Lei Chao, Li Shi-Yuan, Liu Jia-Kun, Zheng Xuan, Zhao Guo-Ping, Wang Jin
Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
University of Chinese Academy of Sciences, Beijing 100049, China.
Nucleic Acids Res. 2017 May 19;45(9):e74. doi: 10.1093/nar/gkx018.
As Cpf1 cleaves double-stranded DNA in a staggered way, it can be used in DNA assembly. However, the Cpf1 cleavage was found to be inaccurate, which may cause errors in DNA assembly. Here, the Cpf1 cleavage sites were precisely characterized, where the cleavage site on the target strand was around the 22nd base relative to the protospacer adjacent motif site, but the cleavage on the non-target strand was affected by the spacer length. When the spacer length was 20 nt or longer, Cpf1 mainly cleaved around the 14th and the 18th bases on the non-target strand; otherwise, with a shorter spacer (i.e. 17-19 nt), Cpf1 mainly cleaved after the 14th base, generating 8-nt sticky ends. With this finding, Cpf1 with a 17-nt spacer crRNA were employed for in vitro substitution of the actII-orf4 promoter in the actinorhodin biosynthetic cluster with a constitutively expressing promoter. The engineered cluster yielded more actinorhodin and produced actinorhodin from an earlier phase. Moreover, Taq DNA ligase was further employed to increase both the ligation efficiency and the ligation accuracy of the method. We expect this CCTL (Cpf1-assisted Cutting and Taq DNA ligase-mediated Ligation) method can be widely used in in vitro editing of large DNA constructs.
由于Cpf1以交错方式切割双链DNA，它可用于DNA组装。然而，人们发现Cpf1切割不准确，这可能会在DNA组装中导致错误。在此，对Cpf1切割位点进行了精确表征，其中靶链上的切割位点相对于原间隔序列相邻基序位点在第22个碱基左右，但非靶链上的切割受间隔序列长度影响。当间隔序列长度为20 nt或更长时，Cpf1主要在非靶链上的第14个和第18个碱基左右切割；否则，间隔序列较短（即17 - 19 nt）时，Cpf1主要在第14个碱基之后切割，产生8 nt的粘性末端。基于这一发现，使用具有17 nt间隔序列的crRNA的Cpf1在体外将放线紫红素生物合成簇中的actII - orf4启动子替换为组成型表达启动子。工程化簇产生了更多的放线紫红素，并在更早阶段产生放线紫红素。此外，进一步使用Taq DNA连接酶来提高该方法的连接效率和连接准确性。我们期望这种CCTL（Cpf1辅助切割和Taq DNA连接酶介导连接）方法能够广泛应用于大型DNA构建体的体外编辑。
