CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.
Mol Cell
; 67(6): 1068-1079.e4, 2017 Sep 21.
Article
en En
| MEDLINE
| ID: mdl-28890334
ABSTRACT
Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.
Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
Codón de Terminación
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Silenciador del Gen
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Proteínas Asociadas a CRISPR
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas
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Sistemas CRISPR-Cas
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Edición Génica
Tipo de estudio:
Prognostic_studies
Límite:
Animals
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Humans
Idioma:
En
Año:
2017
Tipo del documento:
Article