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

Purusothaman, D.-K. , Shackleford, L., Anderson, M. A. E., Harvey-Samuel, T. and Alphey, L. (2021) CRISPR/Cas-9 mediated knock-in by homology dependent repair in the West Nile Virus vector Culex quinquefasciatus Say. Scientific Reports, 11(1), 14964. (doi: 10.1038/s41598-021-94065-z) (PMID:34294769) (PMCID:PMC8298393)

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.

• Item Type: Articles
• Additional Information: LA and THS are supported through strategic funding from the UK Biotechnology and Biological Sciences Research Council (BBSRC) to The Pirbright Institute (BBS/E/I/00007033, BBS/E/I/00007038 and BBS/E/I/00007039). DP is funded by a PhD Studentship from The Pirbright Institute. LS and MAEA are funded by the Bill and Melinda Gates Foundation (INV-008549). This report is based on research funded in part by the Bill & Melinda Gates Foundation.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Purusothaman, Dr Deepak Kumar
• Authors: Purusothaman, D.-K., Shackleford, L., Anderson, M. A. E., Harvey-Samuel, T., and Alphey, L.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
• Journal Name: Scientific Reports
• Publisher: Nature Research
• ISSN: 2045-2322
• ISSN (Online): 2045-2322
• Copyright Holders: Copyright © The Author(s) 2021
• First Published: First published in Scientific Reports 11(1):14964
• Publisher Policy: Reproduced under a Creative Commons license