CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations
CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations
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DOI：
10.1038/s41467-019-09006-2
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发表时间：
2019-03-08
影响因子：
16.6
通讯作者：
Bedel, Aurelie
中科院分区：
文献类型：
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作者：
Cullot, Gregoire;Boutin, Julian;Bedel, Aurelie
CRISPR-Cas9是一种很有前途的基因组编辑技术。本研究利用Cas9核酸酶在UROS位点诱导的双链断裂DNA （DSB）来模拟和纠正先天性红细胞生成性卟啉症。我们证明，与NHEJ途径相比，同源性定向修复是罕见的，NHEJ途径导致靶向索引并导致不需要的功能失调蛋白。此外，我们描述了一种Cas9核酸酶通过p53依赖机制在细胞系和原代细胞中诱导DSB导致的意想不到的染色体截断。总之，这些副作用可能会限制CRISPR-Cas9核酸酶系统用于疾病建模和基因治疗的前景。我们表明，单缺口酶方法可能更安全，因为它可以防止靶向和脱靶索引和染色体截断。这些结果表明，单缺口酶而非核酸酶方法更可取，不仅用于疾病建模，而且更重要的是用于未来crispr - cas9介导的基因治疗的安全管理。
CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on-and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.