CRISPR-Cas9 HDR Optimization: RAD52, Denatured and 5'-Modified DNA Templates in Knock-In Mice Generation

Skryabin, B. V.; Braun, D. A.; Kaiser, H.; Gubar, L.; Seeger, B.; Khanam, T.; Stegemann, A.; Pavenstädt, H.; Rozhdestvensky, T. S.

doi:10.1101/2025.05.05.652162

bioRxiv · 10.1101/2025.05.05.652162

CRISPR-Cas9 HDR Optimization: RAD52, Denatured and 5'-Modified DNA Templates in Knock-In Mice Generation

Skryabin, B. V.·Braun, D. A.·Kaiser, H.·Gubar, L.·Seeger, B.·Khanam, T.·Stegemann, A.·Pavenstädt, H.·Rozhdestvensky, T. S.

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Abstract

AbstractCRISPR/Cas9-mediated genome editing is a powerful tool for producing animal models of human diseases. However, it often encounters challenges related to low efficiency of donor DNA templates insertion through homology-directed repair (HDR) pathway or unwanted insertions and/or multiplications. Here, we present findings from multiple targeting experiments aimed at generating a Nup93 conditional knockout (cKO) mouse model. Injection of CRISPR/Cas9 components into over two thousand zygotes, resulted in 270 founder animals. Our study revealed various obstacles associated with the use of single-stranded (ssDNA) and double-stranded DNA (dsDNA) templates during cKO generation, highlighting the critical role of denaturation of long 5-monophosphorylated dsDNA templates in enhancing precise genome editing and reducing template multiplications. Application of RAD52 protein increased HDR efficiency of ssDNA integration almost 4-fold, albeit with an associated increase in template multiplication. Targeting the antisense strand of DNA using two crRNAs demonstrated better efficacy in HDR-mediated precise genome editing when compared to targeting the sense or sense-antisense strands. In addition, the application of 5-end biotin-modified donor DNA resulted in up to a 8-fold increase in HDR-mediated single-copy template integration compered to unmodified dsDNA donor. Furthermore, application of 5-end C3 spacer modified template resulted in up to a 20-fold increase in correctly HDR modified mice independent from ssDNA or dsDNA template employment. This study underscores potential pitfalls in CRISPR/Cas9-mediated genome editing and offers simple practical solutions to refine this potent tool. These findings highlight various strategies to enhance CRISPR/Cas9 HDR efficiency, providing a framework for improving precision in the generation of conditional knockout models.

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Skryabin, B. V., Braun, D. A., Kaiser, H., Gubar, L., Seeger, B., Khanam, T., Stegemann, A., Pavenstädt, H., Rozhdestvensky, T. S.. 2025-05-07. CRISPR-Cas9 HDR Optimization: RAD52, Denatured and 5'-Modified DNA Templates in Knock-In Mice Generation. https://doi.org/10.1101/2025.05.05.652162

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