Generation of Cas9 Knock-In <i>Culex quinquefasciatus</i> Mosquito Cells
<b>Background/Objectives</b>: <i>Culex</i> species mosquitoes are globally distributed and transmit several pathogens that impact animal and public health, including West Nile virus, Usutu virus, and <i>Plasmodium relictum</i>. Despite their relevance, <i>Cu...
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2025-01-01
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| author | Elizabeth Walsh Tran Zen B. Torres Brian C. Prince Claudia Rückert |
| author_facet | Elizabeth Walsh Tran Zen B. Torres Brian C. Prince Claudia Rückert |
| author_sort | Elizabeth Walsh |
| collection | DOAJ |
| description | <b>Background/Objectives</b>: <i>Culex</i> species mosquitoes are globally distributed and transmit several pathogens that impact animal and public health, including West Nile virus, Usutu virus, and <i>Plasmodium relictum</i>. Despite their relevance, <i>Culex</i> species are less widely studied than <i>Aedes</i> and <i>Anopheles</i> mosquitoes. To expand the genetic tools used to study <i>Culex</i> mosquitoes, we previously developed an optimized plasmid for transient Cas9 and single-guide RNA (sgRNA) expression in <i>Culex quinquefasciatus</i> cells to generate gene knockouts. Here, we established a monoclonal cell line that consistently expresses Cas9 and can be used for screens to determine gene function or antiviral activity. <b>Methods</b>: We used this system to perform the successful gene editing of seven genes and subsequent testing for potential antiviral effects, using a simple single-guide RNA (sgRNA) transfection and subsequent virus infection. <b>Results</b>: We were able to show antiviral effects for the <i>Cx. quinquefasciatus</i> genes <i>dicer-2</i>, <i>argonaute-2b</i>, <i>vago</i>, <i>piwi5</i>, <i>piwi6a</i>, and <i>cullin4a</i>. In comparison to the RNAi-mediated gene silencing of <i>dicer-2</i>, <i>argonaute-2b</i>, and <i>piwi5</i>, our Cas9/sgRNA approach showed an enhanced ability to detect antiviral effects. <b>Conclusions</b>: We propose that this cell line offers a new tool for studying gene function in <i>Cx. quinquefasciatus</i> mosquitoes that avoids the use of RNAi. This short study also serves as a proof-of-concept for future gene knock-ins in these cells. Our cell line expands the molecular resources available for vector competence research and will support the design of future research strategies to reduce the transmission of mosquito-borne diseases. |
| format | Article |
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| institution | DOAJ |
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| language | English |
| publishDate | 2025-01-01 |
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| spelling | doaj-art-bfac506fd06c4c0d857d78e9e7717bf62025-08-20T02:42:42ZengMDPI AGDNA2673-88562025-01-0151110.3390/dna5010001Generation of Cas9 Knock-In <i>Culex quinquefasciatus</i> Mosquito CellsElizabeth Walsh0Tran Zen B. Torres1Brian C. Prince2Claudia Rückert3Department of Biochemistry and Molecular Biology, College of Agriculture, Biotechnology & Natural Resources, University of Nevada, Reno, NV 89557, USADepartment of Biochemistry and Molecular Biology, College of Agriculture, Biotechnology & Natural Resources, University of Nevada, Reno, NV 89557, USADepartment of Biochemistry and Molecular Biology, College of Agriculture, Biotechnology & Natural Resources, University of Nevada, Reno, NV 89557, USADepartment of Biochemistry and Molecular Biology, College of Agriculture, Biotechnology & Natural Resources, University of Nevada, Reno, NV 89557, USA<b>Background/Objectives</b>: <i>Culex</i> species mosquitoes are globally distributed and transmit several pathogens that impact animal and public health, including West Nile virus, Usutu virus, and <i>Plasmodium relictum</i>. Despite their relevance, <i>Culex</i> species are less widely studied than <i>Aedes</i> and <i>Anopheles</i> mosquitoes. To expand the genetic tools used to study <i>Culex</i> mosquitoes, we previously developed an optimized plasmid for transient Cas9 and single-guide RNA (sgRNA) expression in <i>Culex quinquefasciatus</i> cells to generate gene knockouts. Here, we established a monoclonal cell line that consistently expresses Cas9 and can be used for screens to determine gene function or antiviral activity. <b>Methods</b>: We used this system to perform the successful gene editing of seven genes and subsequent testing for potential antiviral effects, using a simple single-guide RNA (sgRNA) transfection and subsequent virus infection. <b>Results</b>: We were able to show antiviral effects for the <i>Cx. quinquefasciatus</i> genes <i>dicer-2</i>, <i>argonaute-2b</i>, <i>vago</i>, <i>piwi5</i>, <i>piwi6a</i>, and <i>cullin4a</i>. In comparison to the RNAi-mediated gene silencing of <i>dicer-2</i>, <i>argonaute-2b</i>, and <i>piwi5</i>, our Cas9/sgRNA approach showed an enhanced ability to detect antiviral effects. <b>Conclusions</b>: We propose that this cell line offers a new tool for studying gene function in <i>Cx. quinquefasciatus</i> mosquitoes that avoids the use of RNAi. This short study also serves as a proof-of-concept for future gene knock-ins in these cells. Our cell line expands the molecular resources available for vector competence research and will support the design of future research strategies to reduce the transmission of mosquito-borne diseases.https://www.mdpi.com/2673-8856/5/1/1Cas9gene editinggene knock-inarbovirusesmosquitoescell lines |
| spellingShingle | Elizabeth Walsh Tran Zen B. Torres Brian C. Prince Claudia Rückert Generation of Cas9 Knock-In <i>Culex quinquefasciatus</i> Mosquito Cells DNA Cas9 gene editing gene knock-in arboviruses mosquitoes cell lines |
| title | Generation of Cas9 Knock-In <i>Culex quinquefasciatus</i> Mosquito Cells |
| title_full | Generation of Cas9 Knock-In <i>Culex quinquefasciatus</i> Mosquito Cells |
| title_fullStr | Generation of Cas9 Knock-In <i>Culex quinquefasciatus</i> Mosquito Cells |
| title_full_unstemmed | Generation of Cas9 Knock-In <i>Culex quinquefasciatus</i> Mosquito Cells |
| title_short | Generation of Cas9 Knock-In <i>Culex quinquefasciatus</i> Mosquito Cells |
| title_sort | generation of cas9 knock in i culex quinquefasciatus i mosquito cells |
| topic | Cas9 gene editing gene knock-in arboviruses mosquitoes cell lines |
| url | https://www.mdpi.com/2673-8856/5/1/1 |
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