CRISPR-Cas9 HDR optimization: RAD52, denatured, and 5'-modified DNA templates in knock-in mice generation.

ABSTRACT: CRISPR-Cas9-mediated genome editing is a powerful method for generating animal disease models, but efficiency of homology-directed repair (HDR) remains a major challenge. To generate conditional knockout (cKO) mouse model of Nup93, we tested strategies to improve HDR precision by injecting CRISPR-Cas9 components into over 2,000 zygotes, producing 270 founders. Our experiments revealed several critical factors. Denaturation of long 5'-monophosphorylated double-stranded DNA (dsDNA) templates enhanced precise editing and reduced unwanted template multiplications. Supplementation with RAD52 increased single-stranded DNA (ssDNA) integration nearly 4 fold, though accompanied by a higher template multiplication. Targeting the antisense strand with two CRISPR RNAs (crRNAs) improved HDR prec

SUBMITTER: Skryabin BV 

PROVIDER: S-EPMC12616026 | biostudies-literature | 2025 Nov

REPOSITORIES: biostudies-literature