Fig 1.
CRISPRa system transgenic line design, genetic crosses, and marker expression in Ae. aegypti.
A) A binary CRISPRa system was designed using two separate transgenic Ae. aegypti lines that when crossed, result in transheterozygous individuals expressing both dCas9-VPR and sgRNAs and have upregulated expression of the target gene. B) The transgenic lines used in this study include one line expressing dCas9-VPR under a polyubiquitin promoter, a sgRNA line targeting eve, and a sgRNA line targeting hh. The sgRNA lines were designed to express two distinct sgRNAs targeting the same promoter region of the respective target gene. C) dCas9 and sgRNA lines were marked with opie2-dsRed and 3xP3-tdTomato, respectively.
Fig 2.
Quantified overexpression of CRISPRa transactivation.
A) Multiple crosses were performed in order to determine if the CRISPRa system could transactivate target genes. Crosses included a wild-type control (+/+ ♀ X +/+ ♂, I), dCas9-VPR Control (+/+ ♀ X Pub:dCas9-VPR/+ ♂, II), sgRNA control (+/U6:sgRNA ♀ X +/+ ♂, III), and a transheterozygous cross (+/U6:sgRNA ♀ X Pub:dCas9-VPR/+ ♂, IV). B) Fold change of expression of targeted genes from CRISPRa transactivation was measured and calculated using qPCR. Differences in mRNA fold change between control crosses and transheterozygous crosses were calculated using a one-way ANOVA and Tukey’s multiple-comparison test, ****p < 0.0001 (F3,8 = 659.7; P = 6.46e-10;F3,8 = 82.67; P = 2.32e-06 for eve and hh respectively). C) Volcano plots of the RNA sequencing data were to compare expression levels of eve and hh as well as other genes potentially affected. Down-regulated genes are colored blue while upregulated genes are in red. Potential off-target genes are in yellow.
Fig 3.
Phenotypic observations of CRISPRa mediated transactivation of eve and hh.
A) Surviving transheterozygous progeny were collected and showed no significant external morphological differences to wild-type larvae. B) The proportions of transheterozygous progeny were calculated from single pair crosses between heterozygous dCas9-VPR and sgRNA parents. Rates of inheritance were compared to an expected rate of Mendelian inheritance, 25% transheterozygous individuals among total offspring, using a Chi-square test (****p < 0.0001). C) Further imaging of HCR in situ hybridization of hh probes showed upregulated and misregulated expression of hh compared to WT, while eve expression stayed relatively the same as that of WT.
Fig 4.
CRISPRa mediated transactivation of Toll immune pathway, RT-PCR validation of AaRel1 overexpression and suppression of viral infection.
A) Schematic representation of the sgRNA AaRel1 construct used to create the U6:sgRNA-AaRel1 lines. B) Schematic representation of the transgenic line expressing Pub:dCas9-VPR and the U6:sgRNA targeting the promoter region of AaRel1 gene and inducing the activation of the Toll immune pathway. C) Quantification by qPCR of AaRel1 gene expression in controls and transactivated lines. A one-way ANOVA and Tukey’s multiple-comparison test was performed between transheterozygous crosses and controls. *P < 0.05, **P < 0.01, ***P < 0.001 D) Antiviral effect of CRISPRa mediated transactivation of AaRel1 in the transheterozygous progeny. sgRNA-AaRel1 line A and B (sgRNA-AaRel1-A and sgRNA-AaRel1-B, or named as U6:sgRNArel1-A and U6:sgRNArel1-B) and transheterozygotes (dCas9-VPR/sgRNA-AaRel1-A and dCas9-VPR/sgRNA-AaRel1-B or named as dCas9-VPR/U6-sgRNArel1-A and dCas9-VPR/U6-sgRNArel1-B) were orally infected with DENV2 as illustrated. E) The viral infection titers and infection prevalence of DENV2 were measured after midgut infection at 7 days post-infectious blood meal (PIBM). Plaque assays were used to determine viral titers and infection prevalence in individual mosquitoes with each dot representing the viral load from one midgut, and pie-chart indicating the infection prevalence. at At least three replicates were pooled for the statistical analyses. Horizontal black lines indicate the median of the viral loads. Considering the non-normal distribution of viral titers, median is used to describe central tendency and non-parametric Mann-Whitney test was used to compare median viral titers and Fisher’s exact test to compare infection prevalence. * P < 0.05, ** P < 0.01, n: total numbers of mosquitoes used in the assays. sgRNA-AaRel1 lines were used as controls. Created with BioRender.com.