Population suppression by release of insects carrying a dominant sterile homing gene drive targeting doublesex in Drosophila

Abstract

CRISPR homing gene drives can suppress pest populations by targeting female fertility genes, converting wild-type alleles into drive alleles in the germline of drive heterozygotes. fsRIDL (female-specific Release of Insects carrying a Dominant Lethal) is a self-limiting population suppression strategy involving continual release of transgenic males carrying female lethal alleles. Here, we propose an improved pest suppression system called "Release of Insects carrying a Dominant-sterile Drive" (RIDD), combining performance characteristics of homing drive and fsRIDL. We construct a split RIDD system in Drosophila melanogaster by creating a 3-gRNA drive disrupting the doublesex female exon. Drive alleles bias their inheritance in males, while drive alleles and resistance alleles formed by end-joining cause dominant female sterility. Weekly releases of RIDD males progressively suppressed and eventually eliminated cage populations. Modeling shows that RIDD is substantially stronger than SIT and fsRIDL. RIDD is also self-limiting, potentially allowing targeted population suppression.

Fig. 1. Gene drive constructs and inheritance rate.

a Our gene drive construct consists of a DsRed fluorescent marker driven by the 3xP3 promoter and three gRNAs driven by the U6:3 promoter that are separated by tRNAs. Exon-4 of dsx on chromosome 3 R is a female-specific exon. gRNAs target the intron 3/exon 4 boundary within the exon. In a drive heterozygote’s germline, Cas9 and gRNA cleaves the wild-type allele. Repair of the cleaved chromosome through homologous directed repair (HDR) leads to the copying of the drive allele (called “drive conversion” or “homing”). b The split Cas9 construct is on chromosome 2 R. Cas9 is under the control of one of the following 5’UTR/promotors: nanos, rcd-1r and CG4415. The 3’ UTR is either Nanos or shu. A 3xP3:EGFP cassette is used to indicate the presence of the Cas9 allele. c The drive inheritance rate of the progeny of dsx drive heterozygous males with heterozygous or homozygous Cas9 (marked by homo. or heter.) controlled by the indicated regulatory elements. Each dot represents progeny from a single drive male in one vial, and the size of each spot shows the number of individuals phenotyped. The mean inheritance rate (± s.e.m.) is shown. Source data are provided in Supplementary Data Set 1.

Fig. 2. Morphology and dsx sex-specific transcript expression.

a Schematic representation of the male- and female-specific dsx transcripts. Red arrows are primers designed for male- or female-specific transcript diagnostic PCR. The PCR product for the female dsx transcript is 713 bp, and the product for the male transcript is 352 bp. b Pictures of wild-type (+/+) flies, female drive carriers (D/+), and nonfunctional (r2) resistance allele females are shown. dominant r2/+ refers to dominant sterile r2 individuals, recessive r2/+ refers to recessive sterile r2 individuals. D/+, dominant r2/+, and recessive r2/+ females were homozygous for the nanos-Cas9-nanos allele. c Diagnostic PCR on cDNA using female-specific primers (top) and male-specific primers (bottom).

Fig. 3. Sequences of three types of nonfunctional resistance alleles.

Each sequence originates from one female carrying a resistance allele with the listed phenotype. Vertical dashed lines indicate the boundaries of the female-specific exon’s coding sequence. Orange highlighting shows the gRNA target sequences, and yellow shows the gRNA PAM sequences.—indicates deletion. “Insertion” refers to a 311 bp region from the right side of the drive that was copied by incomplete homology-directed repair.

Fig. 4. Scheme of Release of Insects carrying a Dominant sterile Drive (RIDD) system.

a Drive inheritance of RIDD. In male drive heterozygotes, germline Cas9 activity converts wild-type alleles to drive alleles by homology-directed repair, while end-joining repair or incomplete homology-directed repair generates nonfunctional resistance (r2) alleles. Females carrying one drive allele are sterile. The most common type of r2 (~ 95%, marked by *) is also dominant sterile in females, though a small fraction are recessive sterile. A small fraction of wild-type alleles (< 1%) in the male germline may also remain uncut. b Concept of RIDD pest control strategy. Drive heterozygous males are continuously released into a wild population. Drive conversion and resistance allele formation take place in the male germline, so nearly all female progeny of drive males are sterile (drive carrier and most r2 carrier females are sterile). Over time, the frequency of drive carriers increases, and with a high enough release level, the population will eventually be suppressed.

Fig. 5. Population dynamics of cages.

Drive heterozygous males were released each week (usually at two evenly spaced time points per week) into continuously maintained fly populations with overlapping generations. For the first 10 weeks, the release size was ~ 5 % of the total population per week. Starting from week 10 (marked by a vertical dashed line), the release size was increased to ~ 23% of the total population per week. a Population size of cages A and B together with the number of males released each week. After the weekly release size was increased, the population steadily declined. b Frequency of individual phenotypes in the cages based on a small weekly sample of flies. Masculine phenotype females include both sterile drive females and those with a nonfunctional resistance allele. c Female population size in cages. This data was in agreement with stochastic simulations of the cage population (yellow lines). d Frequency of drive individuals in cages A and B together with results from twenty simulations. Error bars show estimated standard error of the mean based on random phenotype samples. Source data are provided in Supplementary Data Sets 3–5.

Fig. 6. Comparison of the performance of SIT, fsRIDL, and RIDD system.

A constant number of drive heterozygous males/transgenic males were released each generation at with the number based on the specified ratio of released males to males in the starting population (that is release ratio). The heatmaps show the outcome of stochastic simulations for suppression of a population of 100,000 with overlapping generations and a linear density growth curve. a The blue heat bar shows the average number of weeks to population elimination. b The red heat bar represents the average number of fertile females at equilibrium when population elimination was not successful before 267 weeks. The released RIDD-split drive males are homozygous for the Cas9 allele. Each point in the parameter space had 20 replicates. Note the different vertical axis scale for SIT and fsRIDL. Gray means “not applicable” (NA - where population elimination always occurred for (a), and where it never occurred for (b).

Fig. 7. Self-limiting property of the RIDD system.

Weekly releases of drive males were conducted into an initial population of 2000 at a ratio of 0.8 (RIDD complete drive) and 1.4 (RIDD split drive) for 15 weeks. The drive conversion rate was 0.9. a Total population of fertile females and (b) the drive carrier frequency in the population is shown. 20 simulations are shown for each drive.