BmATAD3A mediates mitochondrial ribosomal protein expression to maintain the mitochondrial energy metabolism of the silkworm, Bombyx mori.

ATAD3A is a mitochondrial membrane protein belonging to the ATPase family that contains the AAA+ domain. It is widely involved in mitochondrial metabolism, protein transport, cell growth, development and other important life processes. It has previously been reported that the deletion of ATAD3A causes growth and development defects in humans, mice and Caenorhabditis elegans. To delve into the mechanism underlying ATAD3A defects and their impact on development, we constructed a Bombyx mori ATAD3A (BmATAD3A) defect model in silkworm larvae. We aim to offer a reference for understanding ATAD3A genetic defects and elucidating the molecular regulatory mechanisms. The results showed that knockout of the BmATAD3A gene significantly affected the weight, survival rate, ATPase production and mitochondrial metabolism of individuals after 24 h of incubation. Combined metabolomics and transcriptomics analysis further demonstrated that BmATAD3A knockout inhibits amino acid biosynthesis through the regulation of mitochondrial ribosomal protein expression. Simultaneously, our findings indicate that BmATAD3A knockout impeded mitochondrial activity and ATPase synthesis and suppressed the mitochondrial oxidative phosphorylation pathway through B. mori mitochondrial ribosomal protein L11 (BmmRpL11). These results provide novel insights into the molecular mechanisms involved in the inhibition of development caused by ATAD3A deficiency, offering a potential direction for targeted therapy in diseases associated with abnormal ATAD3A expression.

CRISPR/Cpf1 multiplex genome editing system increases silkworm tolerance to BmNPV.

The CRISPR/Cas9 genome editing technology is now widely used in insect studies, but the use of CRISPR can be further increased to improve insect genome engineering. We established a direct mutation at multiple loci in several genes simultaneously used by CRISPR/Cpf1 multiplex genome editing technology to target the BmNPV genome. We constructed a transgenic line that can target the BmNPV ie-1, gp64, and DNApoly genes simultaneously, and hybridized this line with an FnCpf1 transgenic line to obtain an FnCpf1 × gNPVM binary hybrid expression system and to activate the FnCpf1 gene editing system. We showed that the multiple gene editing system introduced deletions, mutations, and insertions at three target sites, and that it did not affect the economic traits of transgenic silkworm lines. The antiviral response of multiplexed genome editing lines increased significantly, and viral gene transcription and replication were significantly affected in the transgenic silkworm lines. This study provides innovative resistance materials for silkworm breeding and also provides a simplified platform for efficient insect multi genome engineering and genetic operation.

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.

Gene editing the BmNPV inhibitor of apoptosis protein 2 (iap2) as an antiviral strategy in transgenic silkworm.

Apoptosis is a cellular defense mechanism used for the elimination of host cells infected by viruses. Viruses have evolved corresponding inhibitors of apoptosis genes to promote their replication. Anti-apoptosis-related genes, involved in baculovirus proliferation, have been proposed but it is unclear whether these genes can be manipulated in gene therapy. We constructed a transgenic silkworm, using the CRISPR/Cas9 system to knock out the BmNPV inhibitor of apoptosis 2 (iap2). The sequencing results showed that all the sequences could edit the target site of BmNPV iap2 gene. There were no differences in economic traits and growth tests between the BmNPV iap2 knockout strain transgenic silkworm lines and the control groups. However, the mortality rate was significantly reduced, the median lethal dose (LD50) was about 100 times higher than the control group, and the onset time was prolonged by 1-2 days after knocking out BmNPV iap2. In addition, the expression levels of apoptotic-related genes Bmiap2, BmICE and BmDreed were significantly affected and the activity of caspase 9 was increased after BmNPV iap2 being edited in transgenic silkworm. These results demonstrated that gene editing BmNPV iap2 could significantly inhibit BmNPV replication and proliferation. This approach provides a new strategy for antiviral research.