CRISPR-Cas9 mediated knockout of the white gene in the bluetongue virus vector, Culicoides sonorensis (biting midge)

CRISPR/Cas9-mediated transformation enables functional characterization of the effector Avr4 in the banana pathogen Pseudocercospora fijiensis

It is demonstrated that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors.

Comparative evaluation of pKSE401 and pHSE401 gRNA constructs for CRISPR/Cas9-Mediated resistance to cotton leaf curl virus

Cotton leaf curl disease (CLCuD), caused by begomoviruses and their betasatellites, is a major threat to cotton production, especially in South Asia, where periodic viral outbreaks continue to affect cotton yields, quality, and livelihoods. The advent of CRISPR/Cas9 gene-editing technology has transformed plant biotechnology, offering efficient, accurate, and programmable methods for combating viral pathogens at the genetic level. Here, the antiviral efficacy of two most widely used CRISPR/Cas9 binary plant expression vectors, pKSE401 and pHSE401, was tested in Nicotiana benthamiana against Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan betasatellite (CLCuMuB). The guide-RNAs (gRNAs) were designed to target viral genes that play significant roles in pathogenicity and replication, with pHSE401 encoding a single gRNA and pKSE401 a multiplex of two gRNAs. Agrobacterium-mediated transient transformation and viral inoculation experiments revealed that both CRISPR/Cas9 vectors effectively delayed symptom onset and reduced virus titers relative to infected controls. Remarkably, the multiplex pKSE401 system was more effective at suppressing viral infection, achieving about a 90% reduction in viral accumulation compared with a 75% reduction by the single gRNA pHSE401 construct. pKSE401-treated plants showed delayed symptom development, reduced severity, and partial recovery, demonstrating the improved efficiency of multiplex genome editing. The results demonstrate the cutting-edge potential of CRISPR/Cas9 multiplex approaches as next-generation methods for designing sustainable resistance to multifaceted plant virus diseases. This research not only contributes to our understanding of CRISPR-based antiviral response mechanisms but also provides a promising avenue for designing broad-spectrum, sustainable resistance against viral epidemics in cotton and other commercially valuable crops.

CRISPR/Cas9 gene editing in the heterotrophic protist Acanthamoeba castellanii

Sequence Homology and Tissue Tropism Determine Superinfection Exclusion of Zika virus in Aedes aegypti Mediated by an insect-specific Binjari-Zika Virus Chimera

Arboviruses such as dengue, Zika, and chikungunya viruses cause widespread disease and continue to expand their geographical range due to climate change and vector spread. Insect-specific flaviviruses (ISFs) are promising biocontrol candidates of arboviruses, due to recent studies showing that prior infection with an ISF can reduce arbovirus replication in mosquitoes through superinfection exclusion (SIE). However, the route of infection, tissue tropism, pathogenesis and the mechanisms underlying SIE of ISFs in mosquitoes remain unclear. RNA interference (RNAi) is a potent antiviral response in insects, therefore it is expected that sequence homology between the ISF and the arbovirus will strengthen SIE. Here, we used ISF Binjari virus and a chimera containing the Zika virus structural proteins prME (BinJ-ZIKV) as a model system. Intrathoracic injection of BinJ-ZIKV in Aedes aegypti led to rapid systemic infection that excluded the midgut, subsequently blocking ZIKV dissemination from the midgut. SIE was strongest in tissues where primary-virus replication was highest. This spatial component of SIE was stronger when there was sequence homology between the ISF and arbovirus and displayed a strong 21nt siRNA response, suggesting RNAi contributed to the observed SIE. Upon oral inoculation, BinJ-ZIKV replicated efficiently in mosquitoes, was detected across multiple tissues, and saliva. BinJ-ZIKV also had higher infection establishment than BinJV at lower oral titres. SIE was observed for BinJ-ZIKV infection after oral exposure interfered with subsequent ZIKV midgut infection. Together, these findings support engineered ISF-chimeras as valuable experimental tools to dissect viral determinants of SIE and to optimize mosquito-based arbovirus interference strategies. Importance Annually, over 400 million people are infected with mosquito-transmitted viruses. Insect-specific flaviviruses (ISFs) can interfere with the transmission of clinically important viruses through a phenomenon termed superinfection exclusion (SIE). However, the mechanisms of SIE remain poorly understood. Using a Binjari virus chimera expressing Zika virus (ZIKV) structural proteins, we show that SIE is highly tissue-specific, with exclusion of ZIKV only occurring at sites where the chimera actively replicates and induces the mosquito antiviral RNA interference pathway. We further demonstrate that incorporation of Zika virus prM and E proteins into the ISF backbone enhances infection of the mosquito midgut following oral exposure, which enables direct inhibition of ZIKV infection after a subsequent infectious blood meal. Together, these findings define a replication-dependent, tissue-specific mechanism of ISF-mediated protection and provide a framework for reducing mosquito-borne virus transmission through SIE.

The efficacy of RNA interference against the microsporidia Vairimorpha ceranae in honeybees Apis mellifera. Can it be enhanced to the level of antiviral and anti-mite activity shown for the Remebee-I and Vadescana dsRNA?

Microsporidia Vairimorpha ceranae are virulent pathogens of the European honeybee Apis mellifera. About ten studies have confirmed that RNA interference (RNAi)-based silencing of the parasite’s genes inhibits microsporidia growth. To further evaluate the RNAi effectiveness against V. ceranae and methods for enhancing it, we synthesized in vitro double-stranded RNA (dsRNA) fragments of five genes encoding microsporidian DNA replication enzymes, their chimeric variants composed of predicted small interfering RNAs (siRNAs), and the fragments of four host genes. Fragments of the parasite’s genes encoding the PTP3 and SWP8 proteins, effective against V. ceranae at low concentrations, were used as positive controls. Ten-day feeding of individually infected and individually maintained young bees with dsRNA was followed by spore counting. This demonstrated (1) increased mortality of individually housed bees, (2) more effective infection suppression at lower dsRNA concentrations, and (3) the effectiveness of siRNA-containing chimeras and a gene fragment encoding a putative host apoptosis inhibitor in reducing spore load. Unfortunately, we were unable to enhance the RNAi efficiency by mixing the V. ceranae PTP3 fragment with polyethyleneimine and chitosan polymers, and V. ceranae growth was also unaffected when hive-derived bees of different ages were fed with E. coliexpressed dsRNA, effective in young insects. Analysis of the RNAi application to control bee pathogens indicates the need to further improve the efficiency of V. ceranae inhibition by searching for new target genes, optimizing the fragment concentration in syrup, and using the most effective dsRNA or siRNA, nanoparticles and liposomes.

A red-eye mutant in Nesidiocoris tenuis (Hemiptera: Miridae) is caused by exon skipping due to an indel mutation in the scarlet gene

Nesidiocoris tenuis is an important zoophytophagous mirid bug used as a biological control agent in agriculture, and breeding efforts based on genomic information aim to increase its utility. Visible eye-color mutants are useful genetic markers because they are easily distinguishable and are therefore widely used in insect genetics and genome editing studies. Here, we investigated the genetic basis of a spontaneous red-eye mutant identified in a laboratory strain of N. tenuis. Classical crossing experiments suggested that the red-eye phenotype is controlled by a single recessive locus. RNA-seq and RNA interference (RNAi) analyses identified scarlet and cinnabar as the primary candidate genes associated with the phenotype. Further genomic analysis revealed a large deletion and insertion within exon 5 of the mutant scarlet allele, potentially causing exon skipping and disrupting transporter structure. The insertion pattern is consistent with a microhomology-mediated break-induced replication (MMBIR)/fork stalling and template switching (FoSTeS)-like event that may have been generated through polymerase θ-mediated repair. Together, these findings identify the causative mutation underlying the red-eye phenotype and provide a useful visible marker for future functional genetic studies and genome-assisted breeding in N. tenuis.