由Cas9-sgRNA介導的染色體重排（包括大片段DNA倒位，缺失和重複）對於研究基因組結構變異和發育基因調控十分重要，但人們對其背後的機制知之甚少。

本文中，研究人員報告了剔除在alt-NHEJ途徑中扮演重要角色的CtIP 或FANCD2基因可以促進精確的DNA片段刪除。通過分析缺失、倒位和重複的DNA片段連接處的插入核苷酸並分析切割產物，研究人員在體內、體外實驗中發現Cas9在非互補鏈的PAM位點上游3個鹼基處具有靈活的內切特徵，可產生具有1-3個核苷酸的5"突出末端的雙鏈斷裂。此外，研究人員發現經人工改造的Cas9具有不同的切割特徵。最後，Cas9介導的核苷酸插入並非是隨機的，該插入序列可以看做為兩端PAM上游的核苷酸組合，並且其頻率可以被預測。因此，通過擾亂DNA修復基因並使用適當的PAM組合，可以實現精確且可預測的DNA片段編輯。

Title:

Precise and Predictable CRISPR Chromosomal Rearrangements Reveal Principles of Cas9-Mediated Nucleotide Insertion

Summary:

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′ 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.

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