Patent Application
20220015339
This disclosure describes, in one aspect, a system for controlling population of a biological species. Generally, the system includes a population of genetically-modified individuals of the biological species, where both males and females in the genetically-modified population carry two mutations. The first mutation is a repressible genetic mutation that results in death of a juvenile individual of the first sex when the juvenile individual of the first sex comprises the repressible lethal mutation and is reared in the absence of a repressor or causes an individual to be sterile when reared in the absence of a repressor. The second mutation is an underdominant genetic mutation. These two mutations work in concert so that offspring of a mating between a member of the system and a wild-type member of the biological species are nonviable and offspring of a mating between two members of the system results in viable offspring only of the second sex.
In some embodiments, the repressible mutation can result in female lethality or female sterility.
In some embodiments, the repressible mutation can result in male lethality or male sterility.
In some embodiments, the underdominant mutation can be a synthetic incompatibility mutation.
In another aspect, this disclosure describes a method for controlling the population of a wild biological species. Generally, the method includes releasing into a wild population of the wild biological species a system for controlling the population of the wild biological species, in which the system includes a population of genetically-modified individuals of the biological species. The system can include any embodiment of the genetically-modified population summarized above.
In some embodiments, the combination of the repressible mutation and the underdominant mutation reduces the population the wild biological species synergistically compared to using either the repressible mutation alone or the underdominant mutation alone.
The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.
This disclosure describes a method of combining an underdominance system (e.g., engineered genetic incompatibility, EGI also known as synthetic incompatibility, SI) with a system that precludes the reproduction of a single sex) in sexually reproducing organisms (e.g., female lethality, FL, or a similarly regulated ‘daughterless’ system). The combined platform allows for various strategies for population control of undesirable species. The method described herein generally involve combining synthetic incompatibility with female lethal techniques to produce a platform with improved scalability and evolutionary robustness compared to previous platforms.
Populations of sexually reproducing pest organism such as disease vectoring mosquitoes or ecologically damaging invasive carp can cause substantial human economic losses and or health risks. Many strategies have been used to help reduce the burden of these organisms (e.g., insecticides, netting, sterile insect technique, synthetic incompatibility, female-lethality, gene-drives, etc.). The success of these strategies has been limited by, for example, challenges in scaling, high rates of escape mutations, and/or producing unintended harmful consequences for non-targeted beneficial organisms.
Genetic biocontrol strategies can produce highly specific and effective forms of population control. These strategies rely on the release of genetically engineered versions of the target species, which then mate with their wild counterparts. The product of this mating may result in no offspring, offspring of a single sex, offspring that are sterile, or offspring that carry a selfish genetic element (e.g., gene-drives, Madea elements) that spreads throughout the population and ultimately results in the population's extirpation/extinction.
Despite their potential, current genetic biocontrol strategies suffer multiple weaknesses. Methods that involve only the release of engineered male organisms that cannot produce viable offspring with wild females such as EGI, or Release of Insect carrying Dominant Lethality (RIDL), or some other underdominance approach require the efficient sorting of males from females. This sorting usually requires rearing both males and females to a life stage where they can be distinguished. This wastes resources on organisms that are never released and severely limits the scale at which production is feasible. Female lethal (FL) technologies allow for the control of female viability at young live stages, usually through the repression of a toxic gene by a small molecule (e.g., tetracycline). In some instances, adult females no longer require the small molecule for survival.
FL can be used as a tool to sort males carrying an FL genetic construct for release into the wild. Male carriers of FL will not produce female offspring in the wild. However, the larva of male offspring can still cause damage in the case of many agricultural pests such as, for example, the Diamondback moth. Furthermore, the male offspring can produce some females in subsequent generations as the FL constructs are diluted with each generation. EGI, RIDL, FL, or any other similar method also suffer from the need to release very large numbers of organisms to reduce wild pest population numbers.
Gene-drives (GD) are selfish genetic elements that are inherited in a non-Mendelian manner. In an organism that is hemizygous for a GD, the GD will be copied into the homologous chromosome that lacks the GD. Homozygosity for the GD elements may be lethal when disrupting an essential gene or cause sterility when disrupting a gene necessary for fertility. However, this process may happen in only a subset of cells, such as germ cells, so that the somatic tissue of an organism is hemizygous, yet all of the haploid gametes carry the GD. The advantage of GDs is that a small number of released organisms have the potential to affect large wild populations. A disadvantage of GDs, if they work as intended, is that it may be difficult to curtail their spread beyond a limited area or to stop them if there are unexpected and undesirable consequences of eliminating a pest organism. Current GDs in consideration of population control use programmable nucleases and homology-directed DNA repair as part of their copy mechanism. These suffer from very high rates of failure due to DNA repair via the non-homologous end-joining pathway which introduces mutations that prevent the GDs ability to spread.
In contrast to the conventional genetic biocontrol strategies summarized above, this disclosure describes a method of combining an underdominance system (e.g., synthetic incompatibility) with a system that precludes the reproduction of a single sex (e.g., female lethality) in sexually reproducing organisms. The combined system results in a self-stocking incompatible male system (SSIMS) (
While illustrated in
In some instances, both male and female SSIMS adults can be released into a wild environment (
SSIMS males may be generated in the wild for the duration of the reproductive life of SSIMS females. This produces limited amplification of engineered males (
In other applications, SSIMS is used as a method to easily isolate SSIMS males that are genetically incompatible with wild females (
SSIMS can be applied to any organisms that have male and female sexes. Exemplary animals into which SSIMS may be introduced can include, for example, an insect (e.g., mosquito, tstetse fly, spotted-wing drosophila, diamondback moth, new world screwworm, Mediterranean fruit-fly, olive fly, gypsy moth, codling moth, deer tick, etc.), a fish (e.g., salmon, carp, sea lamprey, etc.), a mammal (e.g., swine, a mouse, a rat, etc.), an amphibian (e.g., a cane toad, a bullfrog, etc.), a reptile (e.g., brown tree snake, etc.), or a crustacean (e.g., rusty crayfish, etc.).
The current examples involve males reaching maturity in the absence of a repressor of a toxic gene. In some embodiments, however, it may be desirable to only release females. In such cases, a male lethality mechanism can be used.
Simulations of population control in common carp (Cyprinus carpio) were performed using an agent-based model specific to common carp biology (Bajer et al., 2015. Oikos 124(11): 1520-1526). These simulations are simply examples that demonstrate the performance of different genetic interventions and should not be interpreted as the only possible scenarios.
All simulations involved the release of sexually mature, three-year-old fish. For the GD simulation, a fish that is heterozygous for the GD will have 100% GD gametes and a fish homozygous for GD will be non-viable. The FL simulation involves the release of males and female fish. Any female born in the wild that carriers the FL system will be non-viable. The EGI and SSIMS fish produce non-viable offspring when crossed with wild-type fish. The SSIMS fish only produce males when crossed with SSIMS fish in the wild; all females die at larval stage. Simulations are performed on a hypothetical 100 ha lake. Simulated mutations in EGI or SSIMS scenarios generated wild-type fish that are capable of hybridizing with the engineered fish due to a mutation in the targeted promoter. Simulated mutations in the GD scenarios resulted in alleles that could not be targeted by the GD nuclease for copying by homology-directed repair. Of these, two-thirds where lethal if homozygous or combined with a GD allele; the remaining one-third of the mutations had no impact on the fish beyond resistance to the GD nuclease. All carp simulations assumed that standard seining practice was performed in addition to a genetic control strategy. Seining reduces the number of engineered fish needed for control, however, it may not be necessary in all cases.
Simulations started following reduction in concentration of fish in the lake to about 1 carp/ha, which can be achieved by conventional management practices such as, for example, netting or the use of a piscicide. An optimum restocking number and male-female ratio was earlier identified by running several simulations with different release numbers and male-female ratio. Data shown in
Results show that in the presence of no mutations, a one-time release of FL, or EGI fish perform marginally better than no genetic management strategy. Genetic conditions that effectively control fish population to extinction are GD or SSIMS. Since GD intervention are susceptible escape mutations from non-homologous end joining (NHEJ), and SSIMS are, in theory, susceptible to escape mutations in the targeted promoter region, these two conditions were simulated with a relatively conservative rate of 1 in a 1000. The simulation showed that SSIMS intervention is more resistant to promoter mutation while GD interventions are very sensitive to NHEJ. SSIMS is therefore a both an effective and robust method for control of wild populations.
An agent-based model was created to simulate a population of Aedes aegypti mosquitoes, based on a previously described model that incorporates density-dependent mortality for eggs and larvae in the first two instars (Dye, C., 1984. Journal of Animal Ecology 53(1):247-268). Values for average egg production per female, gonotrophic cycle length, and adult mortality per time step were obtained from published experimental results (Goindin et al., 2015. PLoS ONE, 10(8)).
The simulations described in
Each of the genetic control strategies were effective when control animals were released at 10× population numbers. Notably, the SSIMS strategy eliminated wild-type adults completely after only 35 time steps, compared to 40 time steps for female lethal and 60 time steps for engineered genetic incompatibility alone. When GMO animals were released at 5× wild-type population numbers, only the SSIMS approach effectively eradicated wild-type animals. The wild-type adult population numbers went to zero after 60 time steps, while wild adult populations grew at a rate that mirrors the no treatment control after an initial suppression period for either single genetic control strategy. Based on the numbers of GMO adults produced in each experiment, it appears that the reason the combined approach outperformed the others is that incompatible adults were present for twice as long. By releasing both sexes of the SSIMS mosquitoes, two generations of incompatible mosquitoes were produced. This simulation allowed for the release of mosquitoes across multiple life stages. Initial SSIMS females survive larval stages via addition of repressor of FL.
Thus, this disclosure describes a system for controlling population of a biological species. The system uses genetically-modified individuals of the biological species designed so that the offspring of matings between the genetically-modified individuals and breed wild individuals of the biological are nonviable.
Generally, the genetic modifications involve two separate genetic modifications that, in concert with one another, can provide synergistic control of the wild population of the biological species compared to systems that use either of the genetic modifications alone. The first genetic modification involves using a repressible genetic mutation that produces lethality or sterility in one sex when an individual of that sex harbors the genetic mutation and is reared in an environment that lacks the repressor. The repressible genetic mutation has no effect on members of the other sex even when reared in the absence of the repressor. In practice, this allows one to rear individuals of both sexes under a controlled environment that includes the repressor—e.g., tetracycline—and then release the mature individuals into a wild environment that lacks the repressor. Since absence of the repressor affects only juveniles, the released individuals are viable and are able to mate with wild individuals (or with other genetically-modified individuals). As used herein, the term “juvenile” refers to an individual who has not yet reached an age of sexual maturity. Offspring of matings that occur in the wild—e.g., in the absence of the repressor—and that harbor the repressible genetic mutation will be affected and will either be sterile or will die, depending on whether the repressible genetic mutation induces sterility or lethality.
The second genetic mutation is an underdominance mutation in which the heterozygous state induces a selective disadvantage (e.g., death, sterility, etc.). Thus, matings in the wild between the genetically-modified individuals of the system population and wild individuals will produce nonviable offspring. Homozygous individuals harboring the underdominance genetic mutation are viable, but since there is no repressor to repress the first mutation, matings in the wild between two individuals of the system population will produce viable offspring of only one sex. This feature of the system can produce self-regulation of the system as the supply of individuals harboring both mutation will eventually die off.
The genetic mutations can be incorporated into any suitable species that reproduces sexually and whose population one wishes to control. The genetic mutations may be introduced into individuals of the biological species using conventional method to introduce each genetic mutation into individuals of the biological species to produce the system population. Generally, the biological species can be a pest species or an invasive species. In certain embodiments, the biological species can be a fish or an insect.
In one exemplary embodiment, the SSIMS organisms can be created using the model eukaryote, D. melanogaster. The exemplary SSIMS flies were created by combining two distinct genetic technologies together: engineered genetic incompatibility (e.g., synthetic incompatibility) and sex sorting (e.g., female lethality). In the exemplary embodiment, flies were created containing a female lethality system (
While exemplified in the context of an embodiment in which sex sorting is accomplished using a female lethality construct, SSIMS organisms and methods described herein can involve any known, conventional sex sorting genetic strategy. Exemplary sex sorting genetic technologies are described in, for example, U.S. Pat. No. 10,376,925; International Publication No. WO 2006/060603 A1; and U.S. Provisional Patent Application No. 62/909,536, (filed Oct. 2, 2019, entitled “SYSTEMS AND METHODS FOR BATCH CULTIVATION OF NON-TRANSGENIC HETEROGAMETES”).
While exemplified in the context of an embodiment in which the SSIMS fail to produce viable progeny due to engineered genetic incompatibility, SSIMS organisms and methods described herein can involve other synthetic incompatibility strategies that make SSIMS organisms incapable of producing viable progeny when crossed with wild-type organisms. Exemplary alternative strategies for producing synthetic incompatibility include, for example, underdominant systems of insect control (Reed, F. A., 2014, PLoS One 9:e97557), precision guided sterile insect technique (Kandul et. al., 2019, Nat Comm 10:84, doi:10.1038/s41467-018-07964-7), and those described in U.S. Patent Publication No. 2018/0327762 A1, International Publication No. WO 2018/209014 A1, U.S. Provisional Patent Application No. 62/928,612 (filed Oct. 31, 2019, entitled “SYSTEMS AND METHODS FOR GENERATING GENETIC INCOMPATIBILITY”),
While exemplified in the context of D. melanogaster, SSIMS organisms may be constructed from any suitable multicellular organism. Exemplary plants into which the SSIMS system may be introduced can include, for example, a field crop (e.g., tobacco, corn, soybean, rice, etc.), a tree (e.g., poplar, rubber tree, etc.), or turfgrass (e.g. creeping bentgrass). Exemplary animals into which the SSIMS system may be introduced can include, for example, an insect (e.g., mosquito, tsetse fly, spotted-wing drosophila, olive fly, gypsy moth, codling moth, deer tick, etc.), a fish (e.g., salmon, carp, sea lamprey, etc.), a mammal (e.g., swine, a mouse, a rat, etc.), an amphibian (e.g., a cane toad, a bullfrog, etc.), a reptile (e.g., brown tree snake, etc.), or a crustacean (e.g., rusty crayfish, etc.).
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
Plasmid expressing dCas9-VPR was constructed by Gibson assembly combining NotI linearized pMBO2744 attP vector backbone with dCas9-VPR PCR amplified from pAct:dCas9-VPR (Addgene #78898) and SV40 terminator for pH-Stinger (BDSC) to generate pMM7-6-1 (SEQ ID NO:1). Gibson assembly was used to clone 5′UTR and approximately 1.5 kb of promoter sequence pFoxO into Nod linearized pMM7-6-1 to create pMM7-6-2 (SEQ ID NO:2).
Plasmid expressing both sgRNAs and dCas9-VPR were generated by assembling amplified sgRNA cassettes targeting pyr (BDSC stock #67537) into KpnI linerarized plasmid pMM7-6-2 to create plasmid pAH1 (SEQ ID NO:3).
The tetracycline female lethal circuit was made by adapting a previously described female-lethal piggybac vector, pB[FL3] (Li et al., 2014, Insect Biochem. Mol. Biol. 51:80-88). The final plasmid, pMM7-8-1 (SEQ ID NO:8), was made by transferring the lethal circuit to pUB-EGFP (Schetelig et al., 2009, Proc. Natl. Acad. Sci. U.S.A. 106:18171-18176), which contains an attB site for ΦC31-mediated integration and ubiquitin promoter driven EGFP expression.
D. melanogaster strains were maintained at 25° C. and 12-hour days in cornmeal agar (NUTRI-FLY, Genesee Scientific Corp., El Cajon, Calif.) supplemented with 10-200 μg/ml tetracycline, as necessary. Experimental crosses were performed at 25° C. and 12-hour days. Existing Cas9 and sgRNA strains were obtained from the Bloomington Drosophila Stock Center (Bloomington, Ind.). All transgenic flies were generated via ΦC31-mediated integration targeted to attP landing sites. Embryo microinjections were performed by BestGene Inc. (Chino Hills, Ca).
Transgenic EGI D. melanogaster strain, DmEGIpyr was generated by microinjection and ΦC31 mediated integration of pAH-1 into the 2nd chromosome attP site of y[1] w[1118]; PBac{y[+]-attP-3B}VK00037; +(BDSC #9752). Transgenic animals were isolated by crossing to balancers present on 2nd (CyO-GFP) and 3rd (Tm2 and Tm6) chromosome, then homozygosed by selecting non-balancer animals to generate true breeding strain.
Transgenic female lethal strains were generated by microinjection and ΦC31 integration of pMM7-8-1 into the X-chromosome at attP sites y[1] w[*] P {y[+t7.7]=CaryIP}su(Hw)attP8 (BDSC #32233) to create DmXFL1tTA or y[1]w[1118]pBac{y[+]-attP-9A}VK00006 (BDSC #9726) to create DmXFL2tTA. Transgenic animals were isolated by crossing to FM6 balancers, then homozygosed by selecting non-balancer animals to generate true breeding strain. DmXFL12tTA flies were created by isolating recombinant chromosome of DmXFL1tTA and DmXFL2tTA and screening for the presence of transgene in both locations of the X-chromosome.
SSIMS flies were generated by mating female lethal flies, DmXFL12tFA and DmEGIpyr together by using balancer chromosome strains that prevent recombination across chromosomes. The following balancer chromosomes were used: FM7-GFP on X, CyO-GFP on 2, Tm2 and Tm6 on 3.
Genetic compatibility was assayed between parental stock homozygous for SSIMS and between SSIMS and wild-type flies. Test crosses were performed by crossing sexually-mature adult males to sexually-mature respective virgin females. The adults were removed from the vials after five days and the offspring were counted after fifteen days.
SSIMS Cyprinus carpio are produced by engineering Female Lethal (FL) components into the fish as previously described (Thomas et al., 2000. Science 287:2474-2476; Concha et al., 2016. BMC Biol. 14:72; Thresher et al., 2014. Nat. Biotechnol. 32:424-427; Fu et al., 2010. Proc. Natl. Acad. Sci. 107:4550-1554).
The transformed fish are further modified by introducing a synthetic incompatibility (SI) components as previously described (Reed, F. A., 2014. PLoS One 9:e97557; Maselko et al., 2017. Nat. Commun. 8:883; Aliota et al., 2016. Sci. Rep. 6:28792).
SSIMS Aedes aegypti are produced by engineering Female Lethal (FL) components into the mosquitoes as previously described (Thomas et al., 2000. Science 287:2474-2476; Concha et al., 2016. BMC Biol. 14:72; Thresher et al., 2014. Nat. Biotechnol. 32:424-427; Fu et al., 2010. Proc. Natl. Acad. Sci. 107:4550-4554).
The transformed mosquitoes are further modified by introducing a synthetic incompatibility (SI) components as previously described (Reed, F. A., 2014. PLoS One 9:e97557; Maselko et al., 2017. Nat. Commun. 8:883; Aliota et al., 2016. Sci. Rep. 6:28792).
In the preceding description and following claims, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended—i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).
In the preceding description, particular embodiments may be described in isolation for clarity. Unless otherwise expressly specified that the features of a particular embodiment are incompatible with the features of another embodiment, certain embodiments can include a combination of compatible features described herein in connection with one or more embodiments.
For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.
Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.
All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.
This application claims the benefit of U.S. Provisional Patent Application No. 62/755,939, filed Nov. 5, 2018, which is incorporated herein by reference in its entirety.
This invention was made with government support under Grant No. HR0011836772 awarded by the Department of Defense/Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/059826 | 11/5/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62928612 | Oct 2019 | US | |
| 62909536 | Oct 2019 | US | |
| 62755939 | Nov 2018 | US |
