Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing.
Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing.
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Cas9-sgRNA 的靶点驻留影响 CRISPR/Cas9 基因组编辑中 DNA 双链断裂修复途径的选择
DOI：
10.1186/s13059-022-02736-5
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发表时间：
2022-08-01
期刊：
影响因子：
12.3
通讯作者：
中科院分区：
文献类型：
--
作者：
由于Cas9 - sgRNA复合物在其靶点处切割后的停留，Cas9诱导的DNA双链断裂（DSBs）必须暴露出来以启动DSB修复途径。Cas9 - sgRNA与靶点的相互作用决定了其靶点结合亲和力，并调节其切割后在靶点的停留时长以及Cas9诱导的DSBs的暴露情况。这种暴露通过不同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，对DSB修复途径选择的这种调控机制了解甚少。
在Cas9诱导的DSBs修复中，不同靶点处的修复途径选择差异很大，在一些位点甚至不涉及经典的非同源末端连接（c - NHEJ）。在小鼠胚胎干细胞中，减弱Cas9 - sgRNA与靶点的相互作用会促使偏向c - NHEJ，增加靶点解离，并降低Cas9 - sgRNAs在体外的靶点停留。作为增强同源定向修复的一种重要策略，c - NHEJ的失活会由于Cas9 - sgRNA与脱靶位点的弱相互作用而加剧其脱靶活性。通过将Cas9 - sgRNA从其切割的靶点上移除，DNA复制改变了DSB末端的构象并抑制c - NHEJ，有利于其他修复途径，而转录对c - NHEJ的参与几乎没有影响。DNA复制使Cas9 - sgRNA从其切割的靶点上解离可能产生三末端DSBs，导致姐妹染色单体的回文融合，这是CRISPR/Cas9诱导的靶向染色体重排的一个潜在来源。
Cas9 - sgRNA在靶点的停留可能通过改变Cas9 - sgRNA从切割的DNA上的解离来调节DSB修复途径的选择，从而拓宽了CRISPR/Cas9基因组编辑中的靶向和脱靶突变谱。
网络版包含补充材料，可在10.1186/s13059 - 2022 - 02736 - 5获取。
BackgroundDue 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.ResultsIn 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.ConclusionsTarget 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.
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