title: Genome Editing in Human Cells Using CRISPR/Cas Nucleases.
abstract: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. This unit describes protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA-guided FokI nucleases.
authors.affiliation: Molecular Pathology Unit, Center for Computational and Integrative Biology and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Wyvekens
authors.affiliation: Molecular Pathology Unit, Center for Computational and Integrative Biology and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts.|Department of Pathology, Harvard Medical School, Boston, Massachusetts.
authors.forename: Shengdar Q
authors.initials: SQ
authors.lastname: Tsai
authors.affiliation: Molecular Pathology Unit, Center for Computational and Integrative Biology and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts.|Department of Pathology, Harvard Medical School, Boston, Massachusetts.
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
mesh_terms: D064113:CRISPR-Cas Systems; D002460:Cell Line; D003584:Cytological Techniques / Q000379:methods*; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D011995:Recombination, Genetic
keywords: CRISPR; Cas9; FokI-dCas9; RNA-guided FokI nucleases; genome editing; human cells; tru-gRNA