Research On The Structure And Function Of CRISPR/C2c1

CRISPR/Cas system is an adaptive immune mechanism for bacteria and archaea to resist exogenous invasion.The system is widely used in various fields because of its high efficiency,simple operation and low cost.It is regarded as a kind of gene editing tool with broad application prospect.CRISPR/Cas system is divided into two classes,of which class 2 systems only need a single effect protein,the operation is more simple,therefore,the class 2 system caused more attention.Currently two class 2 CRISPR/Cas proteins have been used for gene editing: Cas9 and Cpf1.C2c1,a newly discovered class 2 CRISPR/Cas protein,has been shown to be capable of breaking target DNA.At present,there are few studies on the structure and function of C2c1 at home and abroad.The mechanism by which C2c1 recognizes sgRNA and cleaves the target DNA is not yet clear.In this project,X-ray diffraction and other molecular biology techniques were used to analyze the structure of the ternary complex of B thC2c1 protein binding sgRNA and target DNA.Combined with digestion,MST detection and other experiments,the mode of BthC2c1 recognized sgRNA and target DNA was revealed,the mechanism of ds DNA cleavage by sgRNA-mediated BthC2c1 was studied.In this project,the BthC2c1-sgRNA-dsDNA complex was first assembled and purified.The composite structure was analyzed by the crystallization screening,optimization and diffraction data collection.The overall structure of the BthC2c1-sgRNA-DNA complex is a bi-lobed architecture composed of a recognition?REC?lobe and a nuclease?NUC?lobe.The molecular mechanism of BthC2c1 recognition of ds DNA was revealed by structure information c ombined with digestion in vitro,especially stringent PAM recognition pattern.This is in contrast with the relaxed PAM recognition mode seen in Sa Cas9 and Cpf1.The truncation of stem loop1 and tetraloop did not affect the DNA cleavage activity of sgRNA-mediated BthC2c1 in vitro.Sequence alignment of BthC2c1 with Aac C2c1 showed that BthC2c1 possessed 33% sequence identity with Aac C2c1.Structural comparison of the C2c1-sgRNA-DNA ternary complex between BthC2c1 and Aac C2c1 indicated that the overall structure of BthC2c1 adopts a similar fold as that of Aac C2c1,and sgRNA and target DNA recognition pattern were similar in these two structures.Then,the mechanism of ds DNA cleavage by sgRNA-mediated BthC2c1 was further studied by Sanger sequencing,digestion and MST affinity detection.It was shown that BthC2c1-cleaved DNA products had a staggered cleavage site.The BthC2c1 cleavage site on the target strand was located outside the guide:target heteroduplex segment.This is distinct from Cas9 and Cpf1.The results of cleavage assay in vitro showed that Mg²⁺,Mn²⁺ and Ca²⁺activated BthC2c1 activity,and Zn²⁺ and Ni²⁺ might attenuate its activity.The activity of BthC2c1 in different temperature was detected.It was shown that BthC2c1 could cleave target DNA at a temperature range of 37? to 67?.The cleavage activity was not detected at 27? and was very low at 32?.The activity was enhanced at 37 ?,then the activity was much higher at 42? and 47?.BthC2c1 cleavage activity was not detected in 293 T cells,DF-1 cells and mouse embryos.In the MST assay at 25?,37? and 42?,the affinity between BthC2c1 and its substrate ds DNA36/36 increased with the increasing temperature,BthC2c1 has a higher affinity with ds DNA at 42?.BthC2c1 binds more closely to ds DNA at 42?,especially PAM.The stringent PAM recognition pattern of BthC2c1 may be the reason why BthC2c1 is more active at 42?.The structural analysis and functional study of BthC2c1-sgRNA-dsDNA complex can help to clarify the key target site and mechanism of sgRNA-mediated C2c1 recognition and cleavage of ds DNA,and provide experimental basis for the development of C2c1 protein as new gene editing tool.Meanwhile,it has great significance to further understand the class 2 CRISPR/Cas protein family,which provides a new idea for the better development and application of the class 2 CRISPR/Cas protein family for gene editing.