title: Improved genome editing in human cell lines using the CRISPR method.
abstract: The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.
authors.affiliation: Medical Research Council Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, Scotland.
authors.forename: Ivan M
authors.initials: IM
authors.lastname: Munoz
authors.affiliation: Medical Research Council Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, Scotland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Szyniarowski
authors.affiliation: Medical Research Council Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, Scotland.
authors.forename: Rachel
authors.initials: R
authors.lastname: Toth
authors.affiliation: Medical Research Council Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, Scotland.
authors.forename: John
authors.initials: J
authors.lastname: Rouse
authors.affiliation: Medical Research Council Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee, Scotland.
authors.forename: Christophe
authors.initials: C
authors.lastname: Lachaud
mesh_terms: D064130:CRISPR-Associated Proteins / Q000235:genetics; D002460:Cell Line; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D016678:Genome; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems