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。

IF 6.1 2区 医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL
Molecular Therapy. Nucleic Acids Pub Date : 2025-05-19 eCollection Date: 2025-06-10 DOI:10.1016/j.omtn.2025.102568
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
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引用次数: 0

摘要

CRISPR-Cas12a基因编辑为基于cas9的方法提供了另一种选择,可以更好地靶向富含at的区域,简化向导RNA的制造,并且具有高特异性。然而,基于捐赠者的编辑效果受到多种因素的影响,其中模板格式起着至关重要的作用。目前,在核酸酶诱导的DNA断裂后,用于同源定向修复(HDR)的主要非病毒模板格式是双链DNA,当高剂量转染时是有毒的。其他人已经证明,使用单链DNA (ssDNA)和侧翼双链cas靶序列(CTS)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取向和结合要求来研究AsCas12a Ultra的CTS设计。此外,我们排除了在细胞生理Mg2+条件下AsCas12a的ssDNase活性。最后,我们展示了高剂量CTS (ssCTS)的ssDNA供体的优势,用于将临床相关的不同大小的转基因递送到三个TCR-CD3复合物基因(TRAC, CD3ζ, CD3ε)中,在CD3ε位点上实现高达90%的敲入率,插入0.8kb。长读测序证实了更高的HDR率,并显示与未修饰的ssDNA相比,CTS减少了部分整合事件。总的来说,AsCas12a和ssCTS代表了一个在原代人T细胞中以最小毒性高效敲入的平台。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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.

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来源期刊
Molecular Therapy. Nucleic Acids
Molecular Therapy. Nucleic Acids MEDICINE, RESEARCH & EXPERIMENTAL-
CiteScore
15.40
自引率
1.10%
发文量
336
审稿时长
20 weeks
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.
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