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bioRxiv · 10.1101/2023.08.14.553172

Gene editing in the Chagas disease vector Rhodnius prolixus by Cas9-mediated ReMOT Control

Lima, L.·Berni, M.·Mota, J.·Bressan, D.·Julio, A.·Cavalcante, R.·Macias, V.·Li, Z.·Rasgon, J. L.·Bier, E.·Araujo, H. M.
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Abstract

Rhodnius prolixus is currently the model vector of choice for studying Chagas disease transmission, a debilitating disease caused by Trypanosoma cruzi parasites. Despite the broad use of double-stranded RNA interference for the knockdown of gene function in R. prolixus, transgenesis and gene editing protocols are still lacking. Here we tested Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) and direct parental injection of CRISPR (DIPA-CRISPR) for the maternal delivery of CRISPR/Cas9 elements to developing R. prolixus oocytes, and strategies for the identification of insertions/deletions (indels) in target loci of resulting gene-edited G0 nymphs. We demonstrate successful ReMOT Control-mediated gene editing of the eye color markers Rp-scarlet and Rp-white, and the cuticle color marker Rp-yellow, with highest effectiveness obtained using the ovary-targeting BtKV ligand. These results provide proof-of-concepts for generating somatic mutations in R. prolixus and potentially for generating germline edited lines in triatomines. Our studies also suggest that optimal strategies for recovery of mutations include performing multiple gRNA injections and the use of visible phenotypes such as those displayed in the Rp-scarlet, Rp-white and Rp-yellow loci for future Co-CRISPR experiments. These results will lay the foundation for gene editing protocols for triatomines and could lead to the development of novel control strategies for vectors of Chagas disease. Author SummaryRhodnius prolixus is an insect vector of the protozoan Trypanossoma cruzi, causative agent of debilitating Chagas disease. To fight the spread of the disease, it has been suggested that biological control of the insect should be attempted. Gene editing by the novel CRISPR methodology holds great promise in this sense, as it enables to target almost any gene in the genome for mutagenesis, thus allowing the control of insect physiology and reproduction. Here we have tested protocols for the delivery of CRISPR reagents as an attempt to enable genome editing of the vector. Our results show that maternal delivery of CRISPR by the ReMOT Control method is efficient for mutating eye and cuticle color genes in the resulting nymphs, generating edited animals with red eyes, white eyes or a yellow cuticle. This is the first report of gene editing in a vector of Chagas disease and should lay the basis to produce modified animals either unable to carry the T. cruzi parasite or to reproduce.

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Lima, L., Berni, M., Mota, J., Bressan, D., Julio, A., Cavalcante, R., Macias, V., Li, Z., Rasgon, J. L., Bier, E., Araujo, H. M.. 2023-08-15. Gene editing in the Chagas disease vector Rhodnius prolixus by Cas9-mediated ReMOT Control. https://doi.org/10.1101/2023.08.14.553172

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