A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines

Abstract Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very...

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Autores principales:	Kathryn Rozen-Gagnon, Soon Yi, Eliana Jacobson, Sasha Novack, Charles M. Rice
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/9ed171dfbd374e46b51b46fd22866552
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spelling	oai:doaj.org-article:9ed171dfbd374e46b51b46fd228665522021-12-02T14:12:42ZA selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines10.1038/s41598-020-80436-52045-2322https://doaj.org/article/9ed171dfbd374e46b51b46fd228665522021-01-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-80436-5https://doaj.org/toc/2045-2322Abstract Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.Kathryn Rozen-GagnonSoon YiEliana JacobsonSasha NovackCharles M. RiceNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kathryn Rozen-Gagnon Soon Yi Eliana Jacobson Sasha Novack Charles M. Rice A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines
description	Abstract Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.
format	article
author	Kathryn Rozen-Gagnon Soon Yi Eliana Jacobson Sasha Novack Charles M. Rice
author_facet	Kathryn Rozen-Gagnon Soon Yi Eliana Jacobson Sasha Novack Charles M. Rice
author_sort	Kathryn Rozen-Gagnon
title	A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines
title_short	A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines
title_full	A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines
title_fullStr	A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines
title_full_unstemmed	A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines
title_sort	selectable, plasmid-based system to generate crispr/cas9 gene edited and knock-in mosquito cell lines
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/9ed171dfbd374e46b51b46fd22866552
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A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines

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