Application Of CRISPR/Cas9 Technique In Functional Study Of General Odorant Binding Protein 2 Gene Of Spodoptera Litura

CRISPR/Cas9 system is an acquired immune mechanism found in bacteria and archaea.Cas9 protein edits target gene of genome mediated by chimeric double stranded RNA and then silences the expression of the target gene.This system has some advantages:high mutant efficiency,simple operation and time-and cost-saving.Although CRISPR/Cas9 system has been successfully applied in genome editing of many model organisms(such as Drosophila,Bombyx mori),the application in non-model insects was rarely reported.Spodlptera lilura,a noctuid species,is an important agricultural pest worldwide.General odorant binding protein(GOBP)is abundantly existed in sensillar lymph,and is thought to be responsible for binding and transporting the plant volatiles to the olfactory receptor located on the membrane of the sensillar dendrite.However,such physiological function of GOBP2 has not directly evidenced due to the unavailability of the proper technology.The purpose of the present study is to explore the use of CRISPR/Cas9 system as an in vivo functional study technique for SlitGOBP2 in a non-model lepidopteran species.As a result,we found a variety of mutant mosaicism at the generation of sgRNA/Cas9 mRNA injection,and obtained a S.litura strain with mutant SlitGOBP2(three bases deletion and six bases insertion at the garget site)after 3 generations of selection.Electroantennography(EAG)detection showed that,comparing with wild-type moths,mutant moths showed a significantly decreased EAG responses to several plant volatiles.This indicates that the mutant SlitGOBP2 has lower binding affinity to those plant volatiles,and that GOBP2 plays an important role in olfaction of S.litura.The main results were as follows:1.Knocking out SlitGOBP2 by CRISPR/Cas9 systemAccording to the genomic DNA sequence of SlitGOBP2 and the target site selection requirements,we selected the target site on the second exon.Then sgRNA and Cas9 mRNA were synthetized in vitro.Eggs were collected within 2 hours after oviposition and microinjection(about InL/egg)was operated in 6 hours.Twenty four hours later,genomic DNA extracted from 20 randomly selected eggs was examined by the RED assay and agarose gel electrophoresis,which showed that the mutation rate was about 49.3%by estimating the relative intensity of the bands.Further direct sequencing of PCR product showed overlapping peaks on the target site and this also indicated mutations in injected eggs.To know the detailed mutations,20 positive clones were sequenced and 10 mutational types were found.With the remaining injected eggs,a heterozygous mutation strain with three bases deletion and six bases insertion at GOBP2 was obtained after three generations'selection.2.Determination of SlitGOBP2 mutations by electroantennography assayEAG responses to 5 plant volatiles and 3 sex pheromone components were tested,using moths from the heterozygous mutation strain and the wild-type strain.After EAG test,genotype(homozygous mutation,heterozygous mutation,and wild type)of the tested moths from the heterozygous mutation strain were determined by RED assay.The results indicated that females showed no significant differences in EAG responses to tested plant volatiles between mutant(homozygous and heterozygous)and wild-type moths,while to sex pheromone Z9,E 12-14:AC at dosage of 5000 ng,female homozygous mutants showed significantly lower EAG values than that of the wild-type moths.Different to mutant females,mutant males showed significantly reducted EAG responses than wild-type males to several plant volatiles(such as hexanol,Phenylacetaldehyde,methyl salicylate);heterozygous mutants showed significantly lower responses than wild-type moths to sex pheromone component Z9,E11-14:AC and Z9-14:AC at 50 ng usage,but no significant difference was found between homozygous mutant and wild-type males.As a whole,the mutatant GOBP2 had no obvious effect to females,but did reduce the EAG responses of male to some plant volatiles and to sex pheromone components to a certain extent.In summary,the present study indicates the feasibility of CRISPR/Casg system in non-model lepidopteran species,and provides an important reference for use of this system as an in vivo functional study of olfactory genes as well as other genes in lepidopteran species.As the mutant strain screened by the present study was only three amino acid difference with the wild type of GOBP2,and therefore,a further screening for a mutant strain with complete loss of GOBP2 function is needed to elucidation of the physiological function of the GOBP2 in S.litura.