title: The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro.
abstract: 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.
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Lei
authors.affiliation: 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.
authors.forename: Shi-Yuan
authors.initials: SY
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Jia-Kun
authors.initials: JK
authors.lastname: Liu
authors.affiliation: 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.
authors.forename: Xuan
authors.initials: X
authors.lastname: Zheng
authors.affiliation: 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.|State Key Lab of Genetic Engineering & Center for Synthetic Biology, Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200032, China.|Department of Microbiology and Li KaShing Institute of Health Sciences, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China.
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
mesh_terms: D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D005603:Francisella / Q000201:enzymology; D019914:Taq Polymerase / Q000378:metabolism*
chemical_list: D064130:CRISPR-Associated Proteins; D004247:DNA; D019914:Taq Polymerase