The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro.
The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro.
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DOI：
10.1093/nar/gkx018
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发表时间：
2017-05-19
影响因子：
14.9
通讯作者：
Wang J
中科院分区：
文献类型：
--
作者：
Lei C;Li SY;Liu JK;Zheng X;Zhao GP;Wang J
由于Cpf 1以交错方式切割双链DNA，因此可用于DNA组装。然而，发现Cpf 1切割是不准确的，这可能导致DNA组装错误。此处，精确表征了Cpf 1切割位点，其中靶链上的切割位点相对于前间隔区相邻基序位点在第22个碱基附近，但非靶链上的切割受间隔区长度的影响。当间隔区长度为20 nt或更长时，Cpf 1主要在非靶链上的第14和第18个碱基附近裂解;否则，当间隔区长度较短（即17-19 nt）时，Cpf 1主要在第14个碱基之后裂解，产生8-nt粘性末端。根据这一发现，使用具有17-nt间隔crRNA的Cpf 1，用组成型表达启动子体外取代放线菌紫素生物合成簇中的actII-orf 4启动子。工程化的簇产生更多的放线菌紫素，并从早期阶段产生放线菌紫素。此外，进一步使用Taq DNA连接酶以提高该方法的连接效率和连接准确性。我们期望这种CCTL（Cpf 1辅助切割和Taq DNA连接酶介导的连接）方法可以广泛应用于大DNA构建体的体外编辑。
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.