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A Cas12a ortholog with stringent PAM recognition followed by low off-target editing rates for genome editing
Genome Biology ( IF 12.3 ) Pub Date : 2020-03-25 , DOI: 10.1186/s13059-020-01989-2
Peng Chen 1 , Jin Zhou 1 , Yibin Wan 1 , Huan Liu 1 , Yongzheng Li 1 , Zhaoxin Liu 1 , Hongjian Wang 1 , Jun Lei 2 , Kai Zhao 1 , Yiliang Zhang 3 , Yan Wang 3 , Xinghua Zhang 4 , Lei Yin 1
Affiliation  

Background AsCas12a and LbCas12a nucleases are reported to be promising tools for genome engineering with protospacer adjacent motif (PAM) TTTV as the optimal. However, the C-containing PAM (CTTV, TCTV, TTCV, etc.) recognition by Cas12a might induce extra off-target edits at these non-canonical PAM sites. Results Here, we identify a novel Cas12a nuclease CeCas12a from Coprococcus eutactus , which is a programmable nuclease with genome-editing efficiencies comparable to AsCas12a and LbCas12a in human cells. Moreover, CeCas12a is revealed to be more stringent for PAM recognition in vitro and in vivo followed by very low off-target editing rates in cells. Notably, CeCas12a renders less off-target edits located at C-containing PAM at multiple sites compared to LbCas12a and AsCas12a, as assessed by targeted sequencing methods. Conclusions Our study shows that CeCas12a nuclease is active in human cells and the stringency of PAM recognition could be an important factor shaping off-target editing in gene editing. Thus, CeCas12a provides a promising candidate with distinctive characteristics for research and therapeutic applications.

中文翻译:

具有严格 PAM 识别的 Cas12a 直向同源物,以及用于基因组编辑的低脱靶编辑率

背景 据报道,AsCas12a 和 LbCas12a 核酸酶是基因组工程的有前途的工具,以原型间隔区相邻基序 (PAM) TTTV 为最佳选择。然而,Cas12a 对含 C 的 PAM(CTTV、TCTV、TTCV 等)的识别可能会在这些非规范 PAM 位点引起额外的脱靶编辑。结果 在这里,我们从 Coprococcus eutactus 中鉴定了一种新型 Cas12a 核酸酶 CeCas12a,这是一种可编程核酸酶,其基因组编辑效率与人类细胞中的 AsCas12a 和 LbCas12a 相当。此外,CeCas12a 被证明对体外和体内的 PAM 识别更加严格,其次是细胞中非常低的脱靶编辑率。值得注意的是,根据靶向测序方法的评估,与 LbCas12a 和 AsCas12a 相比,CeCas12a 在多个位点的含 C PAM 处呈现的脱靶编辑更少。结论 我们的研究表明,CeCas12a 核酸酶在人类细胞中具有活性,PAM 识别的严格性可能是影响基因编辑中脱靶编辑的一个重要因素。因此,CeCas12a 为研究和治疗应用提供了具有独特特征的有前途的候选者。
更新日期:2020-03-25
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