瑞士联邦理工学院Jacob E. Corn课题组发现，去除TREX1活性可增强CRISPR–Cas9介导的同源重组。相关论文于2024年8月12日在线发表在《自然—生物技术》杂志上。

研究人员在Fanconi贫血患者淋巴母细胞系中进行了全基因组筛选，发现了CRISPR–Cas9介导的同源定向修复（HDR）的抑制因子。研究人员发现，单一外切酶TREX1在修复模板为单链或线性双链DNA时会降低HDR效率。

TREX1的表达可作为CRISPR–Cas9介导HDR的生物标志物，因为在许多不同细胞类型（如U2OS、Jurkat、MDA-MB-231以及原代T细胞和造血干/祖细胞）中存在的高TREX1表达预测了HDR效率低。

研究人员展示了通过TREX1敲除或使用化学保护的单链DNA模板（免受TREX1活性影响），来恢复HDR效率（改善范围从两倍到八倍）。这些数据解释了为什么某些细胞类型比其他细胞更容易编辑，并指出了在TREX1表达背景下提高CRISPR–Cas9介导HDR的途径。

据介绍，CRISPR–Cas9介导的HDR可以在靶向基因组位点引入所需的突变，但在许多细胞类型中，包括DNA修复活性缺陷的细胞，实现高效率的HDR是一个主要障碍。

附：英文原文

Title: Removal of TREX1 activity enhances CRISPR–Cas9-mediated homologous recombination

Author: Karasu, Mehmet E., Toufektchan, Elonore, Chen, Yanyang, Albertelli, Alessandra, Cullot, Grgoire, Maciejowski, John, Corn, Jacob E.

Issue&Volume: 2024-08-12

Abstract: CRISPR–Cas9-mediated homology-directed repair (HDR) can introduce desired mutations at targeted genomic sites, but achieving high efficiencies is a major hurdle in many cell types, including cells deficient in DNA repair activity. In this study, we used genome-wide screening in Fanconi anemia patient lymphoblastic cell lines to uncover suppressors of CRISPR–Cas9-mediated HDR. We found that a single exonuclease, TREX1, reduces HDR efficiency when the repair template is a single-stranded or linearized double-stranded DNA. TREX1 expression serves as a biomarker for CRISPR–Cas9-mediated HDR in that the high TREX1 expression present in many different cell types (such as U2OS, Jurkat, MDA-MB-231 and primary T cells as well as hematopoietic stem and progenitor cells) predicts poor HDR. Here we demonstrate rescue of HDR efficiency (ranging from two-fold to eight-fold improvement) either by TREX1 knockout or by the use of single-stranded DNA templates chemically protected from TREX1 activity. Our data explain why some cell types are easier to edit than others and indicate routes for increasing CRISPR–Cas9-mediated HDR in TREX1-expressing contexts.

DOI: 10.1038/s41587-024-02356-3

Source: https://www.nature.com/articles/s41587-024-02356-3