State Key Laboratory of Virology, Hubei Key Laboratory of Cell Homeostasis, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan, China.
Department of Clinical Oncology, Renmin Hospital of Wuhan University, Wuhan, 430060, China.
Genome Biol. 2020 Mar 25;21(1):78. doi: 10.1186/s13059-020-01989-2.
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.
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.
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.
Cas12a 和 LbCas12a 核酸酶被报道为具有前间隔基序 (PAM) TTTV 作为最佳 PAM 的基因组工程有前途的工具。然而，Cas12a 对含 C 的 PAM (CTTV、TCTV、TTCV 等) 的识别可能会在这些非典型 PAM 位点诱导额外的脱靶编辑。
在这里，我们从 Coprococcus eutactus 中鉴定出一种新型的 Cas12a 核酸酶 CeCas12a，它是一种可编程的核酸酶，在人类细胞中的基因组编辑效率可与 AsCas12a 和 LbCas12a 相媲美。此外，CeCas12a 在体外和体内对 PAM 的识别更为严格，随后在细胞中的脱靶编辑率非常低。值得注意的是，与 LbCas12a 和 AsCas12a 相比，CeCas12a 在多个位点的含 C 的 PAM 处产生的脱靶编辑较少，这是通过靶向测序方法评估的。
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一种有前途的候选工具，具有独特的特性。
