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

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.