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.
Keywords: CRISPR; genome engineering; human stem cells.
Copyright © 2014 John Wiley & Sons, Inc.