CRISPR-Cas9^D10A nickase-based genotypic and phenotypic screening to enhance genome editing
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Chiang, T. W., le, S. C., Larrieu, D., Demir, M., & Jackson, S. (2016). CRISPR-Cas9^D10A nickase-based genotypic and phenotypic screening to enhance genome editing. Scientific Reports, 6 (24356)https://doi.org/10.1038/srep24356
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 “doublenicking” 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 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.
CRISPR-Cas9, nickase, genotypic screening, phenotypic screening, DNA damage response
We thank all members of the S.P.J. laboratory for helpful discussions. We are especially grateful to Y. Galanty, R. Belotserkovskaya and J. Forment for valuable ideas and suggestions, and for critically reading the manuscript, and K. Dry for editorial assistance. In addition, we thank A. Riddell (Flow Cytometry Core Facility at the University of Cambridge Stem Cell Institute) for flow cytometry cell sorting support, and Roger Grand (University of Birmingham, Birmingham, UK) for the gift of HEK293FT cells. The Jackson laboratory is funded by Cancer Research UK program grant C6/A18796 and the European Research Council. Core infrastructure funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, UK, supplemented by CRUK. T-W.C. is supported by a Cambridge International Scholarship. C.S. and M.D. are funded by ERC Advanced Researcher Grant DDREAM. D.L is funded by a Project Grant from the Medical Research Council, UK MR/L019116/1.
Cancer Research UK (A18796)
Cancer Research UK (11224)
Wellcome Trust (092096/Z/10/Z)
Cancer Research UK (A14492)
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External DOI: https://doi.org/10.1038/srep24356
This record's URL: https://www.repository.cam.ac.uk/handle/1810/254946