title: CRISPR-Cas9(D10A) nickase-based genotypic and phenotypic screening to enhance genome editing.
abstract: The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system's specificity. Recently a "double-nicking" strategy using catalytic mutant Cas9(D10A) nickase has been developed to minimise off-target effects. Here, we describe a Cas9(D10A)-based screening approach that combines an All-in-One Cas9(D10A) nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities.
authors.affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK.|Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.
authors.forename: Ting-Wei Will
authors.initials: TW
authors.lastname: Chiang
authors.affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK.
authors.forename: Carlos
authors.initials: C
authors.lastname: le Sage
authors.affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK.
authors.forename: Delphine
authors.initials: D
authors.lastname: Larrieu
authors.affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK.
authors.forename: Mukerrem
authors.initials: M
authors.lastname: Demir
authors.affiliation: Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, UK.|Department of Biochemistry, University of Cambridge, Cambridge CB2 1GA, UK.|The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Jackson
mesh_terms: D000483:Alleles; D001483:Base Sequence; D064113:CRISPR-Cas Systems; D002460:Cell Line; D003850:Deoxyribonuclease I / Q000235:genetics* / Q000378:metabolism*; D055808:Drug Discovery; D000072669:Gene Editing; D055786:Gene Knockout Techniques; D023061:Gene Order; D018390:Gene Targeting; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D023281:Genomics / Q000379:methods; D005838:Genotype; D057166:High-Throughput Screening Assays; D006801:Humans; D016296:Mutagenesis; D010641:Phenotype; D000094704:RNA, Guide, CRISPR-Cas Systems; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D042461:Vascular Endothelial Growth Factor A; D003850:Deoxyribonuclease I