Genome Editing of Wnt-1, a Gene Associated with Segmentation, via CRISPR/Cas9 in the Pine Caterpillar Moth, Dendrolimus punctatus

The pine caterpillar moth, Dendrolimus punctatus, is a devastating forest pest. Genetic manipulation of this insect pest is limited due to the lack of genomic and functional genomic toolsets. Recently, CRISPR/Cas9 technology has been demonstrated to be a promising approach to modify the genome. To investigate gene functions during the embryogenesis, we introduced CRISPR/Cas9 system in D. punctatus to precisely and effectively manipulate gene expressions inmutant embryos. Compared to controls, knocking out of DpWnt-1, a gene well known for its role in the early body planning, led to high embryonic mortality. Among these mutants, 32.9% of the embryos and larvae showed an abnormal development. DpWnt-1 mutants predominantly exhibited abnormal posterior segments. In addition, multiple phenotypes were observed, including the loss of limbs and the head deformation, suggesting that DpWnt-1 signaling pathway is necessary for anterior segmentation and appendage development. Overall, our results demonstrate that CRISPR/Cas9 system is feasible and efficient in inducing mutations at a specific locus in D. punctatus. This study not only lays the foundation for characterizing gene functions in a non-model species, but also facilitates the future development of pest control alternatives for a major defoliator.

Protocol publication

[…] To search for the Wnt-1 homolog, nucleotide sequence of BmWnt-1 (NM_001043850.1) was used as a query to BLAST against a D. punctatus transcriptome (HHL, unpublished data). RACE was used to obtain the full length cDNA of DpWnt-1. The predicted open reading frame (ORF) was subjected to motif search, pattern analysis, and phylogenetic analysis. The MEME online server was used for motif analysis, and parameters were as follows: a minimum width was 6; a maximum width was 12; and a maximum number of motif was 8 (http://meme-suite.org/tools/meme). To understand the phylogenetic relationship of DpWnt-1 with homologs from other animals, a neighbor-joining tree was constructed using MEGA5, http://www.mega-software.net/ (Tamura et al., ). The Wnt-1 ORFs included in the analysis are as follows: B. mori (NM_001043850), H. armigera (KJ206240), Amyelois transitella (XM_013345048), P. xylostella (XM_011569928), M. sexta (Z30280), P. xuthus (XM_013325799), Danio rerio (XP_005162280), Fopius arisanus (XM_011300877), Bombus terrestris (XM_003393116), Nasonia vitripennis (XM_001603338), Bactrocera dorsalis (XM_011204079), Drosophila willistoni (XM_002066877), Drosophila melanogaster (NM_078778), Tribolium castaneum (EFA04660), Periplaneta americana (KC311252), Gryllus bimaculatus (BAB19660), Homo sapiens (NP_005421) and Mus musculus (NP_067254). […]