The CRISPR/Cas9 System Was Used To Mass Knock Out Genes Related To Silkworm Importance

The domestic silkworm,Bombyx mori,is an important commercial holometabolous lepidopteran insect;sericulture is an outstanding invention by Chinese civilization.Nowadays,Chinese silk industry struggles to survive.From the perspective of domestication,under artificial selection,silkworms' many traits pertaining to sericulture's profitability have improved significantly in relation to that of their ancestors,wild silkworms.This reveals the genetic basis of the biological difference between silkworm and wild silkworm,offering some clues not only to explore the mechanism of silkworm domestication but also to study how silkworms' spinning ability enhance over time;This also lays a solid foundation of successful foreign proteins expression and its application in silk production in the future.The rapid development of genome sequencing and gene editing techniques in this big data era enable us to better research the function of genes in silkworm.In particular,the gradually maturing CRISPR/Cas9 genome editing techniques make editing genes relatively easy.In this study,we applied the CRISPR/Cas9 system to edit the gene of a multivoltine silkworm strain named nistari to preliminarily investigate the biological function of fibroin heavy chain(Fib-H),fibrohexamerin protein/P25 gene,and six candidates of domestication genes identified by our previous genome-wide screening on artificial selection signals,namely BGIBMGA011266-TA(storage protein 1),BGIBMGA014418-TA(zinc finger protein 706-like),BGIBMGA012700-TA(glutamate synthase),BGIBMGA011521-TA(asparagine synthetase).BGIBMGA000221-TA(cryptochrome-1),BGIBMGA007789-TA(cryptochrome-2).We tried to knock out P25 gene,BGIBkMGA000612-TA,BGIBMGA000648-TA,BGIBMGA006831-TA,BGIBMGA000601-TA in bivoltine silkworm strain,yun,and for now we obtained preliminary results of genotyping on the effects of gene editing.The results are as follows:1)Fibroin gene:Successfully,we knocked out Fib-H and obtained stable cocoon sericin phenotype,then we implemented transcriptome analysis of the middle silk gland and posterior silk gland.We discovered that knocking out of fibroin heavy chain had little impact on the middle silk glands,but had a great influence on posterior silk glands.Gene ontology(GO)analysis of the differentially expressed genes showed differentially expressed genes enrichment in metabolic process at the posterior silk glands.KEGG pathway analysis of the differentially expressed genes showed that differentially expressed genes mostly present in the Protein processing in endoplasmic reticulum and Proteasome pathway.We predicted that the absence of fibroin heavy chain gene results in the formation of sericin cocoon,a self-degradation pathway caused by protein error translation.Although P25 gene the important part of fibroin,no obvious effect on cocoon silk synthesis was observed after knocking out of this gene.2)Candidate domestication genes:A.Storage protein.We knocked out Storage protein 1(SP1)gene using CRISPR/Cas9 system.And obtained two types of mutants with large deletion,causing severe premature termination in translation.The mutants showed a drastic decrease in egg hatchability,while the larval development and metamorphosis of the mutants were not affected.We suspected that knocking-out of BmSp1 would influence supply of vitellogenin,further reducing silkworm hatchability.The results suggested that artificial selection of BmSp1 might be associated with the improvement of silkworm hatchability during domestication.B.Zinc finger protein.Knocking out zinc finger protein 706-like significantly increased the larval mortality rate and made the cocoon silk thinner,and it would affect the metamorphosis development of silkworm at extreme conditions.C.Genes on amino acid metabolism pathway.By knocking out amino acid metabolism pathway genes which were under strong artificial selection,we found that the lack of glutamate synthase causes abnormal metamorphosis;the lack of asparagine synthetase made cocoon layer thinner and the quantity of silk production produced significantly reduced.D.Cryptochrome genes.Cryptochrome-2 is under strong artificial selection,the efficiency of knocking out cryptochrome-2 was very low and needed further study.After knocking out of cryptochrome-1,we didn't observed consistent mutant phenotype.3)Applications on the in bivoltine silkworm strain.It is an important exploration for the application of the CRISPR/Cas9 system to investigate the biological function in bivoltine silkworm strain,yun,and we found that the knockout efficiency didn't differ to that of multivoltine silkworm strain.In conclusion,This study offering some clues to explore the mechanism of silkworm domestication,to study how silkworms' spinning ability enhance over time,and lays a solid foundation of successful foreign proteins expression and its application in silk production in the future.