Generation of pigs with a Belgian Blue mutation in MSTN using CRISPR/Cpf1-assisted ssODN-mediated homologous recombination

CRISPR/Cpf1 has emerged recently as an effective tool for genome editing in many organisms,but its use in pigs to generate precise genetic modifications has seldom been described.Myostatin(MSTN)is a well-characterized negative regulator of muscle development,and natural mutations in this gene cause a double-muscled phenotype in many species.However,to the best of our knowledge,no naturally occurring mutation in MSTN has been found in pigs.In addition,no living pig models with sophisticated modifications orthologous to natural mutations in MSTN have yet been reported.In this study,we exploited the CRISPR/Cpf1 system to introduce a predefined modification orthologous to the natural MSTN mutation found in Belgian Blue cattle(thus known as the Belgian Blue mutation).Our research demonstrated that the cutting efficiency of CRISPR/Cpf1 was 12.3%in mixed porcine fetal fibroblasts in drug free medium,and 41.7%in clonal colonies obtained using G418 selection.Then,the Cpf1-sgRNA vector,ssODN template,and a self-excision cassette were co-transfected into porcine fetal fibroblasts.After G418 selection,8 clonal colonies were examined and 5 with genetic modification were found.Of these 5,2 harbored the precise 11-bp deletion.Using 1 heterozygous clonal colony,2 cloned Duroc piglets were successfully generated,which was heterozygous for the Belgian Blue mutation.In summary,our results demonstrate that CRISPR/Cpf1 system can be used efficiently to generate double-stranded breaks,and also to mediate homologous recombination to introduce precise genomic modifications in pigs.

Comprehensive silk gland multi-omics comparison illuminates two alternative mechanisms in silkworm heterosis

Heterosis is a common phenomenon in plants and animals with diverse underlying mechanisms.Here,we applied two widely used silkworm hybrid systems and performed multi-omics analysis to identify possible intrinsic associations between different hybrid strategies and epigenetic mechanisms with silkworm heterosis.We found significant differences in the silk gland transcriptomic landscape between the two systems,including differentially expressed genes and expression patterns in the hybrid offspring compared to their parents.In the quaternary hybrid system,hybrid vigor was primarily due to upregulated genes and the parent-dominant upregulated expression pattern,involving multiple transport processes,cellular nitrogen compound catabolism,glucose metabolism,and tricarboxylic acid cycle.In the binary system,hybrid vigor was mainly due to the down-regulated genes and transgressively down-regulated expression pattern,mainly involving basic nitrogen synthesis metabolism and body function.We also demonstrated that DNA methylation may affect hybrid vigor by regulating the expression of several heterosis-related genes.Thus,this study revealed two alternative mechanisms that may contribute to silkworm heterosis,both of which facilitate the efficient utilization of energy and nitrogen for silk production.

Disruption of Zfh3 abolishes mulberry-specific monophagy in silkworm larvae

Feeding behavior is critical for insect survival and fitness.Most researchers have explored the molecular basis of feeding behaviors by identifying and elucidating the function of olfactory receptors(ORs)and gustatory receptors(GRs).Other types of genes,such as transcription factors,have rarely been investigated,and little is known about their potential roles.The silkworm(Bombyx mori)is a well-studied monophagic insect which primarily feeds on mulberry leaves,but the genetic basis of its monophagy is still not un-derstood.In this report,we focused on a transcription factor encoded by the Zfh3 gene,which is highly expressed in the silkworm central and peripheral nervous systems,in-cluding brain,antenna,and maxilla.To investigate its function,Zfh3 was abrogated using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated protein 9(Cas9)mutagenesis.Since Zfh3 knockout homozygotes are not viable,we stud-ied feeding behavior in heterozygotes,and found that disruption of Zfh3 affects both gus-tation and olfaction.Mutant larvae lose preference for mulberry leaves,acquire the ability to consume an expanded range of diets,and exhibit improved adaptation to the Mo artifi-cial diet,which contains no mulberry leaves.These results provide the first demonstration that a transcription factor modulates feeding behaviors in an insect.

Evolutionary and functional analyses of the interaction between the Bombyx mori inhibitor of apoptosis(IAP)and nucleopolyhedrovirus IAPs

As an important insect immune response,apoptosis plays a critical role in the interaction between baculoviruses and insect hosts.Previous reports have identified inhibitor of apoptosis(IAP)proteins in both insects and baculoviruses,but the relationship between these proteins is still not clearly understood.Here,we found that insect IAP proteins were clustered with baculovirus IAP3,suggesting that the baculovirus iap3 gene might be derived from the Lepidoptera or Diptera.We demonstrated that Bombyx mori inhibitor of apoptosis(Bmiap)gene had an inhibitory effect on apoptosis in silkworm cells.Further analysis of the effects of Bmiap genes on the proliferation of B.mori nucleopolyhedrovirus(BmNPV)showed that both the Bmiap and BmNPV iap genes increased BmNPV proliferation after BmNPV infected silkworm cells.Our results also indicated that BmNPV IAP1 and IAP2 directly interacted with BmIAP in silkworm cells,implying that the Bmiap gene might be hijacked by BmNPV iap genes during BmNPV infection.Taken together,our results provide important insights into the functional relationships of iap genes,and improve our knowledge of apoptosis in baculoviruses and insect hosts.

Molecular basis of the silkworm mutant re^(l) causing red egg color and embryonic death

The coloration and hatchability of insect eggs can affect individual and population survival.However,few genetic loci have been documented to affect both traits,and the genes involved in regulating these two traits are unclear.The silkworm recessive mutant re^(l)shows both red egg color and embryo mortality.We studied the molecular basis of the re^(l)phenotype formation.Through genetic analysis,gene screening and sequencing,we found that two closely linked genes,BGIBMGA003497(Bmre)and BGIBMG4003697(Bm-Semala),control egg color and embryo mortality,respectively.Six base pairs of the Bm-re gene are deleted in its open reading frame,and BmSemala is expressed at abnormally low levels in mutant re^(l).BmSemala gene function verification was performed using RNA interference and clustered randomly interspersed palindromic repeats(CRISPR)/CRISPR-associate protein 9.Deficiency of the BmSemala gene can cause the death of silkworm embryos.This study revealed the molecular basis of silkworm re^(l)mutant formation and indicated that the Semala gene is essential for insect embryo development.

Identification of a PP2A gene in Bombyx mori with antiviral function against B.mori nucleopolyhedrovirus

Ser/Thr protein phosphatase 2A(PP2A)is one of the type 2 protein phosphatases,which is required for many intracellular physiological processes and pathogen infection.However,the function of PP2A is unclear in silkworm,Bombyx mori.Here,we cloned and identified BmPP2A,a PP2A gene from B.mori,which has two HEAT domains and a high similarity to PP2A from other organisms.Our results showed that BmPP2A is localized in the cytoplasm and highly expressed in silkworm epidermis and midgut,and that Bombyx mori nucleopolyhedrovirus(BmNPV)infection induces down-regulation of BmPP2A expression.Furthermore,up-regulation of BmPP2A via overexpression significantly inhibited BmNPV multiplication.In contrast,down-regulation of BmPP2A via RNA interference and okadaic acid(a PP2A inhibitor)treatment allowed robust BmNPV replication.This is the first report of PP2A having an antiviral effect in silkworm and provides insights into the function of BmPP2A,a potential anti-BmNPV mechanism,and a possible target for the breeding of silkworm-resistant strains.

Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in Silkworms

Recently the developed single guide(sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease(CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector(pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus(BmNPV) in insect cells.We screened the immediate-early-1 gene(ie-1), the major envelope glycoprotein gene(gp64), and the late expression factor gene(lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector(PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.

Bmmp influences wing morphology by regulating anterior-posterior and proximal-distal axes sdevelopment

Insect wings are subject to strong selective pressure,resulting in the evolution of remarkably diverse wing morphologies that largely determine flight capacity.However,the genetic basis and regulatory mechanisms underlying wing size and shape development are not well understood.The silkworm Bombyx mori micropterous(mp)mutant exhibits shortened wing length and enlarged vein spacings,albeit without changes in total wing area.Thus,the mp mutant comprises a valuable genetic resource for studying wing de-velopment.In this study,we used molecular mapping to identify the gene responsible for the mp phenotype and designated it Bmmp.Phenotype-causing mutations were identified as indels and single nucleotide polymorphisms in noncoding regions.These mutations resulted in decreased Bmmp messenger RNA levels and changes in transcript isoform composition.Bmmp null mutants were generated by clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9 and exhibited changed wing shape,similar to mp mutants,and significantly smaller total wing area.By examining the expression of genes critical to wingdevelopment inwildtype and Bmmp null mutants,we found that Bmmp exerts its function by coordinately modulating anterior-posterior and proximal-distal axes development.We also studied a Drosophila mp mutant and found that Bmmp is functionally conserved in Drosophila.The Drosophila mp mutant strain exhibits curly wings of reduced size and a complete loss of flight capacity.Our results increase our understanding of the mechanisms underpinning insect wing development and reveal potential targets for pest control.

E2F4 regulates the cell cycle and DNA replication in the silkworm,Bombyx mori

The E2F family of transcription factors is crucial for cell cycle progression and cell fate decisions.Although E2Fs have been widely studied in mammals,there have been few studies performed in insects.Here,we determined the function of E2F4 in the silkworm,Bombyx mori.We demonstrate that E2F proteins are highly conserved among species from lower animals to higher mammals.Overexpression of the BmE2F4 gene led to cell cycle arrest in the G1 phase,whereas interfering with the BmE2F4 mRNA led to accumulation of cells in the S phase.These results indicate that BmE2F4 is important in cell cycle regulation.We also demonstrate that the BmE2F4 gene is involved in DNA replication of BmN-SWU1 cells and DNA synthesis in the silk gland.Furthermore,we identified a protein called Bm14-3-3ζthat can interact with BmE2F4 and allow it to localize in the nucleus.Overexpression of the Bm14-3-3ζgene led to cell cycle arrest in the G1 phase,while knocking down the gene increased the proportion of cells in S phase.These findings provide important insights into the function of E2F transcription factors and increase our understanding of their involvement in cell cycle regulation.

Sob gene is critical to wing development in Bombyx mori and Tribolium castaneum

The development of insect appendages requires the expression of multiple genes in a strict spatial and temporal order. The odd-skipped family genes are vital transcriptional factors involved in embryonic development. The development and morphogenesis of the insect wing requires multiple transcription factors to regulate the expression of wing patterning genes at the transcriptional level. However, the function of odd-related genes in insect wing morphogenesis and development during postembryonic stages is unclear. We focused on the roles of the sister of odd and bowl (sob) gene, a member of odd-skipped family genes, during the wing morphopoiesis in Bombyx mori using the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 system and in Tribolium castaneum by RNA interference. The results showed that the wings were significantly smaller and degenerated, and wing veins were indistinct in the sob gene loss-of-function group in both B. mori and T. castaneum. Quantitative real-time polymerase chain reaction revealed that the Tcsob gene regulated the expression of wing development genes, such as the cht 7 and the vg gene. The findings suggest the importance of sob gene in insect wing morphology formation during postembryonic stages.

Bmelo12,an elongase of very long-chain fatty acids gene,regulates silk yield in Bombyx mori

In silkworm,cocoon shell weight(CSW),an index of silk yield,is a quantitative trait(Nagaraju and Goldsmith,2002),and multiple quantitative trait loci(QTLs)located on different chromosomes have been found(Zhan et al.,2009;Zhang et al.,2010;Li et al.,2015;Fang et al.,2020).