A Preliminary Functional Research Of Cpf1 Proteins

Since the CRISPR/Cas systems were discovered,they have been widely used in different areas such as genome editing,expressional regulation,live imaging and so on.Due to the advantage of guide RNAs that direct the gene manipulation of Cas proteins at the target sites without changing the protein sequences,the CRISPR/Cas systems are gradually replacing the position of ZFNs and TALENs with the low cost and easy operation.The most commonly used CRISPR/Cas9 system runs with single guide RNAs guiding the gene manipulation of Cas9 proteins.However,it has some defects about multiplex editing,off-target efficiency and so on.Then,a new CRISPR/Cas system called CRISPR/Cpf1 system was discovered.It recognizes a T-rich PAM sequence that is totally different with the CRISPR/Cas9 system.Cpf1 proteins can process the precursor cr RNA for cr RNA maturation,useful for the design and manipulation of multiplex editing.What's more,it's reported that the CRISPR/Cpf1 system shows high specificity than the CRISPR/Cas9 system,making the former one a promising tool for genome editing.In this thesis,we picked three CRISPR/Cpf1 systems from Acidaminococcus sp.BV3L6(As),Lachnospiraceae bacterium ND2006(Lb)and Francisella novicida U112(Fn)separately for functional research.We designed and constructed a PAM library to confirm and analyze their features of PAM recognition.We also researched the characteristics of the three different systems by the experiments of cleavage in cells,cr RNA compatibility,fragment insertion and transcriptional activation to provide directions and suggestions for the science researchers.First,according to the characteristics of PAM recognition,we designed and constructed a PAM library with 256 kinds of substrates by the random arrangement of four bases at the PAM position.The three kinds of Cpf1 proteins were expressed with prokaryotic expression systems and purified to cleavage every substrate in the PAM library separately.By the gray analysis and data statistics,we confirmed TTTV as the PAM sequence of As Cpf1 and Lb Cpf1,and TTV the PAM sequence of Fn Cpf1.It's also revealed that one or two substitutions of T to C could be tolerated for the PAM recognition.Second,we conducted the cleavage of endogenous genes DNMT1 and CCR5 and confirmed the cleavage activities of all three proteins in human cells,with Fn Cpf1 the weakest one for cleavage.Third,we matched the Cpf1 proteins and cr RNAs randomly to target the EGFP plasmids or the endogenous gene VEGFA.From the different efficiency by different combinations,we concluded that the Cpf1 proteins could exhibit the strongest activity only when the proteins and cr RNAs were matched.However,weaker cleavage could also be observed with the wrong combination.Then,we designed and obtained the double stranded DNA donor fragments with the homologous arms of 100 bp both at the 5' end and the 3' end of the insert fragments.Results of PCR amplifications indicated that all the three proteins could cause the homolog-directed repair.Finally,we fused the transcriptional activation factor VPR(VP64-p65-Rta)with the DNase-deficient Cpf1 proteins,including d As Cpf1(D908A),d Lb Cpf1(D832A)and d Fn Cpf1(D917A),to target the upstream of the transcription initiation sites of MIAT and IL1 RN.d Fn-VPR exhibited the strongest activation ability at all the four sites in the two genes.All in all,we conducted a preliminary research on the three CRISPR/Cpf1 systems,which could provide directions and suggestions for the science researchers and lay the foundations for the further applications such as gene editing in T cells.