CRISPR/Cas12a (Cpf1) is a single RNA-guided endonuclease that provides new opportunities for targeted genome engineering through the CRISPR/Cas9 system. Only AsCas12a has been developed for insect genome editing, and the novel Cas12a orthologs nucleases and editing efficiency require more study on insects. We compared three Cas12a orthologs nucleases, AsCas12a, FnCas12a, and LbCas12a, for their editing efficiencies and antiviral abilities. The three Cas12a efficiently edited the Bombyx mori nucleopolyhedrovirus (BmNPV) genome and inhibited BmNPV replication in BmN-SWU1 cells. The antiviral ability of the FnCas12a system was more efficient than that of the SpCas9 system after infection by BmNPV. We created FnCas12a × gIE1 and SpCas9 × sgIE1 transgenic hybrid lines and evaluated the gene-editing efficiency of different systems at the same target site. We improved the antiviral ability using the FnCas12a system in transgenic silkworm. This study demonstrated the use of the CRISPR/Cas12a system to achieve high editing efficiencies, and increase disease resistance in the silkworm.

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 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 (OB). 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.