Single-Stranded Hdr Templates With Truncated cas12a-Binding Sequences Improve Knock-in Efficiencies in Primary Human T Cells

Authors

• Ana-Maria Nitulescu
• Weijie Du
• Viktor Glaser
• Jonas Kath
• Eric J Aird
• Grégoire Cullot
• Robert Greensmith
• Nanna Steengaard Mikkelsen
• Maik Stein
• Rasmus O Bak
• Michael Kaminski
• Jacob E Corn
• Dimitrios L Wagner

Language

English

Publication Date

6-10-2025

Journal

Molecular Therapy Nucleic Acids

DOI

10.1016/j.omtn.2025.102568

PMID

40520364

PMCID

PMC12166421

PubMedCentral® Posted Date

5-19-2025

PubMedCentral® Full Text Version

Post-print

Abstract

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 Mg2+ 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 (TRAC, CD3ζ, CD3ε), achieving up to 90% knock-in rates for a 0.8kb-insert at the CD3ε 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.

Keywords

MT: RNA/DNA Editing, gene editing, CRISPR-Cas12a, non-viral, electroporation, ssDNA, HDR, CAR-T cells, Cas-target-sequences, AsCas12a

Published Open-Access

yes

Recommended Citation

Nitulescu, Ana-Maria; Du, Weijie; Glaser, Viktor; et al., "Single-Stranded Hdr Templates With Truncated cas12a-Binding Sequences Improve Knock-in Efficiencies in Primary Human T Cells" (2025). Faculty, Staff and Students Publications. 8697.
https://digitalcommons.library.tmc.edu/baylor_docs/8697