title: Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system.
abstract: Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.
authors.affiliation: Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu, China; Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Zhongjie
authors.initials: Z
authors.lastname: Zhang
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 200032, China.
authors.forename: Abu F M
authors.initials: AF
authors.lastname: Aslam
authors.affiliation: Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu, China.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Liu
authors.affiliation: Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu, China.
authors.forename: Muwang
authors.initials: M
authors.lastname: Li
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 200032, China.
authors.forename: Yongping
authors.initials: Y
authors.lastname: Huang
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 200032, China. Electronic address: ajtan01@sibs.ac.cn.
authors.forename: Anjiang
authors.initials: A
authors.lastname: Tan
mesh_terms: D000818:Animals; D012831:Bombyx / Q000196:embryology / Q000235:genetics*; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004625:Embryo, Nonmammalian; D055786:Gene Knockout Techniques; D049750:Genome, Insect; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D010858:Pigmentation / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051155:Wnt1 Protein / Q000235:genetics* / Q000378:metabolism
chemical_list: D019476:Insect Proteins; D012333:RNA, Messenger; D051155:Wnt1 Protein
keywords: Bombyx mori; CRISPR/Cas9; Embryos; Genome editing; Wnt1