A homing suppression gene drive with multiplexed gRNAs maintains high drive conversion efficiency and avoids functional resistance alleles

Gene drives are engineered alleles that can bias inheritance in their favor, allowing them to spread throughout a population. They could potentially be used to modify or suppress pest populations, such as mosquitoes that spread diseases. CRISPR/Cas9 homing drives, which copy themselves by homology-directed repair in drive/wild-type heterozygotes, are a powerful form of gene drive, but they are vulnerable to resistance alleles that preserve the function of their target gene. Such resistance alleles can prevent successful population suppression. Here, we constructed a homing suppression drive in Drosophila melanogaster that utilized multiplexed gRNAs to inhibit the formation of functional resistance alleles in its female fertility target gene. The gRNA target sites were placed close together, preventing reduction in drive conversion efficiency. The construct reached a moderate equilibrium frequency in cage populations without apparent formation of resistance alleles. However, a moderate fitness cost prevented suppression of the cage population. Nevertheless, our results experimentally demonstrate the viability of the multiplexed gRNAs strategy in homing type suppression gene drives.

A CRISPR homing gene drive targeting a haplolethal gene removes resistance alleles and successfully spreads through a cage population

Engineered gene drives are being explored as a new strategy in the fight against vector-borne diseases due to their potential for rapidly spreading genetic modifications through a population. However, CRISPR-based homing gene drives proposed for this purpose have faced a major obstacle in the formation of resistance alleles that prevent Cas9 cleavage. Here, we present a homing drive in Drosophila melanogaster that reduces the prevalence of resistance alleles below detectable levels by targeting a haplolethal gene with two guide RNAs (gRNAs) while also providing a rescue allele. Resistance alleles that form by end-joining repair typically disrupt the haplolethal target gene and are thus removed from the population because individuals that carry them are nonviable. We demonstrate that our drive is highly efficient, with 91% of the progeny of drive heterozygotes inheriting the drive allele and with no functional resistance alleles observed in the remainder. In a large cage experiment, the drive allele successfully spread to all individuals within a few generations. These results show that a haplolethal homing drive can provide an effective tool for targeted genetic modification of entire populations.

A toxin-antidote CRISPR gene drive system for regional population modification

ABSTRACTEngineered gene drives have been suggested as a mechanism for rapidly spreading genetic alterations through a population. One promising type of drive is the CRISPR homing drive, which has recently been demonstrated in several organisms. However, such drives face a major obstacle in the form of resistance against the drive that typically evolves rapidly. In addition, homing-type drives are generally self-sustaining, meaning that a drive would likely spread to all individuals of a species even when introduced at low frequency in a single location. Here, we develop a new form of CRISPR gene drive, the Toxin-Antidote Recessive Embryo (TARE) drive, which successfully limits resistance by targeting a recessive lethal gene while providing a recoded sequence to rescue only drive-carrying individuals. Our computational modeling shows that such a drive will have threshold-dependent dynamics, spreading only when introduced above a frequency threshold that depends on the fitness cost of the drive. We demonstrate such a drive in Drosophila with 88-95% transmission to the progeny of female drive heterozygotes. This drive was able to spread through a large cage population in just six generations following introduction at 24% frequency without any apparent evolution of resistance. Our results suggest that TARE drives constitute promising candidates for the development of effective, regionally confined population modification drives.

Changes in the microbiota cause genetically modified Anopheles to spread in a population

The mosquito’s innate immune system controls both Plasmodium and bacterial infections. We investigated the competitiveness of mosquitoes genetically modified to alter expression of their own anti-Plasmodium immune genes in a mixed-cage population with wild-type mosquitoes. We observed that genetically modified mosquitoes with increased immune activity in the midgut tissue did not have an observed fitness disadvantage and showed reduced microbial loads in both the midgut and reproductive organs. These changes result in a mating preference of genetically modified males for wild-type females, whereas wild-type males prefer genetically modified females. These changes foster the spread of the genetic modification in a mosquito cage population.

Effect of cage population density on plasma corticosterone and peripheral lymphocyte populations of laboratory mice

The effect of different population densities of mice per cage on plasma corticosterone, peripheral lymphocytes and specific lymphocyte subpopulations was investigated. The animals were housed in groups of 2, 4 or 8 mice per cage and the blood samples were taken from each animal of these groups on days one, 7 and 14. A significant elevation ( P<0·05) in plasma corticosterone concentration was observed in the group of 8 mice per cage on days one and 7 as compared with those of 2 or 4 mice per cage. The number of peripheral lymphocytes was significantly decreased in the groups of 2 ( P<0·01) and 8 ( P<0·05) mice per cage as compared with the group of 4 mice per cage on day one. A significantly decreased number of lymphocytes ( P<0·01) in the group of 8 mice per cage continued to day 7. There were no significant differences in specific lymphocyte subpopulations observed among these groups. The results of this study suggest that a population density of 4 mice per cage induced minimal stress compared to that induced by the population densities of 2 or 8 mice per cage. Since stress is known to induce alteration in a variety of biological functions, the population density of mice per cage should be considered in the interpretaion of research data.

Quantitative genetic variability maintained by mutation-stabilizing selection balance: sampling variation and response to subsequent directional selection

SummaryA model of genetic variation of a quantitative character subject to the simultaneous effects of mutation, selection and drift is investigated. Predictions are obtained for the variance of the genetic variance among independent lines at equilibrium with stabilizing selection. These indicate that the coefficient of variation of the genetic variance among lines is relatively insensitive to the strength of stabilizing selection on the character. The effects on the genetic variance of a change of mode of selection from stabilizing to directional selection are investigated. This is intended to model directional selection of a character in a sample of individuals from a natural or long-established cage population. The pattern of change of variance from directional selection is strongly influenced by the strengths of selection at individual loci in relation to effective population size before and after the change of regime. Patterns of change of variance and selection responses from Monte Carlo simulation are compared to selection responses observed in experiments. These indicate that changes in variance with directional selection are not very different from those due to drift alone in the experiments, and do not necessarily give information on the presence of stabilizing selection or its strength.

GENE FLOW IN NATURAL POPULATIONS OF DROSOPHILA MELANOGASTER WITH SPECIAL REFERENCE TO LETHAL ALLELISM RATES AND PROTEIN VARIATION

ABSTRACT A simultaneous survey of 14 protein loci, together with frequencies and within- and between-population allelism rates of lethal chromosomes, was carried out in five (four Japanese and one Korean) natural populations and one cage population of Drosophila melanogaster. It was found that lethal allelism rates decrease rapidly as geographic distance between two populations increases, while variation at protein loci shows a remarkable similarity over all populations examined. These findings suggest that there are very high levels of gene flow in these natural populations and that selection at protein loci which can maintain substantial geographic variation, if present, is overshadowed by gene flow. There is no indication that invasion of D. melanogaster to the Far East occurred so recently that the frequencies of lethal chromosomes are still in nonequilibrium.

Effect of Selection on Oviposition Site Preference in Drosophila melanogaster

A divergent selection program for gregarious oviposition behaviour was carried out for 112 generations on cage population of D. melanogaster. Gregarious oviposition was measured using five different indices: number of occupied tubes, percentage of eggs in the preferred tube, mean variance ratio, Charlier coefficient of disturbance and k parameter of the negative binomial distribution. All these statistics showed similar tendencies, but k seemed to be the most sensitive indicator of intensity of aggregation. The high aggregation line showed a rapid initial response but little change later. In contrast, the low aggregation line showed a steady but slow response to selection. The realized heritability for the low line in generations 67-73 was 0�11 � 0�04.

Further study of DDT resistance in Anopheles gambiae Giles (Diptera: Culicidae) and a cage test of elimination of resistance from a population by male release

Three West African strains of Anopheles gambiae Giles were intensively selected for DDT resistance at the adult stage using both mass selection and family selection with inbreeding. The LT50s for 4% DDT were thereby raised to 2–6 h, i.e. 6–18 × that of a susceptible strain. The selected strains were found to show DDT resistance as first-instar larvae and permethrin resistance as adults. The results of tests for the number of genes involved in causing the resistance and their linkage to genes for dieldrin resistance were equivocal. One of the DDT-resistant strains was bred in population cages with overlapping generations. Releases were made of males of the R70 translocation strain which transmit DDT and dieldrin susceptibility to their female progeny. When DDT selection on the cage population was relaxed, the releases caused a more rapid and complete loss of resistance than occurred in a control population. When selection for DDT resistance was applied to females of the cage population, the releases were able to prevent a build-up of resistance. The use of male releases is discussed as a possible means of countering insecticide resistance in mosquitoes.