The Design Of The Optimized CRISPR-Cpf1 For Gene Transcription Regulation

CRISPR(clustered regularly interspaced short palindromic repeats)-Cas(CRISPR-associated)system has not only provided a revolutionary changes in the field of gene editing,but also in gene expression regulation,including programmable,specific gene suppression and activation regulation techniques:CRISPR interference(CRISPRi)and CRISPR activation(CRISPRa).CRISPRi/a technology based on CRISPR-Cas9 system is the most widely used.Compared to CRISPR-Cas9 system,the recently reported type V-A CRISPR-Cpfl system offers distinct advantages.Cpfl is naturally a single RNA-guided endonuclease and can process its own crRNA array into mature crRNAs to facilitate targeting of multiple genes simultaneously.Moreover,Cpfl has low mismatch tolerance and high levels of on-target specificity.Here,we present type V-A CRISPR-Cpfl system-based CRISPRi.We selected Cpfl from Francisella novicida U112(FnCpfl).First,three DNase-deactivated Cpfl(FndCpfl),FndCpfl(D917A),FndCpfl(E1006A)and FndCpfl(D917A/E1006A),were constracted according to the report,and three types FndCpfl were compared by flow cytometry and the repression effect of FndCpfl(D917A)was found to be optimal.And then use this protein for further study,it showed significant gene repression when it was targeted to promoter and the template strand of the coding DNA sequences,however,the non-template strand of the coding DNA sequences almost no gene repression.FndCpfl requires at least 16 nt of guide sequence to achieve significant repression.When a RNA transcript array expressed multiple guide sequences targeting multiple loci(up to three),the greater the number of targeting loci,the greater the repression effect.Truncation of the direct repeat of the transcript expressed three guide sequences revealed that at least 19 nt is required for efficient repression.In addition,we also constructed a 6 bp-PAM sequence library and analyzed the relation between it and the efficiency of FndCpfl gene repression.We find that in addition to the 5'-T preference,the 3-terminal base of the PAM sequence has a V preference.This study not only guide for the application of CRISPR-FndCpfl technology,but also is significance for gene editing of FnCpfl-sgRNA in sgRNA design and PAM sequence selection.