CRISPR-based innovative genetic tools for control of Anopheles gambiae mosquitoes The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Smidler, Andrea. 2019. CRISPR-based innovative genetic tools for control of Anopheles gambiae mosquitoes. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42029729 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA CRISPR-based innovative genetic tools for control of Anopheles gambiae mosquitoes A dissertation presented by Andrea L. Smidler to The Committee on Higher Degrees in Biological Sciences in Public Health in partial fulfillment of the requirements for the degree of Doctor of Philosophy In the subject of Biological Sciences in Public Health Harvard University Cambridge, Massachusetts April 2019 i © 2019 – Andrea Smidler All rights reserved ii Dissertation Advisor: Dr. Flaminia Catteruccia, Dr. George Church Andrea Smidler CRISPR-based innovative genetic tools for control of Anopheles gambiae mosquitoes ABSTRACT Malaria and other mosquito-borne diseases pose an immense burden on mankind. Since the turn of the century, control campaigns have relied on the use of insecticide-impregnated bed nets and indoor residual sprays to stop Anopheles mosquitoes from transmitting the malaria parasite. Although these are our best strategies to control the spread of disease, wild mosquito populations are developing resistance to insecticides at an alarming rate, making disease control increasingly challenging. In the search for new powerful strategies aimed at controlling malaria-transmitting Anopheles populations, we can now exploit a suite of powerful genome engineering tools to control wild populations and monitor releases. In this dissertation we utilize CRISPR/Cas9 technology and other genetic engineering tools in Anopheles gambiae to generate genetically sterile males for population suppression, to assess the feasibility of developing evolutionarily stable gene drives for population replacement, and to expand the genetic toolkit for field releases of genetically modified mosquitoes. We develop CRISPR/Cas in A. gambiae for male genetic sterilization and for basic biological study. Using this system we generate a line of mutant mosquitoes with deletions in Zero Population Growth (ZPG), a gene critical for germ cell development. Resulting male mutants show no sperm in the testes and sterilize the females with which they mate, demonstrating that similar systems could be adapted for use in Sterile-Insect Technique (SIT)-like release campaigns. We also test whether CRISPR/Cas9 can facilitate the sustainable and stable spread of gene drives in natural mosquito populations. Specifically, we design gene drives that have the potential of being evolutionarily stable by insertion into haplolethal ribosomal genes. To facilitate this goal, we create gene drive docking lines via a novel knockin technology for insertion of complex DNA templates into genetically iii intractable loci. We identify multiple challenges associated with such systems, including the occurrence of Minute-like mutant phenotypes that present severe fitness costs when targeting haploinsufficient genes, a general decay in gRNA function over time that has consequences for all gene drives designed to date, and the critical need for precisely controlling Cas9 expression to avoid large fitness costs. Finally, we develop and validate a novel transgenic tool for monitoring GM field releases. We generate transgenic lines expressing a fusion of a fluorescent marker with a male seminal protein specifically in male reproductive tissues. Incorporation of this fluorescence fusion into the mating plug, which is transferred to the females during mating, allows the visual identification of successful mating events for a few hours after copulation. This transgenic tool enables effective monitoring of GM male mating competitiveness in field trials, overcoming current limitations. The work outlined here significantly expands the genetic toolkit for the manipulation of Anopheles mosquitoes, facilitating the implementation of genetic control strategies aimed at malaria-transmitting vector populations. iv TABLE OF CONTENTS ABSTRACT …………………………………..…………………………………………………………..………………………………………. iii TABLE OF CONTENTS ……………………………..…………………………..………………………………………………………….…. v LIST OF FIGURES AND TABLES …………..……………………………………………………………………………………….…… viii GENOTYPE NOMENCLATURE……………………………………………………………………………………………………………… x ACKNOWLEDGEMENTS ……………..………………..…………………………………………………………………………….…….. xi CHAPTER 1: Introduction …………..……………………………………………………………………………………………………… 1 1.1 The burden of malaria and its current control …………………………………………………………………. 2 1.2 A brief history of malaria and its eradication from Italy and the United States – a precedent for mosquito elimination to control malaria……………………………………………….. 4 1.3 Mosquito genetic control – An introduction …………………………………………..………………………. 6 1.4 Population suppression systems ……………….…………………………………………..……………………….. 8 1.4 A. Sterile Insect Technique for mosquito population suppression ………………..…………………..... 8 1.4 B. RIDL-based transgenic population suppression systems ………………………..………………………… 9 1.5 Population replacement systems ………………………………………………………..…………………….….. 10 1.5 A. Endonuclease gene drives – Mode of action and history of development .………….…..……. 11 1.5 B. TALEN and ZFN-mediated gene drives ………………………………………..………..…………….…………. 12 1.5 C. CRISPR-mediated gene drives …………………………………………………..………..……………………….... 13 1.5 D. Safety considerations concerning gene drive……………………………………….………………………….. 14 1.5 E. Anti-parasite cargoes for population replacement …………………………………………………..……. 15 1.6 The problem of drive-resistant alleles (DRAs) ..…………………………………………………………….. 16 1.7 Learning from other CRISPR gene drives ..……………………………………………………………………… 18 1.8 Fitness considerations for gene drives …………………………………………………………………………… 20 1.8 A. CRISPR off-target mutagenesis ..…………………………………………………………………………………… 21 1.8 B. Gene drive-imposed evolutionary selective pressures ..…………..……………………………………. 22 1.9 Designing evolutionarily stable gene drives for population replacement ..…………………….. 25 1.10 Mosquito genetic control strategies need more tools ..……………………………………………….. 26 1.11 Summary of Aims ..……………………………………………………………………………………………………… 28 1.12 References ..………………………………………………………………………………………………………………… 30 CHAPTER 2: CRISPR-mediated germline knockouts for genetic sterilization of male Anopheles gambiae ………………………………………………………..………………………………. 46 ABSTRACT …………………………………………………………………………………………………………………………… 47 INTRODUCTION ………………………………………………………………………………………………………………….. 48 RESULTS ……………………………………………………………………………………………………………………………… 49 2.1 Generation of transgenic CRISPR-expression lines …………………………………………….. 50 2.2 ZPG mutants fail to develop normal testes ……………………………………………………….. 51 v 2.3 Male ZPG mutants are highly sterile ……………………………………………………………….... 52 2.4 Characterization of fertile ZPG mutant males ……………………………………………………. 53 2.5 Female ZPG mutants display severe ovarian atrophy ………………………………………… 54 DISCUSSION ………………………………………………………………………………………………………………………… 56 FUTURE DIRECTIONS …………………………………………………………………………………………………………… 58 METHODS …………………………………………………………………………………………………………………………… 61 REFERENCES ……………………………………………………………………………………………………………………….. 65 CHAPTER 3: Advancement towards developing evolutionarily stable gene drives for Anopheles gambiae population replacement …………………………………………………………. 69 ABSTRACT …………………………………………………………………………………………………………………………… 70 INTRODUCTION ………………………………………………………………………………………………………………..... 71 RESULTS ………………………………………………………………………………………………………………………………. 72 3.1 Gene drive design ……………………………………………………………………………………………… 72 3.2 Gene drive docking site locus identification ………………………………………………………. 72 3.3 Designing two docking sites in RpL11-Rpt1 – benefits and drawbacks of each design …………………………………………………………….. 74 3.3 A. Designing dRPLT – a gene drive docking site with Rpt1 knocked-out …………..…… 76 3.3 B. Designing dRPLTu – a gene drive docking site with Rpt1 function maintained…. 77 3.4 Knockin of dRPLT docking line into RpL11-Rpt1 by HDR …………………………………….. 77 3.5 Developing transgenic Interlocus Gene Conversion (IGC) for knockin into genetically intractable loci ……………………………………………………….77 3.5 A. Generating dRPLT by CrIGCkid ………………………………………………………………………….. 79 3.5 B. Generating dRPLTu by CrIGCkid …………….…………………………………………………………. 80 3.5 C. CrIGCkid does not occur in females, and infertility phenotypes lessen over time. 84 3.6 Characterization of dRPLT and dRPLTu docking lines …………………………………………. 84 3.6 A. dRPLT is homozygous lethal, experiences proteasome dysfunction, and is likely a Rpt1 knockout ………………………………………………………………………………….. 84 3.6 B. dRPLTu homozygotes are viable but display Minute-like fertility phenotypes ..… 85 3.7 Gene drives in dRPLT and dRPLTu may be lethal ……………………………………………….. 87 3.7 A. Inserting B2GD into dRPLT or dRPLTu results in death of all transgenic individuals ……………………………………………………………………………………. 87 3.7 B. Lethality of gene drives in docking sites may be due to aberrant Cas9 overexpression ………………………………………………………………………….…… 89 3.8 Developing drive-enabling germline-Cas9 promoters independent of docking sites ……………………………………………………………………………. 92