Single-stranded HDR templates with truncated Cas12a-binding sequences improve knock-in efficiencies in primary human T cells.

Fiche publication

Date publication

juin 2025

Journal

Molecular therapy. Nucleic acids

Auteurs

Membres identifiés du Cancéropôle Est :
Dr CULLOT Grégoire

Tous les auteurs :
Nitulescu AM, Du W, Glaser V, Kath J, Aird EJ, Cullot G, Greensmith R, Mikkelsen NS, Stein M, Bak RO, Kaminski M, Corn JE, Wagner DL

Lien Pubmed

https://www.ncbi.nlm.nih.gov/pubmed/40520364

Résumé

CRISPR-Cas12a gene editing offers an alternative to Cas9-based methods, providing better targeting of AT-rich regions, simplified guide RNA manufacturing, and high specificity. However, the efficacy of donor-based editing is subject to various factors, with template format playing a crucial role. Currently, the predominant non-viral template format for homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA, which is toxic when transfected at high doses. Others have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a template for Cas9-mediated gene editing can mitigate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for AsCas12a Ultra by exploring PAM orientation and binding requirements. Additionally, we rule out ssDNase activity of AsCas12a under cell-physiological Mg conditions. Finally, we showcase the advantage of ssDNA donors with CTS (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three TCR-CD3 complex genes (, ), achieving up to 90% knock-in rates for a 0.8kb-insert at the locus. Long-read sequencing confirmed higher HDR rates and revealed that CTS reduced partial integration events compared to unmodified ssDNA. Overall, AsCas12a and ssCTS represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.