title: CRISPR/Cas9-mediated targeted gene mutagenesis in Spodoptera litura.
abstract: Custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) system provide attractive genome editing tools for insect functional genetics. The targeted gene mutagenesis mediated by the CRISPR/Cas9 system has been achieved in several insect orders including Diptera, Lepidoptera and Coleoptera. However, little success has been reported in agricultural pests due to the lack of genomic information and embryonic microinjection techniques in these insect species. Here we report that the CRISPR/Cas9 system induced efficient gene mutagenesis in an important Lepidopteran pest Spodoptera litura. We targeted the S. litura Abdominal-A (Slabd-A) gene which is an important embryonic development gene and plays a significant role in determining the identities of the abdominal segments of insects. Direct injection of Cas9 messenger RNA and Slabd-A-specific single guide RNA (sgRNA) into S. litura embryos successfully induced the typical abd-A deficient phenotype, which shows anomalous segmentation and ectopic pigmentation during the larval stage. A polymerase chain reaction-based analysis revealed that the Cas9/sgRNA complex effectively induced a targeted mutagenesis in S. litura. These results demonstrate that the CRISPR/Cas9 system is a powerful tool for genome manipulation in Lepidopteran pests such as S. litura.
authors.affiliation: College of Life Science, Fujian Agriculture and Forestry University, Fuzhou.|Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Hong-Lun
authors.initials: HL
authors.lastname: Bi
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Xu
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: An-Jiang
authors.initials: AJ
authors.lastname: Tan
authors.affiliation: Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China.
authors.forename: Yong-Ping
authors.initials: YP
authors.lastname: Huang
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004625:Embryo, Nonmammalian; D018390:Gene Targeting; D007814:Larva / Q000254:growth & development; D016296:Mutagenesis; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D018411:Spodoptera / Q000196:embryology / Q000235:genetics* / Q000254:growth & development
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems; D012333:RNA, Messenger
keywords: CRISPR/Cas9 system; Spodoptera litura; abdominal-A gene; genome editing; microinjection