Cas9-sgRNA 的靶位分布影响 CRISPR/Cas9 基因组编辑中 DNA 双链断裂修复途径的选择。

BACKGROUND: Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood. RESULTS: In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements. CONCLUSIONS: Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

背景：由于 Cas9-sgRNA 复合物在其靶标上的切割后停留，因此 Cas9 诱导的 DNA 双链断裂 (DSB) 必须暴露于 DSB 修复途径中。Cas9-sgRNA 的靶标相互作用决定了其靶标结合亲和力，并调节其切割后靶标停留时间和 Cas9 诱导的 DSB 的暴露。这种暴露通过不同的机制，可能引发不同的 DNA 损伤反应，影响 DSB 修复途径的选择，并导致基因组编辑中的突变异质性。然而，这种 DSB 修复途径选择的调控机制尚不清楚。

结果：在 Cas9 诱导的 DSB 的修复中，不同靶位的修复途径选择差异很大，有些靶位甚至不参与经典的非同源末端连接 (c-NHEJ)。在小鼠胚胎干细胞中，削弱 Cas9-sgRNA 的靶标相互作用会促进偏向 c-NHEJ，并增加靶标解离和减少 Cas9-sgRNA 在体外的靶标停留。作为增强同源定向修复的重要策略，c-NHEJ 的失活由于其与脱靶位点的弱相互作用而加剧了 Cas9-sgRNA 的脱靶活性。通过将 Cas9-sgRNA 从其切割的靶标上置换下来，DNA 复制改变了 DSB 末端构型，有利于其他修复途径而抑制 c-NHEJ，而转录对 c-NHEJ 的参与几乎没有影响。DNA 复制使 Cas9-sgRNA 从其切割的靶标上解离，可能产生三末端 DSB，导致姐妹染色单体的回文融合，这是 CRISPR/Cas9 诱导的靶标染色体重排的潜在来源。

结论：Cas9-sgRNA 的靶标停留可能通过改变 Cas9-sgRNA 从切割 DNA 上的解离来调节 DSB 修复途径的选择，从而扩大了 CRISPR/Cas9 基因组编辑中的靶标和脱靶突变谱。