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Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes

Author

Listed:
  • C. D. Richardson

    (Innovative Genomics Initiative, University of California
    University of California)

  • G. J. Ray

    (Innovative Genomics Initiative, University of California
    University of California)

  • N. L. Bray

    (Innovative Genomics Initiative, University of California
    University of California)

  • J. E. Corn

    (Innovative Genomics Initiative, University of California
    University of California)

Abstract

The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9–sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated gene disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous gene disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced gene disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of gene disruption.

Suggested Citation

  • C. D. Richardson & G. J. Ray & N. L. Bray & J. E. Corn, 2016. "Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes," Nature Communications, Nature, vol. 7(1), pages 1-7, November.
    • Handle: RePEc:nat:natcom:v:7:y:2016:i:1:d:10.1038_ncomms12463
    DOI: 10.1038/ncomms12463
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    Cited by:

    1. Stephan Riesenberg & Nelly Helmbrecht & Philipp Kanis & Tomislav Maricic & Svante Pääbo, 2022. "Improved gRNA secondary structures allow editing of target sites resistant to CRISPR-Cas9 cleavage," Nature Communications, Nature, vol. 13(1), pages 1-8, December.
    2. Martin Peterka & Nina Akrap & Songyuan Li & Sandra Wimberger & Pei-Pei Hsieh & Dmitrii Degtev & Burcu Bestas & Jack Barr & Stijn Plassche & Patricia Mendoza-Garcia & Saša Šviković & Grzegorz Sienski &, 2022. "Harnessing DSB repair to promote efficient homology-dependent and -independent prime editing," Nature Communications, Nature, vol. 13(1), pages 1-9, December.
    3. Michael Kosicki & Felicity Allen & Frances Steward & Kärt Tomberg & Yangyang Pan & Allan Bradley, 2022. "Cas9-induced large deletions and small indels are controlled in a convergent fashion," Nature Communications, Nature, vol. 13(1), pages 1-11, December.

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