CRISPR target site design

To confer female lethality and male sterility, target sites for guide RNAs (gRNAs) were chosen inside female-specific exons of sex-determination genes, Sex Lethal (Sxl), Transformer (tra), and Doublesex (dsx), and in male-specific genes, βTubulin 85D (βTub), fuzzy onions (fzo), Protamine A (ProA), and spermatocyte arrest (sa), respectively. CHOPCHOP v261 was used for choosing gRNA target sites from a specified sequence in Drosophila genome (dm6) to minimize the off-target cleavage. Due to the alternative splicing, functional Sxl and Tra proteins are produced only in Drosophila females26,27, while two versions of Dsx protein—female (DsxF) or male (DsxM)—are made each in the corresponding gender28 (Fig. 1b). The gRNA target for βTub was chosen in the vicinity to the βTub85DD (B2tD) mutant allele29. Sequences of gRNA target sites are presented in Supplementary Fig. 1.

Design and assembly of constructs

The Gibson enzymatic assembly method was used to build all constructs62. The previously described plasmid harboring the SpCas9-T2A-GFP with nuclear localization signals (NLS) flanking SpCas9 coding sequence and the Opie2-dsRed transformation marker was used to build Drosophila Cas9 constructs. This plasmid was used for Ae. aegypti transgenesis and had both piggyBac and an attB-docking sites (Addgene #100608)36. The Ae. aegypti promoter was removed from the plasmid by cutting at NotI and XhoI sites and replacing it with Nanos (nos), or Ubiquitin-63E (Ubi), or Vasa (vas) promoter (Supplementary Fig. 1). Promoter fragments were PCR amplified from Drosophila genomic DNA using the following primers: nos-F, nos-R, Ubi-F, Ubi-R, vas-F, and vas-F (Supplementary Table 11). To generate constructs with a single gRNA, Drosophila U6-3 promoter and guide RNA with a target, scaffold, and terminator signal (gRNA) was cloned at the multiple cloning site (MCS) between the white gene and an attB-docking site inside a plasmid used for D. melanogaster transformation63. For the first plasmid in this series, U6-3-gRNAβTub, Drosophila U6-3 promoter was amplified from Drosophila genomic DNA with U6-1F and U6-2R primers, while the complete gRNA was PCR-assembled from two Ultramer® gRNA-3F and gRNA-4R oligos synthesized by Integrated DNA Technology (IDT). To improve the efficiency of termination of gRNA transcription, a termination signal with 11 thymines was used in our design. In the successive plasmids, the U6-3 promoter and gRNA’s scaffold was amplified from the U6-3-gRNAβTub plasmid using the overlapping middle oligos designed to replace 20 bases that constitute a gRNA target (U6-1AF, U6-2A/B/CR, gRNA-3A/B/CF, and gRNA-4AR), and replaced by digesting the same plasmid at AscI and NotI sites. To assemble the set of plasmids with double gRNAs (dsRNAs), the U6-3 promoter and gRNA was amplified as one fragment from the single gRNA (sgRNA) plasmids targeting female sex-determination genes with 2XgRNA-5F and 2XgRNA-6R primers, and cloned inside the U6-3-gRNAβTub plasmid that was linearized at a BamHI site between the white gene and the U6-3 promoter. Each dgRNA plasmid had the same gRNAβTub targeting βTub85D and a different gRNA targeting Sxl, tra, or dsxF expressed independently in the same direction (Supplementary Fig. 1). Drosophila Cas9 plasmids and gRNA plasmids generated for this study were deposited at Addgene (Supplementary Fig. 1). To build the βTub85D-GFP construct, a 481-bp fragment directly upstream of βTub coding sequence was PCR amplified from Drosophila genomic DNA with βTub-F and βTub-R primers and cloned upstream of GFP into the white attB-docking site plasmid described above.

Fly genetics and imaging

Flies were maintained under standard conditions at 25 °C. Embryo injections were carried out at Rainbow Transgenic Flies, Inc. (http://www.rainbowgene.com). The Cas9 and gRNA constructs were inserted at the PBac{y + -attP-3B}KV00033 on the 3rd chromosome (Bloomington #9750) and the P{CaryP}attP1 on the 2nd chromosome (Bloomington #8621), respectively; while βTub-GFP construct was inserted at the M{3XP3-RFP.attP’}ZH-86Fa on the 3rd chromosome (Bloomington #24486). Transgenic flies were balanced with w1118; CyO/snaSco and w1118; TM3, Sb1/TM6B, and Tb1; and double balanced with w1118; CyO/Sp; Dr1/TM6C,Sb,Tb1. The βTub-GFP (on the 3rd chromosome) was double balanced and introgressed with gRNAβTub,Sxl, gRNAβTub,Tra, and gRNAβTub,DsxF, each on the 2nd chromosome, to generate trans-heterozygous balanced stocks (dgRNA/CyO; βTub-GFP/TM6C,Sb,Tb).

To test the efficiency of knockouts and corresponding phenotypes caused by sgRNAs, seven flies of each gender were crossed to generate trans-heterozygous F 1 sgRNA/+; nos-Cas9/+ flies for each combination of sgRNA; and their external morphology and fertility were examined. Both transgenes were identified on a fluorescent stereomicroscope with w+ eyes (sgRNA, dgRNA) and dsRed (Cas9). The sgRNA lines that caused knockout phenotypes were further tested as homozygous stocks with nos-Cas9 flies in both directions using 10♂ and 10♀ flies for each replicate cross. DgRNA lines were tested bidirectionally with homozygous nos-Cas9, vas-Cas9, and Ubi-Cas9 lines. In addition, sgRNA, dgRNA, and Cas9 homozygous lines were crossed to w− flies in both directions to provide the comparison control. To test for the non-Mendelian dominant maternal effect of Cas9 loaded as a protein into embryos38, homozygous dgRNA flies were crossed to heterozygous Cas9 flies; and phenotypes of dgRNA/+;+/TM3, Sb progeny with either maternal Cas9 or paternal Cas9 were compared. The F 1 progeny from crosses with the paternal Cas9 served as a control group to examine the dominant maternal effect of Cas9. To test the fertility of generated knockout flies with and without the Cas9 gene, batches of 10–20 F 1 males and females, or intersexes, were crossed to 15–20 female virgin and male flies, correspondingly, from w− and/or Cantos S stocks. Three or 4 days after the cross, the flies were passaged into fresh vials, and in a week, both vials were examined for the presence of any viable progeny. The fertility of an entire batch was scored as 100% when viable larvae were identified in a vial, or 0% when no progeny hatched in both vials. The vials containing intersexes and wt males were also examined for the presence of laid eggs. All crosses were repeated at the minimum three times to generate means and standard deviations for statistical comparisons and thus measure consistency and robustness of the results.

Flies were scored, examined, and imaged on the Leica M165FC fluorescent stereomicroscope equipped with the Leica DMC2900 camera. To generate images of adult flies, image stacks collected at different focal plates were compiled into single images in Helios Focus 6, and then edited in Adobe Photoshop CS6. To study internal anatomical features of intersex flies and sterile males, their reproductive organs were dissected in PBS buffer, examined, and imaged. To estimate the variation of knockout phenotypes, around 10–20 flies were dissected for each tested genotype.

Developmental stage of Sxl lethality

To identify the developmental stage at which Sxl knockout females die, egg hatching and larval death rates were quantified for the dgRNAβTub,Sxl/+; nos-Cas9/+ trans-heterozygous flies. To quantify the egg hatching rate, three replicate crosses, each with 20–30 homozygous nos-Cas9 female virgins and 10–20 dgRNAβTub,Sxl males, were set up in embryo collection cages (Genesee Scientific 59–100) with grape juice agar plates. Three embryo collection cages with w− flies served as a comparison control. Batches of around 200 laid eggs were counted from each collection cage and followed for over 36 h to count the number of unhatched eggs. To quantify the rate of larval death, two batches of 50 emerged larvae were transferred from each agar plate to separate fly vials with food and raised to adults, and then the number and sex of emerged adults were recorded. To quantify the lethality at a pupal stage, a number of dead pupae were also recorded for each vial.

RT-PCR of female- and male-specific transcripts of Dsx

To assess the effect of tra knockout on dsx splicing, we screened for female- and male-specific mRNA of dsx in tra knockout intersexes. Total RNA was extracted from adult w− male, w− female, and tra knockout (dgRNAβTub,Tra/+; nos-Cas9/+) intersex flies following the standard protocol of the MirVana miRNA isolation kit (Ambion). To remove DNA contamination, 2 µg was treated with TURBOTM DNase using the TURBO DNA-freeTM Kit (Ambion). Dsx female and male splice variants were amplified with the SuperScript® III One-Step RT-PCR Kit (Invitrogen) following the protocol. The same forward primer, Dsx-RT-1F, and two different reverse primers, DsxF-RT-2R and DsxM-RT-3R (Supplementary Table 11), were used to amplify either female or male transcripts, respectively. In total, 10 µL of PCR products were run on a 1% agarose gel to test PCR specificity, and the remaining 40 µL were purified using a QIAquick PCR purification kit (QIAGEN) or, when double bands were identified on a gel, gel-purified with a Zymoclean™ Gel DNA Recovery Kit (Zymo Research), then clean amplicons were sequenced in both directions using Sanger method at Source BioScience (https://www.sourcebioscience.com).

Genotyping loci targeted with gRNAs

To examine the molecular changes that caused female lethality or masculinization and male sterility in the flies carrying Cas9 and gRNAs, four genomic loci that include targets sites for four functional gRNAs (Supplementary Fig. 1) were amplified and sequenced. Single-fly genomic DNA preps were prepared by homogenizing a fly in 30 µl of a freshly prepared squishing buffer (10 mM Tris-Cl pH 8.0, 1 mM EDTA, 25 mM NaCl, and 200 μg/mL Proteinase K), incubating at 37 °C for 35 min, and heating at 95 °C for 2 min. In total, 2 µl of genomic DNA was used as a template in a 40-µL PCR reaction with LongAmp® Taq DNA polymerase (NEB). The following primers (Supplementary Table 11) were used to amplify the loci with the corresponding gRNA targets: βTub-1AF and βTub-2AR for βTubulin 85D; Sxl-3BF and Sxl-4AR for Sex lethal; Tra-5F and Tra-6R for Transformer; Dsx-7F and Dsx-8R for Double sex. PCR products were purified using a QIAquick PCR purification kit (QIAGEN), and sequenced in both directions with Sanger method at Source BioScience. To characterize molecular changes at the targeted sites, sequence AB1 files were aligned against the corresponding reference sequences in SnapGene® 4 and/or Sequencher™ 5.

Competition assay of sterile males

To evaluate the competitiveness of the βTub knockout (gRNAβTub,Sxl/+; nos-Cas9/+) males, their ability to secure matings with females in the presence of wt males was evaluated. The w− males share the same genetic background with the βTub knockout males, and provide an ideal comparison. Two wt, one wt, one wt plus one βTub knockout, or two βTub knockout males were placed into a fly vial with ten w− virgins isolated on yeast paste for 2 days and allowed to court and mate with the females overnight (12 h) in the dark. To increase the male courtship drive, freshly emerged dgRNAβTub,Sxl/+; nos-Cas9/+ and wt males were isolated from females and aged for 4 days before the competition assay. Drosophila females mate with multiple males during a lifespan; and in the absence of sperm transferred to spermatheca after copulation, female abstinence lasts for 1 day post copulation64. Therefore, after 12 h of mating, all males were removed from the vials, while the females were transferred into small embryo collection cages (Genesee Scientific 59–100) with grape juice agar plates. Three batches of eggs were collected within 36 h and unhatched eggs were counted. The decrease in fertility, estimated by a number of unhatched eggs, indicated the ability of a gRNAβTub,Sxl/+; nos-Cas9/+ male to score successful matings with females in the presence of a wt male; and thus provided a readout of the competitiveness of βTub knockout males. A single wt male was used to test its ability to inseminate each of ten females in 12 h, and thus discriminate between a true competition or a dilution effect of two wt males.

Survival curves to estimate longevity of pgSIT males

To compare differences in survival between pgSIT (gRNAβTub,Sxl/+; nos-Cas9/+) and wt males, average longevities for three experimental groups of males were estimated. We treated two types of pgSIT, one carrying paternal Cas9 and the other maternal Cas9, as separate experimental groups. Five replicates per each of three groups were applied to estimate survival curves. Males of each type were collected daily and aged in batches of 20 males per vial. Each replicate had from 40 to 75 males kept in two or four vials, respectively. Numbers of died flies were recorded every third day during the transfer of flies into a new vial with fresh food. We analyzed the interval-censored time-to-event (i.e., death) data for the three experimental groups by computing nonparametric maximum likelihood estimates (NPMLE) of the survival curves for each group, implemented in the R package interval65. The estimation procedure takes into account uncertainty introduced by the 3-day observation period. Bootstrap with 10,000 repetitions was applied to quantify median survival time and standard deviation.

Mathematical modeling

To model the expected performance of pgSIT at suppressing local Ae. aegypti populations in comparison with currently available self-limiting suppression technologies—RIDL, fsRIDL, and IIT—we simulated release schemes for each using the MGDrivE simulation framework43 (https://marshalllab.github.io/MGDrivE/). This framework models the egg, larval, pupal, and adult mosquito life stages (both male and female adults are modeled) implementing a daily time step, overlapping generations and a mating structure in which adult males mate throughout their lifetime, while adult females mate once upon emergence, retaining the genetic material of the adult male with whom they mate for the duration of their adult lifespan. Density-independent mortality rates for the juvenile life stages are assumed to be identical and are chosen for consistency with the population growth rate in the absence of density-dependent mortality. Additional density-dependent mortality occurs at the larval stage, the form of which is taken from Deredec et al.66. The inheritance patterns for the pgSIT, RIDL, fsRIDL, and IIT systems are modeled within the inheritance module of the MGDrivE framework43, along with their impacts on adult lifespan, male mating competitiveness, and pupatory success. We implement the stochastic version of the MGDrivE framework to capture the random effects at low population sizes and the potential for population elimination. We simulated weekly releases over a period of 6 months into a randomly mixing population consisting of 10,000 adult females at equilibrium, with Ae. aegypti life history and intervention parameter values listed in Supplementary Table 10.

Statistical analysis

Statistical analysis was performed in JMP 8.0.2 by SAS Institute Inc. Three to five biological replicates were used to generate statistical means for comparisons. P values were calculated for a two-sample Student’s t test with unequal variance. To test for significance of male sterilization, Pearson’s chi-squared tests for contingency tables were used to calculate P values. To test for differences between the inferred survival curves, we used Sun’s generalization of the log-rank test67. In addition, we performed pairwise post hoc tests of differences between the two pgSIT groups with conservative Bonferroni correction.

Reporting Summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.