Base de dados : MEDLINE
Pesquisa : G02.111.570.080.708.800.325.500 [Categoria DeCS]
Referências encontradas : 1415 [refinar]
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  1 / 1415 MEDLINE  
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[PMID]:29231142
[Au] Autor:Duardo-Sanchez A
[Ad] Endereço:Department of Public Law, Law and the Human Genome Research Group, University of the Basque Country UPV/EHU, 48940, Leioa, Biscay, Spain.
[Ti] Título:CRISPR-Cas in Medicinal Chemistry: Applications and Regulatory Concerns.
[So] Source:Curr Top Med Chem;17(30):3308-3315, 2017.
[Is] ISSN:1873-4294
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:A rapid search in scientific publication's databases shows how the use of CRISPR-Cas genome editions' technique has considerably expanded, and its growing importance, in modern molecular biology. Just in pub-med platform, the search of the term gives more than 3000 results. Specifically, in Drug Discovery, Medicinal Chemistry and Chemical Biology in general CRISPR method may have multiple applications. Some of these applications are: resistance-selection studies of antimalarial lead organic compounds; investigation of druggability; development of animal models for chemical compounds testing, etc. In this paper, we offer a review of the most relevant scientific literature illustrated with specific examples of application of CRISPR technique to medicinal chemistry and chemical biology. We also present a general overview of the main legal and ethical trends regarding this method of genome editing.
[Mh] Termos MeSH primário: Química Farmacêutica
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética
Edição de Genes/ética
Edição de Genes/legislação & jurisprudência
[Mh] Termos MeSH secundário: Animais
Seres Humanos
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Em] Mês de entrada:1803
[Cu] Atualização por classe:180308
[Lr] Data última revisão:
180308
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171213
[St] Status:MEDLINE
[do] DOI:10.2174/1568026618666171211151142


  2 / 1415 MEDLINE  
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[PMID]:29379011
[Au] Autor:Yoon Y; Wang D; Tai PWL; Riley J; Gao G; Rivera-Pérez JA
[Ad] Endereço:Department of Pediatrics, Division of Genes and Development, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA, 01655, USA.
[Ti] Título:Streamlined ex vivo and in vivo genome editing in mouse embryos using recombinant adeno-associated viruses.
[So] Source:Nat Commun;9(1):412, 2018 01 29.
[Is] ISSN:2041-1723
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:Recent advances using CRISPR-Cas9 approaches have dramatically enhanced the ease for genetic manipulation in rodents. Notwithstanding, the methods to deliver nucleic acids into pre-implantation embryos have hardly changed since the original description of mouse transgenesis more than 30 years ago. Here we report a novel strategy to generate genetically modified mice by transduction of CRISPR-Cas9 components into pre-implantation mouse embryos via recombinant adeno-associated viruses (rAAVs). Using this approach, we efficiently generated a variety of targeted mutations in explanted embryos, including indel events produced by non-homologous end joining and tailored mutations using homology-directed repair. We also achieved gene modification in vivo by direct delivery of rAAV particles into the oviduct of pregnant females. Our approach greatly simplifies the generation of genetically modified mice and, more importantly, opens the door for streamlined gene editing in other mammalian species.
[Mh] Termos MeSH primário: Sistemas CRISPR-Cas
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
Dependovirus/genética
Desenvolvimento Embrionário/genética
Edição de Genes/métodos
Engenharia Genética/métodos
[Mh] Termos MeSH secundário: Animais
Proteínas de Bactérias/genética
Proteínas de Bactérias/metabolismo
Blastocisto
Reparo do DNA por Junção de Extremidades
Dependovirus/metabolismo
Endonucleases/genética
Endonucleases/metabolismo
Tubas Uterinas/embriologia
Feminino
Genes Reporter
Proteínas de Fluorescência Verde/genética
Proteínas de Fluorescência Verde/metabolismo
Células HEK293
Seres Humanos
Camundongos
Camundongos Transgênicos
Mutagênese Sítio-Dirigida
Gravidez
Reparo de DNA por Recombinação
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (enhanced green fluorescent protein); 147336-22-9 (Green Fluorescent Proteins); EC 3.1.- (Cas9 endonuclease Streptococcus pyogenes); EC 3.1.- (Endonucleases)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180227
[Lr] Data última revisão:
180227
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180131
[St] Status:MEDLINE
[do] DOI:10.1038/s41467-017-02706-7


  3 / 1415 MEDLINE  
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[PMID]:29377933
[Au] Autor:Sato'o Y; Hisatsune J; Yu L; Sakuma T; Yamamoto T; Sugai M
[Ad] Endereço:Department of Bacteriology, Hiroshima University, Graduate school of Biomedical and Health Sciences, Hiroshima, Hiroshima, Japan.
[Ti] Título:Tailor-made gene silencing of Staphylococcus aureus clinical isolates by CRISPR interference.
[So] Source:PLoS One;13(1):e0185987, 2018.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Preparing the genetically modified organisms have required much time and labor, making it the rate-limiting step but CRISPR/Cas9 technology appearance has changed this difficulty. Although reports on CRISPR/Cas9 technology such as genome editing and CRISPR interference (CRISPRi) in eukaryotes increased, those in prokaryotes especially in Staphylococci were limited. Thus, its potential in the bacteriology remains unexplored. This is attributed to ecological difference between eukaryotes and prokaryotes. Here, we constructed a novel CRISPRi plasmid vector, pBACi for Staphylococcus aureus. The transformation efficiency of S. aureus was ~104 CFU/µg DNA using a vector extracted from dcm negative, which encoded one of DNA modification genes, E. coli. Further, pBACi was introduced into various clinical isolates including that not accepting the conventional temperature-sensitive vector. dcas9 in the vector was expressed throughout the growth phases of S. aureus and this vector decreased various gene mRNA expressions based on the crRNA targeting sequences and altered the knockdown strains' phenotypes. The targeted genes included various virulence and antibiotic resistant genes. Bioinformatics suggest this vector can be introduced into wide range of low-GC Gram-positive bacteria. Because this new CRISPR/Cas9-based vector can easily prepare knockdown strains, we believe the novel vector will facilitate the characterization of the function of genes from S. aureus and other Gram-positive bacteria.
[Mh] Termos MeSH primário: Técnicas de Silenciamento de Genes/métodos
Vetores Genéticos/síntese química
Staphylococcus aureus/genética
[Mh] Termos MeSH secundário: Proteínas de Bactérias/genética
Sistemas CRISPR-Cas/genética
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
Escherichia coli/genética
Edição de Genes
Inativação Gênica
Vetores Genéticos/genética
Genoma Bacteriano
Plasmídeos/genética
RNA Guia/genética
Infecções Estafilocócicas/genética
Staphylococcus aureus/patogenicidade
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Bacterial Proteins); 0 (RNA, Guide)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180226
[Lr] Data última revisão:
180226
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180130
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0185987


  4 / 1415 MEDLINE  
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[PMID]:27773522
[Au] Autor:Tamulaitis G; Venclovas C; Siksnys V
[Ad] Endereço:Institute of Biotechnology, Vilnius University, Sauletekio av. 7, Vilnius 10257, Lithuania.
[Ti] Título:Type III CRISPR-Cas Immunity: Major Differences Brushed Aside.
[So] Source:Trends Microbiol;25(1):49-61, 2017 Jan.
[Is] ISSN:1878-4380
[Cp] País de publicação:England
[La] Idioma:eng
[Ab] Resumo:For a long time the mechanism of immunity provided by the Type III CRISPR-Cas systems appeared to be inconsistent: the Type III-A Csm complex of Staphylococcus epidermidis was first reported to target DNA while Type III-B Cmr complexes were shown to target RNA. This long-standing conundrum has now been resolved by finding that the Type III CRISPR-Cas systems are both RNases and target RNA-activated DNA nucleases. The immunity is achieved by coupling binding and cleavage of RNA transcripts to the degradation of invading DNA. The base-pairing potential between the target RNA and the CRISPR RNA (crRNA) 5'-handle seems to play an important role in discriminating self and non-self nucleic acids; however, the detailed mechanism remains to be uncovered.
[Mh] Termos MeSH primário: Antibiose/genética
Sistemas CRISPR-Cas/genética
Proteínas de Ligação a DNA/genética
DNA/metabolismo
Proteínas de Ligação a RNA/genética
RNA/metabolismo
Staphylococcus epidermidis/genética
[Mh] Termos MeSH secundário: Sistemas CRISPR-Cas/fisiologia
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Nm] Nome de substância:
0 (DNA-Binding Proteins); 0 (RNA-Binding Proteins); 63231-63-0 (RNA); 9007-49-2 (DNA)
[Em] Mês de entrada:1711
[Cu] Atualização por classe:180227
[Lr] Data última revisão:
180227
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:161025
[St] Status:MEDLINE


  5 / 1415 MEDLINE  
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[PMID]:28749735
[Au] Autor:Borges AL; Davidson AR; Bondy-Denomy J
[Ad] Endereço:Department of Microbiology and Immunology, University of California, San Francisco, California 94158; email: joseph.bondy-denomy@ucsf.edu.
[Ti] Título:The Discovery, Mechanisms, and Evolutionary Impact of Anti-CRISPRs.
[So] Source:Annu Rev Virol;4(1):37-59, 2017 Sep 29.
[Is] ISSN:2327-0578
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Bacteria and archaea use CRISPR-Cas adaptive immune systems to defend themselves from infection by bacteriophages (phages). These RNA-guided nucleases are powerful weapons in the fight against foreign DNA, such as phages and plasmids, as well as a revolutionary gene editing tool. Phages are not passive bystanders in their interactions with CRISPR-Cas systems, however; recent discoveries have described phage genes that inhibit CRISPR-Cas function. More than 20 protein families, previously of unknown function, have been ascribed anti-CRISPR function. Here, we discuss how these CRISPR-Cas inhibitors were discovered and their modes of action were elucidated. We also consider the potential impact of anti-CRISPRs on bacterial and phage evolution. Finally, we speculate about the future of this field.
[Mh] Termos MeSH primário: Bactérias/genética
Bacteriófagos/genética
Bacteriófagos/fisiologia
Sistemas CRISPR-Cas
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
Evolução Molecular
[Mh] Termos MeSH secundário: Archaea/genética
Bactérias/virologia
Bacteriófagos/metabolismo
Edição de Genes
Proteínas Virais/genética
Proteínas Virais/fisiologia
[Pt] Tipo de publicação:JOURNAL ARTICLE; REVIEW
[Nm] Nome de substância:
0 (Viral Proteins)
[Em] Mês de entrada:1710
[Cu] Atualização por classe:180217
[Lr] Data última revisão:
180217
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170728
[St] Status:MEDLINE
[do] DOI:10.1146/annurev-virology-101416-041616


  6 / 1415 MEDLINE  
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[PMID]:29293625
[Au] Autor:Kell MJ; Riccio RE; Baumgartner EA; Compton ZJ; Pecorin PJ; Mitchell TA; Topczewski J; LeClair EE
[Ad] Endereço:Department of Pediatrics, Northwestern University Feinberg School of Medicine / Stanley Manne Children's Research Center, Chicago, Illinois, United States of America.
[Ti] Título:Targeted deletion of the zebrafish actin-bundling protein L-plastin (lcp1).
[So] Source:PLoS One;13(1):e0190353, 2018.
[Is] ISSN:1932-6203
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Regulation of the cytoskeleton is essential for cell migration in health and disease. Lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) is a hematopoietic-specific actin-bundling protein that is highly conserved in zebrafish, mice and humans. In addition, L-plastin expression is documented as both a genetic marker and a cellular mechanism contributing to the invasiveness of tumors and transformed cell lines. Despite L-plastin's role in both immunity and cancer, in zebrafish there are no direct studies of its function, and no mutant, knockout or reporter lines available. Using CRISPR-Cas9 genome editing, we generated null alleles of zebrafish lcp1 and examined the phenotypes of these fish throughout the life cycle. Our editing strategy used gRNA to target the second exon of lcp1, producing F0 mosaic fish that were outcrossed to wild types to confirm germline transmission. F1 heterozygotes were then sequenced to identify three unique null alleles, here called 'Charlie', 'Foxtrot' and 'Lima'. In silico, each allele truncates the endogenous protein to less than 5% normal size and removes both essential actin-binding domains (ABD1 and ABD2). Although none of the null lines express detectable LCP1 protein, homozygous mutant zebrafish (-/-) can develop and reproduce normally, a finding consistent with that of the L-plastin null mouse (LPL -/-). However, such mice do have a profound immune defect when challenged by lung bacteria. Interestingly, we observed reduced long-term survival of zebrafish lcp1 -/- homozygotes (~30% below the expected numbers) in all three of our knockout lines, with greatest mortality corresponding to the period (4-6 weeks post-fertilization) when the innate immune system is functional, but the adaptive immune system is not yet mature. This suggests that null zebrafish may have reduced capacity to combat opportunistic infections, which are more easily transmissible in the aquatic environment. Overall, our novel mutant lines establish a sound genetic model and an enhanced platform for further studies of L-plastin gene function in hematopoiesis and cancer.
[Mh] Termos MeSH primário: Deleção de Genes
Glicoproteínas de Membrana/genética
Proteínas dos Microfilamentos/genética
Proteínas de Peixe-Zebra/genética
Peixe-Zebra/genética
[Mh] Termos MeSH secundário: Alelos
Sequência de Aminoácidos
Animais
Clonagem Molecular
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas
Seres Humanos
Camundongos
Homologia de Sequência de Aminoácidos
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (Membrane Glycoproteins); 0 (Microfilament Proteins); 0 (Zebrafish Proteins); 0 (plastin)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180215
[Lr] Data última revisão:
180215
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180103
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pone.0190353


  7 / 1415 MEDLINE  
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[PMID]:29339069
[Au] Autor:Liu H; Sui T; Liu D; Liu T; Chen M; Deng J; Xu Y; Li Z
[Ad] Endereço:Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, Changchun 130062, China.
[Ti] Título:Multiple homologous genes knockout (KO) by CRISPR/Cas9 system in rabbit.
[So] Source:Gene;647:261-267, 2018 Mar 20.
[Is] ISSN:1879-0038
[Cp] País de publicação:Netherlands
[La] Idioma:eng
[Ab] Resumo:The CRISPR/Cas9 system is a highly efficient and convenient genome editing tool, which has been widely used for single or multiple gene mutation in a variety of organisms. Disruption of multiple homologous genes, which have similar DNA sequences and gene function, is required for the study of the desired phenotype. In this study, to test whether the CRISPR/Cas9 system works on the mutation of multiple homologous genes, a single guide RNA (sgRNA) targeting three fucosyltransferases encoding genes (FUT1, FUT2 and SEC1) was designed. As expected, triple gene mutation of FUT1, FUT2 and SEC1 could be achieved simultaneously via a sgRNA mediated CRISPR/Cas9 system. Besides, significantly reduced serum fucosyltransferases enzymes activity was also determined in those triple gene mutation rabbits. Thus, we provide the first evidence that multiple homologous genes knockout (KO) could be achieved efficiently by a sgRNA mediated CRISPR/Cas9 system in mammals, which could facilitate the genotype to phenotype studies of homologous genes in future.
[Mh] Termos MeSH primário: Sistemas CRISPR-Cas/genética
Mutação/genética
[Mh] Termos MeSH secundário: Sequência de Aminoácidos
Animais
Sequência de Bases
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética
Fucosiltransferases/genética
Técnicas de Inativação de Genes/métodos
Fenótipo
RNA Guia/genética
Coelhos
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Nm] Nome de substância:
0 (RNA, Guide); EC 2.4.1.- (Fucosyltransferases)
[Em] Mês de entrada:1802
[Cu] Atualização por classe:180209
[Lr] Data última revisão:
180209
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:180118
[St] Status:MEDLINE


  8 / 1415 MEDLINE  
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[PMID]:28459449
[Au] Autor:Roper J; Tammela T; Cetinbas NM; Akkad A; Roghanian A; Rickelt S; Almeqdadi M; Wu K; Oberli MA; Sánchez-Rivera FJ; Park YK; Liang X; Eng G; Taylor MS; Azimi R; Kedrin D; Neupane R; Beyaz S; Sicinska ET; Suarez Y; Yoo J; Chen L; Zukerberg L; Katajisto P; Deshpande V; Bass AJ; Tsichlis PN; Lees J; Langer R; Hynes RO; Chen J; Bhutkar A; Jacks T; Yilmaz ÖH
[Ad] Endereço:The David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, Massachusetts, USA.
[Ti] Título:In vivo genome editing and organoid transplantation models of colorectal cancer and metastasis.
[So] Source:Nat Biotechnol;35(6):569-576, 2017 06.
[Is] ISSN:1546-1696
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:In vivo interrogation of the function of genes implicated in tumorigenesis is limited by the need to generate and cross germline mutant mice. Here we describe approaches to model colorectal cancer (CRC) and metastasis, which rely on in situ gene editing and orthotopic organoid transplantation in mice without cancer-predisposing mutations. Autochthonous tumor formation is induced by CRISPR-Cas9-based editing of the Apc and Trp53 tumor suppressor genes in colon epithelial cells and by orthotopic transplantation of Apc-edited colon organoids. ApcΔ/Δ;Kras ;Trp53Δ/Δ (AKP) mouse colon organoids and human CRC organoids engraft in the distal colon and metastasize to the liver. Finally, we apply the orthotopic transplantation model to characterize the clonal dynamics of Lgr5 stem cells and demonstrate sequential activation of an oncogene in established colon adenomas. These experimental systems enable rapid in vivo characterization of cancer-associated genes and reproduce the entire spectrum of tumor progression and metastasis.
[Mh] Termos MeSH primário: Neoplasias Colorretais/genética
Modelos Animais de Doenças
Edição de Genes/métodos
Genes Neoplásicos/genética
Neoplasias Hepáticas/genética
Neoplasias Hepáticas/secundário
Transplante de Órgãos/métodos
[Mh] Termos MeSH secundário: Animais
Carcinogênese/genética
Linhagem Celular Tumoral
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética
Feminino
Masculino
Camundongos
Camundongos Transgênicos
Metástase Neoplásica
[Pt] Tipo de publicação:JOURNAL ARTICLE
[Em] Mês de entrada:1708
[Cu] Atualização por classe:180207
[Lr] Data última revisão:
180207
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170502
[St] Status:MEDLINE
[do] DOI:10.1038/nbt.3836


  9 / 1415 MEDLINE  
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[PMID]:29253874
[Au] Autor:Martynov A; Severinov K; Ispolatov I
[Ad] Endereço:Center for Data-Intensive Biomedicine and Biotechnology, Skolkovo Institute of Science and Technology, Moscow, Russia.
[Ti] Título:Optimal number of spacers in CRISPR arrays.
[So] Source:PLoS Comput Biol;13(12):e1005891, 2017 12.
[Is] ISSN:1553-7358
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:Prokaryotic organisms survive under constant pressure of viruses. CRISPR-Cas system provides its prokaryotic host with an adaptive immune defense against viruses that have been previously encountered. It consists of two components: Cas-proteins that cleave the foreign DNA and CRISPR array that suits as a virus recognition key. CRISPR array consists of a series of spacers, short pieces of DNA that originate from and match the corresponding parts of viral DNA called protospacers. Here we estimate the number of spacers in a CRISPR array of a prokaryotic cell which maximizes its protection against a viral attack. The optimality follows from a competition between two trends: too few distinct spacers make host vulnerable to an attack by a virus with mutated corresponding protospacers, while an excessive variety of spacers dilutes the number of the CRISPR complexes armed with the most recent and thus most useful spacers. We first evaluate the optimal number of spacers in a simple scenario of an infection by a single viral species and later consider a more general case of multiple viral species. We find that depending on such parameters as the concentration of CRISPR-Cas interference complexes and its preference to arm with more recently acquired spacers, the rate of viral mutation, and the number of viral species, the predicted optimal number of spacers lies within a range that agrees with experimentally-observed values.
[Mh] Termos MeSH primário: Sistemas CRISPR-Cas
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética
[Mh] Termos MeSH secundário: Imunidade Adaptativa/genética
Archaea/genética
Archaea/imunologia
Archaea/virologia
Bactérias/genética
Bactérias/imunologia
Bactérias/virologia
Biologia Computacional
Simulação por Computador
DNA Intergênico/genética
DNA Viral/genética
Modelos Genéticos
Modelos Imunológicos
Mutação
Células Procarióticas/imunologia
Células Procarióticas/virologia
[Pt] Tipo de publicação:JOURNAL ARTICLE; RESEARCH SUPPORT, NON-U.S. GOV'T
[Nm] Nome de substância:
0 (DNA, Intergenic); 0 (DNA, Viral)
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180123
[Lr] Data última revisão:
180123
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:171219
[St] Status:MEDLINE
[do] DOI:10.1371/journal.pcbi.1005891


  10 / 1415 MEDLINE  
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[PMID]:28745625
[Au] Autor:Merenda A; Andersson-Rolf A; Mustata RC; Li T; Kim H; Koo BK
[Ad] Endereço:Wellcome Trust - Medical Research Council Stem Cell Institute, University of Cambridge; Department of Genetics, University of Cambridge.
[Ti] Título:A Protocol for Multiple Gene Knockout in Mouse Small Intestinal Organoids Using a CRISPR-concatemer.
[So] Source:J Vis Exp;(125), 2017 Jul 12.
[Is] ISSN:1940-087X
[Cp] País de publicação:United States
[La] Idioma:eng
[Ab] Resumo:CRISPR/Cas9 technology has greatly improved the feasibility and speed of loss-of-function studies that are essential in understanding gene function. In higher eukaryotes, paralogous genes can mask a potential phenotype by compensating the loss of a gene, thus limiting the information that can be obtained from genetic studies relying on single gene knockouts. We have developed a novel, rapid cloning method for guide RNA (gRNA) concatemers in order to create multi-gene knockouts following a single round of transfection in mouse small intestinal organoids. Our strategy allows for the concatemerization of up to four individual gRNAs into a single vector by performing a single Golden Gate shuffling reaction with annealed gRNA oligos and a pre-designed retroviral vector. This allows either the simultaneous knockout of up to four different genes, or increased knockout efficiency following the targeting of one gene by multiple gRNAs. In this protocol, we show in detail how to efficiently clone multiple gRNAs into the retroviral CRISPR-concatemer vector and how to achieve highly efficient electroporation in intestinal organoids. As an example, we show that simultaneous knockout of two pairs of genes encoding negative regulators of the Wnt signaling pathway (Axin1/2 and Rnf43/Znrf3) renders intestinal organoids resistant to the withdrawal of key growth factors.
[Mh] Termos MeSH primário: Sistemas CRISPR-Cas/genética
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética
Técnicas de Inativação de Genes/métodos
[Mh] Termos MeSH secundário: Animais
Camundongos
Camundongos Knockout
Organoides
Transfecção
[Pt] Tipo de publicação:JOURNAL ARTICLE; VIDEO-AUDIO MEDIA
[Em] Mês de entrada:1801
[Cu] Atualização por classe:180119
[Lr] Data última revisão:
180119
[Sb] Subgrupo de revista:IM
[Da] Data de entrada para processamento:170727
[St] Status:MEDLINE
[do] DOI:10.3791/55916



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