title: Functional characterization of SlitPBP3 in Spodoptera litura by CRISPR/Cas9 mediated genome editing.
abstract: Functional gene analysis by using genome editing techniques is limited only in few model insects. Here, we reported an efficient and heritable gene mutagenesis analysis in an important lepidopteran pest, Spodoptera litura, using the CRISPR/Cas9 system. By using this system, we successfully obtained the homozygous S. litura strain by targeting the pheromone binding protein 3 gene (SlitPBP3), which allowed us to elucidate the role of this gene in the olfaction of the female sex pheromones. By co-injection of Cas9 mRNA and sgRNA into S. litura eggs, highly efficient chimera mutation in SlitPBP3 loci was detected both in injected eggs (39.1%) and in the resulting individual moths (87.5%). We used the mutant moths as parents to obtain the G1 offspring and the homozygous mutant strain in G2. The function of SlitPBP3 was explored by Electroantennogram (EAG) recordings with a homozygous mutant strain. The result showed that the EAG responses were significantly decreased in mutant males than in control males when treated with the major sex pheromone component (Z9,E11-14:Ac) and a minor component (Z9-14:Ac) at higher dosages. The results demonstrate that s SlitPBP3 gene plays a minor role in the perception of the female sex pheromones. Furthermore, our study provides a useful methodology with the CRISPR/Cas9 system for gene in vivo functional study, particular for lepidopteran species in which the RNAi approach is not efficient.
authors.affiliation: Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Guan-Heng
authors.initials: GH
authors.lastname: Zhu
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, Shanghai 200032, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Xu
authors.affiliation: Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Cui
authors.affiliation: Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Xiao-Tong
authors.initials: XT
authors.lastname: Dong
authors.affiliation: Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Zhan-Feng
authors.initials: ZF
authors.lastname: Ye
authors.affiliation: Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Dong-Juan
authors.initials: DJ
authors.lastname: Niu
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai Institutes for Biological Sciences, Shanghai 200032, China.
authors.forename: Yong-Ping
authors.initials: YP
authors.lastname: Huang
authors.affiliation: Education Ministry Key Laboratory of Integrated Management of Crop Disease and Pests, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address: sldong@njau.edu.cn.
authors.forename: Shuang-Lin
authors.initials: SL
authors.lastname: Dong
mesh_terms: D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002633:Chemotaxis; D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D018076:DNA, Complementary / Q000235:genetics / Q000378:metabolism; D005260:Female; D000072669:Gene Editing; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007814:Larva / Q000235:genetics / Q000502:physiology; D008297:Male; D009154:Mutation; D010063:Ovum / Q000254:growth & development / Q000502:physiology; D011679:Pupa / Q000254:growth & development / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D012724:Sex Attractants / Q000378:metabolism*; D018411:Spodoptera / Q000235:genetics / Q000254:growth & development / Q000502:physiology*
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; D019476:Insect Proteins; D012333:RNA, Messenger; D012724:Sex Attractants; C403713:pheromone binding protein, insect
keywords: CRISPR/Cas9 system; Genome editing; Pheromone binding protein 3; Spodoptera litura