Patent Application
20220330533
This application contains a Sequence Listing electronically submitted to the United States Patent and Trademark Office via EFS-Web as an ASCII text file entitled “Seq_Listing-0110-000629_ST25.txt” having a size of 30 kilobytes and created on Sep. 29, 2020. Due to the electronic filing of the Sequence Listing, the electronically submitted Sequence Listing serves as both the paper copy required by 37 CFR § 1.821(c) and the CRF required by § 1.821(e). The information contained in the Sequence Listing is incorporated by reference herein.
This disclosure describes, in one aspect, a system that includes a first strain of a biological species genetically engineered to include a conditional Y-linked genetic lethal circuit and a second strain of the biological species genetically engineered to include a conditional X-linked genetic lethal circuit. The system may be used to selectively produce non-transgenic males.
In some embodiments, the X-linked genetic lethal circuit is the same as the Y-linked genetic lethal circuit.
In some embodiments, the X-linked genetic lethal circuit is different than the Y-linked genetic lethal circuit.
In some embodiments, the biological species is a pest species.
In some embodiments, the biological species is a species in which one sex has greater commercial value than the other sex.
In another aspect, this disclosure describes a method of selecting non-transgenic males of a biological species. Generally, the method includes providing a first strain of the biological species genetically engineered to have a conditional Y-linked genetic lethal circuit; providing a second strain of the biological species genetically engineered to have a conditional X-linked genetic lethal circuit; performing a first cross mating males of the first strain and females of the first strain under conditions effective to express the conditional Y-linked genetic lethal circuit, thereby producing non-transgenic female progeny, then mating the non-transgenic progeny of the first cross with males of the second strain under conditions effective to express the conditional X-linked genetic lethal circuit, thereby producing non-transgenic males.
In some embodiments, the X-linked genetic lethal circuit is the same as the Y-linked genetic lethal circuit.
In some embodiments, the X-linked genetic lethal circuit is different than the Y-linked genetic lethal circuit.
In some embodiments, the biological species is a pest species.
In some embodiments, the biological species is a species in which one sex has greater commercial value than the other sex.
In some embodiments, the method further includes subjecting the non-transgenic males to a treatment effective to sterilize the males. In some of these embodiments, the males are sterilized by subjecting the males to X-ray irradiation.
In another aspect, this disclosure describes a system that includes a first strain of a biological species genetically engineered to have a conditional W-linked genetic lethal circuit and a second strain of the biological species genetically engineered to have a conditional Z-linked genetic lethal circuit. The system may be used to selectively produce non-transgenic females.
In some embodiments, the Z-linked genetic lethal circuit is the same as the W-linked genetic lethal circuit.
In some embodiments, the Z-linked genetic lethal circuit is different than the W-linked genetic lethal circuit.
In some embodiments, the biological species is a pest species.
In some embodiments, the biological species is a species in which one sex has greater commercial value than the other sex.
In another aspect, this disclosure describes a method of selecting non-transgenic females of a biological species. Generally, the method includes providing a first strain of the biological species genetically engineered to have a conditional W-linked genetic lethal circuit, providing a second strain of the biological species genetically engineered to have a conditional Z-linked genetic lethal circuit, performing a first cross mating males of the first strain and females of the first strain under conditions effective to express the conditional W-linked genetic lethal circuit, thereby producing non-transgenic male progeny, and mating the non-transgenic progeny of the first cross with females of the second strain under conditions effective to express the conditional Z-linked genetic lethal circuit, thereby producing non-transgenic females.
In some embodiments, the Z-linked genetic lethal circuit is the same as the W-linked genetic lethal circuit.
In some embodiments, the Z-linked genetic lethal circuit is different than the W-linked genetic lethal circuit.
In some embodiments, the biological species is a pest species.
In some embodiments, the biological species is a species in which one sex has greater commercial value than the other sex.
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.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
This disclosure describes and demonstrates systems and methods for batch production of the heterogametic sex of a species that are suitable for, for example, sterile pest control. In many species, the heterogametic sex is male (XY,
Insect pests impose a major burden to food production and human health worldwide. The most successful population control method in use today is the sterile insect technique (SIT). SIT relies on mass rearing of pest insects followed by a sterilization treatment (e.g., X-ray irradiation). Sterilized insects are released into the wild where sterile males compete with wild males to seek out and mate with wild females. The female of many pest insects will typically only mate once in her lifetime. Mating with a sterile male therefore prevents successful reproduction. Sufficiently large releases of sterile insects can be used to eliminate wild populations or prevent their introduction in an area at threat of their introduction. Also, SIT is considered safe to humans and the environment, as there are fewer off-target effects compared to the application of chemical pesticides.
Existing SIT programs are used to control several major agricultural pests including the New World Screwworm (Cochliomyia hominivorax), Mediterranean Fruit Fly (Ceratitis capitata), and Queensland Fruit Fly (Bactrocera tryoni). All together, these programs produce and release billions of sterile insects on a weekly basis. SIT for many insects, including C. hominivorax and B. tryoni, currently involves releasing both sterilized males and females. However, the effectiveness of SIT can be substantially increased if only males are released since they will then seek out wild females instead of mating with co-released sterile females. In some insect pests, such as the Yellow Fever Mosquito (Aedes aegypti), SIT programs only release sterile males since sterile females can vector disease.
A variety of sex-sorting techniques have been developed. Mechanical separation of Aedes aegypti pupae based on size differences can be effective and flow cytometric separation of transgene-expressing female Anopholes gambiae has been demonstrated. These approaches can be labor intensive and/or require sophisticated equipment, however. Combining irradiation with temperature-sensitive lethal (tsl) mutant strains of C. capitata is presently in use in SIT programs. Repressible transgenic female-elimination constructs act as genetic biocontrol systems on their own and have been developed for Ae. aegypti, C. hominivorax, Sheep Blow Fly (Lucilia cuprina), Diamondback moth (Plutella xylostella), Pink Bollworm (Pectinophora gossypiella), and Silkworm (Bombyx mori). Despite their effectiveness, specificity, reduction of insecticide use, and safety, public resistance and regulatory hurdles have limited the broad use of released transgenic insects for pest control.
The sex selection methods described herein may have applications beyond controlling pest species. For example, selection of female-only progeny is desirable in egg-layer poultry production since there is little economical need for the male chicks in the egg-layer industry. Current practices of female-only chicken selection use physical characteristics in day-old chicks. In some cases, the separated male chicks are culled and discarded, often subjected to industrial grade mechanical grinders. This practice is banned in several countries as being inhumane. It is also laborious and error-prone. Other methods for in-egg sex selection require individual screening of eggs with sophisticated and time-consuming instrumentation. The methods described herein provide a humane, labor-free, and accurate strategy for sex selection in egg-laying poultry.
This disclosure describes a genetic approach to produce non-transgenic males in Drosophila melanogaster, referred to herein as Subtractive Transgene Sex Sorting (STSS). STSS relies on two transgenic strains, each of which has bi-sex lethal genetic circuit that can be induced or repressed. One of the strains has the lethal circuit on the Y-chromosome (YL strain,
A repressible lethal genetic construct can be designed with a conditionally-expressed promoter driving a toxic gene product. Two exemplary constructs are illustrated in
PhiC31-mediated transgenesis was used to integrate a single copy of the tTA circuit into AttP landing sites on the X and Y chromosomes of two separate strains (referred to as DmXL-tTA and DmYL-tTA from here on). Both of these strains were maintained in the presence of 200 μg/ml tetracycline. The genotype of transgenic flies was confirmed by PCR amplification and Sanger sequencing of engineered loci. The DmXL-tTA were mated with a X-chromosome balancer strain and then selfed to screen for females homozygous for the modified X-chromosome, which was confirmed by PCR. From this point on, DmXL-tTA and DmYL-tTA were maintained as true-breeding lines in the presence of tetracycline.
Similar methods were used to create XFL strain with the exception of there being insertions into two separate locations in the X-chromosome (
To test the efficiency of toxic gene expression, virgin females and males were mated on media lacking tetracycline. In each of three replicate crosses, no DmXL-tTA adults survived to adulthood (
Sub-Stoichiometric Ratio of Mixed-Sex DmXLtTA to Female DmYLtTA Sufficient for Non-Transgenic Male Production
Non-transgenic males can be generated by crossing non-transgenic females produced by the DmYL-tTA strain and males from a mixed-sex true-breeding population of DmXL-tTA flies (
At or below the optimal ratios of DmXL-tTA males to DmYL-tTA females, 100% male offspring (Ncombined=5388 male offspring, 0 female offspring) were observed (
An alternative approach of creating STSS (
Next, the effectiveness of producing only non-transgenic males by the mating scheme in
This disclosure therefore describes a method of Subtractive Transgene Sex Sorting (STSS) using D. melanogaster as a model system. While described in detail in the context of an exemplary embodiment in which species is D. melanogaster, the methods described herein can involve any organism that relies on genetic, as opposed to environmental, sex determination. Exemplary other species in which the method may be employed include, for example, an insect (e.g., mosquito, tstetse fly, spotted-wing drosophila, diamond back moth, fall army worm, soybean gall midge, white fly, Mediterranean fruit fly, olive fly, gypsy moth, codling moth, deer tick, etc.), a fish (e.g., salmon, carp, sea lamprey, etc.), a bird (e.g., poultry), 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 method involves the use of two genetically-engineered strains of a pest species and a mating protocol that produces only non-transgenic males. The males may be subsequently sterilized using, for example, SIT sterilization techniques to produce sterile non-transgenic males. The sterile non-transgenic males may be released to control the population of the pest species.
Each strain is engineered to possess a lethal genetic circuit that can be induced or repressed. While described herein in the context of an exemplary embodiment in which both genetically engineered strains possess a tet-transactivator (tTA) genetic circuit that is lethal in the absence of tetracycline, one or both strains may be constructed to include an alternate lethal genetic circuit (e.g. temperature inducible activation of a gene causing lethality,
The basic genetic architecture has been demonstrated in numerous pest insects and STSS can be readily adapted to improve SIT programs by enabling efficient sex-sorting for male only release. Although described in the context of using PhiC31-mediated integration, CRISPR systems also may be used to target integration to many genomic loci in insects, including the repeat rich Y chromosome. This approach allows one to generate transgene-free males in species where males are heterogametic. For species where the female is heterogametic (i.e., lepidoptera), generation of transgene-free males is simplified and would only require a W′ construct. This approach can be applied to species with homomorphic sex chromosomes, assuming the sex-determining region of the sex chromosome is accessible to transgene integration technology.
Using the STSS system, no transgenic flies survives in the absence of tetracycline. The scale of the experiments described herein support a conclusion that the transgenic fly escape is less than 0.1%. SIT programs generate millions of flies for release on a weekly basis and some small number of transgenic flies may be produced and released using the STSS system, although they are likely to suffer fitness defects.
Production of males incapable of reproduction with wild females does not necessarily require radiation treatment. The incompatible insect technique relies on males infected with certain strains of Wolbachia bacteria. Mating between infected males and uninfected females results in embryonic lethality since the female-produced egg does not contain an antidote to a deubiquitylating enzyme toxin delivered by the sperm. Transgenic approaches have been developed or proposed to generate males that cannot reproduce with wild females. So far only Release of Insects with Dominant Lethal (RIDL) has made it to small scale commercial use for the control of Ae. aegypti. RIDL utilizes a bi-sex lethal genetic circuit where larvae, but not adults, require tetracycline to survive to adulthood. Mechanically sorted male pupae are subsequently used for release and their offspring die in the absence of tetracycline in the wild.
Combining STSS with cytoplasmic incompatibility can eliminate the need for any additional sterilization treatments and could enable the release of eggs/larvae to further reduce costs. Shipping of eggs, particularly of species such as Ae. aegypti that can be stored dry for extended periods, would allow for rearing facilities to be located far from control sites and reduce local infrastructure costs. However, this would require using non-antibiotic control of a lethal circuit such as temperature inducible activation of a lethal gene.
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 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.
The tetracycline repressible lethal circuit was made by adapting a previously described female-lethal piggybac vector, pB[FL3] (Yan et al., 2017, Sci Rep 7:2538). The female specific intron and 5′UTR were replaced with a myosin heavy chain intron and syn21 translational enhancers (Pfeiffer et al., 2012, Proc Natl Acad Sci USA 109: 6626-6631). The final plasmid, pMM7-10-1 (SEQ ID NO:1), was made by transferring the lethal circuit to pUB-EGFP (Schetelig et al., 2009, Proc Natl Acad Sci USA 106: 18171-18176), which contains an attB site for PhiC31 mediated integration and ubiquitin promoter driven EGFP expression.
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:2), was made by transferring the lethal circuit to pUB-EGFP (Schetelig et al., 2009, Proc Natl Acad Sci USA 106:18171-18176), which contains an attB site for PhiC31 mediated integration and ubiquitin promoter driven EGFP expression.
D. melanogaster strains were maintained at 25° C. and 12 hours light in cornmeal agar (FLYSTUFF, Genesee Scientific Corp., San Diego, Calif.) supplemented with 10-200 μg/ml tetracycline, as necessary. Transgenic D. melanogaster strains where generated by microinjection (BestGene Inc, Chino Hills, Calif.) and PhiC31 mediated integration of pMM7-10-1 into the X-chromosome attP site of y[1] w[*] P{y[+t7.7]=CaryIP}su(Hw)attP8 (BDSC #3233; Pfeiffer et al., 2010, Genetics 186: 735-755) to make DmXLtTA and the Y-chromosome attP of y1 w*/Dp(2;Y)G, P{CaryP}attPY (Szabad et al., 2012, Genes/Genomes/Genetics 2: 1095-1102) to make DmYLtTA.
Desired number of male and virgin female flies were moved to new tubes containing media either with or without tetracycline and allowed to lay eggs for five days at 25° C. and 12 hours light protocol. After five days, adults were removed from the tubes and offspring were allowed to develop in the incubator. Adult flies were counted as they emerged from the pupae for a total of 15 days from the start of experiment.
Fly genomic DNA was isolated in a pool by grinding in 25 μl of “Squish Buffer” (10 mM Tris, 1 mM EDTA, 25 mM NaCl, 8 U/ml ProK (New England Biolabs, Inc., Ipswich, Mass.) per adult. ProK was heat inactivated at 98° C. for four minutes. For transgene PCR screen, 1181 STSS males were pooled together as one sample and compared to five male and five female DMXL-tTA flies in a separate pool of genomic DNA as positive control. The positive control samples were diluted in with STSS gDNA in the following ratios: 1:5, 1:25, 1:125, 1:625, and 1:3125. For each reaction, 1 μl, of template gDNA was used in a 20 μL PCR reaction with primers that anneal within the transgene. The following primers were used for amplification of the transgene,
All the experiments were performed with at least two and as high as 10 replicates. Raw offspring numbers from each experiment were converted into percent male/female and averaged across the replicates. Chi-squared test was performed to test difference between observed and expected sex ratio in different mating. Number of flies from each experiment across the different replicates was summed together then converted into percent male/female and used in the Chi-squared test. To test the effect of different parental male-female ratio on adult offspring numbers, One-way ANOVA was performed followed by Bonferroni's post-hoc test. P-value <0.05 was considered significant.
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. 62/909,536, filed Oct. 2, 2019, 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/US2020/053749 | 10/1/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62909536 | Oct 2019 | US |
