Identifying optimal conditions for precise knock-in of exogenous DNA into the zebrafish genome.

ABSTRACT: CRISPR nucleases can be used to insert exogenous DNA into the zebrafish genome by homology-dependent repair (HDR), although germline transmission rates for precise edits remain quite low. Comparative studies to optimize HDR parameters for introducing base pair changes using short-read deep sequencing have been successful, but similar analysis for insertions is challenging due to read-length constraints. Here, we quantified editing outcomes using long-read sequencing to identify optimal template and CRISPR parameters for precise targeted insertion in zebrafish. Through side-by-side comparisons, we found that chemically modified templates out-perform those released in vivo from a plasmid, while Cas9 and Cas12a nucleases performed similarly for targeted insertion. Consistent with previous stu

SUBMITTER: Oikemus S 

PROVIDER: S-EPMC12211562 | biostudies-literature | 2025 Jun

REPOSITORIES: biostudies-literature