CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say

Abstract Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical...

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Autores principales:	Deepak-Kumar Purusothaman, Lewis Shackleford, Michelle A. E. Anderson, Tim Harvey-Samuel, Luke Alphey
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/403f6a669c9d407497a486fafd81472c
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spelling	oai:doaj.org-article:403f6a669c9d407497a486fafd81472c2021-12-02T16:17:34ZCRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say10.1038/s41598-021-94065-z2045-2322https://doaj.org/article/403f6a669c9d407497a486fafd81472c2021-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-94065-zhttps://doaj.org/toc/2045-2322Abstract Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.Deepak-Kumar PurusothamanLewis ShacklefordMichelle A. E. AndersonTim Harvey-SamuelLuke AlpheyNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Deepak-Kumar Purusothaman Lewis Shackleford Michelle A. E. Anderson Tim Harvey-Samuel Luke Alphey CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say
description	Abstract Culex quinquefasciatus Say is a mosquito distributed in both tropical and subtropical regions of the world. It is a night-active, opportunistic blood-feeder and vectors many animal and human diseases, including West Nile Virus and avian malaria. Current vector control methods (e.g. physical/chemical) are increasingly ineffective; use of insecticides also imposes hazards to both human and ecosystem health. Advances in genome editing have allowed the development of genetic insect control methods, which are species-specific and, theoretically, highly effective. CRISPR/Cas9 is a bacteria-derived programmable gene editing tool that is functional in a range of species. We describe the first successful germline gene knock-in by homology dependent repair in C. quinquefasciatus. Using CRISPR/Cas9, we integrated an sgRNA expression cassette and marker gene encoding a fluorescent protein fluorophore (Hr5/IE1-DsRed, Cq7SK-sgRNA) into the kynurenine 3-monooxygenase (kmo) gene. We achieved a minimum transformation rate of 2.8%, similar to rates in other mosquito species. Precise knock-in at the intended locus was confirmed. Insertion homozygotes displayed a white eye phenotype in early-mid larvae and a recessive lethal phenotype by pupation. This work provides an efficient method for engineering C. quinquefasciatus, providing a new tool for developing genetic control tools for this vector.
format	article
author	Deepak-Kumar Purusothaman Lewis Shackleford Michelle A. E. Anderson Tim Harvey-Samuel Luke Alphey
author_facet	Deepak-Kumar Purusothaman Lewis Shackleford Michelle A. E. Anderson Tim Harvey-Samuel Luke Alphey
author_sort	Deepak-Kumar Purusothaman
title	CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say
title_short	CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say
title_full	CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say
title_fullStr	CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say
title_full_unstemmed	CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say
title_sort	crispr/cas-9 mediated knock-in by homology dependent repair in the west nile virus vector culex quinquefasciatus say
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/403f6a669c9d407497a486fafd81472c
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CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say

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