Aedes mosquitoes (Aedes aegypti and Aedes albopictus) are principal vectors for transmitting several arthropod borne viral diseases to humans which includes dengue, zika, yellow fever and chikungunya. These diseases disproportionately affect the poorest populations causing substantial morbidity and mortality. Although currently distributed throughout the tropical and subtropical regions of the world (Kraemer et al., 2015), Aedes mosquitoes are predicted to invade new geographical areas owing to higher adaptability and climate change (Iwamura et al., 2020). Spread of mosquito vectors poses threat of spreading of mosquito borne diseases beyond the tropical regions of the world. Over the past decade, the re-emergence of these viral diseases and epidemics has been a cause of global concern. In absence of effective vaccines or therapy, control of the mosquito vector is vital to reduce the disease burden and protect people from infection. While traditional interventions to control mosquito population have limited success, genetic approaches have shown promising results (Wang et al., 2021). With increasing availability of the “omics” data, it is now feasible to gain mechanistic insights into the role of host factors in virus life cycle and develop rational genetic strategies for vector control and disease management. However, the genetic tools and technologies to manipulate the mosquito genome efficiently are lacking.

The CRISPR-Cas9 system, repurposed from the bacterial adaptive immune system, is a two component RNA-directed DNA cleavage system that consists of a Cas9 endonuclease and a non-coding single guide RNA (sgRNA) containing a 20 nt DNA targeting sequence (Jinek et al., 2012). The Cas9-sgRNA complex binds to the complimentary strand of DNA adjacent to a conserved three nucleotide protospacer adjacent motif (PAM) and creates a blunt-ended double-stranded break that results in indel (insertion-deletion) mutations due to imperfect repair by the non-homologous end joining (NHEJ) DNA repair pathway. In addition to DNA editing, nuclease deficient “dead” Cas9 (dCas9) has been fused with effectors and used as synthetic transcription factors (La Russa and Qi, 2015). For example, fusing dCas9 protein with either transcription activators (VP16, p65, rta, VPR, etc.) or repressor (KRAB, MeCP2, Mxi, etc.) domains led to ectopic activation (CRISPR activation system; CRISPRa) or repression (CRISPR interference system; CRISPRi) of the target genes, respectively (Larson et al., 2013; Gilbert et al., 2014; Ghosh et al., 2016; Liao et al., 2017). In addition, diverse CRISPR systems have been adapted successfully for targeting DNA and RNA for various applications (Pickar-Oliver and Gersbach, 2019). While CRISPR-based strategies have been used to modify the Aedes genome (Kistler et al., 2015; Li et al., 2017; Liu et al., 2019; Rozen-Gagnon et al., 2021), its use in modulating gene expression using dCas9-based systems has not been explored in Aedes cells.

Here, we report construction of a versatile plasmid-based system that allows rapid testing of transcription upregulation of genes in Aedes cells. Using a fluorescent reporter based assay we demonstrate a robust gene over expression, thus validating the CRISPRa system in the mosquitoes.

We set out to establish the CRISPR activation (CRISPRa) system to over-express genes in the Aedes mosquito cells. To implement CRISPRa, we chose dCas9 fused with VPR (VPR is a tripartite activator composed of the VP64, p65, and Rta domains) as transcriptional activator (Chavez et al., 2015) and designed a bi-partite plasmid system for functional evaluation of dCas9-VPR activity using fluorescent reporter assay in A. albopictus C6/36 cells (Figures 1A, B). In this scheme, one of the plasmid (pJVG1) encodes the effector protein dCas9-VPR while the other plasmid (pJVG2) transcribes the 20 nt non-coding sgRNA targeting a heterologous sequence (tertracycline operator sequence from bacteria; tetO). Upon co-transfection, the dCas9-VPR-sgRNA complex is formed in the cells and binds the tetO sequence positioned upstream of a RNA polymerase II promoter on pJVG2 plasmid resulting in transcription upregulation of the fluorescent protein mCherry whose levels can be determined quantitatively (Figure 1C).

The dCas9-VPR encoding plasmid pJVG1 is designed to consist of a multicistronic expression cassette with dual selection markers. As shown in Figure 1A, the dCas9-VPR gene, flanked with NLS sequences and tagged with FLAG epitope at the N-terminal end, is followed by the puromycin acetyltransferase (pac) and eGFP encoding gene. The expression of this cassette is under the control of the strong constitutively active Aedes PUb promoter and SV40 polyadenylation sequence. This architecture allows antibiotic as well as fluorescence-based selection of the transfected cells for transient cell-based assays. In addition, to generate stable cell lines rapidly and efficiently, the transfected cells can be sorted by FACS analysis followed by incubation with puromycin. Finally, the eGFP fluorescence in the transfected cells is indicative of dCas9-VPR expression.

Our vector design allows transcription of a single mRNA encoding simultaneous co-expression of the dCas9-VPR, puromycin acetyltransferase and eGFP proteins. To separate individual gene products, viral 2A peptides were chosen which mediate peptide cleavage due to ribosome skipping between a conserved glycine and proline residues at the C-terminus of the 2A element (Ryan and Drew, 1994). Although 2A peptides have been used to derive multiple proteins from a single transcript (Liu et al., 2017), the efficiency of peptide cleavage varies in different cell types and organisms (González et al., 2011; Daniels et al., 2014; Souza-Moreira et al., 2018). Importantly, the efficiency of 2A peptides in co-translational cleavage of proteins in Aedes has not been determined. Since our plasmid design requires 2A peptides (two in pJVG1 and one in pJVG2), we tested the efficacy of T2A and dT2A elements in Aedes cells. For this we generated a bi-cistronic reporter construct wherein the fluorescent proteins eGFP and mCherry are divided by either T2A or dT2A sequences (Figure 2A). Fluorescence microscopy analysis of the transfected cells showed green (eGFP) and red (mCherry) signals (Figure 2B–D”) showing production of functional proteins. To ascertain if the cleavage is efficient, Western blotting was performed using the extracts prepared from the transfected cells. As shown in Figure 2E, both proteins are cleaved efficiently in presence of T2A and dT2A peptides, thus validating their function in Aedes cells. Both T2A and dT2A sequences were incorporated in the DNA vectors for the dCas9-VPR assay design.

We next designed the transfection vector pJVG2 to express the guide RNA and the fluorescent reporter cassette (Figure 1B). Recruitment of dCas9-VPR protein upstream of a promoter leads to transcription upregulation of the genes regulated by the promoter (Chavez et al., 2015). To accomplish this, we reasoned that the recruitment of dCas9-VPR fusion protein at the 5’ end of a promoter with weak or low basal activity but high inducibility is desirable. However, a comparative analysis of the strength of promoter activity in Aedes cells is not available. Therefore, we performed a fluorescent reporter based promoter activity assay wherein the eGFP coding region was cloned downstream of three constitutive promoters (Figure 3) and the DNA constructs used to transfect Aedes C6/36 cell line individually. Analysis of the eGFP signal strength by flow cytometry analysis revealed that, as compared with the highly active PUb (polyubiquitin) promoter, UBL40 (ubiquitin fusion ribosomal protein L40) promoter showed lower activity while the baculovirus polyhedrin promoter was least active in Aedes cells. We chose to express the mCherry reporter gene under the control of UbL40 promoter to generate the reporter cassette in pJVG2.

To engage dCas9-VPR protein upstream of the pUBL40, we selected the conserved 19 bp tetracycline operator (tetO) sequence which is derived from the tetracycline operon in bacteria. This sequence is heterologous to Aedes cells and likely to have low off-target effects with respect to dCas9-VPR binding. To implement the synergistic effect of dCas9-VPR activity, seven copies of the tetO sequence was cloned at the 5’ end of the UbL40 promoter in pJVG2 (Figure 1B). In addition, the guide RNA expression cassette consisting of the 20 nt tetO targeting sequence along with an optimized guide RNA scaffold sequence was placed under the control of the RNA polymerase III promoter 7SK which showed constitutive and robust expression in A. albopictus cells (Anderson et al., 2020).

The plasmids pJVG1 and pJVG2 (Figures 1A, B) were assembled using Infusion DNA assembly method and validated by Sanger sequencing. To evaluate dCas9-VPR function as a transcription activator, A. albopictus C6/36 cells were transfected with the dCas9-VPR expressing plasmid pJVG1, the sgRNA-reporter containing plasmid pJVG2 or both pJVG1 and pJVG2 together. Cells were imaged 48 h post-transfection under a fluorescent microscope and samples collected to quantify the levels of mCherry mRNA and protein using RT-qPCR and flow cytometry analysis, respectively. The dCas9-VPR gene was expressed in Aedes cells as inferred from the eGFP expression (Figure 4A'). As shown in Figure 4B, we observed substantial mCherry expression in cells transfected with the pJVG2 plasmid. However, the intensity of mCherry appeared to be higher in the cells co-transfected with pJVG1 and pJVG2 suggesting increased protein levels (Figure 4C). Indeed, qPCR analysis revealed a 10-fold increase in the mCherry mRNA levels in the cells co-expressing dCas9-VPR and tetO sgRNA as compared with cells expressing the reporter construct alone (Figure 4D). This data demonstrates transcriptional activation of mCherry gene in a dCas9-VPR dependent manner. Furthermore, flow cytometry analysis shows a similar 10-fold increase in mCherry signal intensity in the co-transfected cells (Figure 4E, Supplementary Figure S1) which co-relates well with the increased mRNA levels. Taken together, this provides the proof of concept of dCas9-VPR based gene over-expression in the Aedes cells.

In this study we describe the generation of plasmids designed for evaluation of CRISPR activation system in Aedes cells. The architecture of both plasmids allows dual selection of the co-transfected cells using fluorescence-based assays and antibiotics-based selection. These features permit rapid generation of stable cells lines. In addition, we show the efficacy of viral 2A peptide sequences in Aedes cells for the first time which should facilitate co-expression and cleavage of multiple gene products from one construct. Finally, our results demonstrate functional validation of dCas9-VPR activity in transcriptional upregulation of genes in Aedes cells. This proof of concept study paves way for gain-of-function CRISPRa genetic screens aimed to identify mosquito host factors and gain mechanistic insights into vector competence. Moreover, the DNA vectors generated in this study are an important resource for up regulating anti-viral host factors in Aedes mosquitoes for vector control and disease management. In the future, CRISPR activation can be used to engineer mosquitoes with desirable traits aimed to minimise the impact of vector-borne diseases in humans (Buchman et al., 2020). However, the implementation of this technology must comply with the appropriate regulatory framework and involve public engagement (Scudellari, 2019; World Health Organization, 2021; Meghani, 2022).