Bui, Michelle and Dalla Benetta, Elena and Dong, Yuemei and Zhao, Yunchong and Yang, Ting and Li, Ming and Antoshechkin, Igor A. and Buchman, Anna and Bottino-Rojas, Vanessa and James, Anthony A. and Perry, Michael W. and Dimopoulos, George and Akbari, Omar S. (2023) CRISPR mediated transactivation in the human disease vector Aedes aegypti. PLOS Pathogens, 19 (1). Art. No. e1010842. ISSN 1553-7374. PMCID PMC9888728. doi:10.1371/journal.ppat.1010842. https://resolver.caltech.edu/CaltechAUTHORS:20230322-367894000.33
Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20230322-367894000.33
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.
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Additional Information: | © 2023 Bui et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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. The views, 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. This project has been funded in part by a DARPA Safe Genes Program Grant under contract number, HR0011-17-2-0047 and NIH awards, R01AI151004, DP2AI152071, and R21AI149161 awarded to OSA, and R01AI141532 awarded to GD. Our funders had no role in study design, data collection and analysis, decision to publish, or the preparation of this manuscript. Data Availability: All plasmids and annotated DNA sequence maps are available at www.addgene.com under accession numbers: #183993, #100581, #184006, #184007, #120363, #190997. Raw sequencing data are available at NCBI Sequence Read Archive, at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA851480. Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: 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. The authors report no other conflict of interest. | ||||||||||||||||||||||
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Issue or Number: | 1 | ||||||||||||||||||||||
PubMed Central ID: | PMC9888728 | ||||||||||||||||||||||
DOI: | 10.1371/journal.ppat.1010842 | ||||||||||||||||||||||
Record Number: | CaltechAUTHORS:20230322-367894000.33 | ||||||||||||||||||||||
Persistent URL: | https://resolver.caltech.edu/CaltechAUTHORS:20230322-367894000.33 | ||||||||||||||||||||||
Usage Policy: | No commercial reproduction, distribution, display or performance rights in this work are provided. | ||||||||||||||||||||||
ID Code: | 120347 | ||||||||||||||||||||||
Collection: | CaltechAUTHORS | ||||||||||||||||||||||
Deposited By: | George Porter | ||||||||||||||||||||||
Deposited On: | 26 Mar 2023 21:09 | ||||||||||||||||||||||
Last Modified: | 26 Mar 2023 21:09 |
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