Yang L, Yang JL, Byrne S, Pan J, Church GM (2014) CRISPR/Cas9-Directed Genome Editing UNIT 31.1 of Cultured Cells. Current Protocols in Molecular Biology 31.1.1-31.1.17. DOI: 10.1002/0471142727.mb3101s107
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. This study describes 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, it also describes 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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