Establishment And Application Of CRISPR/Cpf1 Gene Editing System In Silkworm Cells

Clustered regularly interspaced short palindromic repeats（CRISPR）is a repeating sequence in the genome of prokaryotes.It is an immune system that appears when confronting viruses during the evolution of bacterial life.Using this system,Bacteria can remove viral genes from their chromosomes.In recent years,the development of CRISPR system as a gene editing tool has been applied to model organisms such as mice,human cells,tobacco and microorganisms.Bombyx mori is an important model organism.Bombyx mori nucleopolyhedrovirus（BmNPV）is one of the major silkworm diseases in the sericulture industry,which seriously restricts the development of the sericulture industry.However,with the in-depth exploration of the CRISPR system,it was discovered that the new member of the CRISPR family,Cpf1,has both DNase activity and RNase activity,which makes the Cpf1 editing system have more advantages in structure and mechanism of action.Therefore,there is an urgent need to establish a Cpf1 editing system in silkworm model organisms,and use its advantages to efficiently act on the Bm NPV genome to improve the silkworm’s resistance to viruses.In this study,we first constructed the silkworm AsCpf1,FnCpf1 and LbCpf1 editing systems to detect their editing capabilities;and then constructed a knockout vector of the target BmNPV ie1,analyzed the impact of the BmNPV ie1 gene knockout on virus proliferation and replication and tested its knockout efficiency;comprehensive Results The best FnCpf1 was selected,and the inhibitory effects of FnCpf1 and SpCas9 on BmNPV were analyzed.Using FnCpf1 to simultaneously target multiple sites of BmNPV,a multi-site gie1234 knockout system targeting the BmNPV ie1 gene and a knockdown system targeting multiple genes of BmNPV g13467 were constructed,and the knock-out system to the target site was analyzed,and the Analysis of gene knockout efficiency after knocking out multiple target sites.The main results obtained in the paper are as follows:1.Establishment of CRISPR/Cpf1 editing system in silkwormAccording to the principles of the CRISPR/Cpf1 editing system,the CRISPR/Cpf1and gRNA（hsp60 and atad3a genes）knockout vectors were designed and successfully constructed.It was found that the Cpf1 protein was expressed in the nucleus and could be used for gene editing detection.After the cells were transfected with the knockout vector for 48 h,the genome was extracted,and the knockout effect of the CRISPR/Cpf1system on the target gene was analyzed by T7E1 digestion and cloning sequencing.It was found that the target gene had base knockout and large fragment insertion.Further,Western blotting was used to detect the effect of CRISPR/Cpf1 system on target gene knockout and its protein expression level.It was found that AsCpf1,FnCpf1 and LbCpf1 had knockout effects on different target genes,resulting in down-regulation of protein expression.2.Application of CRISPR/Cpf1 system in single gene editing systemFurther testing the inhibitory effect of CRISPR/Cpf1 on the virus,constructing an expression vector for knocking out BmNPV ie1,sequencing found that the ie1 virus gene showed single base deletion and large fragment knockout.After 48 hours of knocking out the vector-transfected cells,the cells were infected with BmNPV^HSP-EGFPSP-EGFP green light tracer virus.The number of fluorescent cells was observed at 0,12,24,and48 hours after virus infection.The proportion of fluorescent cells in MOCK,AsCpf1,FnCpf1 and LbCpf1 was 25.6%,13.2%,10.8%and 13.2%;the proportion of fluorescent cells in MOCK,AsCpf1,FnCpf1 and LbCpf1 was 48.5%and 30.7%at 48 h after infection,24.1%and 27.8%;a significant decrease in the number of fluorescence can be observed at 24 and 48 h of viral infection.Using flow cytometry to analyze the proliferation of EGFP-positive cells after virus infection,the proportion of EGFP-positive cells of MOCK,AsCpf1,FnCpf1 and LbCpf1 were observed at 24 h after virus infection were 19%,15%,10%and 15%,respectively;48 hours after virus infection,the EGFP-positive cells of MOCK,AsCpf1,FnCpf1 and LbCpf1 were 57%,47%,43%and 44%,respectively.It was learned that AsCpf1,FnCpf1 and LbCpf1 all knocked out the ie1 gene to varying degrees EGFP cell proliferation is reduced.Furthermore,48 hours after viral infection was detected by qRT-PCR and Western Boltting,AsCpf1,FnCpf1,and LbCpf1 knocked out BmNPV ie1,which affected the virus copy number and viral protein expression,and found that the transcription and translation levels of ie1 gene were significantly inhibited.Based on the above results,it is found that the As Cpf1,FnCpf1 and LbCpf1 editing systems can effectively knock out BmNPV ie1,of which FnCpf1 is the most obvious inhibitor of viral proliferation.Using qRT-PCR and Western Boltting to detect the editing of BmNPV ie1 gene by SpCas9 and FnCpf1 knockout system,it was found that both knockout systems can knock out the exogenous genes,and the effect of FnCpf1knockout is better than SpCas9.3.Application of CRISPR/Cpf1 system in multi-gene editing systemIn order to detect the gene editing efficiency of multiple sites of the FnCpf1 editing system,the gie1234 expression vector at multiple sites of BmNPV ie1 knockout at 360bp,597 bp,927 bp and 1200 bp,the g13467 expression vector at multiple genes of BmNPV knockout at lef1,lef3,p143,gp64,p74,ie1 were constructed.After 48 hours of transfection of cells with knockout vectors,fluorescence microscopy was used to observe the changes in the number of fluorescent cells at 0,12,24 and 48 hours after infection with the BmNPV^HSP-EGFP tracer virus.It was found that 24 and 48 hours after infection,compared with the number of fluorescent cells of MOCK,the number of gie1,gie1234 and g13467 fluorescent cells of FnCpf1 system is significantly reduced.Compared with the single target gie1,the fluorescence of the two multi-site knockout systems was significantly reduced.The co-localization of the virus and the knockout vector was analyzed at 48 h after virus infection.Further using sequencing to detect the knockout of the target site,it was found that both the gie1234 and g13467 systems can edit the BmNPV genome.Fluorescent quantitative PCR and Western Boltting were used to detect the changes in viral copy number and viral protein after FnCpf1 knocked out multiple target sites.It was found that 48 hours after infection,FnCpf1 effectively knocked out the viral gene,resulting in the down-regulation of gp41 and viral protein VP39;phase Compared to the single target gie1,the two multi-site knockout system is significantly less.TCID50 analysis calculated the virus titer of the FnCpf1 knockout virus gene.It can be seen that the TCID50 of the FnCpf1 knockout system is one order of magnitude lower than that of the MOCK control group.The results showed that the silkworm CRISPR/Cpf1 gene editing technology system was successfully constructed,and the BmNPV genome was knocked out more efficiently by the multi-gene editing system.This system not only provides a new direction for breeding silkworms for disease resistance,but also lays a good technical platform for the identification of gene functions of organisms.