Engineering gene drive docking sites in a haplolethal locus in Anopheles gambiae.

Abstract

Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing and functional r1 alleles permits the cell to survive. Here we outline the development and characterization of two ΦC31-Recombination mediated cassette exchange gene drive docking lines with these features in Anopheles gambiae, a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired putative haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants in the process reveal a major lethality and infertility hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for development of future gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.

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laboratory

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published

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Conflict statement

Declarations. Competing interests: O.S.A is a founder of Agragene, Inc. and Synvect, Inc. with equity interest. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. G.M.C. has the following patents related to this work: WO2015006290A1 (“Multiplex RNA-guided genome engineering”) and WO2016089866A1 (“RNA-guided systems for in vivo gene editing”). A complete list of G.M.C’s conflict of interest can be found at arep.med.harvard.edu/gmc/tech.html. K.M.E. and A.L.S have pending patents related to the described work: WO2015105928A1 (“RNA-guided gene drives”) and K.M.E. has an additional patent WO2015006294A2 (“Orthogonal Cas9 proteins for RNA-guided gene regulation and editing”). All other authors (E.M., S.S., E.M., D.A., F.C.) declare no competing interests. Ethical conduct of research: All animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals as recommended by the National Institutes of Health and approved by the UCSD Institutional Animal Care and Use Committee (IACUC, Animal Use Protocol #S17187) and UCSD Biological Use Authorization (BUA #R2401).

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