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

Abstract

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.

Keywords: CRISPR/Cas9; Dendrolimus punctatus; Wnt-1; embryogenesis; genome editing; segmentation.

Figure 1

Motif analysis of Wnt-1 primary structure. (A) Approximate location of each motif in the protein sequence. (B) The most conserved motifs. The number in the boxes corresponds to the numbered motifs. The number in parentheses represents the e-values.

Figure 2

Temporal expression of DpWnt-1during embryonic stages. The relative mRNA levels of DpWnt-1 in embryos from day 1 to 8 (E1-8). RP32 was used as a reference gene to normalize target gene expression. The data are presented as mean values ± S.E.M (n = 3).

Figure 3

Cas9/sgRNA-induced DpWnt-1 mutations. (A) Schematic representation of Wnt-1 sgRNA targeting sites. The boxes indicate the three deduced exons of DpWnt-1, and the black line represents the untranslated regions and introns. The sgRNA targeting sites, (A) (74–96 bp) and (B) (151–173 bp), are located on exon 3. Wnt-1-F and Wnt-1-R were annealed to the upstream and downstream regions of the targeted site. (B–D) CRISPR/Cas9-induced mutagenesis of DpWnt-1. (B) Representative electrophoretogram of PCR products. Mutants with defective segments (1), defective legs (2), and malformed head (3) were sequenced. (C) DpWnt-1 protein was undetectable in mutants by Western blotting analysis. (D) Various deletion genotypes. The fragment flanking the two targeted sites were deleted. The indel mutation genotype is noted on the right.

Figure 4

Cas9/sgRNA-induced posterior segment defects in D. punctatus larvae and pupae. (A,E) EGFP-specific sgRNAs/Cas9 mRNA control. (B–D,F–H) Mildly affected larvae resulting from DpWnt-1 sgRNAs/Cas9 mRNA co-injection. Transformation of the abdominal segment from posterior to anterior. (I) Fifth instar larvae, wild type (up) and DpWnt-1 mutant (down), displaying the transformation of A6/7 into A6. (J) Wild type and DpWnt-1 mutant pupae. (B,F) The mutant larvae type I showed a transformation of A3/5 into A3 and a disturbance of the anterior-posterior axis. (C,G) The mutant larvae type II showed a transformation of A2/4 into A3 and a disturbance of the anterior-posterior axis. (D,H) The mutant larvae type III has extra pigmentation at A2. (E–H) Close-up images of the wild type and mutant individuals. The scale bars represent 0.5 mm (A–D), 0.25 mm (E–H), 50.0 mm (I), and 2.0 mm (J).

Figure 5

Embryonic phenotypes in D. punctatus. (A,F) EGFP sgRNAs/Cas9 mRNA injected control embryo. (B–E,G–J) Severely affected embryo resulting from DpWnt-1 sgRNAs/Cas9 mRNA injection. (B,G) Thoracic leg and prolegs missing on one side. (C,H) Compact body with thoracic legs and prolegs missing on both sides. (D,I) Twisted body without thoracic legs or patterning along anterior and posterior axis, with all prolegs missing. (E,J) Deformed body with malformed head, missing thoracic legs and prolegs on one side. All images were taken at the same magnification. Dorsal is on left and ventral is on right. The scale bars represent 1 mm.

Figure 6

Head phenotypes of DpWnt-1 mutants. (A,C,E) Wild type embryo. (B,D,F) Severely affected embryo with malformed head, missing thoracic legs and prolegs on both sides. The scale bars represent 1 mm.

Figure 7

Expression profiling in DpWnt-1 mutants. Compared to the controls, the mRNA expression of Sex combs reduced (Scr), Deformed (Dfd), and Abdominal-b (Abd-b) increased more than 4-fold in the DpWnt-1 mutants. Others, including Labial (Lab), Proboscipedia (Pb), Antennapedia (Antp), Ultrabithorax (Ubx), and Abdominal-a (Abd-a), changed <2-fold. Rp32 was used as reference gene for RT-PCR normalization. The data are presented as mean values ± S.E.M (n = 3).