title: CRISPR/Cas9-Directed Genome Editing of Cultured Cells.
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.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts.
authors.forename: Luhan
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts.|Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts.
authors.forename: Joyce L
authors.initials: JL
authors.lastname: Yang
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts.
authors.forename: Susan
authors.initials: S
authors.lastname: Byrne
authors.affiliation: Biological and Biomedical Sciences Program, Harvard Medical School, Boston, Massachusetts.
authors.forename: Joshua
authors.initials: J
authors.lastname: Pan
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection / Q000379:methods
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: CRISPR; genome engineering; human stem cells