Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands.
Department of Psychiatry, Erasmus Medical Center, Rotterdam, the Netherlands.
Cell Rep. 2023 Feb 28;42(2):112019. doi: 10.1016/j.celrep.2023.112019. Epub 2023 Jan 25.
Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair pathways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (Polϴ) inhibitor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engineering.
通过修复 CRISPR-Cas9 诱导的染色体断裂进行基因编辑为纠正广泛的遗传缺陷提供了一种手段。通过调节 DNA 修复途径来指导修复以产生理想的结果，这为提高基因组工程的效率提供了巨大的潜力。在这里，我们表明，抑制非同源末端连接（NHEJ）或聚合酶θ介导的末端连接（TMEJ）可用于改变 CRISPR-Cas9 的突变结果。我们使用强效聚合酶θ（Polϴ）抑制剂 ART558 显示出对 CRISPR-Cas9 诱导的双链断裂（DSB）处 TMEJ 活性的强大抑制作用。通过靶向测序，我们表明 ART558 抑制了微同源性驱动的缺失的形成，有利于 NHEJ 特异性结果。相反，NHEJ 缺陷会触发大 kb 大小的缺失的形成，我们表明这些缺失是诱变 TMEJ 的产物。最后，我们表明，TMEJ 和 NHEJ 的联合化学抑制可提高同源性驱动修复（HDR）介导的精确基因编辑的效率。我们的工作报告了一种提高基因组工程准确性和安全性的强大策略。
