Rationally engineered Cas9 nucleases with improved specificity.
Rationally engineered Cas9 nucleases with improved specificity.
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DOI：
10.1126/science.aad5227
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发表时间：
2016-01-01
期刊：
影响因子：
--
通讯作者：
Zhang F
中科院分区：
文献类型：
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作者：
Slaymaker IM;Gao L;Zetsche B;Scott DA;Yan WX;Zhang F
RNA引导的核酸内切酶Cas9是一种通用的基因组编辑工具，具有从治疗到基因功能注释的广泛应用。Cas9在与短RNA向导互补的靶向基因组基因座处产生双链断裂（DSB）。然而，Cas9可以切割与向导不完全互补的脱靶位点，这对基因组编辑构成了重大挑战。在这里，我们使用结构指导的蛋白质工程来提高化脓链球菌Cas9（SpCas9）的特异性。使用靶向深度测序和无偏的全基因组脱靶分析来评估Cas9介导的人细胞中的DNA切割，我们证明了“增强特异性”的SpCas9（eSpCas9）变体减少了脱靶效应并保持了稳健的靶向切割。因此，eSpCas9可广泛用于需要高水平特异性的基因组编辑应用。
The RNA-guided endonuclease Cas9 is a versatile genome editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that “enhanced specificity” SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome editing applications requiring a high level of specificity.