带有截短 Cas12a 结合序列的单链 HDR 模板可提高原代人类 T 细胞的基因敲入效率

Ana-Maria Nitulescu, Weijie Du, Viktor Glaser, Jonas Kath, Robert Greensmith, Nanna S Mikkelsen, Maik Stein, Rasmus Bak, Michael Kaminski, Dimitrios Laurin Wagner
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引用次数: 0

摘要

通过CRISPR-Cas12a进行非病毒基因编辑是基于Cas9的方法的一种替代方法,它能更好地靶向富含AT的区域,简化引导RNA的制造,并具有高特异性。然而,编辑结果的有效性受多种因素影响,其中模板格式起着至关重要的作用。目前,在核酸酶诱导 DNA 断裂后诱导同源定向修复(HDR)的主要非病毒模板格式是双链 DNA(dsDNA),这种模板在高剂量转染时具有毒性。之前的研究表明,使用带有双链 Cas-target-sequences(CTS)侧翼的单链 DNA(ssDNA)作为 Cas9 介导的基因编辑的修复模板,可以缓解这种毒性并提高基因敲入效率。在这里,我们通过探索 PAM 方向和 Cas12a-crRNA 复合物的结合要求,研究了 As-Cas12a Ultra 的 CTS 设计。此外,我们还排除了 AsCas12a Ultra 在细胞生理 Mg2+ 条件下的体外 ssDNase 活性。最后,我们展示了高剂量使用具有双链 CTS 端部修饰(ssCTS)的 ssDNA 的优势,它能将不同大小的临床相关转基因传递到三个 T 细胞受体-CD3 复合基因(TRAC、CD3ζ、CD3ϵ)中,CD3ϵ 基因座上 0.8kb 插入物的基因敲入率高达 90%。总之,AsCas12a Ultra 和 ssCTS 供体是在原代人类 T 细胞中实现高效基因敲入的平台,而且毒性极低。
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
Non-viral gene editing via CRISPR-Cas12a 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 editing outcomes is subject to various factors, with tem-plate format playing a crucial role. Currently, the predominant non-viral template format for inducing homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA (dsDNA), which is toxic when transfected at high doses. Previous studies have demonstrated that using single-stranded DNA (ssDNA) with flanking double-stranded Cas-target-sequences (CTS) as a repair template for Cas9-mediated gene editing can miti-gate this toxicity and increase knock-in efficiency. Here, we investigate CTS design for As-Cas12a Ultra by exploring PAM orientation and binding requirements of the Cas12a-crRNA complex. Additionally, we rule out in-vitro ssDNase activity of AsCas12a Ultra under cell-physiological Mg2+ conditions. Finally, we showcase the advantage of using ssDNA with double-stranded CTS end modifications (ssCTS) at high doses for delivering clinically relevant transgenes of varying sizes into three T-cell receptor-CD3 complex genes (TRAC, CD3ζ, CD3ϵ), achieving up to 90% knock-in rates for a 0.8kb insert at the CD3ϵ locus. Overall, AsCas12a Ultra and ssCTS donors represent a platform for highly efficient knock-in in primary human T cells with minimal toxicity.
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