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

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<sup>2+</sup> 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 (<i>TRAC</i>, <i>CD3ζ, CD3ε</i>), achieving up to 90% knock-in rates for a 0.8kb-insert at the <i>CD3ε</i> 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.

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Publication Details

Journal
Molecular Therapy — Nucleic Acids
Published
2025-05-19
DOI
https://doi.org/10.1016/j.omtn.2025.102568
Citations
5
Primary Topic
CRISPR and Genetic Engineering
Type
article
Field-Weighted Citation Impact
4.03

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

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5 citations
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article

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

Dimitrios L. Wagner, Robert Greensmith, Eric J. Aird, Grégoire Cullot, Ana-Maria Nitulescu, Maik Stein, Rasmus Otkjær Bak, Nanna S. Mikkelsen, Michael M. Kaminski, Viktor Glaser, Jonas Kath, Weijie Du, Jacob E. Corn
article en
5 citations

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 Mg<sup>2+</sup> 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 (<i>TRAC</i>, <i>CD3ζ, CD3ε</i>), achieving up to 90% knock-in rates for a 0.8kb-insert at the <i>CD3ε</i> 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.

Molecular Therapy — Nucleic AcidsVol. 36(2)
Baylor College of Medicine (US), Aarhus University (DK), Max Delbrück Center (DE), ETH Zurich (CH), Humboldt-Universität zu Berlin (DE), Berlin Institute of Health at Charité - Universitätsmedizin Berlin (DE), Charité - Universitätsmedizin Berlin (DE)
National Science Foundation, University of Chicago, Lotte und Adolf Hotz-Sprenger Stiftung, European Commission, Deutsche Forschungsgemeinschaft, Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung, Charité – Universitätsmedizin Berlin, NOMIS Stiftung, Berlin Institute of Health, Horizon 2020 Framework Programme, HORIZON EUROPE Framework Programme, European Research Council, H2020 European Research Council
Zero hunger
Openalex Percentile: Top 6%
CRISPR and Genetic Engineering
4.03
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