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.

CRISPR–Cas9 介导的同源定向修复 (HDR) 可以在目标基因组位点引入所需的突变，但实现高效率是许多细胞类型的主要障碍，包括 DNA 修复活性缺陷的细胞。在这项研究中，我们对范可尼贫血患者淋巴母细胞系进行全基因组筛选，以发现 CRISPR-Cas9 介导的 HDR 抑制因子。我们发现，当修复模板是单链或线性化双链 DNA 时，单一核酸外切酶TREX1会降低 HDR 效率。 TREX1表达可作为 CRISPR–Cas9 介导的 HDR 的生物标志物，因为许多不同细胞类型（例如 U2OS、Jurkat、MDA-MB-231 和原代 T 细胞以及造血干细胞和祖细胞）中均存在高TREX1表达预测 HDR 较差。在这里，我们展示了通过TREX1敲除或使用化学保护免受 TREX1 活性影响的单链 DNA 模板来挽救 HDR 效率（提高两倍到八倍）。我们的数据解释了为什么某些细胞类型比其他细胞类型更容易编辑，并指出了在TREX1表达环境中增加 CRISPR–Cas9 介导的 HDR 的途径。