![]() ![]() Our strategy makes it possible to generate refined DNA mutations for improved safety without sacrificing efficiency of genome editing. 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. Counteracting DNA resection was one of the mechanisms perturbing deletion sizes. In addition, templated insertions (the insertion of the nucleotide 4 nt upstream of the protospacer adjacent motif) were increased relative to other insertions. ![]() Importantly, doing so also greatly decreases the generation of long deletions, including those >2 kb. 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. ![]() Methods for generating refined mutations are desirable but currently unavailable. 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. Most insertions or deletions generated by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) endonucleases are short (500 bp) can be observed. ![]()
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