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May 7th, 2025
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Medical Faculty, Core Facility Transgenic animal and genetic engineering Models (TRAM), University of Muenster
molecular biology
biorxiv

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

Skryabin, B. V.Open in Google Scholar•Braun, D. A.Open in Google Scholar•Kaiser, H.Open in Google Scholar•Gubar, L.Open in Google Scholar•Seeger, B.Open in Google Scholar•Khanam, T.Open in Google Scholar•Stegemann, A.Open in Google Scholar•Pavenstädt, H.Open in Google Scholar•Rozhdestvensky, T. S.Open in Google Scholar

CRISPR/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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