Patent Application
20230416783
This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named Sequence_Listing.txt. The ASCII text file, created on Dec. 19, 2022, is 11,045 bytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.
Exploiting post zygotic barriers between otherwise reproductively compatible organisms to ensure reproductive isolation has practical applications to both basic and applied research. It can be utilized to study natural speciation processes (Moreno PLoS One. 7, e39054 (2012)) and is a useful research tool. These barriers can also block gene flow from engineered organisms, such as agricultural plants, to wild varieties by preventing the development of viable hybrid zygotes (Maselko et al., Nat. Commun., 8:883 (2017); Burgos et al., Plant Physiol., 166:1208-1220 (2014)).
This disclosure provides methods and materials for engineering reproductive isolation in an animal.
In one aspect, this disclosure a method of engineering a reproductive barrier in an insect including: (a) introducing into a first insect a first nucleic acid sequence, where the first nucleic acid sequence targets a genomic sequence, where the genomic sequence is proximal to a transcription start site of a gene product; and (b) introducing into a second insect a second nucleic acid sequence encoding a nuclease-deficient endonuclease; and (c) genetically crossing the first insect with the second insect, where the second insect includes a second copy of the genomic sequence where the second copy of the genomic sequence is proximal to a transcription start site of a second copy of the gene product, where the first nucleic acid sequence targets the genomic sequence and/or the second copy of the genomic sequence, and where in the progeny of the genetic cross between the first insect and the second insect the nuclease-deficient endonuclease binds to the genomic sequence and/or the second copy of the genomic sequence, thereby promoting expression of the (i) gene product and/or (ii) the second copy of the gene product. In some embodiments, the nuclease-deficient endonuclease interacts with the genomic sequence and/or the second copy of the genomic sequence via the first nucleic acid sequence. In some embodiments, step (a) includes integrating the first nucleic acid sequence into the genome of the first insect. In some embodiments, step (b) includes integrating the second nucleic acid sequence into the genome of the second insect.
In some embodiments, the genomic sequence includes a regulatory element. In some embodiments, the regulatory element at least partially controls expression of the gene product. In some embodiments, the regulatory element includes a promoter, an enhancer, a silencer, an insulator, a locus control region, or a synthetic promoter. In some embodiments, the regulatory element is a promoter. In some embodiments, expression of the gene product is lethal when misexpressed as compared to native expression.
In some embodiments, the first nucleic acid sequence includes a guide RNA targeting (i) the genomic sequence and/or (ii) the second copy of the genomic sequence. In some embodiments, the nuclease-deficient endonuclease includes a CRISPR-associated sequence 9 (Cas9) endonuclease or a variant thereof, a CRISPR-associated sequence 13 (Cas13) endonuclease or a variant thereof, a CRISPR from Prevotella and Francisella 1 (Cpf1) endonuclease or a variant thereof, a CRISPR from Microgenomates and Smithella 1 (Cms1) endonuclease or a variant thereof, or a CRISPR-associated sequence 6 (Cas6) endonuclease or a variant thereof. In some embodiments, the nuclease-deficient endonuclease includes a Streptococcus pyogenes Cas9 (SpCas9), a Staphylococcus aureus Cas9 (SaCas9), a Francisella novicida Cas9 (FnCas9), or a variant thereof. In some embodiments, the variant thereof includes a protospacer adjacent motif (PAM) SpCas9 (xCas9), high fidelity SpCas9 (SpCas9-FIF1), a high fidelity SaCas9, or a high fidelity FnCas9. In some embodiments, the nuclease-deficient endonuclease includes a Cas fusion nuclease including a Cas9 protein or a variant thereof fused with a Fokl nuclease or variant thereof. In some embodiments, the nuclease-deficient endonuclease includes a CRISPR-associated sequence 9 (Cas9) endonuclease or a variant thereof. In some embodiments, nuclease-deficient Cas9 includes a first amino acid substitution of D10A and a second amino acid substitution of H840A.
In some embodiments, the nuclease-deficient endonuclease is fused to one or more effector domains. In some embodiments, the one or more effector domains including at least one of an activator domain, a repressor domain, a recruitment domain, a transcription factor, or a chromatin modifier. In some embodiments, the one or more effector domains including at least one of a SunTag, a SAM, a VPR, or a VP64. In some embodiments, the effector domain is VP64. In some embodiments, the effector domain is VPR.
In some embodiments, the first nucleic acid sequence includes one or more RNA hairpins, where the RNA hairpins bind one or more RNA binding proteins fused to one or more effector domains. In some embodiments, the one or more effector domains including at least one of an activator domain, a repressor domain, a recruitment domain, a transcription factor, or a chromatin modifier. In some embodiments, the one or more effector domains including at least one of a SunTag, a SAM, a VPR, or a VP64. In some embodiments, the effector domain is VP64. In some embodiments, the effector domain is VPR.
In some embodiments, step (a) further includes introducing into the first insect an endonuclease, where the endonuclease cleaves the genomic sequence, thereby creating a mutation in the genomic sequence. In some embodiments, the endonuclease includes a CRISPR-associated sequence 9 (Cas9) endonuclease or a variant thereof, a CRISPR-associated sequence 13 (Cas13) endonuclease or a variant thereof, a CRISPR from Prevotella and Francisella 1 (Cpf1) endonuclease or a variant thereof, a CRISPR from Microgenomates and Smithella 1 (Cms1) endonuclease or a variant thereof, or a CRISPR-associated sequence 6 (Cas6) endonuclease or a variant thereof. In some embodiments, the endonuclease includes a Streptococcus pyogenes Cas9 (SpCas9), a Staphylococcus aureus Cas9 (SaCas9), a Francisella novicida Cas9 (FnCas9), or a variant thereof. In some embodiments, the variant thereof includes a protospacer adjacent motif (PAM) SpCas9 (xCas9), high fidelity SpCas9 (SpCas9-FIF1), a high fidelity SaCas9, or a high fidelity FnCas9. In some embodiments, the mutation prevents the first nucleic acid sequence from targeting the genomic sequence. In some embodiments, the mutation includes an insertion or a deletion. In some embodiments, the mutation occurs within the first 10 nucleotides of a PAM sequence associated with the first nucleic acid sequence. In some embodiments, step (b) includes transiently introducing the endonuclease. In some embodiments, step (b) includes introducing mRNA encoding the endonuclease. In some embodiments, the mutation in the genomic sequence does not alter expression of the gene product. In some embodiments, the gene product including at least one of even skipped (eve), head involution defective (hid), hedgehog (hh), or wingless (wg), or a combination thereof.
In some embodiments, the first nucleic acid sequences includes a first gRNA and a second gRNA, where the first gRNA targets a first genomic sequence and the second gRNA targets a second genomic sequence. In some embodiments, the first nucleic acid sequence includes a first gRNA, a second gRNA, and a third gRNA, where the first gRNA targets a first genomic sequence, the second gRNA targets a second genomic sequence, and the third gRNA targets a third genomic sequence, where each genomic sequence is proximal to a transcription start site of a gene product. In some embodiments, the combination of gene products including at least one of: (i) eve and hid; (ii) eve, hid, and hh; (iii) eve, hid, and wg; or (iv) hh and wg. In some embodiments, the first insect is a mosquito from the genera Stegomyia, Aedes, Anopheles, or Culex and the second insect is a mosquito from the genera Stegomyia, Aedes, Anopheles, or Culex. In some embodiments, where the first mosquito and/or the second mosquito include Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus), Anopheles stephensi, Anopheles albimanus, Anopheles gambiae, Anopheles quadrimaculatus, Anopheles freebori, Culex species, or Culiseta melanura. In some embodiments, the insect includes a tephritid fruit fly selected from Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens), Oriental fruit fly (Bactrocera dorsalis), Olive fruit fly (Bactrocera oleae), Melon fly (Bactrocera cucurbitae), Natal fruit fly (Ceratitis rosa), Cherry fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyroni), Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), Oriental Fruit Fly (Bactrocera dorsalis), West Indian fruit fly (Anastrepha obliqua), the New World screwworm (Cochliomyia hominivorax), the Old World screwworm (Chrysomya bezziana), Australian sheep blowfly/greenbottle fly (Lucilia cuprina), the pink bollworm (Pectinophora gossypiella), the European Gypsy moth (Lymantria dispar), the Navel Orange Worm (Amyelois transitella), the Peach Twig Borer (Anarsia lineatella), the rice stem borer (Tryporyza incertulas), the noctuid moths, Heliothinae, the Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.), Boll weevil (Anthonomous grandis), the Colorado potato beetle (Leptinotarsa decern lineata), the vine mealybug (Planococcus ficus), Asian citrus psyllid (Diaphorina citri), Spotted wing drosophila (Drosophila suzukii), Bluegreen sharpshooter (Graphocephala atropunctata), Glassy winged sharpshooter (Flomalodisca vitripennis), Light brown apple moth (Epiphyas postvittana), Bagrada bug (Bagrada hilaris), Brown marmorated stink bug (Halyomorpha halys), Asian Gypsy Moth selected from the group of Lymantria dispar asiatica, Lymantria dispar japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba, Asian longhomed beetle (Anoplophora glabripennis), Coconut Rhinoceros Beetle (Oryctes rhinoceros), Emerald Ash Borer (Agrilus planipennis), European Grapevine Moth (lobesia botrana), European Gypsy Moth (Lymantria dispar), False Codling Moth (Thaumatotibia leucotreta), fire ants selected from Solenopsis invicta Buren, and S. richteri Forel, Old World Bollworm (Flelicoverpa armigera), Spotted Lanternfly (Lycorma delicatula), Africanized honeybee (Apis mellifera scutellata), Fruit and shoot borer (Leucinodes orbonalis), corn root worm (Diabrotica spp.), Western corn rootworm (Diabrotica virgifera), Whitefly (Bemisia tabaci), Flouse Fly (Musca domestica), Green Bottle Fly (Lucilia cuprina), Silk Moth (Bombyx mori), Red Scale (Aonidiella aurantia), Dog heartworm (Dirofilaria immitis), Southern pine beetle (Dendroctonus frontalis), Avocado thrip (Thysanoptera Spp.), Botfly selected from Oestridae spp. and Dermatobia hominis), Florse Fly (Tabanus sulcifrons), Florn Fly (Flaematobia irritans), Screwworm Fly selected from Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi, or C. minima, Tsetse Fly (Glossina spp.), Warble Fly selected from Flypoderma bovis or Hypoderma lineatum, Spotted lanternfly (Lycorma delicatula), Khapra beetle (Trogoderma granarium), Honeybee mite (Varroa destructor), Termites (Coptotermes formosanus), Hemlock woolly adelgid (Adelges tsugae), Walnut twig beetle (Pityophthorus juglandis), European wood wasp (Sirex noctilio), Pink-spotted bollworm (Pectinophora scutigera), Two spotted spider mite (Tertanychus urticae), Diamondback moth (Plutella xylostella), Taro caterpillar (Spodoptera litura), Red flour beetle (Tribolium castaneum), Green peach aphid (Myzus persicae), Cotton Aphid (Aphis gossypii), Brown planthopper (Nilaparvata lugens), Beet armyworm (Spodotera exigua), Western flower Thrips (Frankliniella occidentalis), Codling moth (Cydia pomonella), Cowpea weevil (Callosobruchus maculatus), Pea aphid (Acyrthosiphon pisum), Tomato leafminer (Tuta absoluta), Onion Thrips (Thrips tabaci), and Cotton bollworm (Helicoverpa armigera).
In another aspect, this disclosure features a method of engineering a reproductive barrier in an insect including: (a) introducing into a first insect a first nucleic acid sequence, where the first nucleic acid sequence targets a genomic sequence that is proximal to a transcription start site of a gene product; and an endonuclease, where the endonuclease cleaves the genomic sequence, thereby creating a mutation in the genomic sequence; and (b) introducing into a second insect a second nucleic acid sequence encoding a nuclease-deficient endonuclease; and (c) genetically crossing the first insect with the second insect, where the second insect includes a second copy of the genomic sequence, that is proximal to a transcription start site of a second copy of the gene product, where the first nucleic acid sequence targets the second copy of the genomic sequence, and where in the progeny of the genetic cross between the first insect and the second insect the nuclease-deficient endonuclease binds to the second copy of the genomic sequence, thereby promoting expression of the second copy of the gene product.
In another aspect, this disclosure features a genetically modified insect produced by the method of any of the method described herein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
Provided herein are methods for engineering reproductive barriers in an animal (e.g., insect) including (a) introducing into a first insect a first nucleic acid sequence, where the first nucleic acid sequence targets a genomic sequence, where the genomic sequence is proximal to a transcription start site of a gene product; and (b) introducing into a second insect a second nucleic acid sequence encoding a nuclease-deficient endonuclease; and (c) genetically crossing the first insect with the second insect, where the second insect includes a second copy of the genomic sequence where the second copy of the genomic sequence is proximal to a transcription start site of a second copy of the gene product, where the first nucleic acid sequence targets the genomic sequence and/or the second copy of the genomic sequence, and where in the progeny of the genetic cross between the first insect and the second insect the nuclease-deficient endonuclease binds to the genomic sequence and/or the second copy of the genomic sequence, thereby promoting expression of the (i) gene product and/or (ii) the second copy of the gene product.
The term “a” and “an” refers to one or more (i.e., at least one) of the grammatical object of the article. By way of example, “a cell” encompasses one or more cells.
As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example 20%, ±10%, or ±5%, are within the intended meaning of the recited value.
As used herein, an “effector domain” can refer to a protein interaction domain that can function in transcriptional regulation via their ability to (i) interact with the basal transcriptional machinery and general co-activators, (ii) interact with other transcriptional factors to allow cooperative binding, and (iii) directly or indirectly recruit histone and chromatin modifying enzymes.
As used herein, “engineered” or “genetically engineered,” in reference to organisms (e.g., insects), refers to an organism that comprises a nucleic acid sequence (e.g., DNA, RNA, or mRNA) that is not present in, or is present at a different level than, an otherwise similar organisms under similar conditions that is not engineered (an exogenous nucleic acid), or an organism that comprises a polypeptide expressed from said nucleic acid. In some embodiments, a genetically engineered organism has been altered from its native state by the introduction of an exogenous nucleic acid, or is the progeny of such an altered organism. In some embodiments, a genetically engineered organism comprises an exogenous nucleic acid (e.g., DNA, RNA, or mRNA).
The term “exogenous” refers to any material introduced from or originating from outside a cell, a tissue or an organism that is not produced by or does not originate from the same cell, tissue, or organism in which it is being introduced.
As used herein, an “insect” can refer to any member of the largest class of the phylum Arthropoda, which is itself the largest of the animal phyla. Insects have segmented bodies, jointed legs, and external skeletons (e.g., exoskeletons). In some embodiments, an insect can include a bedbug, a housefly, a clothes moth, a Japanese beetle, an aphid, a mosquito, a flea, a horsefly, a hornet, a butterfly, or a moth. In some embodiments, an insect can be a mosquito from the genera Stegomyia, Aedes, Anopheles, or Culex. In some embodiments, the mosquito can include Aedes aegypti, Aedes albopictus, Ochlerotatus triseriatus (Aedes triseriatus), Anopheles stephensi, Anopheles albimanus, Anopheles gambiae, Anopheles quadrimaculatus, Anopheles freebori, Culex species, or Culiseta melanura. In some embodiments, the insect can include a tephritid fruit fly selected from Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens), Oriental fruit fly (Bactrocera dorsalis), Olive fruit fly (Bactrocera oleae), Melon fly (Bactrocera cucurbitae), Natal fruit fly (Ceratitis rosa), Cherry fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyroni), Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), Oriental Fruit Fly (Bactrocera dorsalis), West Indian fruit fly (Anastrepha obliqua), the New World screwworm (Cochliomyia hominivorax), the Old World screwworm (Chrysomya bezziana), Australian sheep blowfly/greenbottle fly (Lucilia cuprina), the pink bollworm (Pectinophora gossypiella), the European Gypsy moth (Lymantria dispar), the Navel Orange Worm (Amyelois transitella), the Peach Twig Borer (Anarsia lineatella), the rice stem borer (Tryporyza incertulas), the noctuid moths, Heliothinae, the Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.), Boll weevil (Anthonomous grandis), the Colorado potato beetle (Leptinotarsa decern lineata), the vine mealybug (Planococcus ficus), Asian citrus psyllid (Diaphorina citri), Spotted wing Drosophila (Drosophila suzukii), Bluegreen sharpshooter (Graphocephala atropunctata), Glassy winged sharpshooter (Flomalodisca vitripennis), Light brown apple moth (Epiphyas postvittana), Bagrada bug (Bagrada hilaris), Brown marmorated stink bug (Halyomorpha halys), Asian Gypsy Moth selected from the group of Lymantria dispar asiatica, Lymantria dispar japonica, Lymantria albescens, Lymantria umbrosa, and Lymantria postalba, Asian longhomed beetle (Anoplophora glabripennis), Coconut Rhinoceros Beetle (Oryctes rhinoceros), Emerald Ash Borer (Agrilus planipennis), European Grapevine Moth (lobesia botrana), European Gypsy Moth (Lymantria dispar), False Codling Moth (Thaumatotibia leucotreta), fire ants selected from Solenopsis invicta Buren, and S. richteri Forel, Old World Bollworm (Flelicoverpa armigera), Spotted Lanternfly (Lycorma delicatula), Africanized honeybee (Apis mellifera scutellata), Fruit and shoot borer (Leucinodes orbonalis), corn root worm (Diabrotica spp.), Western corn rootworm (Diabrotica virgifera), Whitefly (Bemisia tabaci), Flouse Fly (Musca domestica), Green Bottle Fly (Lucilia cuprina), Silk Moth (Bombyx mori), Red Scale (Aonidiella aurantia), Dog heartworm (Dirofilaria immitis), Southern pine beetle (Dendroctonus frontalis), Avocado thrip (Thysanoptera Spp.), Botfly selected from Oestridae spp. and Dermatobia hominis), Florse Fly (Tabanus sulcifrons), Florn Fly (Flaematobia irritans), Screwworm Fly selected from Cochliomyia macellaria (C. macellaria), C. hominivorax, C. aldrichi, or C. minima, Tsetse Fly (Glossina spp.), Warble Fly selected from Flypoderma bovis or Hypoderma lineatum, Spotted lanternfly (Lycorma delicatula), Khapra beetle (Trogoderma granarium), Honeybee mite (Varroa destructor), Termites (Coptotermes formosanus), Hemlock woolly adelgid (Adelges tsugae), Walnut twig beetle (Pityophthorus juglandis), European wood wasp (Sirex noctilio), Pink-spotted bollworm (Pectinophora scutigera), Two spotted spider mite (Tertanychus urticae), Diamondback moth (Plutella xylostella), Taro caterpillar (Spodoptera litura), Red flour beetle (Tribolium castaneum), Green peach aphid (Myzus persicae), Cotton Aphid (Aphis gossypii), Brown planthopper (Nilaparvata lugens), Beet armyworm (Spodotera exigua), Western flower Thrips (Frankliniella occidentalis), Codling moth (Cydia pomonella), Cowpea weevil (Callosobruchus maculatus), Pea aphid (Acyrthosiphon pisum), Tomato leafminer (Tuta absoluta), Onion Thrips (Thrips tabaci), or Cotton bollworm (Helicoverpa armigera).
As used herein, the term “misexpression” or “misexpressed” may refer to the process or result of altering a gene product so as to change its associated character when compared to expression of a gene product in a “native” or “non-perturbed” state.
As used herein, the terms “nucleic acid” and “nucleotide” are intended to be consistent with their use in the art and to include naturally-occurring species or functional analogs thereof. Naturally-occurring nucleic acids generally have a backbone containing phosphodiester bonds. An analog structure can have an alternate backbone linkage including any of a variety of those known in the art. Naturally-occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).
A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include native or non-native nucleotides. In this regard, a native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine (A), thymine (T), cytosine (C), or guanine (G), and a ribonucleic acid can have one or more bases selected from the group consisting of uracil (U), adenine (A), cytosine (C), or guanine (G). Useful non-native bases that can be included in a nucleic acid or nucleotide are known in the art.
The term “nucleic acid” refers to a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination thereof, in either a single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is DNA. In some embodiments of any of the isolated nucleic acids described herein, the isolated nucleic acid is RNA.
Unless otherwise specified, a “nucleotide sequence encoding a protein” includes all nucleotide sequences that are degenerate versions of each other and thus encode the same amino acid sequence.
As used herein the term “promoter” may refer to a DNA sequence recognized by enzymes/proteins in a mammalian cell required to initiate the transcription of an operably linked coding sequence. A promoter typically refers, to e.g. a nucleotide sequence to which an RNA polymerase and/or any associated factor binds and at which transcription is initiated.
The promoter can be constitutive, inducible, or tissue-specific (e.g., a brain-specific promoter). The promoter can be an exogenous promoter operably linked to an isolated nucleic acid. The promoter can also be a genomic sequence where the promoter is proximal to a transcription start site and at least partially controls expression of the associated gene product. A promoter within the genome can be either proximal (e.g., within 2000 nucleotides) or distal (e.g., greater than 2000 nucleotides) from a transcription start site.
The term “proximal” may refer to a distance from a transcription start site to a genomic sequence where “proximal” may include genomic sequences (e.g., a regulatory elements) within at least 2000 nucleotides either upstream or downstream. “Proximal” may also refer to a physical distance between a genomic sequence and the transcription start site. For example, in the context of three-dimensional genome organization, a genomic sequence (e.g., a regulatory element) that is distal (e.g., greater than 2000 nucleotides away from the transcription start site) can be proximal to a transcription start site because the genomic sequence “loops” around to interact with the transcription start site. One example of this is enhancer-promoter interactions where a distal enhancer is brought proximal to the promoter via a “loop” in the three-dimensional organization of the genome.
As used herein, a “reproductive barrier” or “reproductive isolation” can refer to an evolutionary mechanism, behavior, and physiological process that are critical for speciation.
Reproductive barriers prevent members of different species from producing offspring, or ensure that any offspring are sterile. In some embodiments, a reproductive barrier can be a post-zygotic isolation mechanism, wherein the mechanism acts after fertilization preventing successful inter-population crossing. In some embodiments, a post-zygotic isolation mechanism can include zygote mortality, hybrid zygote abnormality, hybrid infertility, or low hybrid viability.
The term “transcription start site” may refer to the genomic location at which transcription begins.
The term “transduced”, “transfected”, or “transformed” refers to a process by which exogenous nucleic acid is introduced or transferred into a cell. A “transduced,” “transfected,” or “transformed” mammalian cell is one that has been transduced, transfected or transformed with exogenous nucleic acid (e.g., a gene delivery vector) that includes an exogenous nucleic acid encoding the first nucleic acid sequence and/or the nuclease-deficient endonuclease). In some embodiments, an exogenous nucleic acid can be introduced into a cell, e.g., by transfection (e.g., using transfectamine, cationic polymers, calcium phosphate or electroporation), by transduction (e.g., using a bacteriophage or recombinant viral vector), by mechanical delivery (e.g., magnetic beads), by lipid (e.g., 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)), or by transporter proteins.
For sequence comparison of polypeptides, typically one amino acid sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g., visual alignment or using publicly available software using known algorithms to achieve maximal alignment.
Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.
Provided herein are isolated nucleic acids that target a genomic sequence. In some embodiments, an isolated nucleic acid can target a genomic sequence, wherein the genomic sequence is proximal to a transcription start site of a gene product. In some embodiments, the isolated nucleic acid can include a guide RNA targeting the genomic sequence. In some embodiments, the isolated nucleic acid can include a single guide RNA (sgRNA).
In some embodiments, the isolated nucleic acid can include one or more RNA hairpins, wherein the RNA hairpins bind one or more RNA binding proteins fused to one or more effector domains. In some embodiments, the one or more effector domains can include at least one of an activator domain, a repressor domain, a recruitment domain, a transcription factor, or a chromatin modifier. In some embodiments, the one or more effector domains can include at least one of a SunTag, a SAM, a VPR, or a VP64. In some embodiments, the effector domain is VP64. In some embodiments, the effector domain is VPR.
Also provided herein are isolated nucleic acids that encode an endonuclease. In some embodiments, an isolated nucleic acid encodes a nuclease-deficient endonuclease. In some embodiments, the isolated nucleic acid encodes a nuclease-deficient endonuclease, wherein the nuclease-deficient endonuclease includes a CRISPR-associated sequence 9 (Cas9) endonuclease or a variant thereof, a CRISPR-associated sequence 13 (Cas13) endonuclease or a variant thereof, a CRISPR from Prevotella and Francisella 1 (Cpf1) endonuclease or a variant thereof, a CRISPR from Microgenomates and Smithella 1 (Cms1) endonuclease or a variant thereof, or a CRISPR-associated sequence 6 (Cas6) endonuclease or a variant thereof. In some embodiments, the nuclease-deficient endonuclease includes a Streptococcus pyogenes Cas9 (SpCas9), a Staphylococcus aureus Cas9 (SaCas9), a Francisella novicida Cas9 (FnCas9), or a variant thereof. In some embodiments, the variant thereof includes a protospacer adjacent motif (PAM) SpCas9 (×Cas9), high fidelity SpCas9 (SpCas9-FIF1), a high fidelity SaCas9, or a high fidelity FnCas9. In some embodiments, the nuclease-deficient endonuclease includes a Cas fusion nuclease comprising a Cas9 protein or a variant thereof fused with a Fokl nuclease or variant thereof. In some embodiments, the nuclease-deficient endonuclease includes a CRISPR-associated sequence 9 (Cas9) endonuclease or a variant thereof. In some embodiments, the nuclease-deficient Cas9 comprises a first amino acid substitution of D10A and a second amino acid substitution of H840A. In some embodiments, the nuclease-deficient endonuclease is fused to one or more effector domains.
In some embodiments of any of the isolated nucleic acid sequences described herein, the isolated nucleic acid sequence is operably linked to a promoter (e.g., any of the exemplary promoters described herein). In some embodiments, the promoter can include a ubiquitous promoter. In some embodiments, the ubiquitous promoter is an ubiquitin-63E promoter. In some embodiments, the promoter can include an early embryonic promoter. In some embodiments, the promoter is a bottleneck/bnk promoter.
In some embodiments of any of the methods described herein, introducing a nucleic acid into an insect is facilitated by vectors. For example, a vector can be an expression vector where the expression vector includes a promoter sequence operably linked to the sequence encoding the molecule (e.g., a nucleic acid molecule). Non-limiting examples of vectors include plasmids, transposons, cosmids, and viral derived vectors (e.g., any adenoviral derived vectors (AV) cytomegaloviral derived (CMV) vectors, simian viral derived (SV40) vectors, adeno-associated virus (AAV) vectors, lentivirus vectors, and retroviral vectors), and any Gateway® vectors. A vector can, for example, include sufficient cis-acting elements for expression where other elements for expression can be supplied by the host mammalian cell or in an in vitro expression system. Skilled practitioners will be capable of selecting suitable vectors and mammalian cells for introducing any of spatial profiling reagents described herein.
In some embodiments, a vector (e.g., gene delivery vector) can include any of the isolated nucleic acids described herein. In some embodiments, the gene delivery vectors are adeno-associated viral (AAV) vectors, lentiviral vectors, adenoviral vectors, or retroviral vectors. AAV vectors are generally described in, e.g., Asokan et al., Mol. Ther. 20: 699-708, 2012, and B. J. Carter, in “Handbook of Parvoviruses”, Ed., P. Tijsser, CRC Press, pp. 155-168, 1990. Adenoviral vectors are generally described in, e.g., Wold and Toth, Curr. Gene Ther. 13(6):421-433, 2013; Baron et al., Curr. Opin. Virol. 29:1-7, 2018; and Barry, Expert Rev. Vaccines 17(2): 163-173, 2018. Lentiviral vectors are generally described in, e.g., Milone and O'Doherty, Leukemia 32(7): 1529-1541, 2018, Zheng et al., Anat. Rec. 301(5): 825-836, 2018; and Cai et al., Curr. Gene Ther. 16(3): 194-206, 2016. Adenoviral vectors are generally described in, e.g., Tatsis et al., Mol. Ther. 10(4):616-629, 2004; Appaiahgari et al., Expert. Opin. Biol. Ther. 15(3):337-351, 2015; Coughlan, Methods Mol. Biol. 1108:23-40, 2014. Retroviral vectors are generally described in, e.g., Miller, Curr. Protoc. Hum. Genet. 80: Unit 12.5, 2014; Kim et al., Adv. Virus Res. 55:545-563, 2000; and Kurian et al., Mol. Pathol. 53(4):173-176, 2000. Some embodiments of any of the gene delivery vectors described herein, can include a promoter and/or enhancer (e.g., any of the exemplary tissue-specific promoter and/or enhancers described herein) that is operably linked to any of the isolated nucleic acids described herein.
In some embodiments, the gene delivery vectors described herein includes one or more (e.g., two, three, four, five, or six) of a promoter (e.g., any of the promoters described herein or known in the art), an enhancer (e.g., any of the enhancers described herein or known in the art), a Kozak sequence (e.g., any of the Kozak sequences described herein or known in the art), an RNA splicing sequence, a polyadenylation (poly(A)) signal sequence (e.g., any of the poly(A) signals described herein), and an internal ribosome entry site (IRES) sequence (e.g., any of the IRES sequences described herein or known in the art).
Also provided are kits that include any of the isolated nucleic acids or any of the gene delivery vectors described herein. In some embodiments, a kit can include a solid composition (e.g., a lyophilized composition including any of the gene delivery vectors described herein) and a liquid for solubilizing the lyophilized composition.
Provided herein are methods of engineering a reproductive barrier in an insect including: (a) introducing into a first insect a first nucleic acid sequence, wherein the first nucleic acid sequence targets a genomic sequence, wherein the genomic sequence is proximal to a transcription start site of a gene product; (b) introducing into a second insect a second nucleic acid sequence encoding a nuclease-deficient endonuclease; and (c) genetically crossing the first insect with the second insect, wherein the second insect comprises a second copy of the genomic sequence wherein the second copy of the genomic sequence is proximal to a transcription start site of a second copy of the gene product, wherein the first nucleic acid sequence targets the genomic sequence and/or the second copy of the genomic sequence, and wherein in the progeny of the genetic cross between the first insect and the second insect the nuclease-deficient endonuclease binds to the genomic sequence and/or the second copy of the genomic sequence, thereby promoting expression of the (i) gene product and/or (ii) the second copy of the gene product.
Also provided herein are methods of engineering a reproductive barrier in an insect including: (a) introducing into a first insect a first nucleic acid sequence, wherein the first nucleic acid sequence targets a genomic sequence that is proximal to a transcription start site of a gene product; and an endonuclease, wherein the endonuclease cleaves the genomic sequence, thereby creating a mutation in the genomic sequence; (b) introducing into a second insect a second nucleic acid sequence encoding a nuclease-deficient endonuclease; and (c) genetically crossing the first insect with the second insect, wherein the second insect comprises a second copy of the genomic sequence, that is proximal to a transcription start site of a second copy of the gene product, wherein the first nucleic acid sequence targets the second copy of the genomic sequence, and wherein in the progeny of the genetic cross between the first insect and the second insect the nuclease-deficient endonuclease binds to the second copy of the genomic sequence, thereby promoting expression of the second copy of the gene product.
In some embodiments, the nuclease-deficient endonuclease can interact with the genomic sequence and/or the second copy of the genomic sequence via the first nucleic acid sequence. In some embodiments, the first nucleic acid sequence includes a guide RNA targeting (i) the genomic sequence and/or (ii) the second copy of the genomic sequence. In some embodiments, step (a) includes integrating the first nucleic acid sequence into the genome of the first insect. In some embodiments, step (b) includes integrating the second nucleic acid sequence into the genome of the second insect. In some embodiments, the genomic sequence includes a regulatory element. In some embodiments, the regulatory element at least partially controls expression of the gene product. In some embodiments, the regulatory element includes a promoter, an enhancer, a silencer, an insulator, a locus control region, or a synthetic promoter. In some embodiments, the regulatory element is a promoter.
In some embodiments, the first nucleic acid sequences comprises a first gRNA and a second gRNA, wherein the first gRNA targets a first genomic sequence and the second gRNA targets a second genomic sequence. In some embodiments, the first nucleic acid sequence comprises a first gRNA, a second gRNA, and a third gRNA, wherein the first gRNA targets a first genomic sequence, the second gRNA targets a second genomic sequence, and the third gRNA targets a third genomic sequence, wherein each genomic sequence is proximal to a transcription start site of a gene product.
As used herein, a “gene product” can refer to the biochemical material (e.g., RNA or protein) that result from expression of a gene. In some embodiments, the gene product can be overexpressed when compared to wild-type gene expression. In some embodiments, expression of the gene product is lethal when misexpressed as compared to native expression.
In some embodiments, the gene product includes at least one of even skipped (eve), head involution defective (hid), hedgehog (hh), or wingless (wg), or a combination thereof. In some embodiments, the combination of gene products comprises at least one of: (i) eve and hid; (ii) eve, hid, and hh; (iii) eve, hid, and wg; or (iv) hh and wg.
In some embodiments, step (a) further includes introducing into the first insect an endonuclease, wherein the endonuclease cleaves the genomic sequence, thereby creating a mutation in the genomic sequence. In some embodiments, the endonuclease comprises a CRISPR-associated sequence 9 (Cas9) endonuclease or a variant thereof, a CRISPR-associated sequence 13 (Cas13) endonuclease or a variant thereof, a CRISPR from Prevotella and Francisella 1 (Cpf1) endonuclease or a variant thereof, a CRISPR from Microgenomates and Smithella 1 (Cms1) endonuclease or a variant thereof, or a CRISPR-associated sequence 6 (Cas6) endonuclease or a variant thereof. In some embodiments, the endonuclease comprises a Streptococcus pyogenes Cas9 (SpCas9), a Staphylococcus aureus Cas9 (SaCas9), a Francisella novicida Cas9 (FnCas9), or a variant thereof. In some embodiments, the variant thereof comprises a protospacer adjacent motif (PAM) SpCas9 (×Cas9), high fidelity SpCas9 (SpCas9-FIF1), a high fidelity SaCas9, or a high fidelity FnCas9. In some embodiments, step (b) includes transiently introducing the endonuclease. In some embodiments, step (b) includes introducing mRNA encoding the endonuclease.
As used herein, a “mutation” can refer to an alteration of the nucleotide sequence of a genomic sequence. In some embodiments, a mutation can include an insertion (e.g., insertion of one or more extra nucleotides into the genomic sequence), a deletion, or a substitution mutation (e.g., a transition or a transversion). In some embodiments, the mutation prevents the first nucleic acid sequence from targeting the genomic sequence. In some embodiments, the mutation comprises an insertion or a deletion. In some embodiments, the mutation occurs within the first 20 (e.g., the first 18, the first 16, the first 14, the first 12, the first 10, the first 8, the first 6, the first 4, or the first 2) nucleotides of a PAM sequence associated with the first nucleic acid sequence. In some embodiments, the mutation occurs within the first 10 nucleotides of a PAM sequence associated with the first nucleic acid sequence. In some embodiments, the mutation in the genomic sequence does not alter expression of the gene product.
It is also possible to construct a synthetic species that is reproductively incompatible with, but otherwise identical to, its wild counterpart for ecosystem engineering (2, 4). Perhaps most interestingly, engineered reproductive isolation can be an innovative means of pest and vector control (2, 4, 5). When released into wild populations in low numbers, the reproductively isolated individuals would principally mate with wild types (WT), leading to inviable hybrid offspring and population suppression. However, releases of engineered individuals above a critical threshold would lead to more frequent intermatings, which would result in viable progeny that could eventually replace the WT populations and function as a gene drive (4-6). Importantly, this type of gene drive represents a safer alternative to certain other types of proposed drives (7, 8), as it is threshold-dependent and reversible via the release of WT individuals, which may be more palatable to both regulators and the public (7, 9).
A number of attempts to generate synthetic species barriers via engineered reproductive isolation, both within and outside the context of gene drives, have been made in the last several decades. For instance, species barriers can be engineered via genetic recoding (2), which has been attempted in bacteria (10, 11) and yeast (12, 13) though is likely infeasible in multicellular organisms. A variety of other techniques have been proposed and attempted in more complex organisms, such as flies. For example, Moreno generated a reproductively isolated strain of D. melanogaster using preexisting transgenes and recessive mutations (1); however, this approach utilized a complex Gal4-UAS-inducible expression system that imparted significant fitness costs on the speciated strain making it impossible for the strain to survive outside the lab, nor function as a gene drive, and also rendered the technique near impossible to transfer to non-model organisms. Several other research groups have successfully engineered under dominance systems, which cause selection against heterozygotes favoring homozygotes of either wild type or mutants, in D. melanogaster (5, 6, 14), although none of these systems achieved complete reproductive isolation such that when mated to WT the progeny survived and reproduced. CRISPR-based genome editing and transcriptional transactivation (CRISPRa) strategies have recently been proposed as a means for engineering synthetic reproductive barriers, and proof-of-concept systems have been constructed in both yeast and flies (2, 4); however, both of these systems failed to achieve complete reproductive isolation, as either escape mutants were readily identified or complete synthetic lethality was not achieved.
As provided herein, this disclosure features the development of synthetic reproductive barriers in D. melanogaster using an approach referred to as “SPECIES” (Synthetic Postzygotic barriers Exploiting CRISPR-based Incompatibilities for Engineering Species). To engineer SPECIES, CRISPR gene editing and CRISPR-based transactivation were utilized, where a nuclease-deficient deactivated Cas9 (dCas9) protein is fused to a transactivation domain that causes dCas9 expression to be lethal in WT individuals. This domain functions by recruiting transcriptional machinery to the site of single guide RNA (sgRNA) binding within the promoter region of a target gene to generate synthetic lethality by the dCas9-mediated lethal overexpression of endogenous target genes (
Altogether, the findings demonstrate that dCas9-induced overexpression can be exploited to build complete synthetic reproductive barriers in vivo that could be used to drive genes through a population in a threshold-dependent manner. The SPECIES approach described here is advantageous over other previously developed technologies for several reasons. CRISPR/Cas9 has been shown to work in most organisms tested so far, and the dCas9-mediated overexpression approach does not rely on any SPECIES-specific mode of incompatibility; instead, it is easily programmable to virtually any suitable target gene and thus should be amenable to any sexually-reproducing organism with appropriate endogenous genes. Additionally, this approach can be used to construct single or multiple “stacked” genetic barriers (i.e., those utilizing more than one sgRNA), which may reduce failure due to natural variation or mutation in the sgRNA target sites, recombination, or transgene silencing/mutation. Furthermore, once a basic engineering toolkit is constructed (e.g., transgenic dCas9-VPR and Cas9-expressing lines; knowledge of suitable genes for targeting), it can be used to build multiple different SPECIES that are reproductively isolated from each other and their WT counterparts.
There are also potential limitations to building the SPECIES system in different organisms, particularly ones with minimal genome characterization. For example, this approach requires a priori knowledge of appropriate gene targets and suitable regulatory targets in these genes as well as an ability to genetically transform the target species. Additionally, there has to be a level of target gene overexpression that would cause this system to be lethal in WT individuals to attain complete reproductive isolation, which may not be easily achievable in all species. Given the potential toxicity of some of the system components, specifically the dCas9-VPR element (17), the expression of these components may need to be optimized for each new context, which may be challenging in some organisms. With the broad applicability of Cas9 tools and the relative simplicity of the SPECIES approach, however, it should be possible to implement this approach in any organism where fundamental genetic knowledge is available.
The gene drive function of the SPECIES technology is particularly useful. The threshold dependence of the SPECIES-based gene drive provides greater control and confinement (7, 18) as well as a reversibility not found in all gene drives (7, 19), which can be accomplished via WT release (6). The simplicity of the SPECIES approach and the near-universal applicability of CRISPR/Cas9 should increase the possibility of engineering organisms of medical or agricultural interest, such as mosquitoes, which also have orthologs to a number of the targeted genes described herein. The SPECIES system could be linked to a useful cargo gene—such as resistance to mosquito-borne viruses like Zika (20) or dengue (21)—to facilitate the spread of disease resistance through a population. Certainly, these cargo genes would also have to be robust enough to function in diverse wild mosquito and virus populations. Finally, given the fitness costs associated with the SPECIES system, such as dCas9 expression or incomplete indel rescue, certain SPECIES systems may not be capable of sustaining a drive in a wild population. In conclusion, the development of SPECIES demonstrates a significant advance in the field of population engineering providing a platform for future development in other organisms to safely control pest populations that pose significant burdens on humanity.
To assemble plasmid OA-986A, the base vector used for generating dCas9-expressing plasmids, several components were cloned into the piggyBac plasmid pBac[3×P3-DsRed] using Gibson assembly/EA cloning. pBac[3×P3-DsRed] was digested with BstBI and NotI, and the following components were cloned in with EA cloning: an attP sequence amplified from plasmid M{3×P3-RFP attP} with primers 986.C1 and 986.C2, a p10 3′UTR fragment amplified from Addgene plasmid #100580 with primers 986.C3 and 986.C4, an opie2 promoter fragment amplified from translocation plasmid B using primers 986.C5 and 986.C6, and an eCFP marker amplified from Addgene plasmid #47917 using primers 986.C7 and 986.C8. The resulting plasmid was then digested with PacI, and the following components were cloned in to generate the final dCas9-expressing vectors: the Ubiquitin-63E promoter fragment amplified with primers 986.C9 and 986.C10 from D. melanogaster genomic DNA and a dCas9-VPR fragment amplified from Addgene plasmid #78898 with primers 986.C11 and 986.C12 to generate plasmid OA-986B (Addgene #124999); the bottleneck promoter fragment amplified with primers 986.C13 and 986.C14 from D. melanogaster genomic DNA and a dCas9-VPR fragment amplified from Addgene plasmid #78898 with primers 986.C15 and 986.C12 to generate plasmid OA-986C (Addgene #125000); the Ubiquitin-63E promoter fragment amplified with primers 986.C9 and 986.C16 from D. melanogaster genomic DNA and a dCas9-VP64 fragment amplified from Addgene plasmid #78897 with primers 986.C17 and 986.C18 to generate plasmid OA-986D (Addgene #125001); and the bottleneck promoter fragment amplified with primers 986.C13 and 986.C19 from D. melanogaster genomic DNA and a dCas9-VP64 fragment amplified from Addgene plasmid #78897 with primers 986.C20 and 986.C18 to generate plasmid OA-986E (Addgene #125002).
To assemble plasmids OA-1045A-E, the multiple-sgRNA containing vectors, several components were cloned into the multiple cloning site (MCS) of a plasmid containing the white gene as a marker and an attB-docking site using Gibson assembly/EA cloning. First, the plasmid was digested with AsiSI and KpnI, and the following components were cloned in with EA cloning to generate base plasmid OA-1045: a D. melanogaster U6:3 promoter fragment sequence amplified from Addgene plasmid #49411 with primers 1045.C1 and 1045.C2, and an sgRNA scaffold fragment amplified from Addgene plasmid #49411 with primers 1045.C3 and 1045.C4. The resulting base plasmid was then used to clone final sgRNA plasmids OA-1045A-OA-1045E. To generate plasmid OA-1045A (Addgene #125003), plasmid OA-1045 was digested with AvrII; then, a fragment containing an 18 base pair (bp) eve sgRNA target site, an sgRNA scaffold, a D. melanogaster U6:1 promoter fragment, and an 18 bp hid sgRNA target site was amplified from a custom gBlocks® Gene Fragment (Integrated DNA Technologies, Coralville, Iowa) with primers 1045.C5 and 1045.C6, and cloned into the digested backbone using EA cloning. To generate plasmid OA-1045B (Addgene #125004), plasmid OA-1045A was digested with XbaI, and a fragment containing a Gypsy insulator, a D. melanogaster U6:1 promoter fragment driving expression of a first hh-targeting sgRNA, and a D. melanogaster U6:3 promoter fragment driving expression of a second hh-targeting sgRNA amplified from plasmid pCFD4-hh with primers 1045.C7 and 1045.C8 was cloned in using EA cloning. To generate plasmid OA-1045C (Addgene #125005), plasmid OA-1045A was digested with XbaI, and a fragment containing a Gypsy insulator, a D. melanogaster U6:1 promoter fragment driving expression of a first wg-targeting sgRNA, and a D. melanogaster U6:3 promoter fragment driving expression of a second wg-targeting sgRNA amplified from plasmid pCFD4-wg with primers 1045.C7 and 1045.C8 was cloned in using EA cloning. To generate plasmid OA-1045D (Addgene #125006), plasmid OA-1045 was digested with AscI and XbaI, and two fragments were cloned in using EA cloning: a first fragment containing a D. melanogaster U6:1 promoter fragment driving expression of a first wg-targeting sgRNA and a D. melanogaster U6:3 promoter fragment driving expression of a second wg-targeting sgRNA amplified from plasmid pCFD4-wg with primers 1045.C9 and 1045.C10, and a second fragment containing a Gypsy insulator, a D. melanogaster U6:1 promoter fragment driving expression of a first hh-targeting sgRNA, and a D. melanogaster U6:3 promoter fragment driving expression of a second hh-targeting sgRNA amplified from plasmid pCFD4-hh with primers 1045.C11 and 1045.C12. Finally, to generate plasmid OA-1045E (Addgene #125007), plasmid OA-1045 was digested with AvrII and NotI, and a fragment comprising D. melanogaster Gly tRNA-flanked sgRNAs targeting, 5′ to 3′, eve, hid, and hh followed by a D. melanogaster U6:3 UTR that was amplified with primers 1045.C13 and 1045.C14 from a gene synthesized vector (GenScript, Piscataway, NJ) was cloned in using EA cloning. All primers used for cloning are listed in Table 1.
Fly husbandry and crosses were performed under standard conditions at 25° C. Rainbow Transgenics (Camarillo, CA) carried out all of the fly injections. Fly strains utilized to generate dCas9-expressing lines were attP lines attP40w (Rainbow Transgenic Flies line; yw P{nos-phiC31\int.NLS}X;P{CaryP}attP40) and 8621 (BSC #8621; y[1] w[67c23]; P{y[+t7.7]=CaryP}attP1). The fly strains used to generate sgRNA-expressing lines were 86Fa (BSC #24486: y[1] M{vas-int.Dm}ZH-2A w[*]; M{3×P3-RFP.attP′}ZH-86Fa), 9732 (BSC #9732: y[1] w[1118]; PBac{y[+]-attP-9A}VK00013), and 8622 (BSC #8622: y[1]w[67c23]; P{y[+t7.7]=CaryP}attP2). For balancing chromosomes, fly stock BSC #39631 (w[*]; wg[Sp-1]/CyO; P{ry[+t7.2]=neoFRT}82B lsn[SS6]/TM6C, Sb[1]) was used. All lines were homozygous-viable.
Single sgRNA lines targeting eve, hid, hh, and wg were utilized. dCas9-VPR- and dCas9-VP64-expressing lines were generated via microinjection as described above; a transgenic line that expressed dCas9-VPR ubiquitously could not be generated, despite numerous attempts, suggesting that such expression was toxic. To test for the ability of all sgRNA lines to induce lethal overexpression (“killing”), five sgRNA males and five virgin females were separately crossed to five dCas9 line individuals of the opposite sex in single vials and were allowed to mate for 7 days. After 7 days, the parents were removed, and the vials were monitored for 7 additional days to assess whether viable larvae were present. No killing was observed in crosses of dCas9-VP64 expressing lines to any of the sgRNA-expressing lines (
To generate protective indel mutations, a Ubiquitin-Cas9 line (BSC #79005) was used. Briefly, ten Ubiquitin-Cas9 virgin females were crossed to ten sgRNA males, and virgin female and male progeny with both transgenes were selected and crossed to each other for at least three generations. Cas9/sgRNA transheterozygous virgins were then outcrossed in groups of 3-5 to homozygous attP40w bnk-dCas9-VPR males, and progeny containing both a sgRNA (identified by the presence of the w+ marker) and bnk-dCas9-VPR (identified by the presence of the opie2-eCFP marker) were isolated as “rescue” individuals that presumably carried protective indel mutations in the target promoter regions that prevented dCas9-induced overexpression (
These “rescue” crosses were also set up in the reverse direction, utilizing 3-5 homozygous attP40w bnk-dCas9-VPR females crossed to Cas9/sgRNA transheterozygous males, to determine whether maternal deposition of Cas9/sgRNAs is required for generating sufficient protective indel mutations to provide rescue of lethality. In particular, it was assumed that, if both copies of the targeted promoter needed to contain protective indel mutations to provide rescue, lack of maternally deposited Cas9/sgRNA (due to Cas9/sgRNA fathers being used) would lead to lack of “rescue” individuals, as all individuals inheriting the sgRNA and bnk-dCas9-VPR transgenes would still have one wildtype copy of the target promoter inherited from the mother available for targeting and would perish.
To further validate whether both copies of the targeted promoter needed to contain protective indel mutations to provide rescue from lethality, “rescue” individuals were also bidirectionally outcrossed in groups of 3-5 and in triplicate to homozygous attP40w bnk-dCas9-VPR individuals, and the resulting progeny were scored for the “rescue” phenotype. Here, it was presumed that the lack of transheterozygous sgRNA/bnk-dCas9-VPR progeny indicated that both copies of the targeted promoter needed to contain protective indel mutations to provide rescue and that the lack of such mutations in the promoter allele inherited from the homozygous attP40w bnk-dCas9-VPR parent led to lethality in the transheterozygous sgRNA/bnk-dCas9-VPR progeny. Here, too, such lethality was observed for crosses with multiple sgRNA transgenes but not for crosses with single sgRNA transgenes, suggesting that, in the case of the latter, one wildtype copy of the targeted promoter was not sufficient to lead to sgRNA/bnk-dCas9-VPR induced lethality.
Double homozygous speciated stocks were generated for all sgRNA combinations by crossing dCas9/sgRNA heterozygotes that lacked the Ubiquitin-Cas9 transgene (as evidenced by lack of red fluorescence) and identifying homozygous progeny by eye color (orange to dark red eyes for homozygotes vs. yellow to light red eyes for heterozygotes, depending on sgRNA insertion site) and opie2-eCFP intensity. Putative double homozygous individuals were then outcrossed to w[1118] individuals of the opposite sex in groups of three per vial to test for reproductive isolation. Flies were allowed to mate and lay eggs for 7 days, and vials were checked daily for hatched embryos. Flies that failed to fruitfully mate with w[1118] were presumed to be reproductively isolated double homozygotes and were then crossed to putative double homozygotes of the opposite sex to generate a double homozygous, reproductively isolated stock for each sgRNA line.
Assessment of Reproductive Isolation from Various Strains
To determine whether double-homozygous speciated lines were reproductively isolated from stocks that were genetically diverse, speciated individuals were outcrossed to various Global Diversity Lines (GDL) isolated from five different continents, and used in previous work examining gene drive function in different genetic contexts. Briefly, 5 double homozygous individuals from each speciated stock were outcrossed to 5 individuals of the opposite sex from each of five Global Diversity Lines (from Beijing, China; Ithaca, NY; the Netherlands; Tasmania, Australia; and Zimbabwe, Africa). All crosses were done bidirectionally with respect to sex, and in triplicate. Flies were allowed to mate and lay eggs for 7 days, and vials were checked daily for hatched embryos for the following 7 days. Lack of embryo hatching was presumed to indicate reproductive isolation.
To assess reproductive isolation between double-homozygous speciated lines, inter-species crosses were performed by crossing 2 speciated virgin females with 2 speciated males from each strain. Flies were allowed to mate for 12-16 hours under standard conditions; following this period, the adult flies were removed and the embryos were counted (
For embryo viability counts (
All genetic experiments were conducted in a high-security Arthropod Containment Level 2 (ACL2) barrier facility, in accordance with protocols approved by the Institutional Biosafety Committee from University of California San Diego. Population cage experiments were carried out at 26° C., 12 hour-12 hour day night cycle, with ambient humidity in 250 ml bottles containing Lewis medium supplemented with live, dry yeast. Starting populations for drive experiments included equal numbers of virgins and males of similar ages, for each genotype. Speciated double homozygotes (dCas9/dCas9; +/+) were introduced at a population frequency of 80% for above threshold drive experiments, and 50% for below threshold drive experiments. OreR virgin females and males (+/+; +/+) of similar age as the translocation-bearing individuals made up the remainder of the population. The total number of flies for each starting population was 100. All experiments were conducted in triplicate. After being placed together, adult flies were removed after seven days. After another seven days, progeny were collected and the fraction of speciated double homozygous individuals was determined (
SPECIES population dynamics were modeled under laboratory conditions assuming random mating and discrete generations. A SPECIES allele, “T”, and a corresponding wildtype allele, “t”, were considered. Since heterozygotes for the SPECIES system are unviable, there are only two viable genotypes-TT and tt. It was denoted such that the proportion of organisms having the genotype TT at generation k by pk, and the proportion having the wildtype genotype at generation k by (1−pk). By considering all possible mating pairs, and assuming a fitness cost for TT individuals relative to wildtype individuals, s, the frequency of TT individuals in the next generation is given by:
p
k+1
=p
k
2(1−s)/(pk2(1−s)+(1−pk)2). (1)
The threshold frequency is an unstable equilibrium that satisfies the condition:
p
k+1
=p
k. (2)
Substituting Eq. (2) into Eq. (1) and solving for pk, two stable equilibria (pk=0 and pk=1) and one unstable equilibrium (pk=1/(2−s)) were found. The latter represents the critical threshold frequency, above which the SPECIES system is more likely to spread to fixation than not, and below which it is more likely to be eliminated than not.
The likelihood of the population data for each SPECIES system was calculated by assuming a binomial distribution of wildtype (CFP−) and SPECIES (CFP+) individuals, and by using the model in Eq. (1) to generate expected proportions for each fitness parameter value, s, i.e., by calculating the log-likelihood:
Here, (1) TTi,k and tti,k are the number of SPECIES (CFP+) and wildtype (CFP−) individuals at generation k in experiment i, respectively, (2) there are a total of j experiments for this SPECIES system, (3) the ith experiment is run for n, generations, and (4) the expected genotype frequencies are dependent on the fitness parameter, s. The initial condition for each experiment is specified by the data. Fitness parameters, including 95% credible intervals, were estimated using a Markov chain Monte Carlo sampling procedure.
The stochastic simulations in
Comparative modeling of other underdominant systems is described in Marshall and Hay (J. Theor. Biol. 294, 153-171 (2012)). Marshall et al uses the mathematical modeling framework described here in addition to two approaches for modeling migration: (1) a “two-population model”, in which reciprocal movement occurs between the two connected populations; and (2) a “source model”, in which the system is initially fixed in the source population, absent from the sink population, and one-way migration occurs from the source to sink population. In Marshall et al., population replacement and confinement dynamics are shown for: (1) extreme underdominance (the SPECIES system modeled here), (2) reciprocal chromosomal translocations, (3) single-locus and two-locus engineered underdominance, (4) Semele, (5) inverse Medea, and (6) Merea (Medea with a recessive antidote). A range of parameter values are compared for each gene drive system, including fitness cost (s, varied between 0 and 30%) and migration rate (m, varied between 0 and 10% per individual per generation for both the source and two-population models).
Results from that analysis suggest that SPECIES-like extreme underdominant systems fare well against other underdominance-based gene drive systems in terms of both confinement and persistence. The most direct comparison can be made to translocations, which also have a 50% release threshold in a single population and in the absence of a fitness cost. Considering a 5% fitness cost for both systems, they still have very similar release thresholds (51.3% for SPECIES-based underdominance cf. 52.8% for translocations); however, for a two-population model with a migration rate of 1% per individual per generation, the SPECIES-based underdominant system spreads to only ˜0.01% in the neighboring population, while the translocations spread to a much higher ˜4.2% in the neighboring population. The migration rate at which the introduced system is lost due to inward migration of wild types is also much higher for the SPECIES-based underdominant system (17.6% per individual per generation cf. 5.8% for translocations, s=0.05). This suggests that SPECIES-like extreme underdominant systems are preferable to translocations for local population replacement since they lead to less contamination of neighboring populations and are less vulnerable to elimination due to inward migration (
Finally, were SPECIES-based underdominant systems to be implemented for local population replacement, strains would likely be used that would have much smaller fitness costs than those observed here (˜30%). Despite that, results from Marshall et al. suggest the population dynamics of the SPECIES system are resilient in the face of these fitness costs. A SPECIES system with a fitness cost of 30% has a release threshold of 58.8%, which could be exceeded through weekly releases over several weeks. Furthermore, in a two-population model, the migration rate at which the SPECIES system would be lost due to inward migration of wild types is 13.3% per individual per generation, which is greater than the movement rate observed between populations of Anopheles gambiae, the main mosquito vector of malaria in Sub-Saharan Africa, and Aedes aegypti, the main mosquito vector of dengue, Zika and Chikungunya viruses.
Embryos were collected from the multiple speciated lines to assess transactivation in the embryo. Male speciated flies were crossed to Oregon R virgin females in glass vials supplemented with Drosophila medium and yeast paste, and incubated at 26° C. for 72 hours. Following this period, the adult flies were transferred to collection chambers containing grape juice agar plates. The flies were allowed to lay for 4-5 hours, after which the embryos were aged for one hour and collected using a paintbrush. 30-50 5-6 hr embryos were collected, washed with ddH2O, and transferred to individual eppendorf tubes. The samples were flash frozen with liquid nitrogen and stored at −80° C. Intra-crosses for Oregon R, Cas-9, dCas-9, dgRNA, and speciated lines were also performed and collected as controls. Each sample was homogenized and processed using the Quick-Start Protocol of the miRNeasy Mini Kit (Qiagen, Hilden, DEU), followed by DNase treatment using the DNA-free™ Kit and protocol (Thermo Fisher Scientific, Waltham, MA, USA).
RNA integrity was assessed using RNA 6000 Pico Kit for Bioanalyzer (Agilent Technologies #5067-1513) and mRNA was isolated from ˜1 μg of total RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB #E7490). RNA-seq libraries were constructed using NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770) following manufacturer's instructions. Briefly, mRNA was fragmented to the average size of 200 nt by incubating at 94° C. for 15 min in first strand buffer, cDNA was synthesized using random primers and ProtoScript II Reverse Transcriptase followed by second strand synthesis using NEB Second Strand Synthesis Enzyme Mix. Resulting DNA fragments were end-repaired, dA tailed and ligated to NEBNext hairpin adaptors (NEB #E7335). After ligation, adaptors were converted to the ‘Y’ shape by treating with USER enzyme and DNA fragments were size selected using Agencourt AMPure XP beads (Beckman Coulter #A63880) to generate fragment sizes between 250 and 350 bp. Adaptor-ligated DNA was PCR amplified followed by AMPure XP bead clean up. Libraries were quantified with Qubit dsDNA HS Kit (ThermoFisher Scientific #Q32854) and the size distribution was confirmed with High Sensitivity DNA Kit for Bioanalyzer (Agilent Technologies #5067-4626). Libraries were sequenced on Illumina HiSeq2500 in single read mode with the read length of 50 nt and sequencing depth of 20 million reads per library following manufacturer's instructions. Base calls were performed with RTA 1.18.64 followed by conversion to FASTQ with bcl2fastq 1.8.4.
Reads were mapped to the Drosophila melanogaster genome (BDGP release 6, GenBank accession GCA_000001215.4) using STAR aligner with default parameters with the addition of ‘--outFilterType BySJout’ filtering option and ‘--sjdbGTFfile Drosophila_melanogaster.BDGP6.22.97.gtf’ GTF file downloaded from ENSEMBL. Expression levels were determined with featureCounts using ‘-t exon -g gene_id -M -O --fraction’ options. Differential expression analyses between homozygous speciation stocks and corresponding heterozygotes outcrossed to wild-type females were performed with DESeq2 using a two factor design formula ‘design=˜line+genotype’. Two independent lines per each target sets (genotype) were used. MA plots (log 2(FoldChange) vs log 10(baseMean)) were generated with ggplot2. All sequencing data can be accessed at NCBI SRA (study accession ID PRJNA578541).
For antibody staining, embryos were collected overnight and then fixed and dechlorinated using standard protocols. Guinea pig anti-Runt polyclonal antibody was used at a concentration of 1:200 and mouse anti-Eve monoclonal 3C10 at 1:20. Nuclei were counterstained with DAPI. Embryos were stained using standard protocols.
Embryos were collected and aged at 27° C. until they were 16-22 h old. Embryos were pipetted onto a slide and excess fluid was removed. Glacial acetic acid mixed 1:1 with Hoyer's solution was added, covered with a coverslip, and allowed to dry for several days in an oven at 65° C. for clearing. After 24 h, the coverslips were weighted to flatten the preps. Cuticles were imaged on an upright Zeiss Axio Imager microscope with bright field illumination, and grayscale images were later inverted and oversaturated for increased contrast using Adobe Photoshop.
To examine the molecular changes that conferred protection from dCas9-mediated overexpression and associated lethality, four genomic loci that include targets sites for four functional gRNAs (
All crosses using gene drives genetics were performed in accordance to an Institutional Biosafety Committee-approved protocol from UCSD in which full gene-drive experiments are performed in a high-security ACL2 barrier facility and split drive experiments are performed in an ACL1 insectary in plastic vials that are autoclaved prior to being discarded in accord with currently suggested guidelines for laboratory confinement of gene-drive systems (Akbari et al. 2015; National Academies of Sciences, Engineering, and Medicine et al. 2016).
D. melanogaster
D. melanogaster
D. melanogaster
D. melanogaster
Flies expressing a dCas9 activator domain fusion (dCas9-VPR) were engineered and evaluated whether these transgenes could drive lethal target overexpression using CRISPRa sgRNA lines each targeting the promoter region of one of four important developmental genes (eve, hid, hh, and wg)(
Importantly, the inherited sgRNA/dCas9-VPR transgenes forced a bottleneck that selected for protective indels which blocked CRISPRa-induced lethality and allowed for endogenous expression levels of the target gene, providing embryonic rescue and survival (
To overcome the incomplete isolation from single-gene overexpression, multiplexed overexpression was tested by engineering flies that simultaneously expressed sgRNAs targeting two or more genes (eve+hid; eve+hid+hh; eve+hid+wg; and hh+wg;
With the selective bottleneck genetic crossing scheme, crosses with multiplexed sgRNA/Cas9-expressing mothers rescued heterozygous dCas9; sgRNA animals through the introduction of indel mutations (
To generate reproductively isolated SPECIES, multiple generations (>5) of dCas9-VPR; sgRNA “rescued” individuals were intercrossed, resulting in homozygous stocks representing eight isolated SPECIES (A1-D2). Each SPECIES was reproductively incompatible with WT (
The extent of target gene overexpression was then determined when outcrossed to WT by visualizing overexpression in embryos via antibody stain, and the effect of misexpression on development was evaluated using cuticle preps of late embryos and young larvae. Target gene overexpression was observed at embryonic stages and segment polarity defects in larvae when the SPECIES lines were mated to WT but not when self-crossed (
To assess whether the SPECIES were capable of reversible WT population replacement via gene drive, population studies were conducted at various release thresholds employing one representative SPECIES, A1 (
To characterize the population dynamics observed in the population studies, a mathematical model was fitted to the observed data, incorporating a fitness cost for reproductively isolated individuals relative to WT individuals. The A1 strain was estimated to have a strong relative fitness cost of 34.84% (95% credible interval [CrI]: 34.82-34.87%), producing a threshold frequency of ˜61%, which corresponds to what was observed in the population studies. Of the seven other SPECIES characterized, two consistently led to population replacement at a release frequency of 80% (A2 and D1), and three led to population replacement at a release frequency of 90% (A2, D1, and D2) (
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a National Stage Application under 35 U.S.C. § 371 and claims the benefit of International Application No. PCT/US2021/038595, filed Jun. 23, 2021, which claims priority to U.S. Application Ser. No. 63/042,614, filed on Jun. 23, 2020. The disclosure of the prior application is considered part of the disclosure of this application, and is incorporated in its entirety into this application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/038595 | 6/23/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63042614 | Jun 2020 | US |
