CRISPR/Cas9 Mediated Gene Editing In Plutella Xylostella(L.)

The diamondback moth(DBM),Plutella xylostella(L.),is one of the most significant worldwide agricultural pest to crucifers.Sufficient control of DBM becomes difficult due to its rapid development of resistance to pesticides.A major contributing factor to resistance issue is that DBM's rapid propagation,short growth cycle and overlapping generations permit rapid selection.Because the shortage of efficient genetic manipulation platform,the genetic research in DBM is relatively rare and insufficient.Recently,CRISPR/Cas9 system had become the dominant technology for genome editing,and provides vast potential for application in studies of functional genomics,transgenic organisms,transcription regulation and gene therapy.Developing efficient and suitable genetic manipulation system is an essential task for the gene functional study and development of novel genetic control strategies in DBM.This research was based on the CRISPR/Cas9 technique and obtained the following results:1.The CRISPR/Cas9 system was utilized for the first time to reveal gene function through targeting Pxabd-A locus in DBM.Pxabd-A has two structurally-similar splicing isoforms(A and B)which only differ in exon ?,with 5 extra amino acids(DFPFP)in isoform A.The expression profile of Pxabd-A showed that the gene was ubiquitous expressed in the entire developmental period and reached the peak expression in pupae and adults stages.One sgRNA target site was designed to target Pxabd-A locus.The mixture solution containing in vitro synthesized sgRNA and different concentration Cas9 mRNA(300 ng/?l or 500 ng/?l)were separately injected into DBM embryos,and the G0 mutation rates were 35%and 91%,respectively.Go chimeric mutations showed varios phenotypes:larvae abdominal segmentation was disrupted,crochets absent from some prolegs,external genitalia deviating from the central axis and testis deformed in adult animals.The indels of target site can be stably transmitted to the progeny,by showing the same phenotype and 9%genetic transformation rate.The results showed that CRISPR/Cas9 could efficiently mediated Pxabd-A knockout,and the mutations and phenotypes were heritable.2.The universal usage of CRISPR/Cas9 system was verified through successfully editing another two endogenous gene PxAntp and PxUbx in DBM.Direct injection of in vitro synthesized Cas9 mRNA and sgRNA for targeting genes induced corresponding phenotypes:under the treatment of PxAntp-sgRNA,mutated larvae exhibited abnormal T1 and T2 thoracic segments andthe PxUbx-sgRNAinduced mutants showed defects of T1-3 thoracic segments.Mutation detection of deformed individuals in the selected target site was showed insertion or deletion mutagenesis.This results suggested that CRISPR/Cas9 can edit other endogenous genes of DBM and induce phenotypic effects.The CRISPR/Cas9 has the potential to replace low efficiency RNAi strategies for genomic analysis in DBM.3.Pol ? U6 promoter for small RNA expression has been characterized in DBM.Six U6 snRNA promoters containing two conserved elements of a proximal sequence element(PSEA)and TATA box,were identified and characterizedfrom the genome.Efficiency of PxU6 expressed shRNAs induced EGFP knockdown was evaluated in cell line.The results showed that the conserved PxU6:3 promoter presented thetranscription capability,indicating that the DNA-dependent shRNA expression plasmid can be used for genome scale shRNA screening in DBM cell line.4.The efficient gene targeting were conducted whereby plasmid DNAs encoding sgRNAs under the control of the PxU6:3 promoter along with Cas9 protein in vitro and in vivo.The PxU6:3:sgRNA and Cas9 expression plasmids were co-transfected into the cells to induce sequence specific mutation.The results showed that efficient disruption of EGFP expression in DBM cell line.The sgRNA drived by PxU6:3 promoter directs Cas9 protein targeting Pxabd-A locusinduced mutagenesis and phenotypic effects in vivo,and stably transmitted to the next generation.The endogenous U6 promoter effectively driven sgRNA transcripts in vivo.The results presented the potential for the establishment of transgenic CRISPR/Cas9 platform in DBM.5.Applification of tRNA-gRNA expression structure and construction of CRISPR derived gene drive systems in DBM.The PxU6:3:tRNA:sgRNA-abd-A expression plasmid was constructed using DmtRNAGIy and OstRNAGly.The sgRNA expression from plasmid directed Cas9 protein to target specific Pxabd-A locus,and induced suddicient mutagenesis.The mutation phenotype was consistent with that of direct injection of sgRNA from in vitro transcription.The establishment of tRNA-sgRNA expression strategy in DBM could break the restriction of targets design,realize the specific expression,make the multi-targeting easier,and lay the foundation for the application of conditional CRISPR/Cas9 in DBM.In addition,the CRISPR derived gene drives system was constructed by using PxU6:3 promoter drive sgRNA transcription for Px016319 targeting in DBM.In summary,the CRISPR/Cas9 system for gene editing was established for the first time in the non-model insect,diamondback moth,and this platform could produce numerous stable mutation lines.The species-specific endogenous PxU6 promoters were identified for small RNA expression in vitro and in vivo.The identified PxU6 promoter makes the application of transgenic CRISPR/Cas9 system and gene drives in DBM possible.This research will further facilitate gene functional studies and development of genetic control of DBM populations.