CRISPR-Cas9-assisted genome editing in E. coli elevates the frequency of unintended mutations.

ABSTRACT: Cas-assisted lambda Red recombineering techniques have rapidly become a mainstay of bacterial genome editing. Such techniques have been used to construct both individual mutants and massive libraries to assess the effects of genomic changes. We have found that a commonly used Cas9-assisted editing method results in unintended mutations elsewhere in the genome in 26% of edited clones. The unintended mutations are frequently found over 200 kb from the intended edit site and even over 10 kb from potential off-target sites. We attribute the high frequency of unintended mutations to error-prone polymerases expressed in response to dsDNA breaks introduced at the edit site. Most unintended mutations occur in regulatory or coding regions and thus may have phenotypic effects. Our findings highlight

SUBMITTER: Widney KA 

PROVIDER: S-EPMC10983943 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature