Transgenic CRISPR/Cas9-Mediated Viral Gene Targeting For Antiviral Therapy Of BmNPV

Bombyx mori is an important economic insect and a model organism for Lepidoptera.At present,varieties of diseases still threaten the sericultural industry.The viral disease is the most serious disease caused by Bombyx mori nuclear polyhedrosis virus(BmNPV)in the sericultural industry,and the losses caused it account for about 80%of the total losses caused by silkworm diseases.However,traditional anti-virus strategies of Bombyx mori are time-consuming and laborious.The anti-virus strategy based on RNAi is the regulation of post transcriptional level,which can not ultimately contribute Bombyx mori completely to resist virus replication.The expression efficiency of over-expressed resistant genes in Bombyx mori is not high.Genome editing technology,a newly developed genetic operation technology,provides a new technological platform for anti-virus researches of Bombyx mori.However,gene therapy for BmNPV is still in infancy.And most of the available gene targets are unknown.Based on this background,we utilized genome editing technology transgenic system Clustered regularly interspaced short palindromic repeats/CRISPR-associtaed 9(CRISPR/Cas9)to research the anti-BmNPV of Bombyx mori by regarding the late viral transcription factors lef-8 and lef-9 as research targets.The main results are as follows:(1)Disruption of the lef-8 and lef-9 as a therapeutic approach to BmNPV in Bombyx mori.Firstly,the transgenic plasmids targeting lef-8 and lef-9 genes were successfully constructed based on CRISPR/Cas9 system.We introduced the transgenic plasmid into the early embryo of Bombyx mori through the genetic transformation technology,and obtained three transgenic Bombyx mori lines.BmNPV mutations were detected in transgenic Bombyx mori,including small base deletion mutations and large deletion mutations.We monitored the percent mortality of each group 10 days postin-fection(dpi).The results showed that the mortality of transgenic Bombyx mori larvae was significantly lower than that of wild-type Bombyx mori larvae in the experimental groups fed with different doses of virus(10~5,10~6 and 10~7 OBs/larvae).We observed that the columnar epithelial cells in the midgut of transgenic Bombyx mori were closely and orderly arranged without obvious pathological changes after infection with BmNPV through tissue sections.At the same time,no polyhedrosis virus was detected in the hemolymph of three transgenic Bombyx mori strains.On the contrary,abundant polyhedrosis virus particles were detected in the hemolymph of wild-type Bombyx mori.Then we detected the relative copy number of BmNPV gene to monitor the virus proliferation in Bombyx mori.The total DNA extracted every 12 h(0 to 72 hpi)from TG-A,TG-B,TG-C,and WT larvae treated with 106 OBs/larva was used as a template with gp64 and lef-3 as the detection indicators for q PCR analysis of the accumulated viral DNA levels.To confirm that the BmNPV-specific CRISPR/Cas9 system could effectively inhibit BmNPV gene expression,we investigated the transcription levels of four BmNPV genes,namely,an immediate early gene(ie-1),a delayed early gene(p143),a late gene(vp39),and a hyperexpressed late gene(p10),which correspond to four different phases in the BmNPV gene temporal expression pattern.Total RNA extracted from larvae in the TG-A,TG-B,TG-C,and WT groups treated with 106 OBs/larva at 60 hpi was used for real-time q PCR analysis.Expression of the four genes in TG-A,TG-B,and TG-C animals decreased to a nearly undetectable level compared to that in WT animals.Knockout of lef-8 and lef-9 can completely block the replication of the virus in Bombyx mori,which provides some new target sites for Bombyx mori antiviral strategy.(2)Application of transgenic-CRISPR/Cas9 based anti-nucleopolyhedrovirus in the practical commercial silkworm strain.We obtained non diapause eggs by adjusting the low temperature and dark diapause releasing method and microinjected the transgenic plasmids targeting ie-1and me53 genes of CRISPR/Cas9 system into the early embryos of jingsong.By using the non-diapause embryo,we successfully obtained two TG Jingsong lines.The results showed that the mortality of transgenic Bombyx mori lines TG-1 and TG-2 was significantly lower than that of wild-type Bombyx mori larvae in the experimental groups fed with different doses of virus(10~5,10~6 and 10~7 OBs/larvae)by calculating the mortality of 10 days after infection.The average mortality of wild-type,TG-1 and TG-3 were 100%,41.71%and 65.81%at the dose of 10~7 OB_S/larva.After 60 hours of oral administration of BmNPV,many free polyhedron viruses were found in the hemolymph of wild-type Bombyx mori.On the contrary,polyhedroviruses were not observed in the hemolymph of transgenic Jingsong.The total DNA extracted every 12 h(0 to 72 hpi)from TG-1,TG-2 and WT larvae treated with 10~6 OBs/larva was used as a template with gp64 and lef-3 as the detection indicators for q PCR analysis of the accumulated viral DNA levels.We investigated the transcription levels of four BmNPV genes,namely,an immediate early gene(ie-1),a delayed early gene(p143),a late gene(vp39),and a hyperexpressed late gene(p10),which correspond to four different phases in the BmNPV gene temporal expression pattern.Total RNA extracted from larvae in the TG-1,TG-2 and WT groups treated with 10~6 OBs/larva at 60 hpi was used for real-time q PCR analysis.These data provide a reasonable explanation for the resistance of transgenic Jingsong to BmNPV at the molecular level.To evaluate whether the CRISPR/Cas9 system will affect the commercial characters,we monitor and analysis data including developmental stages,cocoon weight,silk quality,matting rate and offspring embryo quantity.Compare with the wild-type bombyx mori larvae,the transgenic bombyx mori larvae has a slight growth delay,and the development was about 24 hours slow.But the transgenic Jingsong did not show other bad phenotypes in the whole growth and development process.The results showed that there was no significant difference between transgenic and wild-type Jingsong in the body weight,mating behavior and oviposition rate of larvae on the third day of the fifth instar.At the same time,it was proved that transgenic silkworm improved resistance without affecting the main economic traits from the cocoon quality survey and silk quality survey results.In conclusion,we obtained the transgenic Bombyx mori with high resistance to BmNPV by using transgenic CRISPR/Cas9 system to specifically target lef-8 and lef-9 of BmNPV,which provide an effective candidate genes for Bombyx mori’s disease resistance research.At the same time,the transgenic CRISPR/Cas9system was successfully applied in Bombyx mori product lines,which provided a new idea for Bombyx mori’s disease resistance breeding and technical support for Bombyx mori production to solve the problem of BmNPV.