Optimization of SgRNA expression with RNA pol III regulatory elements in Anopheles stephensi

Gonzalez, Estela, Anderson, Michelle A. E., Ang, Joshua X. D., Nevard, Katherine, Shackleford, Lewis, Larrosa-Godall, Mireia, Leftwich, Philip T. and Alphey, Luke (2025) Optimization of SgRNA expression with RNA pol III regulatory elements in Anopheles stephensi. Scientific Reports, 15. ISSN 2045-2322

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Abstract

Anopheles stephensi, a major Asian malaria vector, is invading Africa and has been implicated in recent outbreaks of urban malaria. Control of this species is key to eliminating malaria in Africa. Genetic control strategies, and CRISPR/Cas9-based gene drives are emerging as promising species-specific, environmentally friendly, scalable, affordable methods for pest control. To implement these strategies, a key parameter to optimize for high efficiency is the spatiotemporal control of Cas9 and the gRNA. Here, we assessed the ability of four RNA Pol III promoters to bias the inheritance of a gene drive element inserted into the cd gene of An. stephensi. We determined the homing efficiency and examined eye phenotype as a proxy for non-homologous end joining (NHEJ) events in somatic tissue. We found all four promoters to be active, with mean inheritance rates up to 99.8%. We found a strong effect of the Cas9-bearing grandparent (grandparent genotype), likely due to maternally deposited Cas9.

Item Type:	Article
Additional Information:	Data availability statement: All data generated for this study is available in the main manuscript and supplementary files. Full plasmid sequences are available from NCBI accession numbers: PQ005639-PQ005643. Funding information: This publication is based on research funded by the Gates Foundation (INV-008549). The conclusions and opinions expressed in this work are those of the author(s) alone and shall not be attributed to the Foundation. This work was also supported by 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). Rights Retention Statement: Under the grant conditions of the Foundation, a Creative Commons Attribution 4.0 License has already been assigned to the Author Accepted Manuscript version that might arise from this submission.
Faculty \ School:	Faculty of Science > School of Biological Sciences
UEA Research Groups:	Faculty of Science > Research Groups > Biosciences Teaching and Education Research
Related URLs:	https://www.nature.com/articles/s41598-0...
Depositing User:	LivePure Connector
Date Deposited:	23 Apr 2025 14:30
Last Modified:	23 Apr 2025 14:30
URI:	https://ueaeprints.uea.ac.uk/id/eprint/99054
DOI:	10.1038/s41598-025-98557-0

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