Construction of a CRISPR/FnCas12a multi-sites editing system for inhibiting proliferation of Bombyx mori nuclearpolyhedrosisvirus.

In recent years, Cas12a, a new member of the CRISPR family, has been found to have both DNase and RNase activities, have a simple structure, and a single promoter can simultaneously initiate multiple crRNAs, making the CRISPR/Cas12a editing system more advantageous in terms of structure and mechanism of action. Our team has successfully constructed Cas12a system that can be used in silkworm. Cas12a can be used to edit the multiple target sites. In production, a lot of factors can affect the production of silk industry. In order to make the silkworm resistant to the virus, using gene editing technology to knock out key genes for replication and proliferation in the Bombyx mori nuclearpolyhedrosisvirus (BmNPV) genome. Multiple sites on the BmNPV genome were selected as the target sites. We constructed the multi-sites expression vector of gie1-M (361 bp, 597 bp, 927 bp of ie-1) that edited multiple sites of BmNPV ie-1. The effects of multi-sites editing system on the proliferation and replication of the virus after the BmNPV genome was knocked out were examined. The results show that compared with CRISPR/FnCas12a single-site editing (gie1), multi-sites editing (gie1-M) can knock out the BmNPV genome more effectively and have a higher inhibitory effect on virus replication and proliferation. This system can provide a new direction for the breeding of silkworm resistant materials, and it can also lay a good technical platform for the identification and research of biological gene function.