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

Skryabin BV, Braun DA, Kaiser H, Gubar L, Seeger B, Khanam T, Stegemann A, Pavenstädt H, Rozhdestvensky TS.

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 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.