Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair.,Communications Biology

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Understanding the diversity of genetic outcomes from CRISPR-Cas generated homology-directed repair.
Communications Biology ( IF 5.2 ) Pub Date : 2019-12-06 , DOI: 10.1038/s42003-019-0705-y
Brett M Sansbury _{1,

2} , Amanda M Hewes ₂ , Eric B Kmiec _{1,

2}

Affiliation

As CRISPR-Cas systems advance toward clinical application, it is essential to identify all the outcomes of gene-editing activity in human cells. Reports highlighting the remarkable success of homology-directed repair (HDR) in the treatment of inherited diseases may inadvertently underreport the collateral activity of this remarkable technology. We are utilizing an in vitro gene-editing system in which a CRISPR-Cas complex provides the double-stranded cleavage and a mammalian cell-free extract provides the enzymatic activity to promote non-homologous end joining, micro-homology mediated end joining, and homology-directed repair. Here, we detail the broad spectrum of gene-editing reaction outcomes utilizing Cas9 and Cas12a in combination with single-stranded donor templates of the sense and nonsense polarity. This system offers the opportunity to see the range of outcomes of gene-editing reactions in an unbiased fashion, detailing the distribution of DNA repair outcomes as a function of a set of genetic tools.

中文翻译：

了解 CRISPR-Cas 遗传结果的多样性产生了同源定向修复。

随着 CRISPR-Cas 系统向临床应用迈进，识别人类细胞中基因编辑活动的所有结果至关重要。强调同源定向修复（HDR）在治疗遗传性疾病方面取得的显着成功的报告可能会无意中低估了这项卓越技术的附带活性。我们正在利用体外基因编辑系统，其中 CRISPR-Cas 复合物提供双链切割，哺乳动物无细胞提取物提供酶活性以促进非同源末端连接、微同源介导的末端连接，以及同源定向修复。在这里，我们详细介绍了利用 Cas9 和 Cas12a 结合有义和无义极性的单链供体模板的广泛基因编辑反应结果。该系统提供了以公正的方式查看基因编辑反应结果范围的机会，详细描述了 DNA 修复结果作为一组遗传工具的函数的分布。

更新日期：2019-12-06

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