Excision of Nucleopolyhedrovirus Form Transgenic Silkworm Using the CRISPR/Cas9 System

Abstract

The CRISPR/Cas9-mediated genome engineering has been shown to efficiently suppress infection by disrupting genes of the pathogen. We recently constructed transgenic lines expressing CRISPR/Cas9 and the double sgRNA target Bombyx mori nucleopolyhedrovirus (BmNPV) immediate early-1 (ie-1) gene in the silkworm, respectively, and obtained four transgenic hybrid lines by G1 generation hybridization: Cas9(-)/sgRNA(-), Cas9(+)/sgRNA(-), Cas9(-)/sgRNA(+), and Cas9(+)/sgRNA(+). We demonstrated that the Cas9(+)/sgRNA(+) transgenic lines effectively edited the target site of the BmNPV genome, and large fragment deletion was observed after BmNPV infection. Further antiviral analysis of the Cas9(+)/sgRNA(+) transgenic lines shows that the median lethal dose (LD50) is 1,000-fold higher than the normal lines after inoculation with occlusion bodies. The analysis of economic characters and off-target efficiency of Cas9(+)/sgRNA(+) transgenic hybrid line showed no significant difference compared with the normal lines. Our findings indicate that CRISPR/Cas9-mediated genome engineering more effectively targets the BmNPV genomes and could be utilized as an insect antiviral treatment.

Keywords: BmNPV; Bombyx mori; CRISPR/Cas9; antiviral therapy; transgenic.

FIGURE 1

Construction of the CRISPR/Cas9 system of transgenic silkworm. Transgenic vector construction of pBac[IE1-Cas9-Ser-PA-3 × P3 EGFP afm] and pBac[U6-sgRNA-3 × P3 DsRed afm] injection with the helper plasmid. G1 generation of Cas9- and sgRNA-positive strains were detected by fluorescence screening, G2 generation four transgenic hybrid lines, namely, Cas9(–)/sgRNA(–), Cas9(+)/sgRNA(–), Cas9(+)/sgRNA(–), and Cas9(+)/sgRNA(+) were generated by G1 hybridization.

FIGURE 2

CRISPR/Cas9-mediated BmNPV site-specific point deletion in transgenic silkworm. (A) DNA sequencing analysis of the sgIE1-53 and sgIE1-352 target sites in the BmNPV genome. The ie-1 gene sequence of the WT is shown on top in bold, the target sequence of sgRNA is indicated in red, and the deletion sequence is indicated by dashes. (B) The ratios of different types of mutations. (C) Determination of editing efficiency of different transgenic lines using T7E1 digestion analysis. The top of the graph represents the transgenic lines, (–) and (+) indicate T7E1 endonuclease. The blue arrow indicates the genome of the target site, and the red arrow shows the mutations induced by T7E1. (D) The efficiency of genome editing at different time points after BmNPV infection based on T7E1 digestion analysis.

FIGURE 3

Survival rate analysis of silkworm transgenic lines. Survival rate analysis of transgenic hybrid silkworm lines after inoculation of fourth instar larvae with: (A) 2 × 10⁵ OBs/larva, (B) 1 × 10⁶ OBs/larva, (C) 1 × 10⁷ OBs/larva, and (D) 1 × 10⁸ OBs/larva. Each transgenic line was screened in triplicate, each replicate included 30 larvae, and mortality statistical analysis was conducted 10 days after inoculation.

FIGURE 4

LD50 analysis of transgenic silkworm lines. LD50 analysis of transgenic hybrid lines Cas9(–)/sgRNA(–) and Cas9(+)/sgRNA(+) after inoculation of fourth instar larvae with BmNPV with 1 × 10³, 1 × 10⁴, 1 × 10⁵, 1 × 10⁶, 1 × 10⁷, and 1 × 10⁸ OBs/larva, respectively. Each transgenic line was screened in triplicate, with each replicate consisting of 30 larvae. The mortality rate was calculated starting from 4 to 8 days after infection using different OB concentrations.

FIGURE 5

Analysis of BmNPV DNA replication of transgenic lines after OB inoculation. BmNPV DNA replication of four transgenic hybrid lines, namely, Cas9(–)/sgRNA(–), Cas9(+)/sgRNA(–), Cas9(+)/sgRNA(–), and Cas9(+)/sgRNA(+) after BmNPV inoculation of fourth instar larvae using: (A) 2 × 10⁵ OBs/larva, (B) 1 × 10⁶ OBs/larva, (C) 1 × 10⁷ OBs/larva, and (D) 1 × 10⁸ OBs/larva. At 0, 12, 24, 48, 72, 96, and 120 h after inoculation, total DNA was isolated from each transgenic line, and Cas9 and sgRNA expression were quantified by Q-PCR. The expression at each time point was determined from the mean of three independent replicates. NS: not significant. ^∗∗Represents statistically significant differences at P < 0.01.

FIGURE 6

Relative expression levels of BmNPV in transgenic lines at different developmental phases. The relative expression levels of four transgenic hybrid lines, namely, Cas9(–)/sgRNA(–), Cas9(+)/sgRNA(–), Cas9(+)/sgRNA(–), and Cas9(+)/sgRNA(+) after BmNPV inoculation of fourth instar larvae with: (A) 2 × 10⁵ OBs/larva, (B) 1 × 10⁶ OBs/larva, (C) 1 × 10⁷ OBs/larva, and (D) 1 × 10⁸ OBs/larva. At 0, 12, 24, 48, 72, 96, and 120 h after inoculation, total RNA from each transgenic line was isolated and BmNPV ie-1, gp64, vp39, and poly gene expression levels were quantified by Q-PCR. The expression level of each gene at each time point was determined from the mean of three independent replicates. NS: not significant. ^∗∗Represents statistically significant differences at the level of P < 0.01.

FIGURE 7

Economic characteristics of transgenic lines. (A) Weight changes in the transgenic hybrid lines Cas9(–)/sgRNA(–), Cas9(+)/sgRNA(–), Cas9(+)/sgRNA(–), and Cas9(+)/sgRNA(+) at the fourth and fifth instar larval stages. IV Represents 4 instar larvae, and V Represents 5 instar larvae. (B) Analysis of the cocoon shell rate of the transgenic hybrid lines. Each value represents the average of 30 repeated measurements.