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.

CRISPR/Cpf1 mediated genome editing enhances Bombyx mori resistance to BmNPV

“…The vectors pBac[IE2-FnCpf1-OPIE2-PA-3×P3 EGFP afm], pBac[U6-gIE1-3×P3 DsRed afm], and pBac[U6-sgIE1-3×P3 DsRed afm] expressed the FnCpf1 protein, gRNA and the sgRNA target sequence, respectively. The SpCas9 transgenic line was studied previously [11].…”

“…The silk industry ( Bombyx mori, B. mori ), suffers great economic losses due to B. mori nucleopolyhedrovirus (BmNPV) infection[10-12]. CRISPR genome editing is an efficient and widely used technology for anti-BmNPV gene therapy, viral gene function research, and screening of potential targets in BmNPV infection[11, 13]. We first reported on highly efficient virus-inducible gene editing system, which demonstrated that CRISPR/Cas9 could edit the BmNPV genome and effectively inhibit virus proliferation[14].…”

“…al effectively inhibited BmNPV proliferation and replication by editing the ie-1 and me53 of BmNPV immediate early genes in transgenic silkworm[15]. We improved the antiviral ability of transgenic silkworm by nearly 1,000 -fold by editing the two target sites of the ie-1 gene to produce a large fragment deletion [11]. The CRISPR/Cas9 gene editing technology has also been used in antiviral resistance breeding by editing host factor and viral key genes in BmNPV infection.…”

CRISPR/Cas9‐mediated disruption of the immediate early‐0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm

“…Since its discovery, the CRISPR/Cas9 system has been developed into a multifunctional genetic modification tool, which can now be used to perform gene deletion, gene insertion, genome‐wide screening, gene therapy and genetic breeding (Khatodia et al , ; Ledford, ; Zhang et al , ; Gebre et al , ). In previous studies, we demonstrated that the system can effectively edit the BmNPV genome by targeting the ie‐1 gene, thereby inhibiting the viral DNA replication in vivo and in vitro (Dong et al , ; Dong et al , ). Here, to screen more efficient antiviral target sites and verify the efficiency of multigene editing, we obtained a transgenic line that could edit ie‐0 and ie‐2 genes of BmNPV and demonstrated that both target sites could be edited efficiently at the same time, which provides evidence for enhanced genetic editing.…”

“…In silkworm, an antiviral effect has also been achieved by editing the ie‐1 and me‐53 genes of the BmNPV in a transgenic CRISPR system (Chen et al , ). Further, we increased gene editing efficiency and antiviral efficacy and reduced off‐target efficiency in this system by optimizing target sites and hybridizing the Cas9 and single guide RNA (sgRNA) double transgenic silkworm lines (Dong et al , ). However, gene therapy for BmNPV is still in its infancy, and there are not many target gene loci applied to this technology for silkworm antiviral research or to further improve the antiviral ability through multigene editing efficiency.…”