iScienceNov 17, 2025

Improving precise gene editing in mice using RAD52 and modified DNA templates

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jun 27, 2026

Abstract

Essence

Specific donor-template and crRNA choices improved the efficiency and precision of CRISPR-Cas9 HDR knock-in mouse generation.

Evidence

This mouse zygote genome-editing optimization experiment injected CRISPR-Cas9 components into over 2,000 zygotes and evaluated 270 founders.

Caveat

The results are platform- and target-context optimization data, and RAD52 increased ssDNA integration while also increasing template multiplication.

Simplified

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, 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 precision compared to other strategies. Importantly, modifying donor DNA 5' ends substantially boosted efficiency: 5'-biotin increased single-copy integration up to 8 fold, while 5'-C3 spacer modification produced up to a 20-fold rise in correctly edited mice, regardless of donor strandness. These findings identify practical approaches to enhance HDR efficiency and precision in CRISPR-Cas9-mediated knock-in model generation. Nup93