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...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2021-07-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-021-94065-z |
| _version_ | 1818430435660660736 |
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| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T15:33:22Z |
| format | Article |
| id | doaj.art-403f6a669c9d407497a486fafd81472c |
| institution | Directory Open Access Journal |
| issn | 2045-2322 |
| language | English |
| last_indexed | 2024-12-14T15:33:22Z |
| publishDate | 2021-07-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj.art-403f6a669c9d407497a486fafd81472c2022-12-21T22:55:48ZengNature PortfolioScientific Reports2045-23222021-07-011111810.1038/s41598-021-94065-zCRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus SayDeepak-Kumar Purusothaman0Lewis Shackleford1Michelle A. E. Anderson2Tim Harvey-Samuel3Luke Alphey4Arthropod Genetics, The Pirbright InstituteArthropod Genetics, The Pirbright InstituteArthropod Genetics, The Pirbright InstituteArthropod Genetics, The Pirbright InstituteArthropod Genetics, The Pirbright InstituteAbstract 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.https://doi.org/10.1038/s41598-021-94065-z |
| spellingShingle | 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 Scientific Reports |
| title | 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_short | 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 |
| url | https://doi.org/10.1038/s41598-021-94065-z |
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