A new DNA seamless splicing method based on the cleavage properties
Nucleic Acids Research online published a research study by Zhao Guoping study group at the Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. The study is titled The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro. This work identifies the exact cleavage site of the Cpf1 protein and develops a new DNA seamless splicing method based on the cleavage properties. There are still more research through recombinant proteins like recombinant horse proteins remains to be conducted.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is an acquired immune system that exists in prokaryotes. CRISPR-related protein Cas9 has been widely used in genome editing and many other applications. The Cpf1 protein belongs to the Class II type V CRISPR system. Compared with Cas9 protein, Cpf1 protein has similar genomic editing efficiency, but it also has a low off-target effect. It has great potential in the application of gene therapy. Different from Cas9, Cpf1 is guided by a single crRNA and cleavage of double-stranded DNA (dsDNA) targets using T-rich protospacer adjacent motif (PAM) sequences to form 5-nt viscous ends. The cleavage properties of Cpf1 make it an effective in vitro DNA splicing tool, and recent studies have reported DNA-splicing standards C-Brick based on Cpf1 digestion and T4 DNA ligase-mediated linkages.
In this study, the researchers found that the Cpf1 cleavage targeting DNA is not as previously reported to cut the 23-position of the complementary strand of the target DNA and the 18-position of the non-complementary strand, but forming multiple cleavage sites at 14 to 18 sites. In addition, the researchers also found that Cpf1 cleavage sites were affected by the length of the crRNA spacer sequence. When the spacer sequence is greater than or equal to 20, Cpf1 tends to cut the 18 bits of the uncompleted chain; and when the spacer sequence length is less than 20, Cpf1 tends to cut 14 bits of the uncompleted chain. Based on the short spacer length of cpf1, the 14-site and 22-site of the target DNA can be specifically cleaved to form the 8-nt long cohesive end, and the Taq DNA ligase binds to high-precision binding properties to develop a new tool for seamless editing of large DNA fragments in vitro. In the application example, the researchers successfully replaced the promoter of the actin-orf4 promoter, which controls the internal control gene of the 30 kb thalassemia synthesis gene cluster. After the optimization of the reaction conditions, the replacement rate was more than 70%. This method provides an efficient tool for large segment in vitro editing. Flarebio provides superior recombinant proteins including recombinant Dsc1 at good prices.
