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

Ananth Pallaseni, Elin Madli Peets, Gareth Girling, Luca Crepaldi, Ivan Kuzmin, Marilin Moor, Núria Muñoz-Subirana, Joost Schimmel, Özdemirhan Serçin, Balca R. Mardin, Marcel Tijsterman, Hedi Peterson, Michael Kosicki () and Leopold Parts ()
Additional contact information
Ananth Pallaseni: Wellcome Genome Campus
Elin Madli Peets: Wellcome Genome Campus
Gareth Girling: Wellcome Genome Campus
Luca Crepaldi: Wellcome Genome Campus
Ivan Kuzmin: University of Tartu
Marilin Moor: University of Tartu
Núria Muñoz-Subirana: Leiden University Medical Center
Joost Schimmel: Leiden University Medical Center
Özdemirhan Serçin: BioMed X Institute (GmbH)
Balca R. Mardin: BioMed X Institute (GmbH)
Marcel Tijsterman: Leiden University Medical Center
Hedi Peterson: University of Tartu
Michael Kosicki: University of Cambridge
Leopold Parts: Wellcome Genome Campus

Nature Communications, 2024, vol. 15, issue 1, 1-14

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-54566-7

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