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Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs

Oberhofer, Georg and Ivy, Tobin and Hay, Bruce A. (2018) Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs. Proceedings of the National Academy of Sciences of the United States of America, 115 (40). E9343-E9352. ISSN 0027-8424. PMCID PMC6176634. https://resolver.caltech.edu/CaltechAUTHORS:20180410-084334530

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Abstract

A gene drive method of particular interest for population suppression utilizes homing endonuclease genes (HEGs), wherein a site-specific, nuclease-encoding cassette is copied, in the germline, into a target gene whose loss of function results in loss of viability or fertility in homozygous, but not heterozygous, progeny. Earlier work in Drosophila and mosquitoes utilized HEGs consisting of Cas9 and a single guide RNA (gRNA) that together target a specific gene for cleavage. Homing was observed, but resistant alleles immune to cleavage, while retaining wild-type gene function, were also created through nonhomologous end joining. Such alleles prevent drive and population suppression. Targeting a gene for cleavage at multiple positions has been suggested as a strategy to prevent the appearance of resistant alleles. To test this hypothesis, we generated two suppression HEGs in Drosophila melanogaster targeting genes required for embryonic viability or fertility, using a HEG consisting of CRISPR/Cas9 and gRNAs designed to cleave each gene at four positions. Rates of target locus cleavage were very high, and multiplexing of gRNAs prevented resistant allele formation. However, germline homing rates were modest, and the HEG cassette was unstable during homing events, resulting in frequent partial copying of HEGs that lacked gRNAs, a dominant marker gene, or Cas9. Finally, in drive experiments, the HEGs failed to spread due to the high fitness load induced in offspring as a result of maternal carryover of Cas9/gRNA complex activity. Alternative design principles are proposed that may mitigate these problems in future gene drive engineering.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1073/pnas.1805278115DOIArticle
http://www.pnas.org/content/suppl/2018/09/15/1805278115.DCSupplementalPublisherSupporting Information
https://doi.org/10.1101/289546DOIDiscussion Paper
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176634PubMed CentralArticle
ORCID:
AuthorORCID
Oberhofer, Georg0000-0003-0930-1996
Additional Information:© 2018 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND). Edited by Dana Carroll, University of Utah School of Medicine, Salt Lake City, UT, and approved August 17, 2018 (received for review March 28, 2018). PNAS published ahead of print September 17, 2018. We thank Marlene Biller for technical assistance and three anonymous reviewers who helped improve and clarify this manuscript. This work was supported by Grant 18-CCB5400-05 from the California Cherry Board (to B.A.H. and G.O.) and by Research Fellowship 428/1-1 from the German Research Foundation (to G.O.). T.I. was supported by NIH Training Grant 5T32GM007616-39. Author contributions: G.O. and B.A.H. designed research; G.O. performed research; G.O., T.I., and B.A.H. analyzed data; and G.O., T.I., and B.A.H. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1805278115/-/DCSupplemental.
Funders:
Funding AgencyGrant Number
California Cherry Board18-CCB5400-05
Deutsche Forschungsgemeinschaft (DFG)428/1-1
NIH Predoctoral Fellowship5T32GM007616-39
Subject Keywords:gene drive; suppression HEG; CRISPR/Cas9; gRNA multiplexing
Issue or Number:40
PubMed Central ID:PMC6176634
Record Number:CaltechAUTHORS:20180410-084334530
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180410-084334530
Official Citation:Behavior of homing endonuclease gene drives targeting genes required for viability or female fertility with multiplexed guide RNAs. Georg Oberhofer, Tobin Ivy, Bruce A. Hay. Proceedings of the National Academy of Sciences Sep 2018, 201805278; DOI: 10.1073/pnas.1805278115
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:85713
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:10 Apr 2018 16:09
Last Modified:09 Mar 2020 13:19

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