The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro

被引：68

作者：

Lei, Chao ^{[1
]}

Li, Shi-Yuan ^{[1
,2
]}

Liu, Jia-Kun ^{[1
,2
]}

Zheng, Xuan ^{[1
,2
]}

Zhao, Guo-Ping ^{[1
,2
,3
,4
,5
,6
]}

Wang, Jin ^{[1
]}

机构：

[1] Chinese Acad Sci, Shanghai Inst Biol Sci, Inst Plant Physiol & Ecol, Key Lab Synthet Biol, Shanghai 200032, Peoples R China

[2] Univ Chinese Acad Sci, Beijing 100049, Peoples R China

[3] Fudan Univ, Sch Life Sci, State Key Lab Genet Engn, Shanghai 200032, Peoples R China

[4] Fudan Univ, Ctr Synthet Biol, Dept Microbiol & Microbial Engn, Sch Life Sci, Shanghai 200032, Peoples R China

[5] Chinese Univ Hong Kong, Prince Wales Hosp, Dept Microbiol, Shatin, Hong Kong, Peoples R China

[6] Chinese Univ Hong Kong, Prince Wales Hosp, Li KaShing Inst Hlth Sci, Shatin, Hong Kong, Peoples R China

来源：

NUCLEIC ACIDS RESEARCH | 2017年 / 45卷 / 09期

基金：

中国国家自然科学基金;

关键词：

STREPTOMYCES-COELICOLOR A3(2); GENE CLUSTERS; HUMAN-CELLS; CRISPR RNA; CPF1; GENOME; CLONING; SPECIFICITIES; ENDONUCLEASE; RECOGNITION;

D O I：

10.1093/nar/gkx018

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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.

引用

页数：7