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
{"title":"截断cas12a结合序列的单链HDR模板提高了原代人T细胞的敲入效率。","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","doi":"10.1016/j.omtn.2025.102568","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":18821,"journal":{"name":"Molecular Therapy. 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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.</p>\",\"PeriodicalId\":18821,\"journal\":{\"name\":\"Molecular Therapy. 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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 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.
期刊介绍:
Molecular Therapy Nucleic Acids is an international, open-access journal that publishes high-quality research in nucleic-acid-based therapeutics to treat and correct genetic and acquired diseases. It is the official journal of the American Society of Gene & Cell Therapy and is built upon the success of Molecular Therapy. The journal focuses on gene- and oligonucleotide-based therapies and publishes peer-reviewed research, reviews, and commentaries. Its impact factor for 2022 is 8.8. The subject areas covered include the development of therapeutics based on nucleic acids and their derivatives, vector development for RNA-based therapeutics delivery, utilization of gene-modifying agents like Zn finger nucleases and triplex-forming oligonucleotides, pre-clinical target validation, safety and efficacy studies, and clinical trials.