CRISPR Mediated Transactivation in the Human Disease Vector Aedes aegypti
As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive ('dead') Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2). This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may lead to the development of innovative vector control tools.
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. We thank Judy Ishikawa for mosquito husbandry assistance. We thank the Johns Hopkins Malaria Research Institute Insectary for providing the mosquito-rearing facility and the Parasitology Core facilities for providing the naïve human blood. Figures were Created with BioRender.com. This work was supported by funding from a DARPA Safe Genes Program Grant (HR0011-17-2-0047), and NIH awards (R01AI151004, DP2AI152071, and R21AI149161) awarded to O.S.A, and NIH award R01AI141532 awarded to G.D. The iews, opinions, and/or findings expressed are those of the authors and should not be interpreted as representing the official views or policies of the U.S. government. AAJ is a Donald Bren Professor at the University of California, Irvine. Author Contributions. O.S.A., A.A.J., and G.D. conceived and designed the experiments. M.B., E.D.B., Y.D., Y.Z., M.L., T.Y., A.B., and V.B. performed molecular and genetic experiments. I.A. performed the RNA sequencing experiments and analysis. M.W.P and Y.Z., performed in situ hybridization experiments. YD performed viral infection experiments. All authors contributed to the writing, analyzed the data, and approved the final manuscript. Competing Interest Statement. O.S.A is a founder of both Agragene, Inc. and Synvect, Inc. with equity interest. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. All other authors declare no competing interests.
Submitted - 2022.08.31.505972v1.full.pdf