CRISPR mediated transactivation in the human disease vector Aedes aegypti

Abstract

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) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.

tgCRISPRi: efficient gene knock-down using truncated gRNAs and catalytically active Cas9

“…As an example of the latter use, including a tgRNA to suppress expression of the FREP1 gene in developing gut cells of Anopheline mosquitoes could help reduce transmission of malarial parasites since the FREP1 protein plays an important role in these parasites transiting the gut epithelium into the body cavity to gain access to salivary glands 51 . Furthermore, Cas9-VPR and tgRNA components could be employed to overexpress immune pathway genes in mosquitos to minimize pathogen survival 52 . Multiple factors need to be considered when developing tgCRISPR-augmented gene-drive systems.…”

CRISPR-Cas9 Genome Editing Technology for Zoonotic Disease Control in Indonesia: A Comprehensive Review

“…The implications of applying CRISPR-Cas9 genome-editing technology for targeting disease vectors are vast and profound, particularly in the realm of public health. This technology has the potential to be utilized for the control of vectorborne diseases [73,74]. These diseases, such as malaria, dengue fever, Zika virus, Lyme disease, Japanese B. encephalitis, and Q fever, pose significant health threats in Indonesia.…”

“…In addition, CRISPR technology helps reduce the dependence on insecticides, larvicides, pesticides, and other vector life-disrupting substances [74]. Traditional vector control methods, such as insecticides, larvicides, and pesticides, can have negative impacts on the environment and contribute to the development of resistance in broad-spectrum vectors that have harmful effects on nontarget organisms and ecosystems [83,84].…”