Research On The Expression And Purification Of S.pyogenes Cas9 And Screening Of Its Inhibitions

CRISPR/Cas systems are adaptive immune systems evolved by bacteria and archaea,The well-characterized S.pyogenes Cas9 system,which belongs to type II-A CRISPR subtype include CRISPR RNA(crRNA),transactivating CRISPR RNA(tracrRNA)and Cas9 protein.The small invading nucleic acids insert in CRISPR locus,then CRISPR locus transcript and process into mature crRNA.Cas9 proteins use crRNA and tracrRNA dsRNA to mediate cleavage of phage nucleic DNA or plasmids which have complementary sequence to the crRNA.To make CRISPR/Cas9 more suitable for gene engineering,crRNA and tracrRNA are fused to form single-giude RNA to mediate the site-directed editing of double-stranded DNA by Cas9.S.pyogenes Cas9 which combines with a synthetic single-guide RNA(sgRNA)has been harnessed as the most common and powerful tool for gene editing and gene regulation in various organisms.However,the activity of S.pyogenes Cas9 cannot be turned off so long as it is turned on.Now,one of the most challenging scientific problem is how to off-target genome edits caused by excessive or prolonged S.pyogenes Cas9 activity,and how to control the activity of S.pyogenes Cas9 precisely.In this project,the wild-type and active site-mutated proteins of S.pyogenes Cas9 were first expressed using a prokaryotic expression systend and purified.Then S.pyogenes Cas9-sgRNA and S.pyogenes Cas9-sgRNA-dsDNA complexes are assembled and purificated in vitro.On this basis,this study expounds 12 kinds of small molecule inhibitors from more than 3 000 compounds which are capable of inhibiting the activity of S.pyogenes Cas9.Through GST-Pulldown and EMSA experiments the inhibition mechanism of inhibitors was expounded.There are six kinds of small molecules inhibiting the binding of S.pyogenes Cas9 protein to DNA(CAS numbers include 107 316-88-1,25 535-16-4,34 157-83-0,545-47-1,519-23-3 and 1 617-53-4),while there are the other six inhibitors inhibiting the activity of S.pyogenes Cas9 protein through different mechanisms(CAS numbers include 518 058-84-9,48 208-26-0,6 902-91-6,2 610-5-1,1 001 645-58-4,and 137-66-6).This study provides small-molecule inhibitors that can inhibit the nuclease activity of S.pyogenes Cas9.It is meaningful for the resesrch of CRISPR/Cas9 inhibitors and great significant for the further promotion of CRISPR/Cas9 technology.