found to be associated with human disease. We also discuss how these synthetic biology tools have helped to elucidate the role of three-dimensional nuclear architecture and higher-order chromatin organization in shaping functional genome and controlling gene expression.},
}

Animals
CRISPR-Cas Systems/*genetics
Chromatin/*chemistry
DNA, Intergenic/genetics
*Gene Expression Regulation
Humans
Mammals/*genetics
Regulatory Sequences, Nucleic Acid/*genetics

@article {pmid28281536,
year = {2017},
author = {Kerr, MC and Gomez, GA and Ferguson, C and Tanzer, MC and Murphy, JM and Yap, AS and Parton, RG and Huston, WM and Teasdale, RD},
title = {Laser-mediated rupture of chlamydial inclusions triggers pathogen egress and host cell necrosis.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14729},
doi = {10.1038/ncomms14729},
pmid = {28281536},
issn = {2041-1723},
mesh = {CRISPR-Cas Systems ; Calpain/*antagonists & inhibitors/genetics/metabolism ; Cell Death/radiation effects ; Chlamydia trachomatis/pathogenicity/physiology ; Cysteine Proteinase Inhibitors/pharmacology ; Gene Editing ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; HeLa Cells ; *Host-Pathogen Interactions ; Humans ; *Laser Therapy ; Leucine/analogs & derivatives/pharmacology ; Luminescent Proteins/genetics/metabolism ; Microscopy, Video ; Necrosis/enzymology/genetics/*parasitology ; Time-Lapse Imaging ; },
abstract = {Remarkably little is known about how intracellular pathogens exit the host cell in order to infect new hosts. Pathogenic chlamydiae egress by first rupturing their replicative niche (the inclusion) before rapidly lysing the host cell. Here we apply a laser ablation strategy to specifically disrupt the chlamydial inclusion, thereby uncoupling inclusion rupture from the subsequent cell lysis and allowing us to dissect the molecular events involved in each step. Pharmacological inhibition of host cell calpains inhibits inclusion rupture, but not subsequent cell lysis. Further, we demonstrate that inclusion rupture triggers a rapid necrotic cell death pathway independent of BAK, BAX, RIP1 and caspases. Both processes work sequentially to efficiently liberate the pathogen from the host cytoplasm, promoting secondary infection. These results reconcile the pathogen's known capacity to promote host cell survival and induce cell death.},
}

CRISPR-Cas Systems
Calpain/*antagonists & inhibitors/genetics/metabolism
Cell Death/radiation effects
Chlamydia trachomatis/pathogenicity/physiology
Cysteine Proteinase Inhibitors/pharmacology
Gene Editing
Genes, Reporter
Green Fluorescent Proteins/genetics/metabolism
HeLa Cells
*Host-Pathogen Interactions
Humans
*Laser Therapy
Leucine/analogs & derivatives/pharmacology
Luminescent Proteins/genetics/metabolism
Microscopy, Video
Necrosis/enzymology/genetics/*parasitology
Time-Lapse Imaging

@article {pmid30429256,
year = {2018},
author = {Strich, JR and Chertow, DS},
title = {CRISPR-Cas Biology and Infectious Diseases Applications.},
journal = {Journal of clinical microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1128/JCM.01307-18},
pmid = {30429256},
issn = {1098-660X},
abstract = {Infectious diseases remain a global threat contributing to excess morbidity and mortality annually with persistent potential for destabilizing pandemics. Improved understanding of the pathogenesis of bacteria, viruses, fungi, and parasites, along with rapid diagnosis and treatment of human infections are essential to improving infectious diseases outcomes worldwide. Genomic loci in bacteria and archea, termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas) proteins, function as an adaptive immune system for prokaryotes protecting against foreign invaders. CRISPR-Cas9 is now routinely applied for efficient gene editing contributing to advances in biomedical science. In the past decade improved understanding of other diverse CRISPR-Cas systems has expanded CRISPR applications, including in the field of infectious diseases. In this review, we summarize the biology of CRISPR-Cas systems and discuss existing and emerging applications to evaluate mechanisms of host-pathogen interactions, to develop accurate and portable diagnostics, and to advance prevention and treatment of infectious diseases.},
}

@article {pmid29986379,
year = {2018},
author = {Belvedere, R and Saggese, P and Pessolano, E and Memoli, D and Bizzarro, V and Rizzo, F and Parente, L and Weisz, A and Petrella, A},
title = {miR-196a Is Able to Restore the Aggressive Phenotype of Annexin A1 Knock-Out in Pancreatic Cancer Cells by CRISPR/Cas9 Genome Editing.},
journal = {International journal of molecular sciences},
volume = {19},
number = {7},
pages = {},
doi = {10.3390/ijms19071967},
pmid = {29986379},
issn = {1422-0067},
mesh = {Annexin A1/*genetics ; CRISPR-Cas Systems ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Disease Progression ; Epithelial-Mesenchymal Transition ; Gene Editing ; Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; MicroRNAs/genetics/*metabolism ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Pancreatic Neoplasms/*genetics/*pathology ; },
abstract = {Annexin A1 (ANXA1) is a Ca2+-binding protein that is involved in pancreatic cancer (PC) progression. It is able to mediate cytoskeletal organization maintaining a malignant phenotype. Our previous studies showed that ANXA1 Knock-Out (KO) MIA PaCa-2 cells partially lost their migratory and invasive capabilities and also the metastatization process appeared affected in vivo. Here, we investigated the microRNA (miRNA) profile in ANXA1 KO cells finding that the modification in miRNA expression suggests the significant involvement of ANXA1 in PC development. In this study, we focused on miR-196a which appeared down modulated in absence of ANXA1. This miRNA is a well known oncogenic factor in several tumour models and it is able to trigger the agents of the epithelial to mesenchymal transition (EMT), like ANXA1. Our results show that the reintroduction in ANXA1 KO cells of miR-196a through the mimic sequence restored the early aggressive phenotype of MIA PaCa-2. Then, ANXA1 seems to support the expression of miR-196a and its role. On the other hand, this miRNA is able to mediate cytoskeletal dynamics and other protein functions promoting PC cell migration and invasion. This work describes the correlation between ANXA1 and specific miRNA sequences, particularly miR-196a. These results could lead to further information on ANXA1 intracellular role in PC, explaining other aspects that are apart from its tumorigenic behaviour.},
}

Annexin A1/*genetics
CRISPR-Cas Systems
Cell Line, Tumor
Cell Movement
Cell Proliferation
Disease Progression
Epithelial-Mesenchymal Transition
Gene Editing
Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
Humans
MicroRNAs/genetics/*metabolism
Neoplasm Invasiveness
Neoplasm Metastasis
Pancreatic Neoplasms/*genetics/*pathology

@article {pmid29440227,
year = {2018},
author = {Choi, JH and Wang, W and Park, D and Kim, SH and Kim, KT and Min, KT},
title = {IRES-mediated translation of cofilin regulates axonal growth cone extension and turning.},
journal = {The EMBO journal},
volume = {37},
number = {5},
pages = {},
doi = {10.15252/embj.201695266},
pmid = {29440227},
issn = {1460-2075},
mesh = {5' Untranslated Regions/genetics ; Animals ; Axons/*physiology ; Brain/embryology ; CRISPR-Cas Systems ; Cell Line ; Cell Proliferation/genetics ; Cofilin 1/*genetics/metabolism ; Growth Cones/*physiology ; Internal Ribosome Entry Sites/*genetics ; Mice ; Neurogenesis/*genetics ; Protein Biosynthesis/genetics ; RNA, Messenger/genetics ; },
abstract = {In neuronal development, dynamic rearrangement of actin promotes axonal growth cone extension, and spatiotemporal translation of local mRNAs in response to guidance cues directs axonal growth cone steering, where cofilin plays a critical role. While regulation of cofilin activity is well studied, regulatory mechanism for cofilin mRNA translation in neurons is unknown. In eukaryotic cells, proteins can be synthesized by cap-dependent or cap-independent mechanism via internal ribosome entry site (IRES)-mediated translation. IRES-mediated translation has been reported in various pathophysiological conditions, but its role in normal physiological environment is poorly understood. Here, we report that 5'UTR of cofilin mRNA contains an IRES element, and cofilin is predominantly translated by IRES-mediated mechanism in neurons. Furthermore, we show that IRES-mediated translation of cofilin is required for both axon extension and axonal growth cone steering. Our results provide new insights into the function of IRES-mediated translation in neuronal development.},
}

5' Untranslated Regions/genetics
Animals
Axons/*physiology
Brain/embryology
CRISPR-Cas Systems
Cell Line
Cell Proliferation/genetics
Cofilin 1/*genetics/metabolism
Growth Cones/*physiology
Internal Ribosome Entry Sites/*genetics
Mice
Neurogenesis/*genetics
Protein Biosynthesis/genetics
RNA, Messenger/genetics

@article {pmid28876032,
year = {2017},
author = {Luo, W and Lin, Q and Li, X and Zhou, Z and Yang, H and Du, R and Li, H},
title = {[Efficient identification of gene knockout mutant mediated by CRISPR/Cas9 by CELⅠ crude extracts].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {33},
number = {5},
pages = {775-784},
doi = {10.13345/j.cjb.160388},
pmid = {28876032},
issn = {1000-3061},
mesh = {*CRISPR-Cas Systems ; *Gene Knockout Techniques ; Oryza/*genetics ; Plant Breeding ; Plant Extracts/*genetics ; },
abstract = {CRISPR/Cas9, emerged as an efficient and powerful gene editing technology, has become the mainstream genome editing technology. Constructing mutants using CRISPR/Cas9 system is of great significance to the functional study and breeding application of useful genes. As the basis of the technology, a method for identification of mutation with efficiency and lower cost is needed. In this report, we studied the factors influencing mutation detected by CEL Ⅰ crude extracts, such as the amount of protein, enzyme incubation time, PCR buffers. Under the optimized conditions, we can integrate the mutation detection steps into one-tube reaction. We used this system to examine the mutation types and frequency of rice stn1 mediated by CRISPR/Cas9. We also used this method to identify different mutation types including homozygous, heterozygous and bi-allelic mutations. The accuracy of this method reached 100% verified by sequencing. Altogether, our results showed that using CELⅠ crude extracts was an efficient and low cost method for identification of CRISPR/Cas9 mediated mutation.},
}

*CRISPR-Cas Systems
*Gene Knockout Techniques
Oryza/*genetics
Plant Breeding
Plant Extracts/*genetics

@article {pmid28262680,
year = {2017},
author = {Sonoiki, E and Ng, CL and Lee, MC and Guo, D and Zhang, YK and Zhou, Y and Alley, MR and Ahyong, V and Sanz, LM and Lafuente-Monasterio, MJ and Dong, C and Schupp, PG and Gut, J and Legac, J and Cooper, RA and Gamo, FJ and DeRisi, J and Freund, YR and Fidock, DA and Rosenthal, PJ},
title = {A potent antimalarial benzoxaborole targets a Plasmodium falciparum cleavage and polyadenylation specificity factor homologue.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14574},
doi = {10.1038/ncomms14574},
pmid = {28262680},
issn = {2041-1723},
mesh = {Amino Acid Sequence ; Animals ; Antimalarials/chemical synthesis/*pharmacology ; Boron Compounds/chemical synthesis/*pharmacology ; CRISPR-Cas Systems ; Catalytic Domain ; Cleavage And Polyadenylation Specificity Factor/antagonists & inhibitors/*chemistry/genetics/metabolism ; Drug Resistance/genetics ; Erythrocytes/drug effects/parasitology ; Gene Editing/methods ; Humans ; Malaria/drug therapy/parasitology ; Malaria, Falciparum/drug therapy/parasitology ; Mice ; Molecular Docking Simulation ; Mutation ; Plasmodium berghei/drug effects/genetics/growth & development/metabolism ; Plasmodium falciparum/*drug effects/genetics/growth & development/metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Structure, Secondary ; Protozoan Proteins/antagonists & inhibitors/*chemistry/genetics/metabolism ; RNA, Messenger/*genetics/metabolism ; Sequence Alignment ; Sequence Homology, Amino Acid ; Trophozoites/drug effects/genetics/growth & development/metabolism ; },
abstract = {Benzoxaboroles are effective against bacterial, fungal and protozoan pathogens. We report potent activity of the benzoxaborole AN3661 against Plasmodium falciparum laboratory-adapted strains (mean IC50 32 nM), Ugandan field isolates (mean ex vivo IC50 64 nM), and murine P. berghei and P. falciparum infections (day 4 ED90 0.34 and 0.57 mg kg-1, respectively). Multiple P. falciparum lines selected in vitro for resistance to AN3661 harboured point mutations in pfcpsf3, which encodes a homologue of mammalian cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3). CRISPR-Cas9-mediated introduction of pfcpsf3 mutations into parental lines recapitulated AN3661 resistance. PfCPSF3 homology models placed these mutations in the active site, where AN3661 is predicted to bind. Transcripts for three trophozoite-expressed genes were lost in AN3661-treated trophozoites, which was not observed in parasites selected or engineered for AN3661 resistance. Our results identify the pre-mRNA processing factor PfCPSF3 as a promising antimalarial drug target.},
}

Amino Acid Sequence
Animals
Antimalarials/chemical synthesis/*pharmacology
Boron Compounds/chemical synthesis/*pharmacology
CRISPR-Cas Systems
Catalytic Domain
Cleavage And Polyadenylation Specificity Factor/antagonists & inhibitors/*chemistry/genetics/metabolism
Drug Resistance/genetics
Erythrocytes/drug effects/parasitology
Gene Editing/methods
Humans
Malaria/drug therapy/parasitology
Malaria, Falciparum/drug therapy/parasitology
Mice
Molecular Docking Simulation
Mutation
Plasmodium berghei/drug effects/genetics/growth & development/metabolism
Plasmodium falciparum/*drug effects/genetics/growth & development/metabolism
Protein Binding
Protein Interaction Domains and Motifs
Protein Structure, Secondary
Protozoan Proteins/antagonists & inhibitors/*chemistry/genetics/metabolism
RNA, Messenger/*genetics/metabolism
Sequence Alignment
Sequence Homology, Amino Acid
Trophozoites/drug effects/genetics/growth & development/metabolism

@article {pmid28262675,
year = {2017},
author = {Lefebure, M and Tothill, RW and Kruse, E and Hawkins, ED and Shortt, J and Matthews, GM and Gregory, GP and Martin, BP and Kelly, MJ and Todorovski, I and Doyle, MA and Lupat, R and Li, J and Schroeder, J and Wall, M and Craig, S and Poortinga, G and Cameron, D and Bywater, M and Kats, L and Gearhart, MD and Bardwell, VJ and Dickins, RA and Hannan, RD and Papenfuss, AT and Johnstone, RW},
title = {Genomic characterisation of Eμ-Myc mouse lymphomas identifies Bcor as a Myc co-operative tumour-suppressor gene.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14581},
doi = {10.1038/ncomms14581},
pmid = {28262675},
issn = {2041-1723},
support = {R01 CA071540/CA/NCI NIH HHS/United States ; },
mesh = {Alleles ; Animals ; B-Lymphocytes/immunology/*metabolism/pathology ; CRISPR-Cas Systems ; Cyclin-Dependent Kinase Inhibitor p16/genetics/immunology ; Disease Models, Animal ; Gene Editing ; *Gene Expression Regulation, Neoplastic ; Gene Frequency ; Janus Kinase 2/genetics/immunology ; Lymphoma, B-Cell/*genetics/immunology/pathology ; Mice ; Mice, Transgenic ; *Mutation ; Proto-Oncogene Proteins c-myc/*genetics/immunology ; Proto-Oncogene Proteins p21(ras)/genetics/immunology ; Repressor Proteins/*genetics/immunology ; STAT5 Transcription Factor/genetics/immunology ; Transcriptome ; Tumor Suppressor Protein p53/genetics/immunology ; Whole Genome Sequencing ; },
abstract = {The Eμ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eμ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eμ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.},
}

Alleles
Animals
B-Lymphocytes/immunology/*metabolism/pathology
CRISPR-Cas Systems
Cyclin-Dependent Kinase Inhibitor p16/genetics/immunology
Disease Models, Animal
Gene Editing
*Gene Expression Regulation, Neoplastic
Gene Frequency
Janus Kinase 2/genetics/immunology
Lymphoma, B-Cell/*genetics/immunology/pathology
Mice
Mice, Transgenic
*Mutation
Proto-Oncogene Proteins c-myc/*genetics/immunology
Proto-Oncogene Proteins p21(ras)/genetics/immunology
Repressor Proteins/*genetics/immunology
STAT5 Transcription Factor/genetics/immunology
Transcriptome
Tumor Suppressor Protein p53/genetics/immunology
Whole Genome Sequencing

@article {pmid30426198,
year = {2018},
author = {Hahn, F and Nekrasov, V},
title = {CRISPR/Cas precision: do we need to worry about off-targeting in plants?.},
journal = {Plant cell reports},
volume = {},
number = {},
pages = {},
doi = {10.1007/s00299-018-2355-9},
pmid = {30426198},
issn = {1432-203X},
support = {BB/P016855/1//Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {The CRISPR/Cas technology has recently become the tool of choice for targeted genome modification in plants and beyond. Although CRSIPR/Cas offers a rapid and facile way of introducing changes at genomic loci of interest, its application is associated with off-targeting, i.e. introduction of unintended mutations at off-target sites within the genome, which has been reported frequently in the mammalian field. Here we summarise the current knowledge on the precision of CRISPR/Cas in plant systems and provide a summary of state-of the-art strategies for avoiding off-target mutations, as well as unintended on-target changes, in plants. These include using natural (e.g. Cas12a) or engineered (e.g. SpCas9-HF) CRISPR/Cas nucleases characterised by higher precision, as compared to the commonly used wild type SpCas9. In addition, we discuss the usage of CRISPR/Cas nucleases in the form of ribonucleoproteins (RNPs) as an option for reducing off-targeting in plants. Finally, we conclude that the most important factor for reducing CRISPR/Cas off-targeting remains careful selection of target sequences, for which we provide an overview of available online software tools and experimental guidance.},
}

@article {pmid30425154,
year = {2018},
author = {Høyland-Kroghsbo, NM and Muñoz, KA and Bassler, BL},
title = {Temperature, by Controlling Growth Rate, Regulates CRISPR-Cas Activity in Pseudomonas aeruginosa.},
journal = {mBio},
volume = {9},
number = {6},
pages = {},
doi = {10.1128/mBio.02184-18},
pmid = {30425154},
issn = {2150-7511},
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (CRISPR-Cas) systems are adaptive defense systems that protect bacteria and archaea from invading genetic elements. In Pseudomonas aeruginosa, quorum sensing (QS) induces the CRISPR-Cas defense system at high cell density when the risk of bacteriophage infection is high. Here, we show that another cue, temperature, modulates P. aeruginosa CRISPR-Cas. Increased CRISPR adaptation occurs at environmental (i.e., low) temperatures compared to that at body (i.e., high) temperature. This increase is a consequence of the accumulation of CRISPR-Cas complexes, coupled with reduced P. aeruginosa growth rate at the lower temperature, the latter of which provides additional time prior to cell division for CRISPR-Cas to patrol the cell and successfully eliminate and/or acquire immunity to foreign DNA. Analyses of a QS mutant and synthetic QS compounds show that the QS and temperature cues act synergistically. The diversity and level of phage encountered by P. aeruginosa in the environment exceed that in the human body, presumably warranting increased reliance on CRISPR-Cas at environmental temperatures.IMPORTANCEP. aeruginosa is a soil dwelling bacterium and a plant pathogen, and it also causes life-threatening infections in humans. Thus, P. aeruginosa thrives in diverse environments and over a broad range of temperatures. Some P. aeruginosa strains rely on the CRISPR-Cas adaptive immune system as a phage defense mechanism. Our discovery that low temperatures increase CRISPR adaptation suggests that the rarely occurring but crucial naive adaptation events may take place predominantly under conditions of slow growth, e.g., during the bacterium's soil dwelling existence and during slow growth in biofilms.},
}

@article {pmid30377319,
year = {2018},
author = {Wight, AJ},
title = {Strict EU ruling on gene-edited crops squeezes science.},
journal = {Nature},
volume = {563},
number = {7729},
pages = {15-16},
doi = {10.1038/d41586-018-07166-7},
pmid = {30377319},
issn = {1476-4687},
mesh = {Agriculture/economics/*legislation & jurisprudence ; Animals ; Belgium ; Brazil ; Breeding/legislation & jurisprudence ; CRISPR-Cas Systems/genetics ; Crops, Agricultural/economics/*genetics ; *European Union ; Evaluation Studies as Topic ; France ; Gene Editing/*legislation & jurisprudence ; Humans ; Plants, Genetically Modified/*genetics ; },
}

Agriculture/economics/*legislation & jurisprudence
Animals
Belgium
Brazil
Breeding/legislation & jurisprudence
CRISPR-Cas Systems/genetics
Crops, Agricultural/economics/*genetics
*European Union
Evaluation Studies as Topic
France
Gene Editing/*legislation & jurisprudence
Humans
Plants, Genetically Modified/*genetics

@article {pmid29141997,
year = {2018},
author = {Zhang, C and Zhou, Y and Xie, S and Yin, Q and Tang, C and Ni, Z and Fei, J and Zhang, Y},
title = {CRISPR/Cas9-mediated genome editing reveals the synergistic effects of β-defensin family members on sperm maturation in rat epididymis.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {32},
number = {3},
pages = {1354-1363},
doi = {10.1096/fj.201700936R},
pmid = {29141997},
issn = {1530-6860},
mesh = {Animals ; *CRISPR-Cas Systems ; *Epididymis ; *Fertility ; *Gene Editing ; Gene Knockout Techniques ; Genome ; Male ; Phenotype ; Rats ; *Sperm Maturation ; Sperm Motility/*physiology ; beta-Defensins/antagonists & inhibitors/*physiology ; },
abstract = {The epididymis is a male reproductive organ involved in posttesticular sperm maturation and storage, but the mechanism underlying sperm maturation remains unclear. β-Defensins (Defbs) belong to a family of small, cysteine-rich, cationic peptides that are antimicrobial and modulate the immune response. A large number of Defb genes are expressed abundantly in the male reproductive tract, especially in the epididymis. We and other groups have shown the involvement of several Defb genes in regulation of sperm function. In this study, we found that Defb23, Defb26, and Defb42 were highly expressed in specific regions of the epididymis. Rats with CRISPR/Cas9-mediated single-gene disruption of Defb23, Defb26, or Defb42 had no obvious fertility phenotypes. Those with the deletion of Defb23/ 26 or Defb23/ 26/ 42 became subfertile, and sperm isolated from the epididymal cauda of multiple-mutant rats were demonstrated decreased motility. Meanwhile, the sperm showed precocious capacitation and increased spontaneous acrosome reaction. Consistent with premature capacitation and acrosome reaction, sperm from multiple-gene-knockout rats had significantly increased intracellular calcium. These results suggest that Defb family members affect sperm maturation by a synergistic pattern in the epididymis.-Zhang, C., Zhou, Y., Xie, S., Yin, Q., Tang, C., Ni, Z., Fei, J., Zhang, Y. CRISPR/Cas9-mediated genome editing reveals the synergistic effects of β-defensin family members on sperm maturation in rat epididymis.},
}

Animals
*CRISPR-Cas Systems
*Epididymis
*Fertility
*Gene Editing
Gene Knockout Techniques
Genome
Male
Phenotype
Rats
*Sperm Maturation
Sperm Motility/*physiology
beta-Defensins/antagonists & inhibitors/*physiology

@article {pmid29092904,
year = {2018},
author = {Kentala, H and Koponen, A and Kivelä, AM and Andrews, R and Li, C and Zhou, Y and Olkkonen, VM},
title = {Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {32},
number = {3},
pages = {1281-1295},
doi = {10.1096/fj.201700604R},
pmid = {29092904},
issn = {1530-6860},
mesh = {Actin Cytoskeleton/*physiology ; CRISPR-Cas Systems ; Cell Adhesion ; *Cell Movement ; Cell Proliferation ; Gene Knockout Techniques ; Hepatocytes/cytology/*physiology ; Humans ; Receptors, Steroid/genetics/*metabolism ; Signal Transduction ; },
abstract = {ORP2 is implicated in cholesterol transport, triglyceride metabolism, and adrenocortical steroid hormone production. We addressed ORP2 function in hepatocytes by generating ORP2-knockout (KO) HuH7 cells by CRISPR-Cas9 gene editing, followed by analyses of transcriptome, F-actin morphology, migration, adhesion, and proliferation. RNA sequencing of ORP2-KO cells revealed >2-fold changes in 579 mRNAs. The Ingenuity Pathway Analysis (IPA) uncovered alterations in the following functional categories: cellular movement, cell-cell signaling and interaction, cellular development, cellular function and maintenance, cellular growth and proliferation, and cell morphology. Many pathways in these categories involved actin cytoskeleton, cell migration, adhesion, or proliferation. Analysis of the ORP2 interactome uncovered 109 putative new partners. Their IPA analysis revealed Ras homolog A (RhoA) signaling as the most significant pathway. Interactions of ORP2 with SEPT9, MLC12, and ARHGAP12 were validated by independent assays. ORP2-KO resulted in abnormal F-actin morphology characterized by impaired capacity to form lamellipodia, migration defect, and impaired adhesion and proliferation. Rescue of the migration phenotype and generation of typical cell surface morphology required an intact ORP2 phosphoinositide binding site, suggesting that ORP2 function involves phosphoinositide binding and transport. The results point at a novel function of ORP2 as a lipid-sensing regulator of the actin cytoskeleton, with impacts on hepatocellular migration, adhesion, and proliferation.-Kentala, H., Koponen, A., Kivelä, A. M., Andrews, R., Li, C., Zhou, Y., Olkkonen, V. M. Analysis of ORP2-knockout hepatocytes uncovers a novel function in actin cytoskeletal regulation.},
}

Actin Cytoskeleton/*physiology
CRISPR-Cas Systems
Cell Adhesion
*Cell Movement
Cell Proliferation
Gene Knockout Techniques
Hepatocytes/cytology/*physiology
Humans
Receptors, Steroid/genetics/*metabolism
Signal Transduction

@article {pmid29089387,
year = {2017},
author = {Casey, AK and Moehlman, AT and Zhang, J and Servage, KA and Krämer, H and Orth, K},
title = {Fic-mediated deAMPylation is not dependent on homodimerization and rescues toxic AMPylation in flies.},
journal = {The Journal of biological chemistry},
volume = {292},
number = {51},
pages = {21193-21204},
doi = {10.1074/jbc.M117.799296},
pmid = {29089387},
issn = {1083-351X},
support = {P40 OD018537/OD/NIH HHS/United States ; R01 EY010199/EY/NEI NIH HHS/United States ; R01 EY021922/EY/NEI NIH HHS/United States ; R01 GM115188/GM/NIGMS NIH HHS/United States ; },
mesh = {Adenosine Monophosphate/*metabolism ; Amino Acid Substitution ; Animals ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Cell Line ; Dimerization ; Drosophila Proteins/chemistry/genetics/*metabolism ; Drosophila melanogaster/genetics ; Endoplasmic Reticulum Stress ; Eye Abnormalities/genetics/metabolism/pathology/veterinary ; Female ; Heat-Shock Proteins/*metabolism ; Homozygote ; Kinetics ; Male ; Mutation ; Nucleotidyltransferases/chemistry/genetics/*metabolism ; Organ Specificity ; *Protein Processing, Post-Translational ; Recombinant Proteins/chemistry/metabolism ; Survival Analysis ; Synthetic Lethal Mutations ; },
abstract = {Protein chaperones play a critical role in proteostasis. The activity of the major endoplasmic reticulum chaperone BiP (GRP78) is regulated by Fic-mediated AMPylation during resting states. By contrast, during times of stress, BiP is deAMPylated. Here, we show that excessive AMPylation by a constitutively active FicE247G mutant is lethal in Drosophila This lethality is cell-autonomous, as directed expression of the mutant FicE247G to the fly eye does not kill the fly but rather results in a rough and reduced eye. Lethality and eye phenotypes are rescued by the deAMPylation activity of wild-type Fic. Consistent with Fic acting as a deAMPylation enzyme, its activity was both time- and concentration-dependent. Furthermore, Fic deAMPylation activity was sufficient to suppress the AMPylation activity mediated by the constitutively active FicE247G mutant in Drosophila S2 lysates. Further, we show that the dual enzymatic activity of Fic is, in part, regulated by Fic dimerization, as loss of this dimerization increases AMPylation and reduces deAMPylation of BiP.},
}

Adenosine Monophosphate/*metabolism
Amino Acid Substitution
Animals
Animals, Genetically Modified
CRISPR-Cas Systems
Cell Line
Dimerization
Drosophila Proteins/chemistry/genetics/*metabolism
Drosophila melanogaster/genetics
Endoplasmic Reticulum Stress
Eye Abnormalities/genetics/metabolism/pathology/veterinary
Female
Heat-Shock Proteins/*metabolism
Homozygote
Kinetics
Male
Mutation
Nucleotidyltransferases/chemistry/genetics/*metabolism
Organ Specificity
*Protein Processing, Post-Translational
Recombinant Proteins/chemistry/metabolism
Survival Analysis
Synthetic Lethal Mutations

@article {pmid28322317,
year = {2017},
author = {Park, JY and Moon, BY and Park, JW and Thornton, JA and Park, YH and Seo, KS},
title = {Genetic engineering of a temperate phage-based delivery system for CRISPR/Cas9 antimicrobials against Staphylococcus aureus.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {44929},
doi = {10.1038/srep44929},
pmid = {28322317},
issn = {2045-2322},
support = {P20 GM103646/GM/NIGMS NIH HHS/United States ; P20 GM103436/GM/NIGMS NIH HHS/United States ; },
mesh = {Bacterial Toxins ; Bacteriophages/*physiology ; *CRISPR-Cas Systems ; *Gene Transfer Techniques ; *Genetic Engineering ; Genome, Viral ; Host Specificity ; Humans ; Plasmids/genetics ; Staphylococcus aureus/*genetics/*virology ; },
abstract = {Discovery of clustered, regularly interspaced, short palindromic repeats and the Cas9 RNA-guided nuclease (CRISPR/Cas9) system provides a new opportunity to create programmable gene-specific antimicrobials that are far less likely to drive resistance than conventional antibiotics. However, the practical therapeutic use of CRISPR/Cas9 is still questionable due to current shortcomings in phage-based delivery systems such as inefficient delivery, narrow host range, and potential transfer of virulence genes by generalized transduction. In this study, we demonstrate genetic engineering strategies to overcome these shortcomings by integrating CRISPR/Cas9 system into a temperate phage genome, removing major virulence genes from the host chromosome, and expanding host specificity of the phage by complementing tail fiber protein. This significantly improved the efficacy and safety of CRISPR/Cas9 antimicrobials to therapeutic levels in both in vitro and in vivo assays. The genetic engineering tools and resources established in this study are expected to provide an efficacious and safe CRISPR/Cas9 antimicrobial, broadly applicable to Staphylococcus aureus.},
}

Bacterial Toxins
Bacteriophages/*physiology
*CRISPR-Cas Systems
*Gene Transfer Techniques
*Genetic Engineering
Genome, Viral
Host Specificity
Humans
Plasmids/genetics
Staphylococcus aureus/*genetics/*virology

@article {pmid28317879,
year = {2017},
author = {Jones, KR and Choi, U and Gao, JL and Thompson, RD and Rodman, LE and Malech, HL and Kang, EM},
title = {A Novel Method for Screening Adenosine Receptor Specific Agonists for Use in Adenosine Drug Development.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {44816},
doi = {10.1038/srep44816},
pmid = {28317879},
issn = {2045-2322},
support = {ZIA AI000988/AI/NIAID NIH HHS/United States ; ZIA AI000989/AI/NIAID NIH HHS/United States ; },
mesh = {Adenosine/metabolism ; CRISPR-Cas Systems ; Calcium/metabolism ; Cells, Cultured ; Drug Discovery/*methods ; Drug Evaluation, Preclinical/methods ; Flow Cytometry ; Gene Expression ; Gene Knockout Techniques ; Gene Targeting ; Humans ; K562 Cells ; Purinergic P1 Receptor Agonists/*chemistry/*pharmacology ; Receptors, Purinergic P1/chemistry/classification/*genetics/*metabolism ; },
abstract = {Agonists that target the A1, A2A, A2B and A3 adenosine receptors have potential to be potent treatment options for a number of diseases, including autoimmune diseases, cardiovascular disease and cancer. Because each of these adenosine receptors plays a distinct role throughout the body, obtaining highly specific receptor agonists is essential. Of these receptors, the adenosine A2AR and A2BR share many sequence and structural similarities but highly differ in their responses to inflammatory stimuli. Our laboratory, using a combination of specially developed cell lines and calcium release analysis hardware, has created a new and faster method for determining specificity of synthetic adenosine agonist compounds for the A2A and A2B receptors in human cells. A2A receptor expression was effectively removed from K562 cells, resulting in the development of a distinct null line. Using HIV-lentivector and plasmid DNA transfection, we also developed A2A and A2B receptor over-expressing lines. As adenosine is known to cause changes in intracellular calcium levels upon addition to cell culture, calcium release can be determined in these cell lines upon compound addition, providing a functional readout of receptor activation and allowing us to isolate the most specific adenosine agonist compounds.},
}

Adenosine/metabolism
CRISPR-Cas Systems
Calcium/metabolism
Cells, Cultured
Drug Discovery/*methods
Drug Evaluation, Preclinical/methods
Flow Cytometry
Gene Expression
Gene Knockout Techniques
Gene Targeting
Humans
K562 Cells
Purinergic P1 Receptor Agonists/*chemistry/*pharmacology
Receptors, Purinergic P1/chemistry/classification/*genetics/*metabolism

@article {pmid28291770,
year = {2017},
author = {Yu, W and Mookherjee, S and Chaitankar, V and Hiriyanna, S and Kim, JW and Brooks, M and Ataeijannati, Y and Sun, X and Dong, L and Li, T and Swaroop, A and Wu, Z},
title = {Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14716},
doi = {10.1038/ncomms14716},
pmid = {28291770},
issn = {2041-1723},
mesh = {Animals ; Basic-Leucine Zipper Transcription Factors/*genetics ; CRISPR-Cas Systems ; Cell Survival/*genetics ; Dependovirus ; Eye Proteins/*genetics ; Gene Editing/methods ; Gene Knockdown Techniques ; Mice ; Retinal Cone Photoreceptor Cells/cytology/*metabolism ; Retinal Degeneration/*genetics ; Retinal Rod Photoreceptor Cells/cytology/*metabolism ; Retinitis Pigmentosa/genetics ; },
abstract = {In retinitis pigmentosa, loss of cone photoreceptors leads to blindness, and preservation of cone function is a major therapeutic goal. However, cone loss is thought to occur as a secondary event resulting from degeneration of rod photoreceptors. Here we report a genome editing approach in which adeno-associated virus (AAV)-mediated CRISPR/Cas9 delivery to postmitotic photoreceptors is used to target the Nrl gene, encoding for Neural retina-specific leucine zipper protein, a rod fate determinant during photoreceptor development. Following Nrl disruption, rods gain partial features of cones and present with improved survival in the presence of mutations in rod-specific genes, consequently preventing secondary cone degeneration. In three different mouse models of retinal degeneration, the treatment substantially improves rod survival and preserves cone function. Our data suggest that CRISPR/Cas9-mediated NRL disruption in rods may be a promising treatment option for patients with retinitis pigmentosa.},
}

Animals
Basic-Leucine Zipper Transcription Factors/*genetics
CRISPR-Cas Systems
Cell Survival/*genetics
Dependovirus
Eye Proteins/*genetics
Gene Editing/methods
Gene Knockdown Techniques
Mice
Retinal Cone Photoreceptor Cells/cytology/*metabolism
Retinal Degeneration/*genetics
Retinal Rod Photoreceptor Cells/cytology/*metabolism
Retinitis Pigmentosa/genetics

@article {pmid28290446,
year = {2017},
author = {Qin, P and Parlak, M and Kuscu, C and Bandaria, J and Mir, M and Szlachta, K and Singh, R and Darzacq, X and Yildiz, A and Adli, M},
title = {Live cell imaging of low- and non-repetitive chromosome loci using CRISPR-Cas9.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14725},
doi = {10.1038/ncomms14725},
pmid = {28290446},
issn = {2041-1723},
support = {R01 GM094522/GM/NIGMS NIH HHS/United States ; R01 GM118773/GM/NIGMS NIH HHS/United States ; U01 EB021236/EB/NIBIB NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems ; Cell Cycle/*genetics ; Cell Line ; Cell Line, Tumor ; Cell Nucleus/*metabolism/ultrastructure ; Chromatin/*metabolism/ultrastructure ; Feasibility Studies ; Genetic Loci ; HEK293 Cells ; HeLa Cells ; Humans ; Intravital Microscopy ; Microscopy, Confocal ; RNA, Guide/*metabolism ; Retinal Pigment Epithelium/cytology ; },
abstract = {Imaging chromatin dynamics is crucial to understand genome organization and its role in transcriptional regulation. Recently, the RNA-guidable feature of CRISPR-Cas9 has been utilized for imaging of chromatin within live cells. However, these methods are mostly applicable to highly repetitive regions, whereas imaging regions with low or no repeats remains as a challenge. To address this challenge, we design single-guide RNAs (sgRNAs) integrated with up to 16 MS2 binding motifs to enable robust fluorescent signal amplification. These engineered sgRNAs enable multicolour labelling of low-repeat-containing regions using a single sgRNA and of non-repetitive regions with as few as four unique sgRNAs. We achieve tracking of native chromatin loci throughout the cell cycle and determine differential positioning of transcriptionally active and inactive regions in the nucleus. These results demonstrate the feasibility of our approach to monitor the position and dynamics of both repetitive and non-repetitive genomic regions in live cells.},
}

CRISPR-Cas Systems
Cell Cycle/*genetics
Cell Line
Cell Line, Tumor
Cell Nucleus/*metabolism/ultrastructure
Chromatin/*metabolism/ultrastructure
Feasibility Studies
Genetic Loci
HEK293 Cells
HeLa Cells
Humans
Intravital Microscopy
Microscopy, Confocal
RNA, Guide/*metabolism
Retinal Pigment Epithelium/cytology

@article {pmid28195176,
year = {2017},
author = {Liu, NQ and Ter Huurne, M and Nguyen, LN and Peng, T and Wang, SY and Studd, JB and Joshi, O and Ongen, H and Bramsen, JB and Yan, J and Andersen, CL and Taipale, J and Dermitzakis, ET and Houlston, RS and Hubner, NC and Stunnenberg, HG},
title = {The non-coding variant rs1800734 enhances DCLK3 expression through long-range interaction and promotes colorectal cancer progression.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14418},
doi = {10.1038/ncomms14418},
pmid = {28195176},
issn = {2041-1723},
mesh = {Alleles ; CRISPR-Cas Systems ; Cell Line, Tumor ; Colonic Neoplasms/*genetics ; Colorectal Neoplasms/*genetics/metabolism ; DNA Methylation ; DNA-Binding Proteins ; *Disease Progression ; Epigenesis, Genetic ; Genetic Predisposition to Disease ; Genome-Wide Association Study ; Humans ; Intracellular Signaling Peptides and Proteins/*genetics/*metabolism ; MutL Protein Homolog 1/genetics ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Protein-Serine-Threonine Kinases/*genetics/*metabolism ; Proteome ; Proteomics ; Transcription Factors ; },
abstract = {Genome-wide association studies have identified a great number of non-coding risk variants for colorectal cancer (CRC). To date, the majority of these variants have not been functionally studied. Identification of allele-specific transcription factor (TF) binding is of great importance to understand regulatory consequences of such variants. A recently developed proteome-wide analysis of disease-associated SNPs (PWAS) enables identification of TF-DNA interactions in an unbiased manner. Here we perform a large-scale PWAS study to comprehensively characterize TF-binding landscape that is associated with CRC, which identifies 731 allele-specific TF binding at 116 CRC risk loci. This screen identifies the A-allele of rs1800734 within the promoter region of MLH1 as perturbing the binding of TFAP4 and consequently increasing DCLK3 expression through a long-range interaction, which promotes cancer malignancy through enhancing expression of the genes related to epithelial-to-mesenchymal transition.},
}

Alleles
CRISPR-Cas Systems
Cell Line, Tumor
Colonic Neoplasms/*genetics
Colorectal Neoplasms/*genetics/metabolism
DNA Methylation
DNA-Binding Proteins
*Disease Progression
Epigenesis, Genetic
Genetic Predisposition to Disease
Genome-Wide Association Study
Humans
Intracellular Signaling Peptides and Proteins/*genetics/*metabolism
MutL Protein Homolog 1/genetics
Polymorphism, Single Nucleotide
Promoter Regions, Genetic
Protein-Serine-Threonine Kinases/*genetics/*metabolism
Proteome
Proteomics
Transcription Factors

@article {pmid28176758,
year = {2017},
author = {Koirala, P and Huang, J and Ho, TT and Wu, F and Ding, X and Mo, YY},
title = {LncRNA AK023948 is a positive regulator of AKT.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14422},
doi = {10.1038/ncomms14422},
pmid = {28176758},
issn = {2041-1723},
support = {R01 CA154989/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Breast/pathology ; Breast Neoplasms/*genetics/mortality/pathology ; CRISPR-Cas Systems ; Class Ia Phosphatidylinositol 3-Kinase/genetics/metabolism ; DEAD-box RNA Helicases/genetics/metabolism ; Disease Progression ; Female ; *Gene Expression Regulation, Neoplastic ; Gene Knockout Techniques ; Humans ; MCF-7 Cells ; Mice, Nude ; Neoplasm Proteins/genetics/metabolism ; Proto-Oncogene Proteins c-akt/*genetics/metabolism ; RNA, Long Noncoding/genetics/*metabolism ; RNA, Small Interfering/metabolism ; Up-Regulation ; Xenograft Model Antitumor Assays ; },
abstract = {Despite the overwhelming number of human long non-coding RNAs (lncRNAs) reported so far, little is known about their physiological functions for the majority of them. The present study uses a CRISPR/Cas9-based synergistic activation mediator (SAM) system to identify potential lncRNAs capable of regulating AKT activity. Among lncRNAs identified from this screen, we demonstrate that AK023948 is a positive regulator for AKT. Knockout of AK023948 suppresses, whereas rescue with AK023948 restores the AKT activity. Mechanistically, AK023948 functionally interacts with DHX9 and p85. Importantly, AK023948 is required for the interaction between DHX9 and p85 to hence the p85 stability and promote AKT activity. Finally, AK023948 is upregulated in breast cancer; interrogation of TCGA data set indicates that upregulation of DHX9 in breast cancer is associated with poor survival. Together, this study demonstrates two previously uncharacterized factors AK023948 and DHX9 as important players in the AKT pathway, and that their upregulation may contribute to breast tumour progression.},
}

Animals
Breast/pathology
Breast Neoplasms/*genetics/mortality/pathology
CRISPR-Cas Systems
Class Ia Phosphatidylinositol 3-Kinase/genetics/metabolism
DEAD-box RNA Helicases/genetics/metabolism
Disease Progression
Female
*Gene Expression Regulation, Neoplastic
Gene Knockout Techniques
Humans
MCF-7 Cells
Mice, Nude
Neoplasm Proteins/genetics/metabolism
Proto-Oncogene Proteins c-akt/*genetics/metabolism
RNA, Long Noncoding/genetics/*metabolism
RNA, Small Interfering/metabolism
Up-Regulation
Xenograft Model Antitumor Assays

@article {pmid30354961,
year = {2018},
author = {Pasricha, SR and Drakesmith, H},
title = {Hemoglobinopathies in the Fetal Position.},
journal = {The New England journal of medicine},
volume = {379},
number = {17},
pages = {1675-1677},
doi = {10.1056/NEJMcibr1809628},
pmid = {30354961},
issn = {1533-4406},
mesh = {Anemia, Sickle Cell/*drug therapy/genetics ; CRISPR-Cas Systems ; Carrier Proteins/genetics ; Erythroid Cells/metabolism ; Fetal Hemoglobin/*genetics ; *Gene Expression Regulation ; Humans ; Molecular Targeted Therapy ; eIF-2 Kinase/*antagonists & inhibitors/genetics/metabolism ; },
}

Anemia, Sickle Cell/*drug therapy/genetics
CRISPR-Cas Systems
Carrier Proteins/genetics
Erythroid Cells/metabolism
Fetal Hemoglobin/*genetics
*Gene Expression Regulation
Humans
Molecular Targeted Therapy
eIF-2 Kinase/*antagonists & inhibitors/genetics/metabolism

@article {pmid30151606,
year = {2018},
author = {Dong, Z and Huang, L and Dong, F and Hu, Z and Qin, Q and Long, J and Cao, M and Chen, P and Lu, C and Pan, MH},
title = {Establishment of a baculovirus-inducible CRISPR/Cas9 system for antiviral research in transgenic silkworms.},
journal = {Applied microbiology and biotechnology},
volume = {102},
number = {21},
pages = {9255-9265},
doi = {10.1007/s00253-018-9295-8},
pmid = {30151606},
issn = {1432-0614},
support = {31472153//National Natural Science Foundation of China/ ; 31572466//National Natural Science Foundation of China/ ; CARS-18//China Agriculture Research System/ ; },
mesh = {Animals ; Animals, Genetically Modified/*genetics ; Baculoviridae/*genetics ; Bombyx/*genetics/virology ; CRISPR-Cas Systems/*genetics ; Gene Targeting/methods ; Genetic Vectors/genetics ; Nucleopolyhedrovirus/genetics ; Pest Control/methods ; Promoter Regions, Genetic/genetics ; Transcription, Genetic/genetics ; },
abstract = {The CRISPR/Cas9 system is a powerful genetic engineering technique that has been widely used in gene therapy, as well as in the development of novel antimicrobials and transgenic insects. However, several challenges, including the lack of effective host target genes and the off-target effects, limit the application of CRISPR/Cas9 in insects. To mitigate these difficulties, we established a highly efficient virus-inducible CRISPR/Cas9 system in transgenic silkworms. This system includes the baculovirus-inducible promoter 39K, which directs transcription of the gene encoding, the Cas9 protein, and the U6 promoter which targets the sgATAD3A site of the ATPase family AAA domain-containing protein 3 (ATAD3A) gene. The double-positive transgenic line sgATAD3A×39K-Cas9 (ATAD3A-KO) was obtained by hybridization; antiviral activity in this hybrid transgenic line is induced only after Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The BmNPV-inducible system significantly reduced off-target effects and did not affect the economically important characteristics of the transgenic silkworms. Most importantly, this novel system efficiently and consistently edited target genes, inhibiting BmNPV replication after the transgenic silkworms were inoculated with occlusion bodies (OBs). The suppression of BmNPV by the virus-inducible system was comparable to that of the stably expressed CRISPR/Cas9 system. Therefore, we successfully established a highly efficient BmNPV-inducible ATAD3A-KO transgenic silkworm line, with improved gene targeting specificity and antiviral efficiency. Our study thereby provides insights into the treatment of infectious diseases and into the control of insect pests.},
}

Animals
Animals, Genetically Modified/*genetics
Baculoviridae/*genetics
Bombyx/*genetics/virology
CRISPR-Cas Systems/*genetics
Gene Targeting/methods
Genetic Vectors/genetics
Nucleopolyhedrovirus/genetics
Pest Control/methods
Promoter Regions, Genetic/genetics
Transcription, Genetic/genetics

@article {pmid29604353,
year = {2018},
author = {Wang, S and Hong, W and Dong, S and Zhang, ZT and Zhang, J and Wang, L and Wang, Y},
title = {Genome engineering of Clostridium difficile using the CRISPR-Cas9 system.},
journal = {Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases},
volume = {24},
number = {10},
pages = {1095-1099},
doi = {10.1016/j.cmi.2018.03.026},
pmid = {29604353},
issn = {1469-0691},
mesh = {Animals ; Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Clostridium difficile/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/*genetics ; Gene Deletion ; Genetic Engineering/methods ; Genome, Bacterial/genetics ; Humans ; Plasmids/genetics ; },
abstract = {OBJECTIVES: Clostridium difficile is a notorious pathogenic species that can cause severe gastrointestinal infections in humans and animals. C. difficile infection (CDI) results in thousands of deaths worldwide every year. The elucidation of related mechanisms of CDI and exploration of potential therapeutic strategies are largely delayed due to the lack of efficient genetic engineering tools for C. difficile strains.

METHODS: Plasmids carrying the CRISPR-Cas9 system were constructed and transformed into C. difficile through conjugation. Mutants were identified using colony PCR with primers annealing to the regions flanking the target gene deletion/integration locus. Heat-survival assay was used to compare the sporulation frequency between the mutant with spo0A deletion and the wild type strain. The fluorescence in the mutant with the insertion of the green fluorescent protein (GFP) gene was inspected under a fluorescent microscope.

RESULTS: An efficient genome editing tool was developed for C. difficile based on the CRISPR-Cas9 system. With this tool, spo0A was deleted with a 100% mutation efficiency. Conversely, an anaerobic GFP gene was successfully inserted into the C. difficile chromosome (with a mutation efficiency of 80%).

CONCLUSIONS: The developed CRISPR-Cas9-based genome engineering tool will facilitate functional genomic studies in C. difficile as well as the elucidation of mechanisms related to host-bacteria interaction and pathogenesis of CDI. This will be highly beneficial for the development of innovative strategies for CDI diagnostics and therapies.},
}

Animals
Bacterial Proteins/genetics
CRISPR-Cas Systems/*genetics
Clostridium difficile/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/*genetics
Gene Deletion
Genetic Engineering/methods
Genome, Bacterial/genetics
Humans
Plasmids/genetics

@article {pmid29540148,
year = {2018},
author = {Vazquez, N and Sanchez, L and Marks, R and Martinez, E and Fanniel, V and Lopez, A and Salinas, A and Flores, I and Hirschmann, J and Gilkerson, R and Schuenzel, E and Dearth, R and Halaby, R and Innis-Whitehouse, W and Keniry, M},
title = {A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone.},
journal = {BMC molecular biology},
volume = {19},
number = {1},
pages = {3},
doi = {10.1186/s12867-018-0105-8},
pmid = {29540148},
issn = {1471-2199},
support = {UTRGV College of Sciences (COS) Seed Grant//UTRGV COS/International ; NIH 1SC3GM11666901/GM/NIGMS NIH HHS/United States ; USDA H.S.I. 2016-38422-25760//National Institute of Food and Agriculture/International ; 1463991//National Science Foundation/International ; NIH 5R25GM10086606/GM/NIGMS NIH HHS/United States ; USDA Step 2 2015-38422-24061//National Institute of Food and Agriculture/International ; NSF 1463991//National Science Foundation/International ; NSF Advance 1209210//National Science Foundation/International ; HHMI 52007568/HHMI/Howard Hughes Medical Institute/United States ; SC3 GM116669/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Cell Line ; Deoxyribonuclease I/metabolism ; Forkhead Box Protein O3/*genetics ; Gene Editing/*methods ; Genetic Vectors ; HEK293 Cells ; Homologous Recombination ; Humans ; Male ; Mutation ; Plasmids/*genetics ; RNA, Guide/metabolism ; },
abstract = {BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone.

RESULTS: We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines.

CONCLUSIONS: Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications).},
}

*CRISPR-Cas Systems
Cell Line
Deoxyribonuclease I/metabolism
Forkhead Box Protein O3/*genetics
Gene Editing/*methods
Genetic Vectors
HEK293 Cells
Homologous Recombination
Humans
Male
Mutation
Plasmids/*genetics
RNA, Guide/metabolism

@article {pmid28917642,
year = {2018},
author = {Timin, AS and Muslimov, AR and Lepik, KV and Epifanovskaya, OS and Shakirova, AI and Mock, U and Riecken, K and Okilova, MV and Sergeev, VS and Afanasyev, BV and Fehse, B and Sukhorukov, GB},
title = {Efficient gene editing via non-viral delivery of CRISPR-Cas9 system using polymeric and hybrid microcarriers.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {14},
number = {1},
pages = {97-108},
doi = {10.1016/j.nano.2017.09.001},
pmid = {28917642},
issn = {1549-9642},
mesh = {*CRISPR-Cas Systems ; Drug Carriers ; Fluorescence ; *Gene Editing ; Gene Transfer Techniques ; Green Fluorescent Proteins/antagonists & inhibitors/genetics/metabolism ; HEK293 Cells ; Humans ; Lycopersicon esculentum/genetics/*metabolism ; Polymers/*chemistry ; Silicon Dioxide/chemistry ; },
abstract = {CRISPR-Cas9 is a revolutionary genome-editing technology that has enormous potential for the treatment of genetic diseases. However, the lack of efficient and safe, non-viral delivery systems has hindered its clinical application. Here, we report on the application of polymeric and hybrid microcarriers, made of degradable polymers such as polypeptides and polysaccharides and modified by silica shell, for delivery of all CRISPR-Cas9 components. We found that these microcarriers mediate more efficient transfection than a commercially available liposome-based transfection reagent (>70% vs. <50% for mRNA, >40% vs. 20% for plasmid DNA). For proof-of-concept, we delivered CRISPR-Cas9 components using our capsules to dTomato-expressing HEK293T cells-a model, in which loss of red fluorescence indicates successful gene editing. Notably, transfection of indicator cells translated in high-level dTomato knockout in approx. 70% of transfected cells. In conclusion, we have provided proof-of-principle that our micro-sized containers represent promising non-viral platforms for efficient and safe gene editing.},
}

*CRISPR-Cas Systems
Drug Carriers
Fluorescence
*Gene Editing
Gene Transfer Techniques
Green Fluorescent Proteins/antagonists & inhibitors/genetics/metabolism
HEK293 Cells
Humans
Lycopersicon esculentum/genetics/*metabolism
Polymers/*chemistry
Silicon Dioxide/chemistry

@article {pmid28803899,
year = {2017},
author = {Yang, N and Wang, R and Zhao, Y},
title = {Revolutionize Genetic Studies and Crop Improvement with High-Throughput and Genome-Scale CRISPR/Cas9 Gene Editing Technology.},
journal = {Molecular plant},
volume = {10},
number = {9},
pages = {1141-1143},
doi = {10.1016/j.molp.2017.08.001},
pmid = {28803899},
issn = {1752-9867},
support = {R01 GM114660/GM/NIGMS NIH HHS/United States ; },
mesh = {CRISPR-Cas Systems/*genetics ; Crops, Agricultural/*genetics/growth & development ; Gene Editing/*methods ; *Genomics ; },
}

CRISPR-Cas Systems/*genetics
Crops, Agricultural/*genetics/growth & development
Gene Editing/*methods
*Genomics

@article {pmid28645639,
year = {2017},
author = {Meng, X and Yu, H and Zhang, Y and Zhuang, F and Song, X and Gao, S and Gao, C and Li, J},
title = {Construction of a Genome-Wide Mutant Library in Rice Using CRISPR/Cas9.},
journal = {Molecular plant},
volume = {10},
number = {9},
pages = {1238-1241},
doi = {10.1016/j.molp.2017.06.006},
pmid = {28645639},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/*genetics ; *Gene Library ; *Genomics ; *Mutation ; Oryza/*genetics ; },
}

CRISPR-Cas Systems/*genetics
*Gene Library
*Genomics
*Mutation
Oryza/*genetics

@article {pmid28645638,
year = {2017},
author = {Lu, Y and Ye, X and Guo, R and Huang, J and Wang, W and Tang, J and Tan, L and Zhu, JK and Chu, C and Qian, Y},
title = {Genome-wide Targeted Mutagenesis in Rice Using the CRISPR/Cas9 System.},
journal = {Molecular plant},
volume = {10},
number = {9},
pages = {1242-1245},
doi = {10.1016/j.molp.2017.06.007},
pmid = {28645638},
issn = {1752-9867},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Genome, Plant/*genetics ; Mutagenesis/*genetics ; },
}

Base Sequence
CRISPR-Cas Systems/*genetics
Genome, Plant/*genetics
Mutagenesis/*genetics

@article {pmid28315751,
year = {2017},
author = {Wang, M and Lu, Y and Botella, JR and Mao, Y and Hua, K and Zhu, JK},
title = {Gene Targeting by Homology-Directed Repair in Rice Using a Geminivirus-Based CRISPR/Cas9 System.},
journal = {Molecular plant},
volume = {10},
number = {7},
pages = {1007-1010},
doi = {10.1016/j.molp.2017.03.002},
pmid = {28315751},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/*genetics ; DNA Repair/*genetics ; Geminiviridae/genetics/*physiology ; Gene Targeting/*methods ; Oryza/*genetics ; *Sequence Homology, Nucleic Acid ; },
}

CRISPR-Cas Systems/*genetics
DNA Repair/*genetics
Geminiviridae/genetics/*physiology
Gene Targeting/*methods
Oryza/*genetics
*Sequence Homology, Nucleic Acid

@article {pmid28272508,
year = {2017},
author = {Mittal, K and Choi, DH and Ogden, A and Donthamsetty, S and Melton, BD and Gupta, MV and Pannu, V and Cantuaria, G and Varambally, S and Reid, MD and Jonsdottir, K and Janssen, EA and Aleskandarany, MA and Ellis, IO and Rakha, EA and Rida, PC and Aneja, R},
title = {Amplified centrosomes and mitotic index display poor concordance between patient tumors and cultured cancer cells.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {43984},
doi = {10.1038/srep43984},
pmid = {28272508},
issn = {2045-2322},
support = {U01 CA179671/CA/NCI NIH HHS/United States ; R01 CA169127/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Breast Neoplasms/metabolism/*pathology ; CRISPR-Cas Systems/genetics ; Cell Hypoxia ; Cell Line, Tumor ; Centrosome/*metabolism ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Male ; Mice ; Mice, Nude ; Microscopy, Fluorescence ; Mitotic Index ; Pancreatic Neoplasms/metabolism/*pathology ; Transplantation, Heterologous ; Urinary Bladder Neoplasms/metabolism/pathology ; },
abstract = {Centrosome aberrations (CA) and abnormal mitoses are considered beacons of malignancy. Cancer cell doubling times in patient tumors are longer than in cultures, but differences in CA between tumors and cultured cells are uncharacterized. We compare mitoses and CA in patient tumors, xenografts, and tumor cell lines. We find that mitoses are rare in patient tumors compared with xenografts and cell lines. Contrastingly, CA is more extensive in patient tumors and xenografts (~35-50% cells) than cell lines (~5-15%), although CA declines in patient-derived tumor cells over time. Intratumoral hypoxia may explain elevated CA in vivo because exposure of cultured cells to hypoxia or mimicking hypoxia pharmacologically or genetically increases CA, and HIF-1α and hypoxic gene signature expression correlate with CA and centrosomal gene signature expression in breast tumors. These results highlight the importance of utilizing low-passage-number patient-derived cell lines in studying CA to more faithfully recapitulate in vivo cellular phenotypes.},
}

Animals
Breast Neoplasms/metabolism/*pathology
CRISPR-Cas Systems/genetics
Cell Hypoxia
Cell Line, Tumor
Centrosome/*metabolism
Female
Humans
Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism
Male
Mice
Mice, Nude
Microscopy, Fluorescence
Mitotic Index
Pancreatic Neoplasms/metabolism/*pathology
Transplantation, Heterologous
Urinary Bladder Neoplasms/metabolism/pathology

@article {pmid28195574,
year = {2017},
author = {Bengtsson, NE and Hall, JK and Odom, GL and Phelps, MP and Andrus, CR and Hawkins, RD and Hauschka, SD and Chamberlain, JR and Chamberlain, JS},
title = {Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14454},
doi = {10.1038/ncomms14454},
pmid = {28195574},
issn = {2041-1723},
support = {R01 AR044533/AR/NIAMS NIH HHS/United States ; U54 AR065139/AR/NIAMS NIH HHS/United States ; U54 HD083091/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Bacterial Proteins/genetics ; CRISPR-Cas Systems/*genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Dependovirus/genetics ; Disease Models, Animal ; Dystrophin/*genetics ; Endonucleases/genetics ; Gene Editing/*methods ; Genetic Therapy/methods ; Genetic Vectors ; High-Throughput Nucleotide Sequencing ; Male ; Mice ; Mice, Inbred mdx ; Muscle, Skeletal/physiopathology ; Muscular Dystrophy, Duchenne/*genetics/*metabolism/*physiopathology/therapy ; Mutation ; Myocardium ; Neuromuscular Diseases/therapy ; RNA, Guide ; Sequence Deletion ; },
abstract = {Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx4cv mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.},
}

Animals
Bacterial Proteins/genetics
CRISPR-Cas Systems/*genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Dependovirus/genetics
Disease Models, Animal
Dystrophin/*genetics
Endonucleases/genetics
Gene Editing/*methods
Genetic Therapy/methods
Genetic Vectors
High-Throughput Nucleotide Sequencing
Male
Mice
Mice, Inbred mdx
Muscle, Skeletal/physiopathology
Muscular Dystrophy, Duchenne/*genetics/*metabolism/*physiopathology/therapy
Mutation
Myocardium
Neuromuscular Diseases/therapy
RNA, Guide
Sequence Deletion

@article {pmid30418590,
year = {2018},
author = {Gordeeva, J and Morozova, N and Sierro, N and Isaev, A and Sinkunas, T and Tsvetkova, K and Matlashov, M and Truncaite, L and Morgan, RD and Ivanov, NV and Siksnys, V and Zeng, L and Severinov, K},
title = {BREX system of Escherichia coli distinguishes self from non-self by methylation of a specific DNA site.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gky1125},
pmid = {30418590},
issn = {1362-4962},
abstract = {Prokaryotes evolved numerous systems that defend against predation by bacteriophages. In addition to well-known restriction-modification and CRISPR-Cas immunity systems, many poorly characterized systems exist. One class of such systems, named BREX, consists of a putative phosphatase, a methyltransferase and four other proteins. A Bacillus cereus BREX system provides resistance to several unrelated phages and leads to modification of specific motif in host DNA. Here, we study the action of BREX system from a natural Escherichia coli isolate. We show that while it makes cells resistant to phage λ infection, induction of λ prophage from cells carrying BREX leads to production of viruses that overcome the defense. The induced phage DNA contains a methylated adenine residue in a specific motif. The same modification is found in the genome of BREX-carrying cells. The results establish, for the first time, that immunity to BREX system defense is provided by an epigenetic modification.},
}

@article {pmid30418108,
year = {2018},
author = {Aung, MS and San, T and San, N and Oo, WM and Ko, PM and Thet, KT and Urushibara, N and Kawaguchiya, M and Sumi, A and Kobayashi, N},
title = {Molecular characterization of Staphylococcus argenteus in Myanmar: identification of novel genotypes/clusters in staphylocoagulase, protein A, alpha-haemolysin and other virulence factors.},
journal = {Journal of medical microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1099/jmm.0.000869},
pmid = {30418108},
issn = {1473-5644},
abstract = {PURPOSE: Staphylococcus argenteus is a novel emerging species of coagulase-positive staphylococcus that is genetically closely related to Staphylococcus aureus. To elucidate the molecular differences in the virulence factors (staphylocoagulase, protein A, alpha-haemolysin, enterotoxin-like toxin and staphylokinase) between these staphylococcal species, S. argenteus that had recently been isolated in Myanmar (five nasal isolates and four clinical isolates) were analysed.

METHODOLOGY: The nucleotide sequences of the virulence factors were determined by PCR and direct sequencing, followed by phylogenetic analysis by mega6 and multiple alignment by clustalw using the published sequence data for S. aureus and S. argenteus.

RESULTS: Six S. argenteus isolates belonged to MLST sequence type (ST) 2250, while others belonged to ST4625, ST2198 and ST2854. The novel staphylocoagulase (coa) genotype XIV and the novel coa-XI subtype (XId) were identified in an ST2198 isolate and all other isolates, respectively. Among the S. argenteus isolates, the protein A and alpha-haemolysin genes showed high sequence identity (96-98 % and >99 %, respectively), while lower identity was observed between S. argenteus and S. aureus (88-91 % and 86 %, respectively), with both species showing phylogenetically distinct clusters. Similar findings were found for the staphylococcal enterotoxin (SE)-like toxin genes selw, selx and sely. In contrast, the staphylokinase genes were almost identical between these two species. All of the coa-XId isolates had a CRISPR/Cas locus at the site of orfX without having SCCmec, whereas an ST2198 isolate lacked this locus.

CONCLUSION: The primary virulence factors (staphylocoagulase, protein A andalpha-haemolysin) as well as the SE-like toxins of S. argenteus were genetically discriminated from those of S. aureus, revealing the presence of the novel coa-type/subtype (coa-IXd, XIV) in S. argenteus.},
}

@article {pmid30413649,
year = {2018},
author = {Li, CJ and Jiang, C and Liu, Y and Bell, T and Ma, W and Ye, Y and Huang, S and Guo, H and Zhang, H and Wang, L and Wang, J and Nomie, K and Zhang, L and Wang, M},
title = {Pleiotropic Action of Novel Bruton's Tyrosine Kinase Inhibitor BGB-3111 in Mantle Cell Lymphoma.},
journal = {Molecular cancer therapeutics},
volume = {},
number = {},
pages = {},
doi = {10.1158/1535-7163.MCT-18-0478},
pmid = {30413649},
issn = {1538-8514},
abstract = {Bruton's tyrosine kinase (BTK) is a key mediator of BCR-dependent cell growth signaling and a clinically effective therapeutic target in mantle cell lymphoma (MCL). The molecular impact of BTK inhibition remains unclear particularly in hematopoietic malignancies. We analyzed the molecular mechanisms of BTK inhibition with the novel inhibitor BGB-3111 (zanubrutinib) in MCL models. The efficacy of BGB-3111 was investigated using growth proliferation/cell viability and apoptosis assays in MCL cell lines and patient-derived xenograft (PDX) MCL cells. The activity and mechanisms of BGB-3111 were further confirmed using a cell line xenograft model, an MCL PDX mouse model, and a human phosphokinase profiler array and reverse phase protein array. Lastly, the mechanisms related to resistance to BTK inhibition were analyzed by creating cell lines with low levels of BTK using CRISPR/Cas 9 genome editing. We found that inhibition of BTK leads to suppression of tumor growth, which was mediated via potent suppression of Akt/mTOR, apoptosis, and metabolic stress. Moreover, targeted disruption of the BTK gene in MCL cells resulted in resistance to BTK inhibition and the emergence of novel survival mechanisms. Our studies suggest a general efficacy of BTK inhibition in MCL and potential drug resistance mechanism via alternative signaling pathways.},
}

@article {pmid30259067,
year = {2018},
author = {Xiang, G and Ren, J and Hai, T and Fu, R and Yu, D and Wang, J and Li, W and Wang, H and Zhou, Q},
title = {Editing porcine IGF2 regulatory element improved meat production in Chinese Bama pigs.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {75},
number = {24},
pages = {4619-4628},
doi = {10.1007/s00018-018-2917-6},
pmid = {30259067},
issn = {1420-9071},
support = {31471215//National Natural Science Foundation of China/ ; 31422038//National Natural Science Foundation of China/ ; 31471395//National Natural Science Foundation of China/ ; 31501188//National Natural Science Foundation of China/ ; XDA16010205//Strategic Priority Research Program of Chinese Academy of Sciences/ ; 2015AA020307//National High-Tech Research and Development Program/ ; 2016ZX08010001//National Special Key Project for Transgenic Breeding/ ; },
mesh = {Alleles ; Animal Husbandry/methods ; Animals ; Breeding/methods ; *CRISPR-Cas Systems ; Female ; Gene Editing/*methods ; Genotype ; Insulin-Like Growth Factor II/*genetics ; *Introns ; Male ; Meat/analysis ; Mutation ; Phenotype ; *Regulatory Sequences, Nucleic Acid ; Swine/*genetics ; },
abstract = {Insulin-like growth factor 2 (IGF2) is an important growth factor, which promotes growth and development in mammals during fetal and postnatal stages. Using CRISPR-Cas9 system, we generated multiple founder pigs containing 12 different mutant alleles around a regulatory element within the intron 3 of IGF2 gene. Crossing two male founders passed four mutant alleles onto F1 generation, and these mutations abolished repressor ZBED6 binding and rendered this regulatory element nonfunctional. Both founders and F1 animals showed significantly faster growth, without affecting meat quality. These results indicated that editing IGF2 intron 3-3072 site using CRISPR-Cas9 technology improved meat production in Bama pigs. This is the first demonstration that editing non-coding region can improve economic traits in livestock.},
}

Alleles
Animal Husbandry/methods
Animals
Breeding/methods
*CRISPR-Cas Systems
Female
Gene Editing/*methods
Genotype
Insulin-Like Growth Factor II/*genetics
*Introns
Male
Meat/analysis
Mutation
Phenotype
*Regulatory Sequences, Nucleic Acid
Swine/*genetics

@article {pmid29079543,
year = {2017},
author = {Li, X and Xie, Y and Zhu, Q and Liu, YG},
title = {Targeted Genome Editing in Genes and cis-Regulatory Regions Improves Qualitative and Quantitative Traits in Crops.},
journal = {Molecular plant},
volume = {10},
number = {11},
pages = {1368-1370},
doi = {10.1016/j.molp.2017.10.009},
pmid = {29079543},
issn = {1752-9867},
mesh = {CRISPR-Cas Systems/genetics ; Crops, Agricultural/*genetics/growth & development ; *Gene Editing ; Genes, Plant/*genetics ; Regulatory Sequences, Nucleic Acid/*genetics ; },
}

CRISPR-Cas Systems/genetics
Crops, Agricultural/*genetics/growth & development
*Gene Editing
Genes, Plant/*genetics
Regulatory Sequences, Nucleic Acid/*genetics

@article {pmid30355950,
year = {2018},
author = {Xu, J and Barone, S and Zahedi, K and Brooks, M and Soleimani, M},
title = {Slc4a8 in the Kidney: Expression, Subcellular Localization and Role in Salt Reabsorption.},
journal = {Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology},
volume = {50},
number = {4},
pages = {1361-1375},
doi = {10.1159/000494596},
pmid = {30355950},
issn = {1421-9778},
mesh = {Animals ; Bicarbonates/metabolism ; CRISPR-Cas Systems/genetics ; Diuresis/drug effects ; Dogs ; Furosemide/pharmacology ; In Situ Hybridization ; Kidney/*metabolism ; Kidney Tubules, Collecting/metabolism ; Madin Darby Canine Kidney Cells ; Mice ; Mice, Knockout ; Oligoribonucleotides, Antisense/metabolism ; Plasmids/genetics/metabolism ; Sodium/urine ; Sodium Chloride/metabolism ; Sodium-Bicarbonate Symporters/deficiency/genetics/*metabolism ; },
abstract = {BACKGROUND/AIMS: The sodium-dependent bicarbonate transporter Slc4a8 (a.k.a NDCBE) mediates the co-transport of sodium and bicarbonate in exchange for chloride. It is abundantly detected in the brain, with low expression levels in the kidney. The cell distribution and subcellular localization of Slc4a8 in the kidney and its role in acid/base and electrolyte homeostasis has been the subject of conflicting reports. There are no conclusive localization or functional studies to pinpoint the location and demonstrate the function of Slc4a8 in the kidney.

METHODS: Molecular techniques, including RT-PCR and in situ hybridization, were performed on kidney sections and tagged epitopes were used to examine the membrane targeting of Slc4a8 in polarized kidney cells. Crispr/Cas9 was used to generate and examine Slc4a8 KO mice.

RESULTS: Zonal distribution and in situ hybridization studies showed very little expression for Slc4a8 (NDCBE) in the cortex or in cortical collecting ducts (CCD). Slc4a8 was predominantly detected in the outer and inner medullary collecting ducts (OMCD and IMCD), and was targeted to the basolateral membrane of osmotically tolerant MDCK cells. Slc4a8 KO mice did not show any abnormal salt or bicarbonate wasting under baseline conditions or in response to bicarbonate loading, salt restriction or furosemide-induced diuresis.

CONCLUSION: Slc4a8 (NDCBE) is absent in the CCD and is predominantly localized on the basolateral membrane of medullary collecting duct cells. Further, Slc4a8 deletion does not cause significant acid base or electrolyte abnormalities in pathophysiologic states. Additional studies are needed to examine the role of Slc4a8 (NDCBE) in intracellular pH and volume regulation in medullary collecting duct cells.},
}

Animals
Bicarbonates/metabolism
CRISPR-Cas Systems/genetics
Diuresis/drug effects
Dogs
Furosemide/pharmacology
In Situ Hybridization
Kidney/*metabolism
Kidney Tubules, Collecting/metabolism
Madin Darby Canine Kidney Cells
Mice
Mice, Knockout
Oligoribonucleotides, Antisense/metabolism
Plasmids/genetics/metabolism
Sodium/urine
Sodium Chloride/metabolism
Sodium-Bicarbonate Symporters/deficiency/genetics/*metabolism

@article {pmid28224990,
year = {2017},
author = {Senturk, S and Shirole, NH and Nowak, DG and Corbo, V and Pal, D and Vaughan, A and Tuveson, DA and Trotman, LC and Kinney, JB and Sordella, R},
title = {Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14370},
doi = {10.1038/ncomms14370},
pmid = {28224990},
issn = {2041-1723},
support = {P30 CA045508/CA/NCI NIH HHS/United States ; R01 CA137050/CA/NCI NIH HHS/United States ; U01 CA168409/CA/NCI NIH HHS/United States ; },
mesh = {A549 Cells ; Animals ; CRISPR-Associated Proteins/chemistry/metabolism ; CRISPR-Cas Systems/*genetics ; Fibroblasts/metabolism ; Gene Editing/*methods ; Humans ; Integrases/metabolism ; Lentivirus/metabolism ; Ligands ; Mice ; Protein Domains ; Protein Stability ; RNA, Guide/metabolism ; Tamoxifen/pharmacology ; Time Factors ; },
abstract = {The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic characterization and identification of essential genes, as well as the investigation of the interactions between functional genes.},
}

A549 Cells
Animals
CRISPR-Associated Proteins/chemistry/metabolism
CRISPR-Cas Systems/*genetics
Fibroblasts/metabolism
Gene Editing/*methods
Humans
Integrases/metabolism
Lentivirus/metabolism
Ligands
Mice
Protein Domains
Protein Stability
RNA, Guide/metabolism
Tamoxifen/pharmacology
Time Factors

@article {pmid28181494,
year = {2017},
author = {Kim, IS and Heilmann, S and Kansler, ER and Zhang, Y and Zimmer, M and Ratnakumar, K and Bowman, RL and Simon-Vermot, T and Fennell, M and Garippa, R and Lu, L and Lee, W and Hollmann, T and Xavier, JB and White, RM},
title = {Microenvironment-derived factors driving metastatic plasticity in melanoma.},
journal = {Nature communications},
volume = {8},
number = {},
pages = {14343},
doi = {10.1038/ncomms14343},
pmid = {28181494},
issn = {2041-1723},
support = {DP2 CA186572/CA/NCI NIH HHS/United States ; K08 AR055368/AR/NIAMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Cell Plasticity/genetics ; Cell Proliferation/genetics ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Humans ; Melanoma/genetics/*pathology ; Models, Biological ; Neoplasm Metastasis ; Phenotype ; *Tumor Microenvironment/genetics ; Zebrafish ; Zebrafish Proteins/metabolism ; },
abstract = {Cellular plasticity is a state in which cancer cells exist along a reversible phenotypic spectrum, and underlies key traits such as drug resistance and metastasis. Melanoma plasticity is linked to phenotype switching, where the microenvironment induces switches between invasive/MITFLO versus proliferative/MITFHI states. Since MITF also induces pigmentation, we hypothesize that macrometastatic success should be favoured by microenvironments that induce a MITFHI/differentiated/proliferative state. Zebrafish imaging demonstrates that after extravasation, melanoma cells become pigmented and enact a gene expression program of melanocyte differentiation. We screened for microenvironmental factors leading to phenotype switching, and find that EDN3 induces a state that is both proliferative and differentiated. CRISPR-mediated inactivation of EDN3, or its synthetic enzyme ECE2, from the microenvironment abrogates phenotype switching and increases animal survival. These results demonstrate that after metastatic dissemination, the microenvironment provides signals to promote phenotype switching and provide proof that targeting tumour cell plasticity is a viable therapeutic opportunity.},
}

Animals
CRISPR-Cas Systems/genetics
Cell Differentiation/genetics
*Cell Plasticity/genetics
Cell Proliferation/genetics
Gene Expression Profiling
Gene Expression Regulation, Neoplastic
Humans
Melanoma/genetics/*pathology
Models, Biological
Neoplasm Metastasis
Phenotype
*Tumor Microenvironment/genetics
Zebrafish
Zebrafish Proteins/metabolism

@article {pmid29907613,
year = {2018},
author = {Pflueger, C and Tan, D and Swain, T and Nguyen, T and Pflueger, J and Nefzger, C and Polo, JM and Ford, E and Lister, R},
title = {A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs.},
journal = {Genome research},
volume = {28},
number = {8},
pages = {1193-1206},
doi = {10.1101/gr.233049.117},
pmid = {29907613},
issn = {1549-5469},
support = {R01 DA036906/DA/NIDA NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Binding Sites ; CRISPR-Cas Systems/*genetics ; Catalytic Domain/genetics ; Chromatin/genetics ; DNA (Cytosine-5-)-Methyltransferases/*genetics ; DNA Methylation/*genetics ; *Epigenesis, Genetic ; Gene Editing ; Promoter Regions, Genetic ; Protein Binding ; Recombinant Fusion Proteins/*genetics ; },
abstract = {Detection of DNA methylation in the genome has been possible for decades; however, the ability to deliberately and specifically manipulate local DNA methylation states in the genome has been extremely limited. Consequently, this has impeded our understanding of the direct effect of DNA methylation on transcriptional regulation and transcription factor binding in the native chromatin context. Thus, highly specific targeted epigenome editing tools are needed to address this. Recent adaptations of genome editing technologies, including fusion of the DNMT3A DNA methyltransferase catalytic domain to catalytically inactive Cas9 (dC9-D3A), have aimed to alter DNA methylation at desired loci. Here, we show that these tools exhibit consistent off-target DNA methylation deposition in the genome, limiting their capabilities to unambiguously assess the functional consequences of DNA methylation. To address this, we developed a modular dCas9-SunTag (dC9Sun-D3A) system that can recruit multiple DNMT3A catalytic domains to a target site for editing DNA methylation. dC9Sun-D3A is tunable, specific, and exhibits much higher induction of DNA methylation at target sites than the dC9-D3A direct fusion protein. Importantly, genome-wide characterization of dC9Sun-D3A binding sites and DNA methylation revealed minimal off-target protein binding and induction of DNA methylation with dC9Sun-D3A, compared to pervasive off-target methylation by dC9-D3A. Furthermore, we used dC9Sun-D3A to demonstrate the binding sensitivity to DNA methylation for CTCF and NRF1 in situ. Overall, this modular dC9Sun-D3A system enables precise DNA methylation deposition with the lowest off-target DNA methylation levels reported to date, allowing accurate functional determination of the role of DNA methylation at single loci.},
}

Binding Sites
CRISPR-Cas Systems/*genetics
Catalytic Domain/genetics
Chromatin/genetics
DNA (Cytosine-5-)-Methyltransferases/*genetics
DNA Methylation/*genetics
*Epigenesis, Genetic
Gene Editing
Promoter Regions, Genetic
Protein Binding
Recombinant Fusion Proteins/*genetics

@article {pmid29720392,
year = {2018},
author = {Yamamoto, M and Nishio, T and Nasrallah, JB},
title = {Activation of Self-Incompatibility Signaling in Transgenic Arabidopsis thaliana Is Independent of AP2-Based Clathrin-Mediated Endocytosis.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {7},
pages = {2231-2239},
doi = {10.1534/g3.118.200231},
pmid = {29720392},
issn = {2160-1836},
mesh = {Adaptor Protein Complex 2/*metabolism ; Amino Acid Motifs ; Amino Acid Sequence ; Arabidopsis/*genetics/*metabolism ; CRISPR-Cas Systems ; Clathrin/*metabolism ; *Endocytosis ; Gene Expression ; Gene Targeting ; Mutation ; Plant Proteins/chemistry/genetics/metabolism ; Plants, Genetically Modified ; Pollination ; Protein Binding ; Protein Interaction Domains and Motifs ; *Self-Incompatibility in Flowering Plants ; *Signal Transduction ; },
abstract = {Internalization of plasma membrane (PM)-localized ligand-activated receptor kinases and their trafficking to sorting endosomes have traditionally been viewed as functioning primarily in the down-regulation of receptor signaling, but are now considered to be also essential for signaling by some receptors. A major mechanism for internalization of PM proteins is clathrin-mediated endocytosis (CME). CME is mediated by the Adaptor Protein Complex 2 (AP2), which is involved in interaction of the AP2 μ-adaptin subunit with a tyrosine-based Yxxϕ motif located in the cytoplasmic domain of the cargo protein. In this study, we investigated the role of AP2-mediated CME for signaling by the S-locus receptor kinase (SRK), a protein localized in the PM of stigma epidermal cells, which, together with its pollen coat-localized S-locus cysteine-rich (SCR) ligand, functions in the self-incompatibility (SI) response of the Brassicaceae. Using Arabidopsis thaliana plants that were made self-incompatible by transformation with an A. lyrata-derived SRK/SCR gene pair, we tested the effect on SI of site-directed mutations in each of the two Yxxϕ motifs in SRK and of a CRISPR/Cas9-induced null mutation in the AP2 μ-adaptin gene AP2M Both in vitro SRK kinase activity and the in planta SI response were abolished by substitution of tyrosine in one of the two Yxxϕ motifs, but were unaffected by elimination of either the second Yxxϕ motif or AP2M function. Thus, AP2-mediated CME is considered to be unnecessary for SRK signaling in the SI response.},
}

Adaptor Protein Complex 2/*metabolism
Amino Acid Motifs
Amino Acid Sequence
Arabidopsis/*genetics/*metabolism
CRISPR-Cas Systems
Clathrin/*metabolism
*Endocytosis
Gene Expression
Gene Targeting
Mutation
Plant Proteins/chemistry/genetics/metabolism
Plants, Genetically Modified
Pollination
Protein Binding
Protein Interaction Domains and Motifs
*Self-Incompatibility in Flowering Plants
*Signal Transduction

@article {pmid30065322,
year = {2018},
author = {Callaway, E},
title = {CRISPR plants now subject to tough GM laws in European Union.},
journal = {Nature},
volume = {560},
number = {7716},
pages = {16},
doi = {10.1038/d41586-018-05814-6},
pmid = {30065322},
issn = {1476-4687},
mesh = {Agriculture/economics/legislation & jurisprudence/methods ; CRISPR-Cas Systems/*genetics ; *European Union ; *Food, Genetically Modified/economics/standards ; Gene Editing/*legislation & jurisprudence/standards/trends ; Humans ; Mutagenesis ; Plant Breeding/economics/legislation & jurisprudence/methods ; Research/*legislation & jurisprudence/trends ; },
}

Agriculture/economics/legislation & jurisprudence/methods
CRISPR-Cas Systems/*genetics
*European Union
*Food, Genetically Modified/economics/standards
Gene Editing/*legislation & jurisprudence/standards/trends
Humans
Mutagenesis
Plant Breeding/economics/legislation & jurisprudence/methods
Research/*legislation & jurisprudence/trends

@article {pmid29882718,
year = {2018},
author = {Lim, D},
title = {Disruption and development: the evolving CRISPR patent and technology landscape.},
journal = {Pharmaceutical patent analyst},
volume = {7},
number = {4},
pages = {141-145},
doi = {10.4155/ppa-2018-0010},
pmid = {29882718},
issn = {2046-8962},
mesh = {Academies and Institutes ; *CRISPR-Cas Systems ; California ; *Patents as Topic ; Technology ; Universities ; },
}

Academies and Institutes
*CRISPR-Cas Systems
California
*Patents as Topic
Technology
Universities

@article {pmid29718583,
year = {2018},
author = {Hiruta, C and Kakui, K and Tollefsen, KE and Iguchi, T},
title = {Targeted gene disruption by use of CRISPR/Cas9 ribonucleoprotein complexes in the water flea Daphnia pulex.},
journal = {Genes to cells : devoted to molecular & cellular mechanisms},
volume = {23},
number = {6},
pages = {494-502},
doi = {10.1111/gtc.12589},
pmid = {29718583},
issn = {1365-2443},
mesh = {Animals ; Arthropod Proteins/genetics/*metabolism ; *CRISPR-Cas Systems ; Daphnia/embryology/*genetics/growth & development/physiology ; Deoxyribonuclease I/metabolism ; Embryo, Nonmammalian/cytology/metabolism ; Gene Targeting/*methods ; Genomics ; Mutagenesis ; Phenotype ; Ribonucleoproteins/genetics/*metabolism ; },
abstract = {The microcrustacean Daphnia pulex is an important model for environmental, ecological, evolutionary and developmental genomics because its adaptive life history displays plasticity in response to environmental changes. Even though the whole-genome sequence is available and omics data have actively accumulated for this species, the available tools for analyzing gene function have thus far been limited to RNAi (RNA interference) and TALEN (the transcription activator-like effector nuclease) systems. The development of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) system is thus expected to further increase the genetic tractability of D. pulex and to advance the understanding of this species. In this study, we developed a genome editing system for D. pulex using CRISPR/Cas9 ribonucleoprotein complexes (Cas9 RNPs). We first assembled a CRISPR single-guide RNA (sgRNA) specific to the Distal-less gene (Dll), which encodes a homeodomain transcription factor essential for distal limb development in invertebrates and vertebrates. Then, we injected Cas9 RNPs into eggs and evaluated its activity in vivo by a T7 endonuclease I assay. Injected embryos showed defective formation of the second antenna and disordered development of appendages, and indel mutations were detected in Dll loci, indicating that this technique successfully knocked out the target gene.},
}

Animals
Arthropod Proteins/genetics/*metabolism
*CRISPR-Cas Systems
Daphnia/embryology/*genetics/growth & development/physiology
Deoxyribonuclease I/metabolism
Embryo, Nonmammalian/cytology/metabolism
Gene Targeting/*methods
Genomics
Mutagenesis
Phenotype
Ribonucleoproteins/genetics/*metabolism

@article {pmid29444903,
year = {2018},
author = {Wei, Y and Yang, L and Zhang, X and Sui, D and Wang, C and Wang, K and Shan, M and Guo, D and Wang, H},
title = {Generation and Characterization of a CYP2C11-Null Rat Model by Using the CRISPR/Cas9 Method.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {46},
number = {5},
pages = {525-531},
doi = {10.1124/dmd.117.078444},
pmid = {29444903},
issn = {1521-009X},
mesh = {Animals ; Aryl Hydrocarbon Hydroxylases/*genetics ; CRISPR-Cas Systems/drug effects/*genetics ; Cytochrome P450 Family 2/*genetics ; Female ; Inactivation, Metabolic/physiology ; Male ; Microsomes, Liver/drug effects ; Models, Animal ; Rats ; Rats, Sprague-Dawley ; Steroid 16-alpha-Hydroxylase/*genetics ; Tolbutamide/pharmacology ; },
abstract = {CYP2C11 is involved in the metabolism of many drugs in rats. To assess the roles of CYP2C11 in physiology and drug metabolism, a CYP2C11-null rat model was generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9method. A 2-base pair insertion was added to exon 6 of CYP2C11 in Sprague-Dawley rats. CYP2C11 was not detected by western blotting in liver microsomes of CYP2C11-null rats. No off-target effects were found at 11 predicted sites of the knockout model. The CYP2C11-null rats were viable and had no obvious abnormalities, with the exception of reduced fertility. Puberty in CYP2C11-null rats appeared to be delayed by ∼20 days, and the average litter size fell by 43%. Tolbutamide was used as a probe in this drug metabolism study. In the liver microsomes of CYP2C11-null rats, the Vmax and intrinsicclearance values decreased by 22% and 47%, respectively, compared with those of wild-type rats. The Km values increased by 47% compared with that of wild types. However, our pharmacokinetics study showed no major differences in any parameters between the two strains, in both males and females. In conclusion, a CYP2C11-null rat model was successfully generated and is a valuable tool to study the in vivo function of CYP2C11.},
}

Animals
Aryl Hydrocarbon Hydroxylases/*genetics
CRISPR-Cas Systems/drug effects/*genetics
Cytochrome P450 Family 2/*genetics
Female
Inactivation, Metabolic/physiology
Male
Microsomes, Liver/drug effects
Models, Animal
Rats
Rats, Sprague-Dawley
Steroid 16-alpha-Hydroxylase/*genetics
Tolbutamide/pharmacology

@article {pmid28418037,
year = {2017},
author = {Yang, Y and Xiong, S and Cai, B and Luo, H and Dong, E and Li, Q and Ji, G and Zhao, C and Wen, Y and Wei, Y and Yang, H},
title = {Mitochondrial C11orf83 is a potent Antiviral Protein Independent of interferon production.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {44303},
doi = {10.1038/srep44303},
pmid = {28418037},
issn = {2045-2322},
mesh = {2',5'-Oligoadenylate Synthetase/genetics/*immunology ; Animals ; CRISPR-Cas Systems ; Carrier Proteins/genetics/*immunology ; Cercopithecus aethiops ; Endoribonucleases/genetics/*immunology ; Gene Deletion ; Gene Expression Regulation ; Genes, Reporter ; Green Fluorescent Proteins/genetics/metabolism ; HEK293 Cells ; Hep G2 Cells ; *Host-Pathogen Interactions ; Human Umbilical Vein Endothelial Cells ; Humans ; Immunity, Innate ; Interferon-gamma/genetics/immunology ; Luciferases/genetics/metabolism ; Mitochondria/genetics/*immunology ; Signal Transduction ; Vero Cells ; Vesicular stomatitis Indiana virus/genetics/*immunology ; Virus Replication ; },
abstract = {Mitochondria have a central position in innate immune response via the adaptor protein MAVS in mitochondrial outer membrane to limit viral replication by inducing interferon production. Here, we reported that C11orf83, a component of complex III of electronic transfer chain in mitochondrial inner membrane, was a potent antiviral protein independent of interferon production. C11orf83 expression significantly increased in response to viral infection, and endows cells with stronger capability of inhibiting viral replication. Deletion of C11orf83 permits viral replication easier and cells were more vulnerable to viral killing. These effects mainly were mediated by triggering OAS3-RNase L system. C11orf83 overexpression induced higher transcription of OAS3, and knockdown either OAS3 or RNase L impaired the antiviral capability of C11orf83. Interestingly, the signaling from C11orf83 to OAS3-RNase L was independent of interferon production. Thus, our findings suggested a new antiviral mechanism by bridging cell metabolic machinery component with antiviral effectors.},
}

2',5'-Oligoadenylate Synthetase/genetics/*immunology
Animals
CRISPR-Cas Systems
Carrier Proteins/genetics/*immunology
Cercopithecus aethiops
Endoribonucleases/genetics/*immunology
Gene Deletion
Gene Expression Regulation
Genes, Reporter
Green Fluorescent Proteins/genetics/metabolism
HEK293 Cells
Hep G2 Cells
*Host-Pathogen Interactions
Human Umbilical Vein Endothelial Cells
Humans
Immunity, Innate
Interferon-gamma/genetics/immunology
Luciferases/genetics/metabolism
Mitochondria/genetics/*immunology
Signal Transduction
Vero Cells
Vesicular stomatitis Indiana virus/genetics/*immunology
Virus Replication

@article {pmid28287132,
year = {2017},
author = {Wrona, D and Siler, U and Reichenbach, J},
title = {CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {44187},
doi = {10.1038/srep44187},
pmid = {28287132},
issn = {2045-2322},
mesh = {Base Sequence ; *CRISPR-Cas Systems ; Genetic Therapy/*methods ; *Genetic Vectors ; *Granulomatous Disease, Chronic/genetics/metabolism/pathology/therapy ; HL-60 Cells ; Humans ; NADPH Oxidases/genetics/metabolism ; Sequence Deletion ; },
abstract = {Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47phox-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47phox-subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47phox-deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.},
}

Base Sequence
*CRISPR-Cas Systems
Genetic Therapy/*methods
*Genetic Vectors
*Granulomatous Disease, Chronic/genetics/metabolism/pathology/therapy
HL-60 Cells
Humans
NADPH Oxidases/genetics/metabolism
Sequence Deletion

@article {pmid28281657,
year = {2017},
author = {Pazhakh, V and Clark, S and Keightley, MC and Lieschke, GJ},
title = {A GCSFR/CSF3R zebrafish mutant models the persistent basal neutrophil deficiency of severe congenital neutropenia.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {44455},
doi = {10.1038/srep44455},
pmid = {28281657},
issn = {2045-2322},
mesh = {Animals ; Base Sequence ; CRISPR-Cas Systems ; Disease Models, Animal ; Embryo, Nonmammalian ; Exons ; Gene Editing/*methods ; Gene Expression ; Granulocyte Colony-Stimulating Factor/*genetics/immunology ; Haploinsufficiency ; Heterozygote ; Humans ; Kidney/immunology/*pathology ; Leukocyte Count ; Morpholinos/genetics/metabolism ; Neutropenia/*congenital/genetics/immunology/pathology ; Neutrophils/immunology/*pathology ; Phenotype ; Receptors, Colony-Stimulating Factor/antagonists & inhibitors/deficiency/*genetics/immunology ; Zebrafish ; },
abstract = {Granulocyte colony-stimulating factor (GCSF) and its receptor (GCSFR), also known as CSF3 and CSF3R, are required to maintain normal neutrophil numbers during basal and emergency granulopoiesis in humans, mice and zebrafish. Previous studies identified two zebrafish CSF3 ligands and a single CSF3 receptor. Transient antisense morpholino oligonucleotide knockdown of both these ligands and receptor reduces neutrophil numbers in zebrafish embryos, a technique widely used to evaluate neutrophil contributions to models of infection, inflammation and regeneration. We created an allelic series of zebrafish csf3r mutants by CRISPR/Cas9 mutagenesis targeting csf3r exon 2. Biallelic csf3r mutant embryos are viable and have normal early survival, despite a substantial reduction of their neutrophil population size, and normal macrophage abundance. Heterozygotes have a haploinsufficiency phenotype with an intermediate reduction in neutrophil numbers. csf3r mutants are viable as adults, with a 50% reduction in tissue neutrophil density and a substantial reduction in the number of myeloid cells in the kidney marrow. These csf3r mutants are a new animal model of human CSF3R-dependent congenital neutropenia. Furthermore, they will be valuable for studying the impact of neutrophil loss in the context of other zebrafish disease models by providing a genetically stable, persistent, reproducible neutrophil deficiency state throughout life.},
}

Animals
Base Sequence
CRISPR-Cas Systems
Disease Models, Animal
Embryo, Nonmammalian
Exons
Gene Editing/*methods
Gene Expression
Granulocyte Colony-Stimulating Factor/*genetics/immunology
Haploinsufficiency
Heterozygote
Humans
Kidney/immunology/*pathology
Leukocyte Count
Morpholinos/genetics/metabolism
Neutropenia/*congenital/genetics/immunology/pathology
Neutrophils/immunology/*pathology
Phenotype
Receptors, Colony-Stimulating Factor/antagonists & inhibitors/deficiency/*genetics/immunology
Zebrafish

@article {pmid30397647,
year = {2018},
author = {Chatterjee, P and Jakimo, N and Jacobson, JM},
title = {Minimal PAM specificity of a highly similar SpCas9 ortholog.},
journal = {Science advances},
volume = {4},
number = {10},
pages = {eaau0766},
doi = {10.1126/sciadv.aau0766},
pmid = {30397647},
issn = {2375-2548},
abstract = {RNA-guided DNA endonucleases of the CRISPR-Cas system are widely used for genome engineering and thus have numerous applications in a wide variety of fields. CRISPR endonucleases, however, require a specific protospacer adjacent motif (PAM) flanking the target site, thus constraining their targetable sequence space. In this study, we demonstrate the natural PAM plasticity of a highly similar, yet previously uncharacterized, Cas9 from Streptococcus canis (ScCas9) through rational manipulation of distinguishing motif insertions. To this end, we report affinity to minimal 5'-NNG-3' PAM sequences and demonstrate the accurate editing capabilities of the ortholog in both bacterial and human cells. Last, we build an automated bioinformatics pipeline, the Search for PAMs by ALignment Of Targets (SPAMALOT), which further explores the microbial PAM diversity of otherwise overlooked Streptococcus Cas9 orthologs. Our results establish that ScCas9 can be used both as an alternative genome editing tool and as a functional platform to discover novel Streptococcus PAM specificities.},
}

@article {pmid30397343,
year = {2018},
author = {Özcan, A and Pausch, P and Linden, A and Wulf, A and Schühle, K and Heider, J and Urlaub, H and Heimerl, T and Bange, G and Randau, L},
title = {Type IV CRISPR RNA processing and effector complex formation in Aromatoleum aromaticum.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41564-018-0274-8},
pmid = {30397343},
issn = {2058-5276},
abstract = {Type IV CRISPR-Cas modules belong to class 1 prokaryotic adaptive immune systems, which are defined by the presence of multisubunit effector complexes. They usually lack the known Cas proteins involved in adaptation and target cleavage, and their function has not been experimentally addressed. To investigate RNA and protein components of this CRISPR-Cas type, we located a complete type IV cas gene locus and an adjacent CRISPR array on a megaplasmid of Aromatoleum aromaticum EbN1, which contains an additional type I-C system on its chromosome. RNA sequencing analyses verified CRISPR RNA (crRNA) production and maturation for both systems. Type IV crRNAs were shown to harbour unusually short 7 nucleotide 5'-repeat tags and stable 3' hairpin structures. A unique Cas6 variant (Csf5) was identified that generates crRNAs that are specifically incorporated into type IV CRISPR-ribonucleoprotein (crRNP) complexes. Structures of RNA-bound Csf5 were obtained. Recombinant production and purification of the type IV Cas proteins, together with electron microscopy, revealed that Csf2 acts as a helical backbone for type IV crRNPs that include Csf5, Csf3 and a large subunit (Csf1). Mass spectrometry analyses identified protein-protein and protein-RNA contact sites. These results highlight evolutionary connections between type IV and type I CRISPR-Cas systems and demonstrate that type IV CRISPR-Cas systems employ crRNA-guided effector complexes.},
}

@article {pmid29950430,
year = {2018},
author = {Chen, X and Liao, S and Huang, X and Xu, T and Feng, X and Guang, S},
title = {Targeted Chromosomal Rearrangements via Combinatorial Use of CRISPR/Cas9 and Cre/LoxP Technologies in Caenorhabditis elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {8},
pages = {2697-2707},
doi = {10.1534/g3.118.200473},
pmid = {29950430},
issn = {2160-1836},
support = {P40 OD010440/OD/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Caenorhabditis elegans/genetics ; *Chromosome Inversion ; Gene Editing/*methods ; Gene Targeting/*methods ; Integrases/genetics/metabolism ; },
abstract = {Rearranged chromosomes have been applied to construct genetic balancers to manipulate essential genes in C. elegans Although much effort has been put into constructing balancer chromosomes, approximately 6% (map units) of the C. elegans genome has not been covered, and this area lies mostly in pairing centers (PCs). Here, we developed a method for conditional chromosomal engineering through combinatorial use of the CRISPR/Cas9 and Cre/LoxP technologies. Functional DNA fragments containing LoxP sequences were inserted into designated genomic loci using a modified counterselection (cs)-CRISPR method. Then, heat-shock-induced Cre recombinase induced an inversion of the chromosomal region between the two LoxP sites. The chromosomal inversions were subsequently detected by the appearance of pharyngeal GFP. Through this method, we have successfully generated several chromosomal inversion lines, providing valuable resources for studying essential genes in pairing centers.},
}

Animals
*CRISPR-Cas Systems
Caenorhabditis elegans/genetics
*Chromosome Inversion
Gene Editing/*methods
Gene Targeting/*methods
Integrases/genetics/metabolism

@article {pmid29934375,
year = {2018},
author = {Ewen-Campen, B and Perrimon, N},
title = {ovoD Co-selection: A Method for Enriching CRISPR/Cas9-Edited Alleles in Drosophila.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {8},
pages = {2749-2756},
doi = {10.1534/g3.118.200498},
pmid = {29934375},
issn = {2160-1836},
support = {R01 GM084947/GM/NIGMS NIH HHS/United States ; F32 GM113395/GM/NIGMS NIH HHS/United States ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Alleles ; Animals ; *CRISPR-Cas Systems ; Drosophila/*genetics ; Female ; Gene Editing/methods ; Gene Targeting/*methods ; Oogonia/metabolism ; Phenotype ; *Selection, Genetic ; },
abstract = {Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of Drosophila genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, ovoD1 In this system ("ovoD co-selection"), the only functional germ cells in injected females are those that have been edited at the ovoD1 locus, and thus all offspring of these flies have undergone editing of at least one locus. We demonstrate that ovoD co-selection can be used to enrich for knock-out mutagenesis via nonhomologous end-joining (NHEJ), and for knock-in alleles via homology-directed repair (HDR). Altogether, our results demonstrate that ovoD co-selection reduces the amount of screening necessary to isolate desired CRISPR events in Drosophila.},
}

Alleles
Animals
*CRISPR-Cas Systems
Drosophila/*genetics
Female
Gene Editing/methods
Gene Targeting/*methods
Oogonia/metabolism
Phenotype
*Selection, Genetic

@article {pmid29884615,
year = {2018},
author = {Pauwels, L and De Clercq, R and Goossens, J and Iñigo, S and Williams, C and Ron, M and Britt, A and Goossens, A},
title = {A Dual sgRNA Approach for Functional Genomics in Arabidopsis thaliana.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {8},
pages = {2603-2615},
doi = {10.1534/g3.118.200046},
pmid = {29884615},
issn = {2160-1836},
mesh = {Arabidopsis/*genetics ; Arabidopsis Proteins/genetics/metabolism ; CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA-Directed DNA Polymerase/genetics/metabolism ; Gene Editing/*methods ; Genomics/*methods ; *Mutagenesis ; RNA, Guide/*genetics/metabolism ; },
abstract = {Reverse genetics uses loss-of-function alleles to interrogate gene function. The advent of CRISPR/Cas9-based gene editing now allows the generation of knock-out alleles for any gene and entire gene families. Even in the model plant Arabidopsis thaliana, gene editing is welcomed as T-DNA insertion lines do not always generate null alleles. Here, we show efficient generation of heritable mutations in Arabidopsis using CRISPR/Cas9 with a workload similar to generating overexpression lines. We obtain for several different genes Cas9 null-segregants with bi-allelic mutations in the T2 generation. While somatic mutations were predominantly generated by the canonical non-homologous end joining (cNHEJ) pathway, we observed inherited mutations that were the result of synthesis-dependent microhomology-mediated end joining (SD-MMEJ), a repair pathway linked to polymerase θ (PolQ). We also demonstrate that our workflow is compatible with a dual sgRNA approach in which a gene is targeted by two sgRNAs simultaneously. This paired nuclease method results in more reliable loss-of-function alleles that lack a large essential part of the gene. The ease of the CRISPR/Cas9 workflow should help in the eventual generation of true null alleles of every gene in the Arabidopsis genome, which will advance both basic and applied plant research.},
}

Arabidopsis/*genetics
Arabidopsis Proteins/genetics/metabolism
CRISPR-Cas Systems
DNA End-Joining Repair
DNA-Directed DNA Polymerase/genetics/metabolism
Gene Editing/*methods
Genomics/*methods
*Mutagenesis
RNA, Guide/*genetics/metabolism

@article {pmid29703785,
year = {2018},
author = {Zhang, XR and He, JB and Wang, YZ and Du, LL},
title = {A Cloning-Free Method for CRISPR/Cas9-Mediated Genome Editing in Fission Yeast.},
journal = {G3 (Bethesda, Md.)},
volume = {8},
number = {6},
pages = {2067-2077},
doi = {10.1534/g3.118.200164},
pmid = {29703785},
issn = {2160-1836},
mesh = {Base Sequence ; CRISPR-Cas Systems/*genetics ; Cloning, Molecular ; DNA Repair/genetics ; Gene Editing/*methods ; Gene Knock-In Techniques ; Point Mutation/genetics ; Schizosaccharomyces/*genetics ; Sequence Deletion ; Temperature ; },
abstract = {The CRISPR/Cas9 system, which relies on RNA-guided DNA cleavage to induce site-specific DNA double-strand breaks, is a powerful tool for genome editing. This system has been successfully adapted for the fission yeast Schizosaccharomyces pombe by expressing Cas9 and the single-guide RNA (sgRNA) from a plasmid. In the procedures published to date, the cloning step that introduces a specific sgRNA target sequence into the plasmid is the most tedious and time-consuming. To increase the efficiency of applying the CRISPR/Cas9 system in fission yeast, we here developed a cloning-free procedure that uses gap repair in fission yeast cells to assemble two linear DNA fragments, a gapped Cas9-encoding plasmid and a PCR-amplified sgRNA insert, into a circular plasmid. Both fragments contain only a portion of the ura4 or bsdMX marker so that only the correctly assembled plasmid can confer uracil prototrophy or blasticidin resistance. We show that this gap-repair-based and cloning-free CRISPR/Cas9 procedure permits rapid and efficient point mutation knock-in, endogenous N-terminal tagging, and genomic sequence deletion in fission yeast.},
}

Base Sequence
CRISPR-Cas Systems/*genetics
Cloning, Molecular
DNA Repair/genetics
Gene Editing/*methods
Gene Knock-In Techniques
Point Mutation/genetics
Schizosaccharomyces/*genetics
Sequence Deletion
Temperature

@article {pmid29542087,
year = {2018},
author = {Gandhi, S and Razy-Krajka, F and Christiaen, L and Stolfi, A},
title = {CRISPR Knockouts in Ciona Embryos.},
journal = {Advances in experimental medicine and biology},
volume = {1029},
number = {},
pages = {141-152},
doi = {10.1007/978-981-10-7545-2_13},
pmid = {29542087},
issn = {0065-2598},
support = {K99 HD084814/HD/NICHD NIH HHS/United States ; R00 HD084814/HD/NICHD NIH HHS/United States ; R01 GM096032/GM/NIGMS NIH HHS/United States ; R01 HL108643/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; CRISPR-Cas Systems ; Ciona intestinalis/embryology/*genetics ; Electroporation ; Embryo, Nonmammalian/cytology/metabolism ; Gene Knockout Techniques/*methods ; INDEL Mutation ; Mutagenesis ; Plasmids ; RNA, Guide/administration & dosage/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has emerged as a revolutionary tool for fast and efficient targeted gene knockouts and genome editing in almost any organism. The laboratory model tunicate Ciona is no exception. Here, we describe our latest protocol for the design, implementation, and evaluation of successful CRISPR/Cas9-mediated gene knockouts in somatic cells of electroporated Ciona embryos. Using commercially available reagents, publicly accessible plasmids, and free web-based software applications, any Ciona researcher can easily knock out any gene of interest in their favorite embryonic cell lineage.},
}

Animals
CRISPR-Cas Systems
Ciona intestinalis/embryology/*genetics
Electroporation
Embryo, Nonmammalian/cytology/metabolism
Gene Knockout Techniques/*methods
INDEL Mutation
Mutagenesis
Plasmids
RNA, Guide/administration & dosage/genetics

@article {pmid28256588,
year = {2017},
author = {Li, C and Unver, T and Zhang, B},
title = {A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.).},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {43902},
doi = {10.1038/srep43902},
pmid = {28256588},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; DNA, Plant/genetics/metabolism ; Gene Targeting/*methods ; Gossypium/*genetics ; Recombination, Genetic ; },
abstract = {The complex allotetraploid genome is one of major challenges in cotton for repressing gene expression. Developing site-specific DNA mutation is the long-term dream for cotton breeding scientists. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as a robust biotechnology for targeted-DNA mutation. In this study, two sgRNAs, GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, were designed in the identical genomic regions of GhMYB25-like A and GhMYB25-like D, which were encoded by cotton A subgenome and the D subgenome, respectively, was assembled to direct Cas9-mediated allotetraploid cotton genome editing. High proportion (14.2-21.4%) CRISPR/Cas9-induced specific truncation events, either from GhMYB25-like A DNA site or from GhMYB25-like D DNA site, were detected in 50% examined transgenic cotton through PCR amplification assay and sequencing analyses. Sequencing results also demonstrated that 100% and 98.8% mutation frequency were occurred on GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2 target site respectively. The off-target effect was evaluated by sequencing two putative off-target sites, which have 3 and 1 mismatched nucleotides with GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, respectively; all the examined samples were not detected any off-target-caused mutation events. Thus, these results demonstrated that CRISPR/Cas9 is qualified for generating DNA level mutations on allotetraploid cotton genome with high-efficiency and high-specificity.},
}

*CRISPR-Cas Systems
DNA, Plant/genetics/metabolism
Gene Targeting/*methods
Gossypium/*genetics
Recombination, Genetic

@article {pmid28240315,
year = {2017},
author = {Sanz, MA and González Almela, E and Carrasco, L},
title = {Translation of Sindbis Subgenomic mRNA is Independent of eIF2, eIF2A and eIF2D.},
journal = {Scientific reports},
volume = {7},
number = {},
pages = {43876},
doi = {10.1038/srep43876},
pmid = {28240315},
issn = {2045-2322},
mesh = {Base Sequence ; CRISPR-Cas Systems ; Cell Line ; Eukaryotic Cells/cytology/virology ; Eukaryotic Initiation Factor-2/genetics/*metabolism ; Humans ; Phosphorylation ; *Protein Biosynthesis ; RNA Interference ; RNA, Messenger/*genetics ; RNA, Viral/*genetics ; Sindbis Virus/*genetics/physiology ; Viral Proteins/genetics/metabolism ; },
abstract = {Translation of Sindbis virus subgenomic mRNA (sgmRNA) can occur after inactivation of eIF2 by phosphorylation in mammalian cells. Several studies have suggested that eIF2 can be replaced by eIF2A or eIF2D. HAP1 human cell lines knocked-out for eIF2A, eIF2D or both by CRISPR/Cas9 genome engineering were compared with wild-type (WT) cells to test the potential role of eIF2A and eIF2D in translation. Sindbis virus infection was comparable between the four cell lines. Moreover, synthesis of viral proteins during late stage infection was similar in all four cell lines despite the fact that eIF2α became phosphorylated. These findings demonstrate that eIF2A and eIF2D are not required for the translation of sgmRNA when eIF2α is phosphorylated. Moreover, silencing of eIF2A or eIF2D by transfection of the corresponding siRNAs in HAP1 WT, HAP1-eIF2A- and HAP1-eIF2D- cells had little effect on the synthesis of viral proteins late in infection. Modification of AUGi to other codons in sgmRNA failed to abrogate translation. Sindbis virus replicons containing these sgmRNA variants could still direct the synthesis of viral proteins. No significant differences were found between the cell lines assayed, suggesting that neither eIF2A nor eIF2D are involved in the translation of this sgmRNA bearing non-AUG codons.},
}

Base Sequence
CRISPR-Cas Systems
Cell Line
Eukaryotic Cells/cytology/virology
Eukaryotic Initiation Factor-2/genetics/*metabolism
Humans
Phosphorylation
*Protein Biosynthesis
RNA Interference
RNA, Messenger/*genetics
RNA, Viral/*genetics
Sindbis Virus/*genetics/physiology
Viral Proteins/genetics/metabolism

@article {pmid30394685,
year = {2018},
author = {Li, P and Fu, X and Zhang, L and Li, S},
title = {CRISPR/Cas-based screening of a gene activation library in Saccharomyces cerevisiae identifies a crucial role of OLE1 in thermotolerance.},
journal = {Microbial biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1751-7915.13333},
pmid = {30394685},
issn = {1751-7915},
support = {21506113//National Natural Science Foundation of China/ ; 31470229//National Natural Science Foundation of China/ ; 31600067//National Natural Science Foundation of China/ ; 2016YFE0108500//National Key R&D Program of China/ ; 2017T100064//China Postdoctoral Science Foundation/ ; 2015M581074//China Postdoctoral Science Foundation/ ; },
abstract = {CRISPR/Cas-based (clustered regularly interspaced short palindromic repeats/CRISPR-associated) screening has been proved to be an efficient method to study functional genomics from yeast to human. In this study, we report the development of a focused CRISPR/Cas-based gene activation library in Saccharomyces cerevisiae and its application in gene identification based on functional screening towards improved thermotolerance. The gene activation library was subjected to screening at 42°C, and the same library cultured at 30°C was set as a control group. After five successive subcultures, five clones were randomly picked from the libraries cultured at 30 and 42°C, respectively. The five clones selected at 30°C contain the specificity sequences of five different single guide RNAs, whereas all the five clones selected at 42°C contain the specificity sequence of one sgRNA that targets the promoter region of OLE1. A crucial role of OLE1 in thermotolerance was identified: the overexpression of OLE1 increased fatty acid unsaturation, and thereby helped counter lipid peroxidation caused by heat stress, rendering the yeast thermotolerant. This study described the application of CRISPR/Cas-based gene activation screening with an example of thermotolerant yeast screening, demonstrating that this method can be used to identify functional genes in yeast.},
}

@article {pmid30394652,
year = {2018},
author = {Borton, MA and Daly, RA and O'Banion, B and Hoyt, DW and Marcus, DN and Welch, S and Hastings, SS and Meulia, T and Wolfe, RA and Booker, AE and Sharma, S and Cole, DR and Wunch, K and Moore, JD and Darrah, TH and Wilkins, MJ and Wrighton, KC},
title = {Comparative genomics and physiology of the genus Methanohalophilus, a prevalent methanogen in hydraulically fractured shale.},
journal = {Environmental microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/1462-2920.14467},
pmid = {30394652},
issn = {1462-2920},
abstract = {About 60% of natural gas production in the United States comes from hydraulic fracturing of unconventional reservoirs, such as shales or organic-rich micrites. This process inoculates and enriches for halotolerant microorganisms in these reservoirs over time, resulting in a saline ecosystem that includes methane producing archaea. Here, we survey the biogeography of methanogens across unconventional reservoirs, and report that members of genus Methanohalophilus are recovered from every hydraulically fractured unconventional reservoir sampled by metagenomics. We provide the first genomic sequencing of three isolate genomes, as well as two metagenome assembled genomes. Utilizing six other previously sequenced genomes, we perform comparative analysis of the 11 genomes representing this genus. This genomic investigation revealed distinctions between surface and subsurface derived genomes that are consistent with constraints encountered in each environment. Genotypic differences were also uncovered between isolate genomes recovered from the same well, suggesting niche partitioning among closely related strains. These genomic substrate utilization predictions were then confirmed by physiological investigation. Fine-scale microdiversity was observed in CRISPR-Cas systems of Methanohalophilus, with genomes from geographically distinct unconventional reservoirs sharing spacers targeting the same viral population. These findings have implications for augmentation strategies resulting in enhanced biogenic methane production in hydraulically fractured unconventional reservoirs. This article is protected by copyright. All rights reserved.},
}

@article {pmid30394044,
year = {2018},
author = {Ali, S and Park, SK and Kim, WC},
title = {The pragmatic introduction and expression of microbial transgenes in plants.},
journal = {Journal of microbiology and biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.4014/jmb.1808.08029},
pmid = {30394044},
issn = {1738-8872},
abstract = {Several genetic strategies have been proposed for the successful transformation and expression of microbial transgenes in model and crop plants. Here, we bring into focus the prominent applications of microbial transgenes in plants for the development of disease resistance; mitigation of stress conditions; augmentation of food quality; and use of plants as "bioreactors" for the production of recombinant proteins, industrially important enzymes, vaccines, antimicrobial compounds, and other valuable secondary metabolites. We discuss the applicable and cost-effective approaches of transgenesis in different plants, as well as the limitations thereof. We subsequently present the contemporary developments in targeted genome editing systems that have facilitated the process of genetic modification and manifested stable and consumer-friendly genetically modified plants and their products. Finally, this article presents the different approaches and demonstrates the introduction and expression of microbial transgenes for the improvement of plant resistance to pathogens and abiotic stress conditions and the production of valuable compounds, together with the promising research progress in targeted genome editing technology. We include a special discussion on the highly efficient CRISPR-Cas system helpful in microbial transgene editing in plants.},
}

@article {pmid30393777,
year = {2018},
author = {Zhang, F and Zhao, S and Ren, C and Zhu, Y and Zhou, H and Lai, Y and Zhou, F and Jia, Y and Zheng, K and Huang, Z},
title = {CRISPRminer is a knowledge base for exploring CRISPR-Cas systems in microbe and phage interactions.},
journal = {Communications biology},
volume = {1},
number = {},
pages = {180},
doi = {10.1038/s42003-018-0184-6},
pmid = {30393777},
issn = {2399-3642},
abstract = {CRISPR-Cas systems not only play key roles in prokaryotic acquired immunity, but can also be adapted as powerful genome editing tools. Understanding the native role of CRISPR-Cas systems in providing adaptive immunity can lead to new CRISPR-based technologies. Here, we develop CRISPRminer, a knowledge base and web server to comprehensively collect and investigate the knowledge of CRISPR-Cas systems and generate instructive annotations, including CRISPR arrays and Cas protein annotation, CRISPR-Cas system classification, self-targeting events detection, microbe-phage interaction inference, and anti-CRISPR annotation. CRISPRminer is user-friendly and freely available at http://www.microbiome-bigdata.com/CRISPRminer.},
}

@article {pmid30393194,
year = {2018},
author = {Hussain, W and Mahmood, T and Hussain, J and Ali, N and Shah, T and Qayyum, S and Khan, I},
title = {CRISPR/Cas system: A game changing genome editing technology, to treat human genetic diseases.},
journal = {Gene},
volume = {685},
number = {},
pages = {70-75},
doi = {10.1016/j.gene.2018.10.072},
pmid = {30393194},
issn = {1879-0038},
abstract = {Genes, are the functional units of heredity that used as an instructors to make proteins either to become the functional or structural part of the cell. Hence, the proteins get more attention because most of the life functions depends on it. Any mutation or alteration in genome sequences results in complete loss of function or formation of abnormal protein which leads to hereditary disorder. Gene therapy on the other hand, used as a remedy, a process that make correction in the gene which is responsible for genomic disorders. The treatment of disease state depends on the understanding of their genetic basis. While, numerous molecular genome editing tools have been developed and are being utilized to translate the abstract of gene therapy into reality, but the problem is still a mystery. The genome editing molecular scissors can be applied to dictate the selected genetic products that can have the therapeutic power. Thus, editing the specific sequences depends on the type of strategies being used by a molecule such is HDR or NHEJ. CRISPR/Cas9 editing technology can use in disease model to study the genitival disorders. One side the CRISPR technology seemed to be extremely accurate but on the other side it has some harmful effects i.e. Cas9 proteins sometimes cuts the similar sequences other than the specific targeted and Off-targeting Sequences etc. Urgent attention and improvement are needed for various implication of CRISPR/Cas9 technology, including the delivery, precision and control over the mention system. This review presents the current scenario of genome editing in vivo and its implications for the future of human genetic disease treatment as well as genome throughput potency.},
}

@article {pmid30391232,
year = {2018},
author = {Chen, H and Li, Y and Du, C and Li, Y and Zhao, J and Zheng, X and Mao, Q and Xia, H},
title = {Aptazyme-mediated direct modulation of post-transcriptional sgRNA level for conditional genome editing and gene expression.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2018.10.011},
pmid = {30391232},
issn = {1873-4863},
abstract = {RNA-guided endonuclease Cas9 derived from microbial CRISPR-Cas adaptive immune systems is a powerful tool for genome editing, which has been widely used in eukaryotic systems, prokaryotic systems, and plants. However, the off-target effects caused by Cas9/sgRNA remain a major concern. Currently, the efforts to reduce the off-target effects mainly focus on improving the targeting specificity of sgRNA/Cas9, regulating the activity of the Cas9 protein or the sgRNA, and controlling the time window of their expression. In this study, a novel system was established to regulate the post-transcriptional sgRNA level by small molecule-controlled aptazyme. This system was shown to reduce the off-target effects caused by Cas9/sgRNA, while enabling precise temporal control over gene editing and regulatory activity. This new system could provide a potentially safer and more powerful tool for genome editing and therapeutic application.},
}

@article {pmid30389787,
year = {2018},
author = {Lynch, JM and Li, B and Katoli, P and Xiang, C and Leehy, B and Rangaswamy, N and Saenz-Vash, V and Wang, YK and Lei, H and Nicholson, TB and Meredith, E and Rice, D and Prasanna, G and Chen, A},
title = {Binding of a glaucoma-associated myocilin variant to the αB-crystallin chaperone impedes protein clearance in trabecular meshwork cells.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1074/jbc.RA118.004325},
pmid = {30389787},
issn = {1083-351X},
abstract = {Myocilin (MYOC) was discovered more than 20 years ago and is the gene whose mutations are most commonly observed in individuals with glaucoma. Despite extensive research efforts, the function of wild-type (wt) MYOC has remained elusive and how mutant MYOC is linked to glaucoma is unclear. Mutant MYOC is believed to be misfolded within the endoplasmic reticulum (ER), and under normal physiological conditions misfolded MYOC should be retro-translocated to the cytoplasm for degradation. To better understand mutant MYOC pathology, we CRISPR-engineered a rat to have a MYOC Y435H substitution which is the equivalent of the pathologic human MYOC Y437H mutation. Using this engineered animal model, we discovered that the chaparone αB-crystallin (CRYAB) is a MYOC-binding partner and that co-expression of these two proteins increases protein aggregates. Our results suggest that the misfolded mutant MYOC aggregates with cytoplasmic CRYAB and thereby compromises protein clearance mechanisms in trabecular meshwork cells and this process represents the primary mode of mutant MYOC pathology. We propose a model by which mutant MYOC causes glaucoma, and we propose that therapeutic treatment of patients having a MYOC mutation may focus on disrupting the MYOC-CRYAB complexes.},
}