The Study Of Screening And Complex Crystallization Of Small Molecular Inhibitors Of Cpf1

CRISPR/Cpf1 system is one of the most researched CRISPR systems after CRISPR/Cas9 system,and has been deeply studied and widely used by many researchers.Compared with Cas9,Cpf1 has the advantages of small protein molecuLar weight,simple composition,and the production of sticky ends and has both DNA and RNA endonuclease activity at the same time.It is expected to become one of the most effective and most widely used gene editing tools in the future.In this study,Lachnospiraceae bacterium ND2006 Cpf1(Lb Cpf1)protein was used as the research object,and a large number of LbCpf1 proteins were expressed and purified by molecular biological methods such as molecular cloning.We obtained the crRNA,which plays an important role in the activity of DNA endonuclease for LbCpf1 protein,through the in vitro transcription method.Then we successfully selected 14 small molecule inhibitors from small molecule libraries purchased by the laboratory.To explore how the screened small molecule inhibitors inhibits the cleavage activity of LbCpf1 protein on target DNA,we performed molecular biology experiments such as GST pull down and EMSA.The results showed that there is one small molecule of the14 small molecules inhibited the binding of LbCpf1 protein to crRNA and thus inhibited the DNA cleavage activity of Lb Cpf1;there are 4 small molecules inhibited the target DNA combined with LbCpf1-cr RNA binary complex.Finally,in order to further explore the molecular mechanism of inhibition of LbCpf1 by the small molecule inhibitors,we mixed every small molecule inhibitors with their corresponding proteins or protein-nucleic acid complexes and screened the crystals with a mass of crystal kits.And after screening and optimization,we successfully selected three kinds of small molecules and protein mixture crystals,laid the foundation for the subsequent crystal optimization researching.