Study Of The DNA Binding Domain Of Cas9 Protein And The SgRNA-shRNA Array Technique From CRISPR/Cas9 System

CRISPR/Cas9 system has been emerging as a powerful genome engineering tool.It consists of two components,non-specific endonuclease Cas9 and a short fragment of guide RNA.Due to its high efficiency and simplicity,the CRISPR/Cas9 has been widely employed for genome engineering studies in varieties of organisms,including microbes,plants and animals.After binding guide RNA,Cas9 protein specifically targets genome target site and makes double strand breaks beside PAM site.Understanding and elucidation of Cas9 DNA binding domain will help to improve Cas9 nuclease activity and further provide useful information for constructing new genome engineering tools.Thus,we fused the non-specific nuclease Fok1 gene to the Cas9 gene and a series of deletion mutations.These constructs were co-transfected into 293 T cells with our dual-reporter system for dissection of Cas9 DNA binding domain.We also optimized our recently developed sgRNA-shRNA array technique by introducing the endoribonuclease Csy4 based on the previous reports.Firstly,we designed eight Cas9 truncated mutants.These deletion mutants and full-length Cas9 gene were fused with FokI gene resulting in the corresponding sgRNA/Cas9-FokI vectors.As Fok1 protein functions in the form of dimer,we designed two target sites on the reporter vector,SSA-DsRed/eGFP,(both have/don't have PAM site).To dissect the Cas9 DNA binding domain,the 9 sgRNA/Cas9-FokI expression vectors were co-transfected into 293 T cell with the SSA-DsRed/eGFP reporter vectors.By examining the eGFP expression,we can determine the mutant Cas9 DNA binding activity and its dependency on PAM.In preliminary experiments,we did not observed any activity from the experiment groups of Cas9 truncated mutants except for the wild type Cas9 gene group.This result suggested that Cas9 protein functions as a whole and any deletion mutants either from 5'end or 3'end will kill its DNA binding activity.Csy4 is a CRISPR-associated endoribonuclease.It binds and cleaves at specific repetitive RNA sequences.Therefore,we incorporated Csy4 cleavage sites into our multiplex sgRNA-shRNA array expression design.We constructed a new sgRNA-shRNA array expression vector VLC-Csy4/Cas9(contains VEGF.sgRNA,LIG4.shRNA and CCR5.sgRNA)and the control vectors VCC-Csy4/Cas9(contains VEGF.sgRNA,control vector CON.shRNA and CCR5.sgRNA).By co-transfected with the SSA-DsRed/eGFP reporter vectors regarding the VEGF.sgRNA and CCR5.sgRNA,we found that sgRNAs expressed by VLC-Csy4/Cas9 both have high activities.The activity of LIG4.shRNA expressed by VLC-Csy4/Cas9 was further examined uing qRT-PCR experiment,and the result shows that the LIG4 gene's expression has been knocked down for more than 40%,which is significantly improved compared to our previous Drosha-mediated sgRNA-shRNA system.The results suggested preliminarily the feasibility of Csy4-mediated sgRNA-shRNA expression strategy and established a basis for further optimization and application study.