CRISPR/Cas9-Directed Genome Editing of Cultured Cells.
Curr Protoc Mol Biol
; 107: 31.1.1-31.1.17, 2014 Jul 01.
Article
em En
| MEDLINE
| ID: mdl-24984853
ABSTRACT
Human genome engineering has been transformed by the introduction of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system found in most bacteria and archaea. Type II CRISPR/Cas systems have been engineered to induce RNA-guided genome editing in human cells, where small RNAs function together with Cas9 nucleases for sequence-specific cleavage of target sequences. Here we describe the protocol for Cas9-mediated human genome engineering, including construct building and transfection methods necessary for delivering Cas9 and guide RNA (gRNA) into human-induced pluripotent stem cells (hiPSCs) and HEK293 cells. Following genome editing, we also describe methods to assess genome editing efficiency using next-generation sequencing and isolate monoclonal hiPSCs with the desired modifications for downstream applications.
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Base de dados:
MEDLINE
Assunto principal:
Genoma Humano
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Células-Tronco Pluripotentes Induzidas
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Sistemas CRISPR-Cas
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article