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Updated: Jun 6, 2025
Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
Published on: March 31, 2022
The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations
Ananth Pallaseni1, Elin Madli Peets1, Gareth Girling1
1Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, UK.
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.
Insights
CRISPR/Cas9 DNA repair outcomes depend on flanking sequences. This study reveals specific gene roles in mutation generation, enabling more precise Cas9 editing predictions.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- CRISPR/Cas9 gene editing introduces double-stranded DNA breaks.
- DNA repair mechanisms dictate the mutational outcomes at the break site.
- Flanking DNA sequences significantly influence the choice of repair pathway.
Purpose of the Study:
- To comprehensively analyze Cas9-induced mutational outcomes.
- To elucidate the roles of specific DNA repair genes in outcome determination.
- To develop predictive models for Cas9-generated mutations.
Main Methods:
- Systematic measurement of over 236,000 mutational outcomes across 2800 synthetic target sequences.
- Utilizing 18 DNA repair-deficient mouse embryonic stem cell lines.
- Unbiased classification of mutation types and analysis of gene involvement.
Main Results:
- Identified specific roles for Prkdc (DNA-PKcs) and Polm in 1 bp insertions.
- Demonstrated variable involvement of Nbn and Polq in deletion formation.
- Characterized uni-directional deletions influenced by end-protection and end-resection processes.
Conclusions:
- DNA repair gene function significantly impacts CRISPR/Cas9 editing outcomes.
- Developed predictive models that surpass current standards for mutation prediction.
- Provides a foundation for enhanced precision in modulating Cas9-induced mutations.
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