Infected Mosquitoes Have Altered Behavior to Repellents: A Systematic Review and Meta-analysis.

Here we conducted a systematic review and meta-analysis to reach a consensus on whether infected and uninfected mosquitoes respond differently to repellents. After screening 2,316 published studies, theses, and conference abstracts, we identified 18 studies that tested whether infection status modulated the effectiveness of repellents. Thirteen of these studies had outcomes available for meta-analysis, and overall, seven repellents were tested (typically DEET with 62% of outcomes), six mosquito species had repellence behaviors measured (typically Aedes aegypti (L.) (Diptera: Culicidae) mosquitoes with 71% of outcomes), and a broad diversity of infections were tested including Sindbis virus (Togaviridae: Alphavirus) (33% of outcomes), Dengue (Flaviviridae: Flavivirus) (31%), malaria (Plasmodium berghei Vincke & Lips (Haemospororida: Plasmodiidae) or P. falciparum Welch (Haemospororida: Plasmodiidae); 25%), Zika (Flaviviridae: Flavivirus) (7%), and microsporidia (4%). Pooling all outcomes with meta-analysis, we found that repellents were less effective against infected mosquitoes-marking an average 62% reduction in protective efficacy relative to uninfected mosquitoes (pooled odds ratio = 0.38, 95% confidence interval = 0.22-0.66; k = 96). Older infected mosquitoes were also more likely to show altered responses and loss of sensitivity to repellents, emphasizing the challenge of distinguishing between age or incubation period effects. Plasmodium- or Dengue-infected mosquitoes also did not show altered responses to repellents; however, Dengue-mosquito systems used inoculation practices that can introduce variability in repellency responses. Given our findings that repellents offer less protection against infected mosquitoes and that these vectors are the most dangerous in terms of disease transmission, then trials on repellent effectiveness should incorporate infected mosquitoes to improve predictability in blocking vector-human contact.

Frequency of off-targeting in genome edited pigs produced via direct injection of the CRISPR/Cas9 system into developing embryos.

BACKGROUND: The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined. RESULTS: First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5' side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5' side, indicating unpredictability of the events. CONCLUSIONS: The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics.