Establishment Of CRISPR/Cas9 Technology And Functional Research Of Doublesex Gene In Bactrocera Dorsalis

Bactrocera dorsalis is an important global fruit fly pests.The CRISPR/Cas9technology has shown the enormous application prospects in the control of pests.In recent years,this technology has been the hottest research hot spot in biology,and its technical methods are endless.Among insects,CRISPR/Cas9 technology is widely used in model insects,such as fruit flies,silkworms and mosquitoes,however it is rarely reported in Tephritidae.Doublesex gene is a key gene in the regulation of insect sex determination and plays a major role in sexual differentiation,reproduction,and maintenance of sexual characteristics.In this study,efficient CRISPR/Cas9 target sites to dsx gene were screened.We compared the efficiency of Cas9 mRNA/sgRNA and Cas9protein/sgRNA RNP complexes for optimizing the CRISPR/Cas9 technology system in B.dorsalis.In addition,we explored the function of the doublesex gene based on the optimized gene editing method in B.dorsalis.We aimed to establish and optimize the CRISPR/Cas9 gene editing system in Tephritidae,including B.dorsalis,and cleared the role of doublesex gene in regulation of development and reproduction.We provided the theoretical and technological foundations for the genetic transformation and control of fruit flies.1.Cloning and analysis of Bddsx geneWe cloned the Bddsx gene and complemented the 475 bp sequence of the B.dorsalis genome,ranging from NW₀11876346.1,1203133 to NW₀11876346.1,1203607.We analyzed the homology between Bddsx and other species,and we found that the Bddsx gene had the highest homology with B.oleae dsx,up to 98%.Besides,we clarified the splicing pattern of the Bddsx gene which includes two male-specific regions and one female-specific region.Bddsx^F mainly spliced in exon skipping?ES?mode on the exon 3of Bddsx gene,and Bddsx^M skipped through the second exon in the 5'UTR region,and its3'CDS region spliced by exon 3 via alternative 5'splice Site?A5SS?.We found and labeled splicing recognition signals,which were the alternative 5'splice site?A5SS?,purine-rich enhancer?PRE?sequences and four tra/tra-2 binding sites.In addition,analysis of Bddsx gene spatial and temporal expression profile showed that Bddsx^F was the most highly expressed in female adults,which was higher expression in the tissues,thorax,head,gut,and fat body,and Bddsx^M was the highest expression in male adults,which specifically expressed in testis.2.Target site designing and screeningWe designed seven target sites in the female-specific region of the Bddsx gene using on-line tools.To assess the quality of the sgRNAs,we analyzed their direction,cleaving location,sgRNA transcription efficiency and cleaving efficiency.In terms of the location and direction of the targets,there are four targets?sg-f2,sg-f4,sg-f5 and sg-f6?and three sites?sg-f1,sg-f3 and sg-7?in the sense strand and antisense strand respectively,which order and position of the sgRNAs'cutting sites are sg-f1?+1?,sg-f3?+127?,sg-f4?+152?,sg-f2?+182?,sg-f5?+330?,sg-f6?+365?and sg-f7?+405?.To evaluate transcription efficiency,we calculated the band gray value of each sgRNAs.The results showed that sg-f7 has the highest transcription efficiency with a band gray value of 94.0,and the second sg-f3 had a gray value of 81.9,and the next sg-f4 had a gray value of 42.3.Besides,the transcription efficiency of sg-f1,sg-f2 and sg-f5 was poor the gray values of6.4,16.7 and 5.4,respectively,and sg-f6 was not transcribed successfully.Furthermore,in terms of cleaving efficiency,sg-f1 and sg-f4 had the highest efficiency with 74.3%and72.9%respectively,and the others sgRNAs had a lower efficiency or non-activity including sg-f3 of 28.9%,sg-f5 of 28.3%,sg-f7 of 15.1%,and sg-f2 of non-activity.Finally,taking into all consideration of factors listed above,we selected sg-f4 and sg-f7 to do the ensuing experiments.3.Evaluation of CRISPR/Cas9 system efficiency based on Cas9 mRNA and proteinWe used Cas9 mRNA/sgRNA and Cas9 protein/sgRNA complexes to inject the B.dorsalis embryos,and recorded the F₀ survival rate,Bddsx^F gene deletion efficiency,rate of mutations,and off-target to compare gene editing efficiency of Cas9 mRNA/sgRNA and Cas9 protein/sgRNA complex.The results showed that the final survival rates of embryos injected with Cas9 mRNA/sgRNA and Cas9 protein/sgRNA complex were 7.7%and 2.0%,respectively;Cas9/sgRNA-mediated gene fragment deletion efficiency was higher than that of Cas9 mRNA/sgRNA?47.8%and 28.1%respectively,Student's t test,P<0.05?.In F₀,rates of mutations in Cas9 mRNA/sgRNA group and Cas9 protein/sgRNA group are 56.2%and 67.7 respectively.In addition,we found that no noticeable off-target was detected in both methods,and indicated that both methods show a high site specificity.In conclusion,microinjection of Cas9 mRNA/sgRNA is the best method to knock out the Bddsx gene in B.dorsalis.4.The function of Bddsx geneWe explored the physiology function of Bddsx gene by utilizing the established CRISPR/Cas9 technology system of B.dorsalis.The results showed that the reproductive ability of B.dorsalis in mutation group decreased significantly compared to the control group,and the eggs'hatching rate of dsx^F-females and males was 5.9%and 28.2%respectively,and the sex ratio of offspring shows no significant difference(Chi-square test,P(?²_C<?²_0.05,1=3.84)>0.05).Compared with the wild type,the color of the pupa shells showed lighter in mutation groups.What's more,a few of the males showed deeper colors on the 4^thh to 5^thh segment of abdomen,and appeared some yellow spots on the ventral side.Normal females have two similar seminal vesicles,whereas dsx^F-individual appeared more than two,and showed a significant difference in size,which was smaller than normal.Dsx^F-flies with abnormal seminal vesicles accounted for 25.0%of the total number of females.