Improved cloning-free one-step CRISPR-Cas12a-assisted tagging of mammalian genes using PCR generated reagents (PCR tagging)

Lou, D.; Kirrmaier, D.; Krause, M.; Li, S.; Gubicza, K.; Herbst, K.; Meurer, M.; Yerlici, T. V.; Wagih, O. M.; Steinmetz, L. M.; Knop, M.

doi:10.64898/2025.12.02.690677

bioRxiv · 10.64898/2025.12.02.690677

Improved cloning-free one-step CRISPR-Cas12a-assisted tagging of mammalian genes using PCR generated reagents (PCR tagging)

Lou, D.·Kirrmaier, D.·Krause, M.·Li, S.·Gubicza, K.·Herbst, K.·Meurer, M.·Yerlici, T. V.·Wagih, O. M.·Steinmetz, L. M.·Knop, M.

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Abstract

Precise DNA integration in mammalian genomes using CRISPR/Cas based strategies remains challenging due to competing DNA repair pathways. We present an update for our CRISPR/Cas12a-assisted PCR-tagging method using linear donor cassettes and self-targeting gRNAs for gene tagging. By incorporating 2A-linked selection markers and chemically modifying PCR cassettes with phosphorothioate and biotin, along with the inhibition of NHEJ, we improve precise insertions while reducing unwanted integration products and we devise a variant of the method for seamless gene tagging. To profile integration outcomes, we further developed our integration site sequencing protocol (Tn5-Anchor-Seq, Meurer et al., 2018) to obtain a protocol using long-read (ONT) sequencing coupled with a custom computational pipeline. This enables high-resolution quantification of all integration events with respect to integration by HDR or NHEJ, as well as the presence of concatemers and off-target integrations. Our data reveal gene-specific editing profiles and demonstrate that combining cassette design with DNA repair modulation yields up to 5-fold increases in HDR and suppression of concatemers and indels. Together, our improvements outline an robust strategy for efficient and high-fidelity gene tagging in mammalian cells, facilitating functional genomics and cell engineering applications.

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Lou, D., Kirrmaier, D., Krause, M., Li, S., Gubicza, K., Herbst, K., Meurer, M., Yerlici, T. V., Wagih, O. M., Steinmetz, L. M., Knop, M.. 2025-12-02. Improved cloning-free one-step CRISPR-Cas12a-assisted tagging of mammalian genes using PCR generated reagents (PCR tagging). https://doi.org/10.64898/2025.12.02.690677

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