Establishment Of SpyCas9 Inhibitors Activity Screening System And Directed Evolution Of Low Activity Inhibitor

CRISPR-Cas is an adaptive immune system present in bacteria and archaea to defend against foreign DNA invasion.According to this feature,scientists have developed a gene editing system based on the Cas9 of Streptococcus pyogenes.Due to the advantages of simple design,simple operation,short cycle and low cost,SpyCas9 has quickly become popular in biological,medical and other related laboratories around the world in a short period of time.At present,SpyCas9 has been used as a powerful gene editing tool in many fields such as medicine,agriculture,and animal husbandry.However,this tool has problems such as off-target editing,certain cytotoxicity,and uncontrollable activity after entering cells.Therefore,there is an urgent need for a method to control the activity of SpyCas9,which can be turned off after gene editing is completed,so that cells can be protected from damage by SpyCas9.The discovery of Anti-CRISPR proteins undoubtedly provides an effective idea for this method.Anti-CRISPR proteins are the evolutionary products of bacteriophages in order to resist the CRISPR-Cas system.Different Anti-CRISPR proteins can inhibit the corresponding types of CRISPR-Cas to varying degrees.system.Therefore,in this paper,three SpyCas9 inhibitors,AcrⅡA2,AcrⅡA4 and AcrⅡA5,whose mechanism of action has been reported,were selected to conduct in-depth research on their functions and activities.First,in order to verify the activity of existing inhibitors,this study constructed an efficient dual-plasmid functional verification system based on antibiotic screening.The system consists of a plasmid with editing function and another plasmid containing inhibitor genes.The DNA cleavage effect of SpyCas9 and the inhibitory effect of the inhibitor are determined by whether the antibiotic resistance is repaired or not.The results showed that on the basis of determining the efficient cleavage target of SpyCas9,AcrⅡA4 efficiently inhibited the editing function of SpyCas9,while AcrⅡA2 had no obvious inhibitory effect on SpyCas9.Second,an efficient LacZ gene-based SpyCas9 for Escherichia coli genome DNA editing and inhibitory effects test system was established.Since the target of the above-mentioned dual-plasmid functional verification system is designed on a plasmid,the plasmid has a certain copy number in bacteria.To the LacZ gene of the BL21(DE3)genome,and design the corresponding homology repair template according to the target.The repaired LacZ gene will undergo frameshift mutation,and the editing and repairing status can be judged by the color and number of transformed bacteria.Then,the editing system that can effectively edit the LacZ gene is transferred into the bacteria containing the inhibitor,and the activity of the inhibitor is judged by the number and color of the colony.The experiment again showed that AcrⅡA4 has excellent inhibitory effect,while AcrⅡA2 has no obvious inhibitory effect.Third,the inhibitory effect of AcrⅡA2 on SpyCas9 was enhanced by a directed evolution strategy.The AcrⅡA2 with no obvious inhibitory effect was molecularly modified by error-prone PCR to construct an AcrⅡA2 mutant library and perform high-throughput functional screening.After primary and secondary screening,an AcrⅡA2 mutant with increased activity was screened from 850 samples.Fourth,the effect of the fusion protein of the inhibitor AcrⅡA5 and APOBEC base editing enzyme was explored.One of the main reasons for the low editing efficiency of existing single-base editors is the low binding rate of deaminase to d Cas9(or nCas9).To solve this problem,the fusion protein of AcrⅡA5 and APOBEC base editing enzyme was designed in this study.AcrⅡA5 can effectively bind to nCas9 but does not affect the complex formation between nCas9 and DNA.This study expects that with the help of AcrⅡA5,the binding effect of APOBEC and nCas9 will be improved,and the editing efficiency will be further improved.The target of APOBEC is located in the highly conserved region of LacZ in the BL21(DE3)genome,which can be judged by the color of the colony.The experimental results show that the base editor has no editing effect in the case of the fusion inhibitor AcrⅡA5,but the construction of the fusion protein provides a new idea for the improvement of the base editor.In conclusion,in this paper,two effective SpyCas9 inhibitor activity screening systems were constructed,and the molecular modification of AcrⅡA2 with no obvious inhibitory effect was carried out to improve its activity.In addition,a fusion protein of inhibitor and deaminase was constructed,and the effect of single-base editing of the fusion protein synergistically with nCas9 was preliminarily explored.This paper not only provides an important reference value for the research on the activity of SpyCas9 inhibitors,but also provides an idea for the control of SpyCas9 activity.