Nat Biotechnol：CRISPR-Cas9所引起的雙鏈斷裂修復可造成大範圍的刪除和重排

CRISPR-Cas9有望成為臨床背景下的基因編輯工具。到目前為止，人們對於Cas9誘導突變的探索僅限於靶位點和脫靶序列的附近，從而得出CRISPR-Cas9具有良好特異性的結論。本文中，研究人員在小鼠胚胎幹細胞、小鼠造血祖細胞和人分化細胞系中報道了其在靶位點附近引發的例如大範圍缺失、複雜基因組重排等重要變異。通過使用long-read測序和long-range PCR基因分型，研究人員發現由sgRNA / Cas9引入的DNA斷裂經常產生數千鹼基的缺失。此外，研究人員還觀察到切割部位遠端的損傷和交叉事件。由CRISPR-Cas9編輯引起的基因組損傷在具備有絲分裂活性的細胞中可能具有致病後果。

Title:

Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements

Abstract:

CRISPR–Cas9 is poised to become the gene editing tool of choice in clinical contexts. Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR–Cas9 was reasonably specific. Here we report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line. Using long-read sequencing and long-range PCR genotyping, we show that DNA breaks introduced by single-guide RNA/Cas9 frequently resolved into deletions extending over many kilobases. Furthermore, lesions distal to the cut site and crossover events were identified. The observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.

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