Establishment Of CRISPR/Cas9 System And Function Research Of Y-link Gene Spermless In Bactrocera Dorsalis

CRISPR/Cas9 is a gene editing system mediated by sg RNA and Cas9 protein,which could specifically cleave ds DNA and induce target gene mutation.The technology,with the characteristics of universality,high specificity and high efficiency,has been widely used in many fields such as medicine,biology and agriculture.In insects,CRISPR/Cas9-based gene editing has also been developed rapidly,mainly used in basic research of insect genomics and pest control.Bactrocera dorsalis is an important agricultural pest in China.It brings huge economic losses to the fruit and vegetable industry every year.Because of the limitation of using traditional methods to control the pest,it is promising to utilize the molecular biotechnology to develop a new environmentally friendly pest control method.Here,we established a high efficient and fast gene editing system in B.dorsalis.We constructed a plasmid and screened the optimized protocol which is suitable for B.dorsalis gene editing.Through single embryo RNA-seq,we revealed the key pathways of sex differentiation at embryo stage and identified several Y-linked genes.We elucidated the role of Y-linked gene spermless in spermatogenesis and testicular development,which provided the target gene for future genetic modification and pest control.1. We knocked-out the bdmew gene successfully in B.dorsalis using CRISPR/Cas9-based gene editing.The bdmew gene was cloned successfully,and its full length is 4051bp,encoding 1168 amino acids.The MEW protein contains four conversed Integrin-?domains and one Integrin-?2 domain.The spatial and temporal expression profile showed the mew has a high expression at thorax tissue and pupae stage.Moreover,we synthesized and screened sg RNA with the highest activity for bdmew knock-out.At G0 generation,we detected genotype mutations and the mutation rate was from 12.1%to30.2%by RED method,while no obvious phenotype changes were observed compared to control group.The G1 generations were generated by G0 out-crossed and 73.8%individuals were mutational,including 20%fail-to-eclosion and 53.8%flightlessness.In the flightless individuals,the wings appeared fold and could not extend normally,and the length was only 75%of the wild type.Since the mutation caused a serious effect on the flies'normal behaviours such as foraging,mating and reproductive,no G2 individual was generated.Furthermore,the thorax and leg muscles were relaxed compared to the control group.A lot of fat drop were enriched in mutant thorax muscle tissue between sarcomere,and the volume of mitochondria was decreased significantly.The genotype detection revealed three main types of mutations,including two base-deletion mutations and a complex type?base-deletion,insertion and replacement?.The off-target detection results showed no off-target mutations occurred,suggesting CRISPR/Cas9 technology can be applied efficiently and specifically in B.dorsalis gene editing.2. We constructed a rapid knockout plasmid suitable for B.dorsalis,and optimized the knockout technology by comparison and screening.Two bd U6 promoters,bd U6.1 and bd U6.2,were cloned which could be used to endogenously express non-coding RNA including sg RNAs.We demonstrated the PUB promoter has high activity in B.dorsalis embryos,and it could be used to initiate expression of downstream genes.We constructed all-in-one plasmid which could to express cas9 m RNA and sg RNA simultaneously,thereby achieving the function of integrated expression and knockout of target genes.We synthesized and screened the sg RNA with the highest activity to knock-out bdtra.Bdtra gene was knocked-out successfully using all-in-one plasmid,and the mosaic rate was43.75%in G0 generation.In tra mutated flies,the reproductive system development was in disorder and the black pigment deposited in the accessory glands.Meanwhile.We compared the efficiency of mutations induced by four Cas9/sg RNA delivery methods including Cas9 plasmid/sg RNA,Cas9 protein/sg RNA,Cas9 m RNA/sg RNA and Cas9/sg RNA plasmid,and the results indicated the Cas9 protein/sg RNA delivery method showed the highest mutation rate in B.dorsalis.3. The early embryo sexual different expression gene database was built through single embryo RNA-seq with reference genome analysis.In total,we obtained 1613differentially expressed genes?DEGs??|log₂?foldchange?|?1,p<0.05?,including 1395male-bias genes and 217 female-bias genes.GO function cluster analysis of these DEGs showed that the main process in biological process included neurogenesis,oogenesis and m RNA splicing via spliceosome,and the main process in molecular function included protein binding,DNA binding and m RNA binding.The results indicated the sexual DEGs were mainly clustered in cell replication and differentiation,especially the process related to nucleotide pathway such as DNA binding,m RNA binding and m RNA splicing,suggesting the sex differentiation is initiated by gene expression regulation at early embryo.The sexual differentiation mainly reflected in the formation of nervous system and the transcription and splicing of m RNA.The results made us focus on the development of the nervous system and the splicing of m RNA in the future study of sex differentiation.KEGG pathway cluster analysis of the database was mainly clustered in RNA transport,spliceosome,m RNA monitoring pathway and RNA degradation.These results suggested that multiple pathways of m RNA played a key role in the process of sex determination,from m RNA splicing,regulation to m RNA transport and degradation.At early embryo stage in B.dorsalis,35 known sex determination related genes were detected,and 8 of them were significantly different in sexual embryos and all were male-bias.The RNA-seq database of sexual embryos were de novo assembled respectively,and 189 possible Y-linked contigs were screened by CQ?Chromosome Quotient?.With PCR verification,we eventually obtained 4 Y-linked contigs.4. We proved that the Y-lined gene spermless played a key role in testis development and spermatogenesis by using CRISPR/Cas9.The spermless gene,with a length of 1056bp,encodes 91 amino acids and has a complete exon without any intron.The protein analysis results showed that spermless contained three transmembrane domains and a signal peptide at N'terminal,and mainly located at the cell membrane and plasma membrane.The spatial and temporal expression profile showed the spermless had a high expression level at 5-6 h post-oviposition embryos.It specifically expressed in testis tissue,and its expression level increased as the testis developed.The spermless gene was successfully knocked-out using CRISPR/Cas9,and the mutation efficiency was 53.85%.The G0 was sterile,and testis and comb development were in disorder,while no sex reversal was detected.The spermless RNAi in embryos induced infertility,testicular dysplasia,albinism and disappearance of male combs.Spermless RNAi in newly emerged males showed that their sperm development was significantly retarded,sperm activity was significantly reduced,and sperm number was reduced.These results indicated that spermless gene plays an important role in testis development and spermatogenesis in B.dorsalis.