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

, 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.