CRISPR/Cas9 System Based Functional Characterization Of PBP And BLOS2 Genes In Spodoptera Litura

Pheromone binding proteins(PBPs)play important roles in sex pheromone perception of Lepidopteran insects.Recently,it has been found there are at least three different PBP genes in one moth species,but there is no in vivo study on the functional differentiation among the genes.The CRISPR/Cas9 system from an acquired immune mechanism of bacteria and archaea was customal designed by scientists in recent years.As a newly developed and powerful genome engineering technique,the CRISPR/Cas9 system has been used in some insect species.In the present study,we used CRISPR/Cas9 system to knock out PBP genes of Spodoptera litura,and obtained the PBP mutant males,and further in vivo verified function of each of the three PBPs.The results were not only important for clarification of the mechanisms of sex pheromone perception,and also for development of novel olfaction based pest control strategy.In addition,we molecularly and functionally identified BLOS2 gene by CRISPR/Cas9,providing a good mark gene for genetic engineering in S.litura.Our study also provided an important methodological reference for application of CRISPR/Cas9 system in gene functional study in others lepidopteran species.The main result were as follows:1.Knocking out and in vivo functional analysis of SlitPBP1 geneAccording to the criteria of 5'-GG-(N)18-NGG-3',a DNA fragment within exon 2 of the SlitPBP1 was selected as the target site.A restriction enzyme(HpyCH4 ?)cutting site adjacent to the TGG(PAM sequence)was selected to analyze the putative mutations by restriction enzyme digestion(RED)assay.To detect the mutation,20 injected eggs were collected and pooled 24 hours after injection.The genomic DNA extracted from these eggs was used as template for PCR amplification,and then the PCR products were analyzed by RED assay for potential mutation.According to the result of RED assay,the indel frequency was calculated as 51.4%.The detailed indel sequences were obtained by TA cloning and sequencing.The top 8 potential off target sequences were checked and no mutation was found,indicating the target specificity in the study.By single pair mating strategy,a homozygous SlitPBP1 mutant line was set up with 2 bases inserted.This insertion resulted in a stop codon,leading to a truncated protein of 104 amino acid in length,with only 3 conserved cysteines(165 amino acids and 6 conserved cysteines for the wild type protein).As the 6 cysteines are essential for the functions of a PBP,the mutant SlitPBPl with only 3 conserved cysteines should completely lost the function.This SlitPBP1-/-strain was used for the further functional study.The EAG response significantly decreased by>50%to the main component(Z9,E11-14:Ac)at dose of 100,500 and 1000 ng,by 40%to Z9,E12-14:Ac at dose of 500 ng,and by 30%to Z9-14:Ac at dose of 500 and 1000 ng,indicating that PBP1 plays more important role in perception of the main component.The behavioral response was further tested to the main component,using Y-tube choice experiment and direct stimulation method.Results showed that mutant males showed obvious reduction in response percentages,in terms of selection for sex pheromone in Y-tube test(Wild vs mutant:83.3 vs 33.3),hair-pencil expansion(100 vs 50)and mating attempt(33 vs 17)in direct stimulation test.To explore the biochemical processes that PBP1 involved in,transcriptomic expression profiling of antennae in response to SlitPBP1 knocked out was performed by means of RNA-seq quantification.According to FPKM result,the expression of 103 genes were up-regulated,and 398 genes down-regulated.KEGG date showed that SlitPBPl was mostly related to pathway of nicotine addiction,taste transduction and sulfur relay system.COG analysis indicated that lose-of-function in SlitPBPl mainly affected the signal transduction mechanism.2.Knocking out and in vivo functional analysis of SlitPBP2 and SllitPBP3 genesThe DNA fragments located at the exon 2 of SlitPBP2 and SlitPBP3 were selected as the target sites.The restriction enzyme BseR I and Xcm I adjacent to the PAM sequence were selected to analyze the potential mutation of SlitPBP2 and SlitPBP3 respectively.By co-injected site specific sgRNA and Cas9 mRNA into the new laying eggs,the target mutagenesis were estimated by RED assay.The chimeric frequency of SlitPBP2 and SlitPBP3 were 46.8%and 39.15%respectively.The potential off target sequences were searched by CasOT from a S.litura transcriptome.As a result,there were no off target effecting of the two genes.According to single pair mate strategy,the homozygous lines of SlitPBP2 was set up with 15 base pair deleted and 5 base pair inserted.A terminator codon will lead to a truncated protein of 114 amino acid length,containing only 5 conserved cysteines(171 amino acids and 6 conserved cysteines for the wild type strain).The EAG recording and behavior experiments were performed to analysis the function of SlitPBP2 in vivo.The behaviors response of SlitPBP2-/-male moths to Z9,E11-14:Ac at 50 ng were significantly inertia and the EAG values of SlitPBP2-/-male moths to three compounds have significantly decreased.Finally,the homozygous lines of SlitPBP3 gene was bred with 10 base pair inserted and 4 base pair deleted.A terminator codon will lead to a truncated protein of 76 amino acid length,containing only 2 conserved cysteines(164 amino acids and 6 conserved cysteines for the wild type strain).By measuring the moth's electrophysiological responses(EAG values)to these three sex pheromones,we found that SlitPBP3-/-male moths could still respond to all three components,but showed a significant decrease in response to the major component Z9,E11-14:Ac at higher stimulant dosages.It means that the SlitPBP3 play a minor role in the sex pheromone perception.3.Molecular and functional identification of SlitBLOS2 by CRISPR/Cas9 systemA fragment of SlitBLOS2 was identified by analyzing a S.litura transcriptome database by local basic BLAST,and then the full length cDNA was acquired by RACE strategy.Temporal expression pattern by qPCR showed that SlitBLOS2 was highly expressed in egg,first and second instar larvae,but weakly expressed in larvae of third to 6th instar and adults.To clarify the function of SlitBLOS2,CRISPR/Cas9 based targeted mutagenesis of SlitBLOS2 was induced.The 62.3-70.6%GO larvae displayed mosaic translucent integument.A mutant individual was obtained in G1 generation,in which the yellow strips and white spots on the larval integument completely disappeared.Our study clearly demonstrate the important function of SlitBLOS2 in the integument formation,and provide an important marker gene for genome editing.