CRISPR mediated transactivation in the human disease vector Aedes aegypti
Abstract
As a major insect vector of multiple arboviruses,Aedes aegyptiposes 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 inAe.aegyptito 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 engineeredAe.aegyptimosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes,even-skipped (eve)andhedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known asAaRel1, 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 inAe.aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Jan 19, 2023
- Source ID
- 10.1371/journal.ppat.1010842
Entities
People
- Anna B Buchman
- Anthony A. James
- Elena Dalla Benetta
- George Dimopoulos
- Igor A. Antoshechkin
- Michael W Perry
- Michelle Bui
- Ming Li
- Omar S. Akbari
- Ting Yang
- Vanessa Bottino-rojas
- Yuemei Dong
- Yunchong Zhao
Organizations
- Division of Intramural Research, National Institute of Allergy and Infectious Diseases