Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion
Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion
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精确且可预测的 CRISPR 染色体重排揭示了 Cas9 介导的核苷酸插入原理
DOI：
10.1016/j.molcel.2018.06.021
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发表时间：
2018-08-16
期刊：
影响因子：
16
通讯作者：
Wu, Qiang
中科院分区：
文献类型：
--
作者：
Shou, Jia;Li, Jinhuan;Wu, Qiang
染色体重排，包括大的DNA片段倒位，缺失，和复制的Cas9与配对的sgRNA是重要的研究基因组结构变异和发育基因调控，但鲜为人知的基本机制。在这里，我们报告说，破坏CtIP或FANCD 2，这在替代非同源末端连接的作用，提高了精确的DNA片段删除。通过分析在缺失、倒位和重复的DNA片段编辑的连接处插入的核苷酸并表征切割的产物，我们发现Cas9在体内和体外以PAM位点的-3位置上游的柔性易切割轮廓内切核酸切割非互补链，产生具有1-3个核苷酸的50个突出端的双链断裂末端。此外，我们发现工程化的Cas9核酸酶具有不同的切割谱。最后，Cas9介导的核苷酸插入是非随机的，并且等于具有预测频率的两个PAM位点上游的组合序列。因此，可以通过干扰DNA修复基因和使用适当的PAM配置来实现精确和可预测的DNA片段编辑。
Chromosomal rearrangements including large DNA-fragment inversions, deletions, and duplications by Cas9 with paired sgRNAs are important to investigate genome structural variations and developmental gene regulation, but little is known about the underlying mechanisms. Here, we report that disrupting CtIP or FANCD2, which have roles in alternative non-homologous end joining, enhances precise DNA-fragment deletion. By analyzing the inserted nucleotides at the junctions of DNA-fragment editing of deletions, inversions, and duplications and characterizing the cleaved products, we find that Cas9 endonucleolytically cleaves the noncomplementary strand with a flexible scissile profile upstream of the -3 position of the PAM site in vivo and in vitro, generating double-strand break ends with 5 0 overhangs of 1-3 nucleotides. Moreover, we find that engineered Cas9 nucleases have distinct cleavage profiles. Finally, Cas9-mediated nucleotide insertions are nonrandom and are equal to the combined sequences upstream of both PAM sites with predicted frequencies. Thus, precise and predictable DNA-fragment editing could be achieved by perturbing DNA repair genes and using appropriate PAM configurations.