Nitulescu Ana-Maria, Du Weijie, Glaser Viktor, Kath Jonas, Aird Eric J, Cullot Grégoire, Greensmith Robert, Mikkelsen Nanna Steengaard, Stein Maik, Bak Rasmus O, Kaminski Michael, Corn Jacob E, Wagner Dimitrios L
Berlin Center for Advanced Therapies, Charité - Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 13353 Berlin, Germany.
Berlin Institute of Health Center for Regenerative Therapies, Berlin Institute of Health at Charité-Universitätsmedizin Berlin, 13353 Berlin, Germany.
Mol Ther Nucleic Acids. 2025 May 19;36(2):102568. doi: 10.1016/j.omtn.2025.102568. eCollection 2025 Jun 10.
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.
CRISPR-Cas12a基因编辑为基于Cas9的方法提供了一种替代方案，能够更好地靶向富含AT的区域，简化向导RNA的制备，且具有高特异性。然而，基于供体的编辑效率受多种因素影响，其中模板形式起着关键作用。目前，核酸酶诱导DNA断裂后用于同源定向修复（HDR）的主要非病毒模板形式是双链DNA，高剂量转染时具有毒性。其他人已证明，使用带有侧翼双链Cas靶向序列（CTS）的单链DNA（ssDNA）作为Cas9介导的基因编辑模板可以减轻这种毒性并提高敲入效率。在这里，我们通过探索PAM方向和结合要求来研究AsCas12a Ultra的CTS设计。此外，我们排除了AsCas12a在细胞生理镁条件下的ssDNase活性。最后，我们展示了高剂量下带有CTS的ssDNA供体（ssCTS）的优势，即能将不同大小的临床相关转基因导入三个TCR-CD3复合基因（，），在 位点对0.8kb插入片段实现高达90%的敲入率。长读长测序证实了更高的HDR率，并表明与未修饰的ssDNA相比，CTS减少了部分整合事件。总体而言，AsCas12a和ssCTS代表了一个在原代人T细胞中高效敲入且毒性最小的平台。
