The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations

Repair of double-stranded breaks generated by CRISPR/Cas9 is highly dependent on the flanking DNA sequence. To learn about interactions between DNA repair and target sequence, we measure frequencies of over 236,000 distinct Cas9-generated mutational outcomes at over 2800 synthetic target sequences in 18 DNA repair deficient mouse embryonic stem cells lines. We classify the outcomes in an unbiased way, finding a specialised role for Prkdc (DNA-PKcs protein) and Polm in creating 1 bp insertions matching the nucleotide on the protospacer-adjacent motif side of the break, a variable involvement of Nbn and Polq in the creation of different deletion outcomes, and uni-directional deletions dependent on both end-protection and end-resection. Using our dataset, we build predictive models of the mutagenic outcomes of Cas9 scission that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of Cas9-generated mutations.

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Publication Details

Journal
Nature Communications
Published
2024-11-26
DOI
https://doi.org/10.1038/s41467-024-54566-7
Citations
12
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
2.81

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article

The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations

Núria Muñoz-Subirana, Ananth Pallaseni, Joost Schimmel, Michael Kosicki et al.
12 citations
Nature Communications
CRISPR and Genetic Engineering
2.81
article

The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations

Núria Muñoz-Subirana, Ananth Pallaseni, Joost Schimmel, Michael Kosicki, Marcel Tijsterman, Leopold Parts, Luca Crepaldi, Özdemirhan Serçin, Elin Madli Peets, Gareth Girling, Hedi Peterson, Ivan Kuzmin, Balca R. Mardin, Marilin Moor
article en
12 citations

Abstract

Repair of double-stranded breaks generated by CRISPR/Cas9 is highly dependent on the flanking DNA sequence. To learn about interactions between DNA repair and target sequence, we measure frequencies of over 236,000 distinct Cas9-generated mutational outcomes at over 2800 synthetic target sequences in 18 DNA repair deficient mouse embryonic stem cells lines. We classify the outcomes in an unbiased way, finding a specialised role for Prkdc (DNA-PKcs protein) and Polm in creating 1 bp insertions matching the nucleotide on the protospacer-adjacent motif side of the break, a variable involvement of Nbn and Polq in the creation of different deletion outcomes, and uni-directional deletions dependent on both end-protection and end-resection. Using our dataset, we build predictive models of the mutagenic outcomes of Cas9 scission that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of Cas9-generated mutations.

Nature CommunicationsVol. 15(1)
Leiden University (NL), Lawrence Berkeley National Laboratory (US), Merck KGaA, Darmstadt (Germany) (DE), University of Cambridge (GB), Leiden University Medical Center (NL), Wellcome Sanger Institute (GB), BioMed X Institute (DE), University of Tartu (EE)
Wellcome, Eesti Teadusagentuur, KWF Kankerbestrijding, National Institutes of Health
Openalex Percentile: Top 9%
CRISPR and Genetic Engineering
2.81
DOIOpen Access PDFOpenAlex
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