Method for suppressing an insect population
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-02-03
- Publication Date
- 2026-08-13
Smart Images
Figure EP2026052795_13082026_PF_FP_ABST
Abstract
Description
[0001] METHOD FOR SUPPRESSING AN INSECT POPULATION
[0002] Field of the invention
[0003] The present invention relates to a method for suppressing a population of insects, a set of genetic constructs and genetically modified insects with which to implement said method.
[0004] Prior art
[0005] Numerous insect species are vectors for the transmission of diseases or harmful pests (e.g., agricultural pests) capable of infecting or harming humans, animals or plants. For example, mosquitoes are the main vector of many diseases caused by parasites (e.g. the Plasmodium genus for malaria) or viruses (e.g. dengue, zika, chikungunya), while flies, such as Ceratitis capitata, various species of Drosophila, and grasshoppers, such as Anacridium aegyptium and Locusta migratoria, are just a few examples of pests harmful to agriculture.
[0006] In order to control insect populations, particularly harmful ones, several approaches have been adopted, such as killing by application of insecticides, trapping with traps containing chemical attractants, but also reducing reproduction by releasing sterile male insects generated by radiation, or genetic techniques into the environment. However, these approaches are limited in their effectiveness: the action of insecticides is variable and can also eliminate beneficial insects, specific chemical attractants are not available for every insect species, and the release of sterile insects is complicated and costly by the need to breed large numbers of such insects.
[0007] To overcome these disadvantages of known techniques, it is possible to employ the most modern genetic engineering techniques through which new, more effective methods of controlling insect populations can be developed. These techniques include the so-called 'gene drive', a genetic engineering technique that allows a particular set of genetic elements to be propagated among the members of a given target population. The gene drive technique, by genetically modifying the entire insect population, can thus be used to suppress pathogen-carrying insects, control pests and / or invasive species, or even eliminate resistance to herbicides or pesticides.
[0008] For example, the CRISPR-Cas-based gene drive technique has recently beenused in some mosquito species that are malaria vectors in humans, in order to develop a method of genetic population control of these insects (Hammond, A. et al. "A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae" Nat Biotechnol 34, 78-83, 2016 and Kyrou, K. et al, “A CRISPR-Cas9 gene drive targeting doublesex causes complete population suppression in caged Anopheles gambiae mosquitoes” Nat Biotechnol 36, 1062-1066, 2018). However, the use of gene drive involves ethical and environmental issues that are still unresolved at the regulatory level.
[0009] There is therefore a need for further and increasingly effective systems for the genetic control of an insect population based on the gene drive technique that can nevertheless be controlled in terms of their range of action and are at the same time reversible, i.e. do not leave any trace of genetic material in the target population after discontinuation of the treatment, in a manner ethically, environmentally and regulatory acceptable.
[0010] The aim of the present invention is therefore to provide a method for controlling an insect population that overcomes the aforementioned disadvantages of the prior art.
[0011] Summary of the invention
[0012] These objects and others which will become clearer hereafter, are achieved by a method for suppressing an insect population comprising the steps of:
[0013] (i) providing:
[0014] (a) a genetically modified male insect, carrying on the Y chromosome:
[0015] a transcription unit A comprising a nucleotide sequence encoding a first Cas nuclease, under transcriptional control of a first insect germlinespecific promoter; and
[0016] a transcription unit B comprising a nucleotide sequence encoding one or more guide RNAs (gRNAs) comprising a binding region for the first Cas nuclease and a sequence that is capable of hybridising to a sequence encoding an Acr protein inhibiting a second Cas nuclease that is different from the first Cas nuclease, under transcriptional control of a promoter selected from a Polymerase I (Pol-I) promoter and a Polymerase III (Pol-III) promoter;
[0017] and
[0018] (b) a genetically modified female insect carrying on the autosomal loci of a gene necessary for spermatogenesis:
[0019] a transcription unit C inserted in a site within the gene necessary for spermatogenesis, wherein said transcription unit C comprises a nucleotide sequence, under transcriptional control of a promoter selected from a Pol-I type promoter and a Pol-Ill type promoter encoding a gRNA comprising a binding region of the first Cas nuclease and a nucleotide sequence that is capable of hybridising to a corresponding nucleotide region of the gene necessary for spermatogenesis upstream and downstream of the insertion site of transcription unit C
[0020] and also carrying on both X chromosomes:
[0021] a transcription unit D comprising a nucleotide sequence encoding the second Cas nuclease, and a further nucleotide sequence, operatively linked upstream of the nucleotide sequence encoding the second Cas nuclease by a TA sequence, encoding the Acr protein inhibiting the second Cas nuclease, under transcriptional control of a promoter active in insects;
[0022] a transcription unit E comprising one or more nucleotide sequences encoding a gRNA comprising a binding region for the second Cas nuclease and a nucleotide sequence capable of hybridising to a sequence of a gene necessary for female development, under transcriptional control of a promoter selected from a Pol-I type promoter and a Pol-Ill type promoter; and
[0023] a transcription unit F comprising a nucleotide sequence encoding a copy of the gene necessary for spermatogenesis lacking the sequence to which the gRNA encoded by the nucleotide sequence comprised in transcription unit C is capable of hybridising, under transcriptional control of a second insect germline-specific promoter;(ii) releasing trans-heterozygous male insects obtained by crossing the genetically modified male insect of (i)(a) with the genetically modified female insect of (i)(b) in an environment where suppression of the insect population is desired, wherein said trans-heterozygous male insects carry the Y chromosome of the genetically modified male insect of (i)(a), the X chromosome of the genetically modified female insect of (i)(b), and the autosomal loci of the gene necessary for spermatogenesis in which the transcription unit C of the genetically modified female insect of (i)(b) is inserted.
[0024] Preferably, the first Cas nuclease is Cas12a nuclease.
[0025] Preferably, the first insect germline-specific promoter is the Vasa2 promoter. Preferably, the second Cas nuclease is Cas9 nuclease and the Acr protein is AcrllA4.
[0026] Preferably, the promoter active in insects is the polyubiquitin (PUBc) promoter. Preferably, the sequence of the gene necessary for female development is selected from the group consisting of the female-specific exon of the doublesex gene (dsx) and a sequence of the femaleless gene (fie).
[0027] More preferably, the sequence of the gene necessary for female development is the female-specific exon of the dsx gene.
[0028] Preferably, the gene necessary for spermatogenesis is selected from the group consisting of the protamine and beta2-tubulin genes.
[0029] More preferably, the gene necessary for spermatogenesis is the beta2-tubulin gene.
[0030] In a particularly preferred embodiment of the method of the invention:
[0031] - the first insect germline-specific promoter is the Vasa2 promoter;
[0032] - the first Cas nuclease is Cas12a nuclease;
[0033] - the promoter active in insects is the PUBc promoter;
[0034] - the second Cas nuclease is Cas9 nuclease;
[0035] - the Acr protein is Acrl IA4;
[0036] - the gene required for spermatogenesis is the beta2-tubulin gene;
[0037] - the second insect germline-specific promoter is the Vasa2 promoter; and- the sequence of a gene necessary for female development is the femalespecific exon of the dsx gene.
[0038] The aims and objects of the present invention are also achieved by a set of CRISPR-Cas-based gene drive constructs comprising:
[0039] - a first construct comprising transcription unit A and transcription unit B as defined at point (i)(a) of the method of the invention;
[0040] - a second construct comprising transcription unit C as defined at point (i)(b) of the method of the invention; and
[0041] - a third construct comprising transcription unit D, transcription unit E, and transcription unit F as defined at point (i)(b) of the method of the invention.
[0042] Furthermore, the aims and objects of the present invention are also achieved by a pair of insects comprising:
[0043] (a) a genetically modified male insect as defined at point (i)(a) of the method of the invention; and
[0044] (b) a genetically modified female insect as defined at point (i)(b) of the method of the invention.
[0045] Finally, the aims and objects of the invention are also achieved by a genetically modified trans-heterozygous male insect carrying the Y chromosome of the genetically modified male insect as defined at point (i)(a) of the method of the invention, the X chromosome of the genetically modified female insect as defined at point (i)(b) of the method of the invention, and the autosomal loci of the gene necessary for spermatogenesis in which the transcription unit C of the genetically modified female insect as defined at point (i)(b) of the method of the invention is inserted.
[0046] Detailed description
[0047] Further features and advantages of the invention will become clearer from the following detailed description and the accompanying drawings, in which:
[0048] - Figure 1 is an illustrative diagram of the parental lines "trigger" and "effector" according to an embodiment of the invention;
[0049] - Figure 2 is an illustrative diagram of an embodiment of the method according to the invention;- Figure 3 shows specific nucleotide sequences which may be used in accordance with preferred embodiments of the present invention.
[0050] The present invention provides a method, a set of genetic constructs and genetically modified insects by which a wild-type population of insects can be suppressed by reducing their reproduction.
[0051] To this end, the present invention is based in part on the well-known CRISPR-Cas system, a genome editing tool that makes it possible to add, destroy or modify the sequence of specific genes in an organism's genome. As is known, this system employs two elements, a Cas nuclease and a guide polynucleotide that is usually a guide RNA (gRNA). Cas nucleases are endonucleases, enzymes that cut a phosphodiester bond in a polynucleotide (e.g. DNA). gRNAs are polynucleotide sequences that form a complex with an endonuclease, allowing the latter to recognise and cut a target polynucleotide sequence. The sequence of a gRNA has complementarity to the target sequence in order to hybridise with it and thus allow sequence-specific binding of a Cas nuclease to the target sequence. In other words, the gRNA directs the Cas nuclease to the target sequence in an organism's DNA based on sequence homology, and the Cas nuclease then cuts the organism's genome at the desired location. After this targeted cut, one of two alternative DNA repair mechanisms, non-homologous end joining (NHEJ) and homology-directed repair (HDR), is activated to restore the integrity of the cut DNA chain. Further aspects of the CRISPR-Cas system, known to the skilled person, are for example described in PCT Publication No. WO 2017 / 049266.
[0052] In particular, the present invention is based on a genetic system comprising a trans-heterozygous male insect obtained by crossing two genetically modified insect parental lines.
[0053] In the trans-heterozygous male insect, there are two distinct CRISPR-Cas systems, inserted on the X and Y chromosome respectively, targeting key male and female genes. These two CRISPR-Cas systems act in a criss-cross mode in order to reduce the reproduction of the target population and achieve its suppression after repeated releases of the trans-heterozygous male into the environment, while at the same time facilitating the production of parental lines of genetically modified males andfemales. The insertion of CRISPR-Cas systems at the level of sex chromosomes leads to the transmission of the desired male and female sterility phenotype, while achieving complete self-extinction of the genetic modification in the target population within two generations.
[0054] The parental lines from which the trans-heterozygous male insect is obtained are represented by a pair of genetically modified male and female insects, preferably by the insertion of appropriate constructs, as described below.
[0055] First parental line
[0056] With reference to Figure 1, the first parental line trigger line) consists of genetically modified male insects carrying the following gene elements on the Y chromosome
[0057] - a nucleotide sequence encoding an early Cas nuclease, e.g. Cas12a nuclease, placed under transcriptional control of an early insect germline-specific promoter, e.g. the Vasa2 promoter known to be transcriptionally active on the Y chromosome in early germ cells; and
[0058] - a nucleotide sequence encoding for a gRNA comprising a binding region for the first Cas nuclease and a sequence capable of hybridising to a sequence encoding for an Acr protein inhibiting a second Cas nuclease other than the first Cas nuclease, placed under transcriptional control of a Pol-I or Pol-Ill promoter, e.g. a U6 promoter; for example, when the second Cas nuclease is Cas9 nuclease, the Acr protein may be AcrllA4, which is known to completely inhibit Cas9 nuclease activity.
[0059] Preferably, the male insects of the first parental line also carry, on the Y chromosome, a sequence encoding a first fluorescent marker, e.g. green fluorescent protein (GFP) or red fluorescent protein (RFP), under transcriptional control of an appropriate promoter, e.g. the Vasa2 or PAX promoter. Advantageously, the expression of such a fluorescent marker allows, by means of techniques known to the skilled person, the isolation of first-line male insects from a population of insects comprising them.
[0060] First-line male insects can also be engineered to carry an autosomal sex ratio distorter system, in order to produce a progeny consisting only of males.The skilled person will appreciate that in the male insect of the first parental line, the CRISPR-Cas system is not active, as the genome of this insect does not contain any target sequences for the first Cas nuclease, such sequences (e.g. the sequence encoding the Acr protein) being present only in the genome of the female parental line insects described below.
[0061] Second parental line
[0062] With reference to Figure 1 , the second parental line (effector line) consists of female insects engineered to carry two modifications in homozygosis: the first affects the autosomal loci of a gene necessary for spermatogenesis, such as the protamine gene or the beta2-tubulin gene, while the second is on the X chromosomes.
[0063] Female insects of the second line therefore carry, on both loci of the gene necessary for spermatogenesis, a transcription unit inserted in a site within the gene, necessary for spermatogenesis that destroys its function. Such a transcriptional unit comprises a nucleotide sequence encoding a gRNA comprising a binding region for the first Cas nuclease and a nucleotide sequence capable of hybridising to a sequence of the gene necessary for spermatogenesis, preferably capable of hybridising to a corresponding nucleotide region of the gene necessary for spermatogenesis upstream and downstream of the insertion site of the transcription unit. The coding sequence of this gRNA is preferably placed under transcriptional control of a promoter selected for example from a Pol-I type promoter and a Pol-Ill type promoter, such as a U6 promoter.
[0064] In addition, female insects of the second parental line carry the following gene elements on both X chromosomes
[0065] - a nucleotide sequence encoding a second Cas nuclease, e.g. Cas9 nuclease, placed under transcriptional control of a promoter active in insects, e.g. the polyubiquitin promoter (PUBc); and
[0066] - a nucleotide sequence encoding the Acr protein inhibiting the second Cas nuclease, e.g. AcrllA4 when the second Cas nuclease is Cas9 nuclease, and operatively linked upstream of the nucleotide sequence encoding the second Cas nuclease by means of a TA sequence, so as to permit equimolar production of the second Cas nuclease and the Acr protein inhibiting the second Cas nuclease;- one or more nucleotide sequences encoding a gRNA comprising a binding region for the second Cas nuclease and a nucleotide sequence capable of hybridising to a sequence of a gene necessary for female development, placed under transcriptional control of a promoter selected, for example, between a Pol-I type promoter and a Pol-Ill type promoter, such as a U6 promoter. For example, the gene sequences necessary for female development could be that of the female exon of the doublesex (dsx) gene or a sequence of the femaleless fie) gene;
[0067] - a nucleotide sequence encoding a gene necessary for spermatogenesis lacking the sequence to which, the gRNA encoded by the nucleotide sequence within the transcription unit associated with the aforementioned autosomal modification, is capable of hybridising, but functionally active, placed under the transcriptional control of a second insect germline-specific promoter, such as the Vasa2 promoter. This sequence encoding the gene necessary for spermatogenesis is resistant to the action of the first Cas nuclease, as it lacks the sequence necessary for sequence cleavage by the first Cas nuclease to occur.
[0068] Preferably, the insects of the second parental line also carry, on the X chromosomes, a sequence encoding a second fluorescent marker (e.g. GFP or RFP), which must be different from that which may be present in the male insects of the first line and the expression of which allows the female insects of the second line to be isolated. If present, the fluorescent marker sequence is placed under transcriptional control of an appropriate promoter, e.g. the Pax promoter or the actin promoter.
[0069] The female insects of the second line can also be engineered to be autosomal carriers of a sex distorter, in order to obtain progeny consisting only of females.
[0070] The skilled person will appreciate that even in the female insect of the second parental line, the CRISPR-Cas system is not active. In fact, the activity of the second Cas nuclease is completely inhibited by the action of the Acr protein, thus leaving intact one or more genes necessary for female development, while the absence in this line of a coding sequence for the first Cas nuclease means that the sequences that would be targeted by the first Cas nuclease also remain intact.
[0071] Trans- heterozygous Male insectCrossing the male insects of the trigger line with the females of the effector line results in a population of insects comprising trans-heterozygous male (illustrated in Figure 2), which inherit from the male insect of the trigger line the Y-chromosome modifications described above, and from the female insect of the effector line the X-chromosome modifications described above, as well as the modification at the autosomal loci of the gene necessary for spermatogenesis, also as described above.
[0072] When released into the environment where they mate with wild-type females, the trans-heterozygous males of the present invention are able to exert a powerful suppressive effect, underlying which are the molecular mechanisms described below.
[0073] At the level of the germ cells of trans-heterozygous males, the two CRISPR-Cas systems present in the genome of these insects are active. Expression of the first Cas nuclease and gRNA targeting the sequence encoding the Acr protein, both encoded on the Y chromosome, thus leads to the destruction of the sequence encoding the Acr protein present on the X chromosome, thereby ceasing Acr protein expression. This allows the second Cas nuclease to be activated, as the inhibitory effect on it previously exerted by the Acr protein is lost.
[0074] In the absence of the inhibitor, the second Cas nuclease is guided by gRNA targeting the sequence of a gene necessary for female development (e.g. dsx or alternatively fie), destroying the sequence of that gene. Since this gene is only essential for female development, its loss does not affect the development and ability to reproduce of the trans-heterozygous male.
[0075] The first Cas nuclease also works in concert with the gRNA encoded by the transcription unit within the gene necessary for spermatogenesis located on the autosomal chromosome inherited from the effector line, also modifying the allele of the same gene supplied by the germ cells of the wild female with which the transheterozygote male crosses, thus inactivating any endogenous copies of the gene (e.g. the beta2-tubulin gene). Although the endogenous copies of the gene necessary for spermatogenesis are compromised, the trans-heterozygous male is nevertheless fertile, since his genome contains, on the X chromosome, the nucleotide sequence encoding a functional copy of the gene necessary for spermatogenesis (e.g., a copy of the beta2-tubulin gene) that is resistant to the action of the first Cas nuclease. This copy of the gene, as explained above, lacks the necessary gRNA targeting sequence to form a complex with the first Cas nuclease.
[0076] Suppressive effect of trans-heterozygous males
[0077] Once released into an environment where it is desired to suppress an insect population, the crossing of trans-heterozygous males with wild females has a suppressive effect in that generates a progeny consisting of:
[0078] (i) female insects incapable of reproducing, as at least both copies of a gene necessary for female development are compromised, since these insects inherit, by means of the X chromosome from the trans-heterozygous male the activated second Cas nuclease and associated gRNA by means of which the gene necessary for female development is destroyed in the early stages of development. For example, these females will be sterile if the compromised gene is dsx, alternatively they will undergo death during the larval stage if the compromised gene is fie; and
[0079] (ii) sterile male insects, the endogenous copies of the gene necessary for spermatogenesis (e.g., the beta2-tubulin gene) being compromised, since these insects inherit, via the Y chromosome from the trans-heterozygous male, the first Cas nuclease that also inactivates the endogenous copy of the gene necessary for spermatogenesis transmitted from the wild female, but they do not inherit the X chromosome from the trans-heterozygous male in which the sequence encoding the gene necessary for spermatogenesis resistant to the action of the first Cas nuclease is present. Although sterile, these males are nevertheless able to exert an additional suppressive effect when they interbreed with wild females of the next generation. In fact, females mate only once during their life cycle and therefore by mating with sterile males they do not give rise to a further generation of insects. Advantageously, this implies that the genetic modifications introduced in accordance with the present invention are self-extinguishing within the target population within two generations.
[0080] Target insects of the method of the invention
[0081] In principle, any insect can be the target of the method of the present invention. However, preferably the target insect is a vector carrying a disease or a pest insect (e.g.an agricultural pest), which can infect, or cause harm to, or kill a human being, an animal, or a plant of agricultural interest. Preferably, the insect may be a mosquito, more specifically a mosquito belonging to an Anopheles species, e.g. Anopheles gambiae, or a mosquito belonging to an Aedes species, such as Aedes aegypti. In an equally preferred alternative, the insect can be a fly, such as Ceratitis capitata, or a midge belonging to a Drosophila species, such as Drosophila suzukii.
[0082] Gene elements used in the invention
[0083] Hereinafter, where a sequence is identified by an access number of the GenBank® database, it is understood to refer to the release version 262 of 15 August 2024 of said database. Where a sequence is identified by means of a VectorBase database access number, it is understood to refer to the release version no. 68 of 7 May 2024 of that database.
[0084] As described above, the genetic modifications introduced into the parental lines and inherited by the trans-heterozygote male consist of the introduction into the genome of these insects of specific gene elements encoding for, among others, two distinct Cas nucleases, gRNAs directing the Cas nucleases to specific target sequences, an Acr protein inhibiting one of the two Cas nucleases, and preferably fluorescent markers. These nucleotide sequences are placed under the transcriptional control of appropriate promoters, some of which can only direct transcription at specific cell lines or tissues.
[0085] According to the present invention, the first and second Cas nuclease can be Cas9 nuclease and Cas12a nuclease (formerly known as Cpf1 ). As will become clear to the skilled person, the first and second Cas nuclease must be different from each other in order to ensure the cross action of the two CRISPR-Cas systems present in the transheterozygous male insect. Another necessary condition for the implementation of the method of the invention is the availability of an inhibitor capable of blocking the activity of the second Cas nuclease. Preferably, in the present invention the first Cas nuclease is Cas12a nuclease and the second Cas nuclease is Cas9 nuclease. The sequences of the aforementioned nuclease are publicly available and therefore known to the skilled person. As an example only, the sequence encoding Cas12a nuclease may be that corresponding to nucleotides 709390 to 713169 of the sequence having referencenumber GenBank® AP018536.1 (SEQ ID NO:1), while the sequence encoding Cas9 nuclease may be that corresponding to nucleotides 763869 to 767975 of the sequence having reference number GenBank® NZ CP010450.1 (SEQ ID NO:2).
[0086] Acr proteins are natural inhibitors of Cas nucleases, which evolved in bacteriophages to counteract the activity of CRISPR-Cas systems. Multiple Acr proteins are known and have been assigned names based on the CRISPR-Cas system (l-C, l-D, l-E, l-F, I l-A, I l-C, V-A, Vl-B) they inhibit and the order in which they were discovered. Examples of Cas9 nuclease-inhibiting Acr proteins are AcrllA2, AcrllA4 and AcrllC2. Examples of Acr proteins inhibiting Cas12a nucleases are AcrVAI , AcrVA4 and AcrVA5. According to the present invention, when the second Cas nuclease is Cas9 nuclease then the Acr protein is preferably AcrllA4. The sequences encoding the aforementioned Acr proteins are publicly available and therefore known to the skilled person. As an example only, the sequence encoding the AcrllA4 protein may be that corresponding to nucleotides 4740 to 5003 of the sequence annotated in GenBank® reference NZ QAAZ01000027.1 (SEQ ID NO:3).
[0087] The sequence encoding the first Cas nuclease (transcription unit A), the sequences encoding the second Cas nuclease and the Acr protein (transcription unit D), and the sequence encoding the gene necessary for spermatogenesis that is resistant to the action of the first Cas nuclease (transcription unit F) are placed under the transcription control of a (first) insect germline-specific promoter, a promoter active in insects, and a (second) insect germline-specific promoter, respectively. Insect germlinespecific promoters essentially limit the expression of sequences under their control to insect germ cells. The promoters active in insects substantially limit the expression of sequences placed under their control to insect cells.
[0088] In accordance with the present invention, the first and / or second insect germ line specific promoter may be the Vasa2 promoter and the promoter active in insects may be the PLIBc promoter. Preferably, in the present invention the first insect germ line specific promoter is the Vasa2 promoter, the second promoter active in insects is the PUBc promoter, and the second insect germ line specific promoter is the Vasa2 promoter.
[0089] In accordance with the present invention, each of the gRNA encoding sequencesthat direct Cas nuclease to the specific target sequences are placed under the control of a promoter selected from a Polymerase I (Pol-I) promoter and a Polymerase III (Pol-Ill) promoter. Preferably, the promoter is a Pol-Ill type promoter, e.g. a U6 promoter.
[0090] The sequences of these transcription promoters are publicly available and therefore known to the skilled person. As an example only, the sequence of the Vasa2 promoter may be that corresponding to nucleotides 4674 to 6964 of the sequence with GenBank® reference number MT270142 (SEQ ID NO:4). The PLIBc promoter sequence may consist of the regions corresponding to nucleotides 12994045 to 12996049 (PUBc5) and 12999279 to 12999686 (PUBc3) of the reference sequence of chromosome 2R of the Anopheles gambiae PEST genome (SEQ ID NO:5-6). The sequence of this genome can be accessed in the Vactorbase database under 'Data / Download Data File' by searching with the keyword 'Anopheles gambiae PEST' and selecting the release version number 68. The Pol-Ill type promoter sequence may be that of the U6 promoter corresponding to nucleotides 10734 to 10876 of the sequence having reference number GenBank®MH541847 (SEQ ID NO:7).
[0091] In accordance with the present invention, the sequence of the gene necessary for female development can be selected from the group consisting of female-specific exon of the dsx gene and a sequence of the fie gene. The sequences of such genes as present in various insect species are publicly available and therefore known to the skilled person. As an example only, the sequence of the dsx gene may be that of Anopheles gambiae having VectorBase reference number AGAP004050 (SEQ ID NO:8).
[0092] According to the present invention, the gene necessary for spermatogenesis is preferably selected from the group consisting of the protamine and beta2-tubulin genes. More preferably, the gene required for spermatogenesis is the beta2-tubulin gene. The sequences of these genes as found in various insect species are publicly available and therefore known to the skilled person. As an example only, the sequence of the beta2-tubulin gene may be that of Anopheles gambiae with VectorBase reference number AGAP008622 (SEQ ID NO:9).
[0093] As described above, in order to facilitate the isolation of genetically modified insects of the first and second parental lines, a sequence encoding a fluorescent markercan also be introduced into the genome of such insects, provided that different markers are used in the two parental lines. As an example, the marker can be selected between GFP and RFP. If present, the sequence encoding for the fluorescent marker is placed under the transcriptional control of an appropriate promoter, e.g. a Pax promoter.
[0094] It is noted that any variant, derivative, analogue or fragment of any of the above nucleotide sequences having at least 40% sequence identity with the above nucleotide sequences, for example 40% identity with one of the sequences identified as SEQ ID NO:1-9, may be used in the context of the present invention. Included are nucleotide sequences having >50%, preferably >65%, more preferably >70%, even more preferably >75%, and still more preferably >80% sequence identity with any of the above nucleotide sequences. Preferably, said nucleotide sequences have at least 85%, more preferably at least 90%, still more preferably at least 95%, still more preferably at least 97%, and still more preferably at least 99% identity with any of the above nucleotide sequences.
[0095] Methods for calculating the percentage of sequence identity are known to the skilled person. Preferably, calculation of the percentage identity between two nucleotide sequences comprises the steps of:
[0096] (i) aligning the sequences using the programme ClustalW using an appropriate set of parameters (e.g. Gap Open Penalty = 15.0; Gap Extension Penalty = 6.66; Matrix = Identity; ENDGAP = -1 , and GAPDIST = 4); and
[0097] (ii) calculate the identity percentage using the formula (N / T)*100, where N is the number of positions where the aligned sequences have the exact same residue, and T is the total number of positions compared, including any gaps and including or excluding any overhangs, preferably including overhangs.
[0098] The present invention will now be described with reference to the following nonlimiting example.
[0099] Example
[0100] A parental trigger line is generated from male mosquitoes whose Y chromosome has been engineered by inserting a construct containing the following elements:
[0101] (a) a nucleotide sequence encoding the fluorescent marker GFP under transcriptional control of the Pax promoter;(b) a nucleotide sequence encoding Cas12a nuclease under transcriptional control of the Vasa2 promoter;
[0102] c) a nucleotide sequence encoding a gRNA recognised by Cas12a and capable of hybridising to the sequence encoding the AcrllA4 protein under transcriptional control of the U6 promoter.
[0103] An effector parental line is generated consisting of female mosquitoes carrying two genetic modifications in homozygosis, one autosomal modification and one modification on the X chromosome.
[0104] The autosomal modification involves the beta2-tubulin gene, which is modified by introducing the encoding sequence of a gRNA recognised by Cas12a and capable of hybridising to the corresponding nucleotide region of beta2-tubulin upstream and downstream of the insertion site of this modification, where this gRNA sequence is placed under transcriptional control of the U6 promoter.
[0105] The modification on the X chromosome, on the other hand, consists of the insertion of a construct containing the following elements:
[0106] (a) a nucleotide sequence encoding the fluorescent marker RFP under transcriptional control of the Pax promoter;
[0107] (b) a nucleotide sequence encoding Cas9 nuclease under transcriptional control of the PLIBc promoter;
[0108] c) a nucleotide sequence encoding the AcrllA4 protein operatively linked upstream of the nucleotide sequence encoding Cas9 via a TA sequence, so as to allow equimolar production of Cas9 and AcrllA4 or excess production of the inhibitor;
[0109] (d) a nucleotide sequence encoding a gRNA recognised by Cas9 and capable of hybridising to the highly conserved female-specific exon sequence of the dsx gene under transcriptional control of the U6 promoter; and
[0110] (e) a nucleotide sequence coding the beta2-tubulin lacking the sequence to which is capable of hybridising the gRNA encoded by the nucleotide sequence within the transcriptional unit referred to in the above autosomal modification, placed under transcriptional control of the Vasa2 promoter. This sequence coding for beta2-tubulin is resistant to the action of the first Cas nuclease, as it lacks the sequence necessary forsequence cleavage by the first Cas nuclease to occur.
[0111] Crossing males of the trigger line with females of the effector line generates trans-heterozygous male mosquitoes. At the germ cells level of these transheterozygous males, Cas12a nuclease interacts with the gRNA targeting the AcrllA4 coding sequence and destroys the AcrllA4 coding sequence. At this point, Cas9 nuclease is no longer inhibited and interacts with gRNA targeting the female exon sequence of the dsx gene, which is in turn destroyed. In addition, Cas12a nuclease interacts with gRNA targeting the beta2-tubulin gene, which is inactivated. Trans-heterozygous males are fertile, as the destruction of the female exon of the dsx gene does not impair the ability to reproduce in male insects, and the destruction of the endogenous beta2-tubulin genes is 'recovered' by the presence on the X chromosome of the Cas12a nuclease-resistant copy of the beta2-tubulin gene.
[0112] Mating trans-heterozygous males with wild-type female mosquitoes generates a progeny consisting of:
[0113] (a) females unable to reproduce, as they are sterile because they inherit, via the X chromosome of the trans-heterozygous male, an active Cas9 through which the female exon sequence of the dsx gene inherited maternally is also destroyed; and (b) infertile males, because they inherit, via the Y chromosome of the transheterozygote male, an active Cas12a through which the maternally inherited beta2-tubulin allele is also destroyed, but lack the Cas12a nuclease-resistant copy of the beta2-tubulin gene on the paternal X chromosome which is not passed on to them.
[0114] Sterile males of this first generation are able to exert a further suppressive effect by interbreeding with wild females of the next generation, preventing them from giving birth to a further generation of mosquitoes.
[0115] In practice, it was observed that the method according to the invention fully fulfils the intended aim, in that it provides a tool capable of exerting a powerful suppressive effect on an insect population, but at the same time self-extinguishing itself over two generations, so as to avoid any risks associated with prolonged propagation of the CRISPR-Cas-based gene drive systems on which the present invention is based.
[0116] The present invention, thus conceived, is susceptible to numerous modificationsand variations, all within the scope of the inventive concept; moreover, all details may be replaced by technically equivalent elements.
[0117] This application claims the priority of Italian Patent Application No.
[0118] 102025000001989, filed on February 4, 2025, the subject matter of which is incorporated herein by reference.
Claims
CLAIMS1. A method for suppressing an insect population, comprising the steps of:(i) providing:(a) a genetically modified male insect, carrying on the Y chromosome:a transcription unit A comprising a nucleotide sequence encoding a first Cas nuclease, under transcriptional control of a first insect germlinespecific promoter; and- a transcription unit B comprising a nucleotide sequence encoding one or more guide RNAs (gRNAs) comprising a binding region of the first Cas nuclease and a sequence that is capable of hybridising to a sequence encoding an Acr protein inhibiting a second Cas nuclease that is different from the first Cas nuclease, under transcriptional control of a promoter selected from a Pol-I type promoter and a Pol-Ill type promoter;and(b) a genetically modified female insect carrying on the autosomal loci of a gene necessary for spermatogenesis:a transcription unit C inserted in a site within the gene necessary for spermatogenesis, wherein said transcription unit C comprises a nucleotide sequence, under transcriptional control of a promoter selected from a Pol-I type promoter and a Pol-Ill type promoter encoding a gRNA comprising a binding region of the first Cas nuclease and a nucleotide sequence that is capable of hybridising to a corresponding nucleotide region of the gene necessary for spermatogenesis upstream and downstream of the insertion site of transcription unit C,and also carrying on both X chromosomes:a transcription unit D comprising a nucleotide sequence encoding the second Cas nuclease and a further nucleotide sequence encoding the Acr protein inhibiting the second Cas nuclease, operatively linked upstream of the nucleotide sequence encoding the second Cas nuclease by means of a TA sequence, under transcriptional control of a promoter active ininsects;a transcription unit E comprising one or more nucleotide sequences encoding a gRNA comprising a binding region for the second Cas nuclease and a nucleotide sequence capable of hybridising to a sequence of a gene necessary for female development, under transcriptional control of a promoter selected from a Pol-I type promoter and a Pol-Ill type promoter; anda transcription unit F comprising a nucleotide sequence encoding a copy of the gene necessary for spermatogenesis lacking the sequence to which the gRNA encoded by the nucleotide sequence comprised in transcription unit C is capable of hybridising, under transcriptional control of a second insect germline-specific promoter;(ii) releasing trans-heterozygous male insects obtained by crossing the genetically modified male insect of (i)(a) with the genetically modified female insect of (i)(b) in an environment where suppression of the insect population is desired, wherein said trans-heterozygous male insects carry the Y chromosome of the genetically modified male insect of (i)(a), the X chromosome of the genetically modified female insect of (i)(b), and the autosomal loci of the gene necessary for spermatogenesis in which is inserted the transcription unit C of the genetically modified female insect of (i)(b).
2. The method according to claim 1, wherein the first Cas nuclease is Cas12a nuclease.
3. The method according to claim 1 or 2, wherein the first insect germline-specific promoter is the Vasa2 promoter.
4. The method according to any one of the preceding claims, wherein the second Cas nuclease is Cas9 nuclease and the Acr protein is AcrllA4.
5. The method according to any one of the preceding claims, wherein the promoter active in the insects is the PUBc promoter.
6. The method according to any one of the preceding claims, wherein the sequence of the gene necessary for female development is selected from the groupconsisting of female-specific exon of the doublesex gene dsx) and a sequence of the femaleless gene (fie).
7. The method according to any one of the preceding claims, wherein the gene necessary for spermatogenesis is selected from the group consisting of the protamine and beta2-tubulin genes, preferably the beta2-tubulin gene.
8. The method according to claim 1 , wherein:- the first insect germline-specific promoter is the Vasa2 promoter;- the first Cas nuclease is Cas12a nuclease;- the promoter active in insects is the PUBc promoter;- the second Cas nuclease is Cas9 nuclease;- the Acr protein is Acrl I A4;- the gene necessary for spermatogenesis is the beta2-tubulin gene;- the second insect germline-specific promoter is the Vasa2 promoter; and - the sequence of the gene required for female development is the femalespecific exon of the doublesex dsx) gene.
9. A set of CRISPR-Cas-based gene drive genetic constructs comprising:- a first construct comprising the transcription unit A and transcription unit B as defined at point (i)(a) of claim 1 ;- a second construct comprising the transcription unit C as defined at point (i)(b) of claim 1 ; and- a third construct comprising the transcription unit D, transcription unit E, and transcription unit F as defined at point (i)(b) of claim 1.
10. A pair of insects consisting of:(a) a genetically modified male insect as defined at point (i)(a) of claim 1 ;(b) a genetically modified female insect as defined at point (i)(b) of claim 1.
11. A genetically modified trans-heterozygous male insect, carrying the Y chromosome of the genetically modified male insect as defined at point (i)(a) of claim 1 , the X chromosome of the genetically modified female insect as defined at point (i)(b) of claim 1 , and of the autosomal loci of the gene necessary for spermatogenesis in which is inserted the transcription unit C of the genetically modified female insect as defined in point (i)(b) ofclaim 1.