Journal
NUCLEIC ACIDS RESEARCH
Volume 50, Issue 7, Pages 3944-3957Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkac186
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Funding
- U.S. Department of Defense [W81XWH2010265]
- North Carolina General Assembly [30053]
- State of North Carolina
- U.S. Department of Defense (DOD) [W81XWH2010265] Funding Source: U.S. Department of Defense (DOD)
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Fusing Escherichia coli DNA polymerase I or the Klenow fragment to Cas9 increases the frequencies of 1-bp deletions and decreases >1-bp deletions or insertions. This strategy also greatly reduces the generation of long deletions (>2 kb) and increases templated insertions. Targeting DNA polymerase to double-strand breaks does not increase off-targets or base substitution rates, but improves editing efficiency in primary cells.
Most insertions or deletions generated by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) endonucleases are short (<25 bp), but unpredictable on-target long DNA deletions (>500 bp) can be observed. The possibility of generating long on-target DNA deletions poses safety risks to somatic genome editing and makes the outcomes of genome editing less predictable. Methods for generating refined mutations are desirable but currently unavailable. Here, we show that fusing Escherichia coli DNA polymerase I or the Klenow fragment to Cas9 greatly increases the frequencies of 1-bp deletions and decreases >1-bp deletions or insertions. Importantly, doing so also greatly decreases the generation of long deletions, including those >2 kb. In addition, templated insertions (the insertion of the nucleotide 4 nt upstream of the protospacer adjacent motif) were increased relative to other insertions. Counteracting DNA resection was one of the mechanisms perturbing deletion sizes. Targeting DNA polymerase to double-strand breaks did not increase off-targets or base substitution rates around the cleavage sites, yet increased editing efficiency in primary cells. Our strategy makes it possible to generate refined DNA mutations for improved safety without sacrificing efficiency of genome editing.
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