Artificial gene drive system

Abstract

The present invention belongs to the technical field of biology. Specifically, the present invention relates to an artificial gene drive system. More specifically, the present invention relates to an artificial gene drive system based on a toxin-antidote mechanism that can be used in plants, wherein a gene editing system that can deprive the function of a protein essential for pollen tube development is used as a toxin, a recoding sequence encoding the protein essential for pollen tube development is used as an antidote, and the recoding sequence encoding the wild-type protein is essential for pollen tube development and cannot be targeted by the gene editing system. The artificial gene drive system of the present invention can be used for transmitting features that are beneficial for human to wildlife populations.

Description

Artificial gene drive system Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to an artificial gene drive system. More specifically, the present invention relates to an artificial gene drive system based on a poison-antidote mechanism that can be applied to plants. Background Art

[0002] Genetic manipulation of wild populations is crucial for addressing diverse challenges, including controlling disease vectors such as mosquitoes, conserving biodiversity, and mitigating agricultural pests. However, under the influence of classical Mendelian inheritance and Darwinian selection, traits beneficial to humans are often difficult to widespread in these populations because they are often selectively neutral or even deleterious to the organism itself. Despite this, selfish genetic elements are widespread in nature, capable of being transmitted to offspring at frequencies exceeding Mendelian laws (>50%, for example, in diploid heterozygotes) to achieve their own advantages. Inspired by these natural processes, artificial gene drive systems have been designed to propagate genetic alterations within populations without regard for the potential fitness costs to individual organisms. Therefore, this technology has the potential to spread traits beneficial to humans into wild populations, providing a compelling tool for addressing these global challenges.

[0003] In yeast 1-2 ,mosquito 3-7 , fruit flies 8-10 and mice 11 In the field of genetics, a variety of artificial gene drive systems have been realized for population modification or population suppression. These gene drive systems are mostly based on positioning endonucleases (hence called homing-based drives), which function through a "copy-paste" mechanism, using CRISPR-Cas9-mediated DNA double-strand breaks (DSBs) and subsequent homologous recombination repair (HDR) processes to convert heterozygotes into homozygotes, so that the gene drive elements are inherited to offspring at a ratio of more than 50%. If the gene drive system carries a cargo chain within it, 3 As gene drive spreads, population improvement can be achieved; if the gene drive system itself is located in an essential gene related to fertility, 4,5If the DSB is repaired internally and continues to cut its homologous genes, population suppression can be achieved. However, if HDR repair is not successfully achieved, the DSB will be repaired by non-homologous end joining (NHEJ), introducing Indel, which will produce a resistance allele that can no longer be targeted and cut by gRNA. The NHEJ method is particularly common in plants, so the continued spread of gene drives in plant populations poses a major challenge. Therefore, in the pursuit of more efficient artificial gene drive systems, mechanisms that do not rely on the HDR repair pathway are crucial.

[0004] The toxin-antidote (TA) mechanism, exemplified by the naturally occurring t-haplotype in mice, offers a promising insight. Toxins are typically expressed before meiosis and are therefore present in all four formed gametes, interfering with normal gamete formation. Antidotes, on the other hand, are activated after meiosis and can mitigate and neutralize the damage caused by the poison, thereby providing an evolutionary advantage to their carriers. Although these natural poison-antidote systems cannot be directly replicated across species, the advent of CRISPR-Cas9 has provided a more universal approach to mimicking natural poison-antidote strategies. In artificially designed TA systems, CRISPR / Cas9 cleaves and NHEJ repairs an essential gene, resulting in loss-of-function (LOF) function as a poison, while a recoded sequence of the essential gene, which is not targeted by the guide RNA, serves as an antidote to rescue the effects of the poison.

[0005] Developed Toxin-Antidote Recessive Embryo (TARE) drive system 10 Also known as Cleave and Rescue or ClvR 9 , targeting an essential gene for zygote development, when both alleles of the gene are inactivated (LOF) in an individual and no gene drive element is inherited, the individual dies. In contrast, those individuals that inherit the gene drive element survive. This system has achieved a gene drive transmission rate of 88-95% in female heterozygotes of Drosophila melanogaster. 10 Although highly effective, the efficiency of this system relies on the presence of Cas9 activity carried over from the egg cell to the zygote (to cut the paternally contributed WT allele) or requires the target gene to be located on the sex chromosome, thus hindering its application to a wide range of species.

[0006] In addition, another design principle that has not yet been implemented is the Toxin-Antidote Dominant Sperm (TADS) drive 12 , which aims to interfere with essential genes in spermatogenesis, theoretically has a higher driving efficiency by destroying only one copy of the target gene, rather than two alleles like the TARE system, thus circumventing the above-mentioned limitations of TARE. However, its technical implementation has been hindered by the problem of determining whether to affect only essential genes for spermatogenesis.

[0007] Summary of the Invention

[0008] In this work, the inventors developed a gene drive system named CAIN (CRISPR-Assisted Inheritance utilizing NPG1). This is a carefully designed gene drive system based on the poison-antidote mechanism that can be applied to plants. It takes advantage of the longer male gametophyte development process of plants and targets an essential gene related to pollen grain germination, NPG1 (No Pollen Germination 1). Experimental verification has shown that the expected 50% can be increased to 88-99% in two consecutive generations in Arabidopsis, successfully achieving biased inheritance and significant super-Mendelian inheritance. The success of CAIN heralds its potential for application in various plant species and provides solutions to important challenges - slowing the spread of invasive species by affecting the inheritance ratio of sterility genes, and managing weed populations by spreading genes sensitive to certain herbicides, thus ushering in a new era of ecological management and sustainable agriculture.

[0009] The present invention includes but is not limited to the following embodiments:

[0010] Embodiment 1. An artificial gene drive system for a plant, comprising:

[0011] a first nucleic acid comprising a coding sequence for a gene editing system component, wherein the gene editing system can target a gene encoding a protein essential for pollen tube development in the plant and cause the protein essential for pollen tube development to lose function, wherein the coding sequence for the gene editing system component is operably linked to a promoter that mediates expression specifically during pollen formation;

[0012] a second nucleic acid comprising a recoded coding sequence of a protein essential for pollen tube development, wherein the recoded sequence encodes a wild-type protein essential for pollen tube development and cannot be targeted by the gene editing system, and is operably linked to a native promoter of the gene essential for pollen tube development; and

[0013] A third nucleic acid comprises a coding sequence for a cargo, eg, a cargo to be propagated among a population of said plant.

[0014] Embodiment 2. The artificial gene drive system of embodiment 1, wherein the first nucleic acid, the second nucleic acid, and the third nucleic acid are located on the same expression construct.

[0015] Embodiment 3. The artificial gene drive system of embodiment 1 or 2, wherein the protein essential for pollen tube development is No Pollen Germination 1 (NPG1).

[0016] Embodiment 4. The artificial gene drive system of embodiment 3, wherein the NPG1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO:1.

[0017] Embodiment 5. The artificial gene drive system of embodiment 3, wherein the coding sequence of endogenous NPG1 in the plant comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO: 2.

[0018] Embodiment 6. The artificial gene drive system of embodiment 3, wherein the coding sequence of the recoded NPG1 comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity with SEQ ID NO: 3, and the coding sequence of the recoded NPG1 cannot be targeted by the gene editing system, thereby not losing function due to expression of the gene editing system.

[0019] Embodiment 7. The artificial gene drive system of embodiment 3, wherein the natural promoter of NPG1 comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO:4.

[0020] Embodiment 8. The artificial gene drive system of any one of Embodiments 1-7, wherein the promoter that mediates specific expression during pollen formation is the promoter of the DMC1 (Disruption of Meiotic Control 1) gene.

[0021] Embodiment 9. The artificial gene drive system of embodiment 8, wherein the DMC1 promoter comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO: 5.

[0022] Embodiment 10. The artificial gene drive system of any one of embodiments 1-7, wherein the promoter that mediates expression specifically during pollen formation is the promoter of the TPD1 (Tapetum Determinant 1) gene.

[0023] Embodiment 11. The artificial gene drive system of embodiment 10, wherein the TPD1 promoter comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO: 6.

[0024] Embodiment 12. The artificial gene drive system of any one of Embodiments 1-11, wherein the gene editing system is selected from a gene editing system based on CRISPR, ZFN or TALEN, preferably, the gene editing system is a CRISPR-based gene editing system.

[0025] Embodiment 13. The artificial gene drive system of embodiment 12, wherein the CRISPR gene editing system comprises a CRISPR nuclease and at least one guide RNA, preferably, the CRISPR nuclease is a Cas9 nuclease.

[0026] Embodiment 14. The artificial gene drive system of embodiment 13, wherein the coding sequence of the CRISPR nuclease is operably linked to the promoter that mediates specific expression during pollen formation, preferably, is operably linked to the TPD1 promoter.

[0027] Embodiment 15. The artificial gene drive system of embodiment 13, wherein the gene editing system comprises a Cas9 nuclease and at least one gRNA targeting endogenous NPG1.

[0028] Embodiment 16. The artificial gene drive system of embodiment 15, wherein the at least one gRNA targeting endogenous NPG1 targets a nucleotide sequence selected from any one of SEQ ID NOs: 7-10.

[0029] Embodiment 17. The artificial gene drive system of any one of embodiments 1-16, wherein expression of the cargo is harmful or beneficial to the plant when the plant is exposed to a specific compound or condition, for example, the cargo is a herbicide-sensitive gene, a gene that destroys herbicide resistance, a gene that improves environmental adaptability, or a gene that improves disease resistance.

[0030] Embodiment 18. A method for producing a modified plant for gene drive modification of a plant population, the method comprising introducing the artificial gene drive system of any one of embodiments 1-17 into at least one plant, thereby obtaining at least one modified plant, the genome of which is integrated with the first nucleic acid, the second nucleic acid, and the third nucleic acid.

[0031] Embodiment 19. The method of embodiment 18, wherein the first nucleic acid, the second nucleic acid, and the third nucleic acid integrated into the genome of the modified plant are closely linked, eg, located at the same locus.

[0032] Embodiment 20. Use of a modified plant for gene drive modification of a plant population, wherein the modified plant is obtained by the method of embodiment 18 or 19 or the modified plant is introduced with the artificial gene drive system for a plant according to any one of embodiments 1 to 17, whereby the first nucleic acid, the second nucleic acid, and the third nucleic acid are integrated into its genome.

[0033] Embodiment 21. A modified plant for gene drive modification of a plant population, wherein the modified plant is obtained by the method of embodiment 18 or 19 or the artificial gene drive system for a plant according to any one of embodiments 1 to 17 is introduced into the modified plant, whereby the first nucleic acid, the second nucleic acid, and the third nucleic acid are integrated into its genome.

[0034] Embodiment 22. A method for modifying a plant population via gene drive, the method comprising placing at least one modified plant of embodiment 21 into the plant population, and allowing the at least one modified plant to hybridize with other plants in the plant population.

[0035] Embodiment 23. The method of embodiment 22, wherein the method allows progeny of hybrids of the at least one modified plant with other plants in the plant population to hybridize with other plants and / or progeny in the population.

[0036] Embodiment 24. The method of embodiment 22 or 23, which results in the modified plant population comprising an increased proportion of plants carrying the cargo compared to a non-modified plant population. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1. CAIN gene drive in Arabidopsis and its theoretical genetic behavior. a, Components of the CAIN gene drive. The left and right boundaries are shown, and the corresponding stages of Arabidopsis male gametophyte development are depicted. TPD1: Tapetum Determinant 1. DMC1: Disruption of Meiotic Control 1. NPG1: No Pollen Germination 1. b, Predicted proportion of CAIN-carrying F1 progeny from crosses between wild-type females and CAIN-carrying males, assuming loss of function of two, one, or none of the NPG1 alleles in male germline cells. Salmon, sky blue, and gray boxes represent the female, male, and progeny, respectively. Dashed boxes represent gametophytes. Red crosses represent non-germinating pollen grains.

[0038] Figure 2. Transmission efficiency of the CAIN gene drive from the T1 to the F1 generation in a testcross. a, Transformation of T1 plants and subsequent hybridization test steps. Transgenic T1 plants were transformed using Agrobacterium carrying either a control vector (FAST only) or one of the gene drive vectors (DMC-CAIN and TPD-CAIN). The F1 generation was generated using T1 plants with a single insertion as the male parent and wild-type Col-0 as the female parent. b, Transmission efficiency of the CAIN gene drive is the ratio of FAST+ F1 seeds to all F1 seeds. Each red dot represents the transmission efficiency in a single silique.

[0039] Figure 3. Genotypes of FAST+F1 plants at the NPG1 locus in the TPD-CAIN experiment. a, Schematic diagram of genotyping of F1 somatic tissues (rosette leaves and inflorescences). b, Summary of genotyping results of 16 FAST+F1 plants at four gRNA target sites. The symbols "+" and "-" before the numerical value and base number indicate insertion and deletion events, respectively. The numerical value after the symbol indicates the number of nucleotides in the indel greater than 2. The symbol "A>C" represents a base substitution from adenine (A) to cytosine (C). c, Genotyping results at the gRNA11 target site based on Illumina sequencing.

[0040] Fig. 4. Transmission efficiency of TPD-CAIN from F1 to F2 generations in backcrosses. Average transmission efficiency of TPD-CAIN in progeny when FAST+F1 plants served as male (a) or female (b) parents, respectively.

[0041] Figure 5. Transmission efficiency of DMC-CAIN from F1 to F2 generations. a, Genotype identification of the inflorescence of F1 plants. b, Summary of genotypes of 12 FAST+F1 plants at four gRNA target sites. c, Average transmission efficiency of DMC-CAIN in F2 seeds produced by FAST+F1 as the male parent.

[0042] Figure 6. Genotypes of FAST-F1 and FAST+ / -F2 plants at the NPG1 locus. a. Genotype identification of the leaf part of the FAST-F1 plant produced by the T1 plant carrying TPD-CAIN as the male parent. b. Summary of the genotypes of 11 FAST-F1 plants at the four gRNA target sites. The mechanism corresponding to each F1 plant (incomplete cutting efficiency or incomplete penetrance) is marked below by a check mark. c. Summary of the genotypes of FAST+ and FAST-F2 plants at the four gRNA target sites based on Sanger sequencing. The F2 plant is produced by reciprocal crosses between F1 carrying TPD-CAIN (TPD-CAIN / +) and the wild type (+ / +).

[0043] Figure 7. Simulations of the spread dynamics of modified and suppressor CAIN drivers. a. Computational simulations illustrating the effects of different settings for male germline cleavage efficiency (empirical value: 98.4%, artificial settings: 50.0% and 100.0%) and penetrance (empirical value: 96.0%, artificial settings: 50.0% and 100.0%) on CAIN spread dynamics. b. Computational simulations of the spread dynamics of homing type, TARE, and CAIN drivers, with an initial release ratio of 1%. The cleavage efficiency of both homing and TARE was set to the maximum. For CAIN, the penetrance was set to the empirical value of 96.0%. Different settings were also used for male (empirical value 98.4% or artificial setting of 50.0%) and female (empirical value 94.1% and artificial settings of 0.0% and 50.0%) germline cleavage efficiencies. c. Spread dynamics of suppressor CAIN drivers. This figure shows the changes in the number and frequency of CAIN carriers and wild-type individuals across generations.

[0044] Figure 8. Four gRNAs involved in CAIN gene drive. a. The CAIN vector contains four tandem gRNAs. Based on the principle of synonymous codons, the NPG1 sequence is altered without changing the encoded amino acid, as Recoded NPG1, and the mutated nucleotides are marked with red boxes. b. The positions of the four gRNA target sequences on the genomic sequence are shown. The primers used for genotyping have been marked. The primer pairs NPG-gDNA-F1 and NPG-gDNA-R1_2 were first used to amplify the genomic region covering the four target sites, and the PCR products were subjected to Sanger sequencing. The primers used for sequencing are: NPG-gDNA-F1, NPG-gDNA-F2, NPG-gDNA-R1_1, and NPG-gDNA-R2.

[0045] Figure 9. CAIN gene drive vector maps. Maps of the control vector FAST only (a), DMC-CAIN (b), and TPD-CAIN (c) gene drive vectors are shown, with the total sequence length and main features annotated.

[0046] Figure 10. Overview of the experimental process. Step 1: Infect Arabidopsis thaliana Col-0 background with a control vector (FAST only), DMC-CAIN, or TPD-CAIN gene-driven vector. Successfully transformed seeds are directly selected based on the FAST phenotype (red fluorescence). T1 plants with a single insertion site are screened by TAIL-PCR and whole-genome sequencing. Step 2: Hybridize the T1 as the male parent with the Col-0 female parent. The percentage of FAST+ seeds in the F1 seeds is used as the transmission rate (drive%) of the CAIN gene drive. Step 3: Plant the F1 seeds to obtain F1 plants. Genotype each F1 plant as described in the methods section. Step 4: Hybridize the F1 plant with known genotype as the male or female parent with the Col-0 plant. Similarly, calculate the drive transmission rate in the F2 seeds. Step 5: Plant the F2 seeds to obtain F2 plants. Genotype the F2 plants similarly.

[0047] Figure 11. Mutation types detected in FAST+ F1 plants. This figure illustrates the insertions, deletions, and single nucleotide polymorphisms (SNPs) generated at the target sites of (a) gRNA2 and (b) gRNA11, along with their locations. * indicates possible alignments, as underlined bases can be located on either side of the deletion.

[0048] Figure 12. Reversible TPD-CAIN gene drive. In the new version of the gene drive TPD-CAIN n+1 Designing gRNAs targeting novel sites in NPG1 n+1 As a new poison, gRNAs n and gRNAs n+1 are all recoded with resistance (cannot be targeted for cleavage) n+1 When Cas9 is active in germ cells and cuts, the new poison destroys NPG1 and the recoded n , so it can only be recoded n+1 In this way, if the new version of TPD-CAIN n+1 Compared with the old version TPD-CAIN n In the same source position, the new version will eliminate and replace the old version. n+1 It also spread.

[0049] Figure 13. Effects of male germ cell cutting efficiency, incomplete penetrance, and female germ cell cutting efficiency on the TPD-CAIN system. a. Estimation of male germ cell cutting efficiency and penetrance. In the F1 progeny, 94.3% (526 / 558) of the seeds were FAST+ (TPD-CAIN / +), and all of them were NPG1 at the gRNA11 target site.- Genotype. In addition, 2.6% (5.7% × 5 / 11) were genotype + / +; NPG1 + / - , 3.1% (5.7% × 6 / 11) were genotype + / +; NPG1 + / + In the F2 generation, 94.8% (3868 / 4080) were FAST+ (TPD-CAIN / +), and all of them were NPG1 at the gRNA11 target site. - Genotype, the remaining 5.2% were + / +; NPG1 + / - . According to the statistical results in the F1 and F2 generations, the average cleavage failure rate was estimated to be 1.6%, that is, the male germ cell cleavage efficiency was 98.4%. The average penetrance was estimated to be 96.0%. b, Estimation of female germ cell cleavage efficiency (r). Since no further cleavage of the target site occurs in FAST-F2 plants, the cleavage efficiency r was estimated based on the NPG1 genotype at its gRNA11 target site. Since only one of the 34 FAST-F2 plants was + / +; NPG1 + / + genotype, thus estimating r to be 94.1%.

[0050] Figure 14. Potential applications of TPD-CAIN gene drive. TPD-CAIN has two potential applications. a. Improving plant adaptability: By disseminating genes that promote the adaptability of specific endangered species to their environments, TPD-CAIN can rapidly rescue and improve the adaptation of target species to their habitats. b. Weed management: By disseminating genes that confer herbicide sensitivity to target weeds, combined with subsequent localized application of herbicides, TPD-CAIN can achieve efficient regional weed management. DETAILED DESCRIPTION

[0051] 1. Definition

[0052] In the present invention, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, and microbiology used herein are terms and routine procedures widely used in the corresponding fields. For example, standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following literature: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). At the same time, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0053] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0054] When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0055] "Exogenous" with respect to a sequence refers to a sequence that is from a foreign species, or, if from the same species, has been significantly altered from its native form in composition and / or locus through deliberate human intervention.

[0056] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid" are used interchangeably and are single-stranded or double-stranded polymers of RNA or DNA that optionally contain synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single-letter names as follows: "A" is adenosine or deoxyadenosine (RNA or DNA, respectively), "C" is cytidine or deoxycytidine, "G" is guanosine or deoxyguanosine, "U" is uridine, "T" is deoxythymidine, "R" is a purine (A or G), "Y" is a pyrimidine (C or T), "K" is G or T, "H" is A or C or T, "D" is A, T, or G, "I" is inosine, and "N" is any nucleotide.

[0057] Codon optimization refers to a process of modifying a nucleic acid sequence to enhance expression in a host cell of interest by replacing at least one codon of the native sequence (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with codons that are more frequently or most frequently used in the genes of the host cell, while maintaining the native amino acid sequence. Different species exhibit specific preferences for certain codons for specific amino acids. Codon bias (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which is believed to depend on the nature of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell generally reflects the codons that are most frequently used for peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the Codon Usage Database available at www.kazusa.orjp / codon / , and these tables can be adapted in various ways. See, Nakamura et al., 2002, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044 Y. et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000. Nucl. Acids Res., 28:292 (2000).

[0058] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0059] Sequence "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. (See, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0060] In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Generally, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0061] As used herein, an "expression construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, expression of a nucleotide sequence can refer to the transcription of the nucleotide sequence (e.g., transcription to produce mRNA or functional RNA) and / or translation of RNA into a precursor or mature protein.

[0062] The "expression construct" of the present invention can be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, can be RNA (such as mRNA) that can be translated, for example, RNA generated by in vitro transcription.

[0063] An "expression construct" of the present invention may comprise regulatory sequences and a nucleotide sequence of interest from different sources, or regulatory sequences and a nucleotide sequence of interest from the same source but arranged in a manner different from that normally found in nature.

[0064] "Regulatory sequence" and "regulatory element" are used interchangeably to refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence and that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include, but are not limited to, promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0065] "Promoter" refers to a nucleic acid fragment that is capable of controlling the transcription of another nucleic acid fragment. In some embodiments of the present invention, a promoter is a promoter that is capable of controlling the transcription of a gene in a cell, whether or not it is derived from the cell. A promoter can be a constitutive promoter, a tissue-specific promoter, a developmentally regulated promoter, or an inducible promoter.

[0066] As used herein, the term "operably linked" refers to the connection of a regulatory element (e.g., but not limited to, a promoter sequence, a transcription termination sequence, etc.) to a nucleic acid sequence (e.g., a coding sequence or an open reading frame) such that transcription of the nucleotide sequence is controlled and regulated by the transcriptional regulatory element. Techniques for operably linking regulatory element regions to nucleic acid molecules are known in the art.

[0067] "Introducing" a nucleic acid molecule (e.g., a plasmid, a linear nucleic acid fragment, RNA, etc.) or a protein into an organism refers to transforming the cells of the organism with the nucleic acid or protein so that the nucleic acid or protein can function in the cell. "Transformation" as used in the present invention includes stable transformation and transient transformation. "Stable transformation" refers to the introduction of an exogenous nucleotide sequence into the genome, resulting in the stable inheritance of the exogenous gene. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof. "Transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell to perform a function without the stable inheritance of the exogenous gene. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.

[0068] As used herein, the term "plant" includes whole plants and any offspring, cells, tissues, or parts of plants. The term "plant part" includes any part of a plant, including, for example, but not limited to: seeds (including mature seeds, immature embryos without seed coats, and immature seeds); plant cuttings; plant cells; plant cell cultures; plant organs (e.g., pollen, embryos, flowers, fruits, buds, leaves, roots, stems, and related explants). Plant tissues or plant organs can be seed, callus, or any other plant cell colony organized into structural or functional units. Plant cells or tissue cultures can regenerate plants with the physiological and morphological characteristics of the plant from which the cells or tissues were derived, and can regenerate plants with substantially the same genotype as the plant. In contrast, some plant cells cannot regenerate to produce plants. The regenerable cell in plant cell or tissue culture can be an embryo, protoplast, meristematic cell, callus, pollen, leaf, anther, root, root tip, silk, flower, kernel, ear, cob, husk, or stem.

[0069] Plant "progeny" include any subsequent generation of the plant.

[0070] 2. Artificial gene drive

[0071] In one aspect, the present invention provides an artificial gene drive system for a plant, comprising:

[0072] a first nucleic acid comprising a coding sequence for a gene editing system component, wherein the gene editing system can target a gene encoding a protein essential for pollen tube development in the plant and cause the loss of function of the protein essential for pollen tube development, wherein the coding sequence for the gene editing system component is operably linked to a promoter that mediates expression specifically during pollen formation;

[0073] a second nucleic acid comprising a recoded coding sequence of the pollen tube development essential protein, wherein the recoded sequence encodes a functional pollen tube development essential protein and cannot be targeted by the gene editing system, and is operably linked to a native promoter of a gene encoding the pollen tube development essential protein; and

[0074] A third nucleic acid comprising a coding sequence for a cargo to be propagated among a population of said plants.

[0075] In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid are located on the same expression construct.

[0076] In some embodiments, the gene encoding a protein essential for pollen tube development is an endogenous gene of the plant. In some embodiments, the gene encoding a protein essential for pollen tube development is an exogenous gene that has been introduced into the plant.

[0077] In some embodiments, the protein essential for pollen tube development is No Pollen Germination 1 (NPG1). NPG1 is associated with male gametophyte development but does not affect female gametophyte development and is required for the later stages of pollen germination. NPG1 is highly conserved across different plants.

[0078] In some embodiments, NPG1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO:1.

[0079] In some embodiments, the coding sequence of endogenous NPG1 in the plant comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO:2.

[0080] In some embodiments, the coding sequence of the recoded NPG1 comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO: 3. The coding sequence of the recoded NPG1 cannot be targeted by the gene editing system, and thus will not lose function due to expression of the gene editing system.

[0081] Generally speaking, a gene promoter refers to a sequence of about 100 bp to about 5 kb, such as about 500 bp to about 3 kb, such as about 2 kb, upstream of the translation start site or transcription start site of the gene coding sequence on the genome.

[0082] In some embodiments, the native promoter of NPG1 comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO:4.

[0083] In some embodiments, the promoter that mediates specific expression during pollen formation is the promoter of the DMC1 (Disruption of Meiotic Control 1) gene. In some embodiments, the DMC1 promoter comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO: 5. The DMC1 promoter is capable of driving expression of a nucleotide sequence operably linked thereto in pollen mother cells.

[0084] In some preferred embodiments, the promoter that mediates specific expression during pollen formation is the promoter of the TPD1 (Tapetum Determinant 1) gene. In some embodiments, the TPD1 promoter comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO: 6. The TPD1 promoter is capable of driving the continuous expression of the nucleotide sequence operably linked thereto during the development of the progenitor of the pollen mother cell, i.e., the archosporium, into the pollen mother cell.

[0085] The gene editing system that can be used in the present invention can be various gene editing systems known in the art, as long as it can perform targeted genome editing in plants. The gene editing system can be a gene editing system based on CRISPR, ZFN or TALEN. Preferably, the gene editing system is a gene editing system based on CRISPR.

[0086] The CRISPR gene editing system can include a CRISPR nuclease and at least one guide RNA. The CRISPR nuclease and guide RNA can form a complex that targets and / or cleaves a genomic target sequence based on complementarity between the guide RNA and the genomic target sequence.

[0087] "CRISPR nuclease" can be derived from a Cas9 nuclease, including a Cas9 nuclease or a functional variant thereof. The Cas9 nuclease can be a Cas9 nuclease from a different species, such as spCas9 from Streptococcus pyogenes (S. pyogenes) or SaCas9 derived from Staphylococcus aureus (S. aureus). "Cas9 nuclease" and "Cas9" are used interchangeably herein and refer to an RNA-guided nuclease comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active DNA cleavage domain of Cas9 and / or a gRNA binding domain of Cas9). Cas9 is a component of the CRISPR / Cas (clustered regularly interspaced short palindromic repeats and related systems) genome editing system that can target and cut a DNA target sequence to form a DNA double-strand break (DSB) under the guidance of a guide RNA.

[0088] "CRISPR nuclease" can also be derived from Cpf1 nuclease, including Cpf1 nuclease or functional variants thereof. The Cpf1 nuclease can be Cpf1 nuclease from different species, such as Cpf1 nuclease from Francisella novicida U112, Acidaminococcus sp. BV3L6, and Lachnospiraceae bacterium ND2006.

[0089] Useful "CRISPR nucleases" can also be derived from Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, Csn2, Cas4, C2c1, C2c3 or C2c2 nucleases, for example, including these nucleases or functional variants thereof.

[0090] In some embodiments, the coding sequence of the CRISPR nuclease is operably linked to the promoter that mediates specific expression during pollen formation, preferably, is operably linked to the TPD1 promoter.

[0091] As used herein, “guide RNA” and “gRNA” are used interchangeably and refer to an RNA molecule that can form a complex with a CRISPR effector protein and can target the complex to a target sequence due to a certain homology with the target sequence. The guide RNA targets the target sequence by base pairing with the complementary strands of the target sequence. For example, the gRNA used by the Cas9 nuclease or its functional variants is generally composed of crRNA and tracrRNA molecules that are partially complementary to form a complex, wherein the crRNA comprises a guide sequence (also known as a seed sequence) that has sufficient homology to the target sequence to hybridize with the complementary strand of the target sequence and guide the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to the target sequence sequence. However, it is known in the art that single guide RNA (sgRNA) can be designed, which contains the features of both crRNA and tracrRNA. The gRNA used by the Cpf1 nuclease or its functional variants is generally composed only of mature crRNA molecules, which may also be referred to as sgRNA. It is within the capabilities of those skilled in the art to design a suitable gRNA based on the CRISPR nuclease used and the target sequence to be edited. In some embodiments, the guide RNA can be expressed, such as transcribed, by a constitutive promoter. In some embodiments, the guide RNA can be expressed, such as transcribed, by a U6 or U3 promoter.

[0092] The gene editing system can target any region of the endogenous gene encoding a protein essential for pollen tube development, as long as it can cause the loss of function of the endogenous protein. For example, the gene editing system can target the endogenous coding sequence of a protein essential for pollen tube development, resulting in mutation or incomplete translation of the protein. Alternatively, the gene editing system can target the endogenous regulatory sequence of a protein essential for pollen tube development, resulting in the inability to express the protein.

[0093] Methods for recoding the coding sequence of the protein essential for pollen tube development so that it expresses a functional protein but is no longer targeted by the gene editing system are well known in the art. For example, the nucleotide sequence can be altered by codon degeneracy to remove the target sequence of the gene editing system without changing the encoded protein sequence. However, if the gene editing system targets the endogenous regulatory sequence of the protein essential for pollen tube development, the coding sequence of the protein essential for pollen tube development contained in the second nucleic acid can also be the same as the wild-type coding sequence, and can also be referred to as recoded herein because it cannot be targeted by the gene editing system.

[0094] In some embodiments, the gene editing system comprises a Cas9 nuclease and at least one gRNA targeting endogenous NPG1. In some embodiments, the at least one gRNA targeting endogenous NPG1 targets a nucleotide sequence selected from any one of SEQ ID NOs: 7-10.

[0095] The "goods to be spread among the plant population" described herein can be any sequence that is desired to be spread among the plant population, such as a wild population. For example, the expression of the goods is harmful to the plant when the plant is exposed to specific compounds or conditions. For example, the goods can be herbicide-sensitive genes, or genes that can destroy existing herbicide resistance. By coordinating with subsequent manual spraying of a certain herbicide or specific compound, effective localized weed management with controllable range can be achieved. The goods can also be genes that affect the development of megaspore mother cells or embryos, thereby achieving control of population size. The goods can also be genes that can improve environmental adaptability, disease resistance, etc., thereby improving the adaptability of endangered plants to their natural environment.

[0096] In another aspect, the present invention provides a method for producing modified plants for gene drive modification of a plant population, the method comprising introducing the artificial gene drive system for plants of the present invention into at least one plant, thereby obtaining at least one modified plant whose genome is integrated with the first nucleic acid, the second nucleic acid and the third nucleic acid.

[0097] In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid integrated into the genome of the modified plant are tightly linked, e.g., located at the same locus. In some embodiments, the first nucleic acid, the second nucleic acid, and / or the third nucleic acid are present in a single copy in the genome of the plant.

[0098] In the methods of the present invention, the artificial gene drive system can be introduced into plants using various methods well known to those skilled in the art. Methods for introducing the artificial gene drive system of the present invention into plants include, but are not limited to, biolistic methods, PEG-mediated protoplast transformation, Agrobacterium-mediated transformation, plant virus-mediated transformation, pollen tube passage, and ovary injection.

[0099] In another aspect, the present invention provides a modified plant for gene drive modification of a plant population, produced by the method of the present invention.

[0100] In another aspect, the present invention provides a modified plant for gene drive modification of a plant population, into which the artificial gene drive system for plants of the present invention is introduced, so that the first nucleic acid, the second nucleic acid and the third nucleic acid are integrated into its genome.

[0101] In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid integrated into the genome of the modified plant are tightly linked, e.g., located at the same locus. In some embodiments, the first nucleic acid, the second nucleic acid, and / or the third nucleic acid are present in a single copy in the genome of the plant.

[0102] In another aspect, the present invention provides a method for modifying a plant population via gene drive, the method comprising placing at least one modified plant of the present invention into a population of plants and allowing the at least one modified plant to hybridize with other plants in the plant population. In some embodiments, the method allows offspring from hybrids of the at least one modified plant with other plants in the plant population to hybridize with other plants and / or offspring in the population. In some embodiments, the method results in the modified plant population comprising an increased proportion of plants carrying the cargo compared to an unmodified plant population. For example, the modified plant population comprises at least 1%-100%, such as at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% of plants carrying the cargo.

[0103] In another aspect, the present invention provides a method for modifying a plant population by gene drive, the method comprising:

[0104] i) introducing the artificial gene drive system for plants of the present invention into at least one plant, thereby obtaining at least one modified plant, the genome of which is integrated with the first nucleic acid, the second nucleic acid and the third nucleic acid;

[0105] ii) placing the at least one modified plant obtained in step i) into said population of plants and allowing said at least one modified plant to crossbreed with other plants in said population of plants.

[0106] In some embodiments, the first, second, and third nucleic acids integrated into the genome of the modified plant are closely linked, for example, located at the same locus. In some embodiments, the first, second, and / or third nucleic acids are present in the genome of the plant as a single copy. In some embodiments, the method allows the offspring of hybrids between the at least one modified plant and other plants in the plant population to hybridize with other plants in the population. In some embodiments, the method allows the offspring of hybrids between the at least one modified plant and other plants in the plant population to hybridize with other plants and / or offspring in the population. In some embodiments, the method results in the modified plant population comprising an increased proportion of plants carrying the cargo compared to an unmodified plant population. For example, the modified plant population comprises at least 1% to 100%, for example, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or even 100% of plants carrying the cargo.

[0107] The plants of various aspects of the present invention may be monocotyledonous or dicotyledonous plants, preferably plants that mainly reproduce by outcrossing. For example, the plants may be Arabidopsis, corn, rapeseed, tobacco, grass weeds, etc.

[0108] Example

[0109] Experimental Materials and Methods

[0110] Plant materials and growth conditions

[0111] All Arabidopsis strains used in this study were Columbia-0 (Col-0) ecotypes. Seeds were surface sterilized in 10% sodium hypochlorite for 10 minutes, rinsed 3 times with sterile water, and then sown on germination medium (2.2 g / L Murashige and Skoog medium, 10 g / L sucrose, and 7.6 g / L plant agar, pH=5.7). After treatment at 4°C for 2 days, the agar plates were transferred to a growth chamber at 22°C with a 16-hour light / 8-hour dark photoperiod. 7-day-old seedlings were transferred to soil and continuously cultivated in a greenhouse under the above-mentioned temperature and light conditions for subsequent experiments.

[0112] Plasmid construction

[0113] All restriction enzymes and the enzyme for Gibson ligation were from New England Biolabs. High-fidelity polymerase (Phanta Max Super-Fidelity DNA Polymerase, P505) and gel extraction kit (FastPure Gel DNA Extraction Mini Kit, DC301) for polymerase chain reaction (PCR) were from Vazyme. All plasmids used in this study were cloned using standard molecular biology techniques and purified using the StarPrep Fast Plasmid Mini Kit (Genstar, D201).

[0114] To construct Fast only vectors, Gibson ligation was used 26 FAST tag sequence 17 The promoter and partial coding sequence of the OLE1 (AT4G25140) gene were amplified from genomic DNA (gDNA), and the sequences of TagRFP and Nos terminator were obtained according to the reference 17 The XF675 plasmid digested with HindIII and the two fragments were assembled together by Gibson ligation.

[0115] To construct the DMC-CAIN and TPD-CAIN vectors, all components were cloned into the XF675 binary vector in five consecutive steps. Specifically, the sgRNA cassette (pU6-SmR-gRNA scaffold-U6 terminator) was amplified from the template pHEE401E (Addgene#71287) and inserted into the XF675 after double enzyme digestion (EcoRI and HindIII) using Gibson ligation. 27Four gRNAs were introduced. The above plasmid was digested with HindIII again, and the FAST marker sequence was cloned in by Gibson ligation, and the HindIII recognition site was filled in. The NPG1 promoter (about 2kb) was amplified from gDNA, and the Recoded NPG1 sequence was obtained by mutation PCR, and then inserted into the HindIII-digested plasmid in the previous step through Gibson ligation. Finally, the plasmid in the previous step was digested with KpnI, and the following fragments were connected by Gibson ligation: the promoter sequences of DMC1 (AT3G22880) and TPD1 (AT4G24972) amplified from gDNA, the plant codon-optimized SpCas9 sequence with two nuclear localization signals (NLS) amplified from the template pHEE401E, and the Nos terminator amplified from the template XF675.

[0116] Assessing editing efficiency at potential target sites in protoplasts

[0117] According to CRISPR-P 2.0 24 (http: / / crispr.hzau.edu.cn / CRISPR2 / ) and CRISPR-GE 28 (http: / / skl.scau.edu.cn / home / ) predicted candidate gRNAs for the NPG1 (AT2G43040) coding sequence, and screened 12 to detect cutting efficiency in Arabidopsis protoplasts.

[0118] Each 20nt gRNA sequence was inserted into the pAtU6-sgRNA vector (Addgene plasmid #119775) by BsaI digestion and Gibson ligation. Arabidopsis protoplasts were prepared according to the reference 29 , 30 After co-transformation with pAtU6-sgRNA and p2X35S-Cas9, protoplasts were cultured at room temperature for 48 hours and then harvested. Simultaneously, one tube of protoplasts was transformed with p2X35S-GFP alone and used to estimate transformation efficiency approximately 16 hours after transformation. Transformation efficiencies of 41% and 45% were found in the two biological replicates, respectively.

[0119] Genomic DNA from each tube of protoplasts was extracted using a DNA extraction kit (Plant Genomic DNA Kit, DP305, TIANGEN). A genomic region of 180–220 bp surrounding each target region was amplified and purified by PCR. A 6-nt barcode ('ATGCAG') was introduced into the primers to distinguish PCR products generated from two biological replicates of the same target site. All 24 purified PCR product samples were quantified using a Nanodrop 2000, then mixed in equal amounts and sent to Novogene for library construction and Illumina PE150 sequencing.

[0120] gRNA editing efficiency is calculated as the number of reads generating edits divided by the total number of reads that can be mapped to that site. Mismatched reads within 10 bp upstream of the PAM (NGG) are considered edited reads. Because Cas9 primarily edits insertions and deletions, single-base substitutions (SNPs) were excluded from the edit type calculation.

[0121] Generation and confirmation of single-locus insertion-driven heterozygosity

[0122] Agrobacterium GV3101 containing DMC-CAIN, TPD-CAIN or FAST only vector was used to culture the plants by floral dipping method. 31 Wild-type Col-0 Arabidopsis thaliana was transformed. Successful primary transformants (T1) were directly selected from the harvested dry seeds based on the presence or absence of red fluorescence under a handheld fluorescence detector (LUYOR 3415RG).

[0123] Agrobacterium-mediated transformation sometimes introduces exogenous DNA sequences into multiple sites of the plant genome. To this end, 48-50 T1 strains obtained from DMC-CAIN and TPD-CAIN were randomly selected and analyzed using thermal asymmetric interleaved polymerase chain reaction (TAIL-PCR). 32 The number of T-DNA insertion sites in each plant was examined and the whole genome was sequenced (Novogene, PE150) using the software TDNAscan 33 Data analysis was performed using a semi-automatic pipeline developed by the inventors to further confirm the T1 plants with single-site insertion, resulting in 3 DMC-CAIN T1s, 5 TPD-CAIN T1s, and 2 FAST-only T1s.

[0124] Cross-pollinate and evaluate drive percentages

[0125] To test the transmission rate of the gene drive, the unopened flowers of the wild-type female parent (several Col-0 plants) were emasculated and pollinated with pollen from T1 plants of DMC-CAIN and TPD-CAIN. To simulate natural hybridization, pollen from multiple flowers of the male parent was smeared on each stigma. The red fluorescence in the F1 dry seeds was identified using a handheld fluorescence detector, and the percentage of red fluorescence in the F1 seeds was used as the transmission rate of the gene drive in the F1 generation. To test whether the TPD-CAIN gene drive is transmitted through the male parent, FAST+F1 plants were used as the female or male parent and crossed with wild-type Col-0 plants to obtain the F2 generation. The method for phenotyping the F2 generation is the same as that for the F1 generation.

[0126] Statistical analysis

[0127] To test the significance of the CAIN transmission ratio, an exact binomial test was performed using the binom.test() function in R to test the ratio of FAST+ seeds to FAST- seeds, with the null hypothesis of 1:1. Because heterogeneity in the proportion of FAST+ seeds existed among different siliques, a replicated G-test was performed using DescTools in R (https: / / cran.r-project.org / package=DescTools) to quantify heterogeneity. Because multiple comparisons may increase type I error (false positives), the false discovery rate (FDR) was calculated using the p.adjust() function in the R package.

[0128] Genotyping

[0129] Genotyping of the target gene NPG1 was accomplished by amplifying a genomic fragment of approximately 2 kb including the four target sites and then performing Sanger sequencing (Figure 8b). The reverse amplification primer was located within the intron region to avoid interference with the recoded NPG1 sequence in the driver element. Genomic DNA (gDNA) extracted from rosette leaves and inflorescences was used as a template for polymerase chain reaction (PCR). The PCR products were directly subjected to Sanger sequencing. Consistent Sanger sequencing results were usually obtained from leaf and inflorescence samples of the same plant. If the sequencing results of the leaf sample showed multiple peaks (indicating possible mosaicism or heteroplasmy) and the genotype could not be determined, the inflorescence sample from the same plant was used to determine the genotype. FAST-F1 and F2 plants were genotyped using only leaf samples.

[0130] For three F1 plants, the gRNA11 target site genotype was determined using Illumina sequencing on leaf and inflorescence samples. PCR products from different tissues, each with a unique barcode sequence introduced during PCR, were mixed in equal amounts and sequenced using Illumina PE150. Clean reads, differentiated by barcode sequence, were aligned back to the genomic sequence surrounding the gRNA11 target site using BWA. The read depth covering the gRNA11 region ranged from 107,207 to 150,422. Reads with mismatches within the 23-nt target site region were considered as edit types, and the editing efficiency of each sample was determined as the ratio of reads containing mismatches to the total reads aligned to the target site. Single-base substitutions with a frequency higher than 0.5% and all indels were considered as edited types.

[0131] Population dynamics simulation

[0132] The population dynamics of CAIN were simulated using an individual-based stochastic model based on the Wright-Fisher model, which assumes a constant population size and non-overlapping generations. For the modified CAIN driver, the genotypes of two unlinked loci, CAIN and NPG1, were considered, and the initial population consisted of 9900 wild-type individuals and 100 heterozygotes carrying CAIN (TPD-CAIN / +; NPG1 + / + ). Refer to previous research 25 , a density-adjusted strategy was employed. Briefly, the scaling factor (S) was calculated using the formula S = 10 / (9 × N / K + 1), where N represents the population size of the current generation and K represents the environmental carrying capacity (i.e., 10,000). The population size (X) for each generation was calculated using the binomial distribution X~B(50, 0.02 × S). For each generation, pairs of individuals were randomly selected to produce offspring, and sex was randomly assigned. This process was repeated X times, with the resulting offspring overwriting the parental data.

[0133] For each generation, CAIN-carrying male parents were able to undergo CRISPR-mediated cleavage at the NPG1 locus, with the cleavage efficiency set to an empirical value (98.4%, Figure 13a) or fixed values ​​(50% or 100%). This cleavage resulted in loss of NPG1 gene function. The penetrance of the phenotypic failure of pollen germination due to loss of male gamete function was set to an empirical value (96.0%, Figure 13a) or an artificial value (100%). The cleavage efficiency of NPG1 in TPD-CAIN / + female germ cells was also considered and set to an empirical value (94.1%, Figure 13b) or an artificial value (0%, 50%, 100%).

[0134] For homing-type drives, the driver allele in the heterozygous state can convert the wild-type allele into the driver allele with 100% efficiency. For TARE drives, the target gene is a haplosufficient gene essential for embryonic development, and Cas9 will cut the wild-type target gene in germ cells with 100% probability. After fertilization, Cas9 carried by the egg cell will further cut the wild-type target gene of paternal origin, with the same cutting efficiency of 100%. Embryos with two destroyed target gene alleles but without inherited TARE driver elements will not be able to continue development.

[0135] For the suppressor CAIN driver, CAIN is located within a haploid-sufficient male fertility gene, rendering it nonfunctional. Settings were identical to those for the modified CAIN driver, except that the initial population size and K were both set to 100,000. Males homozygous for CAIN are unable to produce viable pollen.

[0136] The simulation was implemented using a custom Python script, which is available at https: / / github.com / QianLabWebsite / GeneDrive.

[0137] Example 1: Design of CAIN, a CRISPR-based poison-antidote gene drive system targeting pollen germination

[0138] CAIN consists of three tightly linked components: a poison, an antidote, and a cargo. The poison, a guide RNA-Cas9 complex, introduces an inactivating mutation in an essential gene involved in pollen germination by initiating a DSB followed by NHEJ repair. The antidote is a recoded version of this essential gene, expressed from its native promoter (Figure 1a). Theoretically, the poison could disrupt both alleles of the essential gene before meiosis, thereby affecting the germination of all four pollen grains. However, this effect can only be neutralized if the antidote is present in the pollen grains. Therefore, when plants carrying CAIN are mated with wild-type plants, only the two pollen grains carrying CAIN will successfully germinate, resulting in a 100% transmission rate of CAIN (Figure 1b). Even if only one of the two alleles of the essential gene is disrupted by the poison, the transmission rate of CAIN will be two-thirds (Figure 1b). In either case, the transmission rate of CAIN is higher than the 50% expected by Mendelian genetics.

[0139] This process can be propagated over successive generations, spreading CAIN throughout the population through continuous hybridization. Although the gene drive system targets outcrossing populations, Arabidopsis thaliana was chosen as the experimental subject because it is a model plant that primarily self-pollinates. Artificial hybridization was performed throughout the experiment, and strict experimental procedures and management were implemented to further enhance the ecological safety of the gene drive system before its official release.

[0140] The implementation of CAIN requires the selection of a gene that is essential for pollen germination. To identify a suitable target gene, a list of genes that are related to male gametophyte development but do not affect female gametophyte development was searched from a previous study. 13 , No Pollen Germination (NPG1) was selected as the target gene, which is required for the late stage of pollen germination 14 Choosing it as a target gene can make the Cas9 cutting period relatively longer, because Cas9 cutting needs to be completed before it can function (Figure 1a).

[0141] Twelve gRNAs were screened within the NPG1 CDS sequence and their cleavage efficiency was initially tested in Arabidopsis protoplasts. Ultimately, four gRNAs (gRNA2, gRNA6, gRNA11, and gRNA23) with varying DNA cleavage efficiencies were selected and incorporated into the drive element (Figure 1a).

[0142] In order to mitigate the negative impact that Cas9 expression in somatic cells may have on plant development and adaptability, we selected a promoter that is mainly active in reproductive cells to drive the expression of Cas9 (Figure 1a). We constructed two gene drive systems using promoters with different spatiotemporal expression patterns (Figure 1a). One is the DMC1 (Disruption of Meiotic Control 1) promoter, which can express Cas9 in pollen mother cells in anthers, and the other is the TPD1 (Tapetum Determinant 1) promoter, which is continuously expressed during the process of the archesporial cells, the progenitor cells of pollen mother cells, gradually developing into pollen mother cells. 16 The latter provides a longer cleavage time window (Figure 1a), potentially leading to higher overall Cas9 cleavage activity before NPG1 takes effect.

[0143] The antidote is a recoded version of the NPG1 gene sequence, driven by its original promoter to restore the gene function. To ensure that the antidote is not cut by Cas9, we mutated the target site of the gRNA (based on synonymous codons, so it does not affect the amino acid composition, Figure 8a). In addition, the intron region of the gene was deleted in this version, making the structure more streamlined and not interfering with the subsequent genotyping of NPG1 in the genome (Figure 8b). Regarding cargo, a red fluorescent protein expressed in the seed drying stage was selected, named FAST 17 , in order to observe the propagation of drive.

[0144] In summary, two CAIN vectors, TPD-CAIN and DMC-CAIN (Figure 1a and Figure 9), were constructed and named according to their respective promoters driving Cas9 expression. In addition, a vector containing only the FAST marker was constructed as a negative control (Figure 9). The above vectors were introduced into wild-type Arabidopsis Col-0 using the Agrobacterium-mediated floral dip method and randomly inserted into a certain genomic location (Figure 10). Transgenic plants T1 (i.e., the first generation of transgenic plants) with single-site insertion were screened for subsequent analysis. In the absence of gene drive activity, according to Mendelian genetics, the drive element will be transmitted to the offspring at a ratio of 50%.

[0145] Example 2: Significantly increased transmission rate of TPD-CAIN to F1 offspring

[0146] To assess whether the constructed gene drive CAIN could be successfully transmitted through the male parent, a T1 Arabidopsis plant carrying CAIN was used as the male parent and crossed with the wild-type Col-0 female parent (Figure 2a). Since the female parent Col-0 always transmits a wild-type (WT) allele to the F1 generation, the dominant phenotype conferred by FAST (i.e., red fluorescence in the F1 seeds) can be used to determine the proportion of CAIN transmitted from the male parent.

[0147] For DMC-CAIN, the results showed that only one of the three crosses (i.e., with D31 as the male parent) showed a significant increase in the transmission rate of CAIN (exact binomial test, Figure 2b). In contrast, for TPD-CAIN, all four crosses showed transmission rates ranging from 89.6% to 96.9%, significantly deviating from Mendelian inheritance (Figure 2b). In contrast, the negative control (i.e., FAST alone) showed transmission at a rate of approximately 50% (Figure 2b), consistent with Mendelian inheritance.

[0148] Because the observations in each silique can be considered an independent test event, the repeated goodness-of-fit test (G-test, Figure 2b) was further used to determine the deviation between the experimental results and 50% to explain the differences between different siliques. It was observed that in the cross with the D31 plant as the male parent, DMC-CAIN had significant heterogeneity between different siliques. This was the only cross that showed a transmission ratio exceeding 50%, which may be due to the low efficiency of Cas9 cutting and the inconsistency between different siliques due to randomness. In contrast, no significant heterogeneity in transmission ratio between different siliques was observed in TPD-CAIN. In addition, all four cross combinations showed a transmission efficiency close to 100% (Figure 2b), indicating the strong and consistent performance of TPD-CAIN.

[0149] Example 3: Segregation bias in TPD-CAIN determined by parents

[0150] To assess whether TPD-CAIN segregates by disrupting NPG1 as designed (Figure 1), the NPG1 genotype in F1 individuals was identified. Rosette leaves and inflorescences of the plants were sampled, and the genotypes were identified by extracting genomic DNA, amplifying the target site, and Sanger sequencing (Figure 3a). The results showed that all F1 plants (n=16) that were FAST+ (i.e., containing the drive element, TPD-CAIN / +) tested carried the disrupted NPG1 allele (NPG1 - )(Figure 3b, Figure 11), various types of indels were observed in 88% and 100% of F1 offspring at the gRNA2 and gRNA11 target sites, respectively. In theory, indels that are multiples of three in length can generate CRISPR-resistant alleles without affecting the subsequent reading frame, but the rarity of their occurrence at both gRNA sites (i.e., 0%) indicates that the CAIN design has a lower rate of resistance allele formation with normal gene function, especially when multiple gRNAs are connected in series. NPG1 - It is ubiquitous in almost all F1 generation plants, indicating that CRISPR-based NPG1 knockout is highly efficient and would theoretically further impair pollen germination, leading to distorted segregation.

[0151] To further investigate the mechanism of segregation distortion, reciprocal crosses were performed between FAST+F1 plants and Col-0 to determine whether the segregation distortion of TPD-CAIN was affected by the direction of hybridization. When FAST+F1 plants served as male parents (n=13), the transmission rate of TPD-CAIN was significantly higher than that expected by Mendelian inheritance (i.e., 50%, Figure 4a). In contrast, when FAST+F1 plants served as female parents (n=8), the transmission rate of TPD-CAIN did not significantly deviate from 50% (Figure 4b). These results suggest that the segregation distortion of TPD-CAIN is caused by defects in male gametophyte function caused by NPG1 gene knockout.

[0152] Compared with DMC-CAIN, TPD-CAIN has a higher transmission rate (Figure 2), which is speculated to be caused by the TPD1 promoter's ability to produce more active Cas9, resulting in a higher efficiency of NPG1 cutting. To verify this hypothesis, the FAST+F1 plants generated by DMC-CAIN were also identified for NPG1 genotype (n=12). The results showed that only two F1 plants had two KO alleles (NPG1 - / - ), one strain has a KO allele (NPG1 + / - ), the remaining 9 strains (NPG1 + / + ) without any KO allele (Figure 5a-b). This also indicates that DMC-CAIN has a lower cleavage efficiency at the NPG1 locus than TPD-CAIN (Figure 3b).

[0153] Although the cutting efficiency of DMC-CAIN was low, we further evaluated whether DMC-CAIN could produce segregation deviation in the next generation. To this end, two DMC-CAIN / +;NPG1 - / - The F1 plants were pollinated by wild-type female parents, and the red fluorescence ratios in their F2 seeds were counted (Figures 5b and 5c). It was found that the DMC-CAIN transmission rates reached 95.9% and 99.5%, respectively. + / - In the F2 offspring produced by the F1 plant as the male parent, the drive% was 63.7% (Figures 5b and 5c). + / + The drive% in the F2 progeny produced by F1 plants was close to 50% (Figures 5b and 5c). On average, the transmission rate of DMC-CAIN from F1 to F2 was 57.5% (3367 / 5857). This limited transmission rate indicates the key role of cutting efficiency in affecting the efficacy of the CAIN system.

[0154] Example 4: Insufficient DNA cleavage and incomplete penetrance may result in TPD-CAIN transmission rates not reaching 100%

[0155] When T1 plants carrying the gene drive element served as male parents, TPD-CAIN transmission rates ranged from 89.6% to 96.9% (Figure 2b). This suggests that a subset (3.1%-10.4%) of offspring remained free of TPD-CAIN (i.e., FAST-). Similarly, among the F2 generations produced by F1 plants carrying TPD-CAIN, a subset (1.0%-12.2%) also remained free of TPD-CAIN (Figure 4a). We further investigated the mechanism by which these FAST-progeny emerged.

[0156] To confirm whether these FAST-F1 plants were caused by the failure of NPG1 to be successfully cut, the NPG1 genotypes of 11 FAST-F1 plants were identified (Figure 6a). Notably, 6 of them were homozygous for the WT allele at all four target sites (Figure 6b). This indicates that the male gametes provided by the father contributed the WT allele and that these FAST-F1 plants were indeed produced by escaping Cas9 cutting (i.e., insufficient DNA cutting).

[0157] On the other hand, five FAST-F1 strains showed a WT / KO genotype at the gRNA2 and gRNA11 target sites (Figure 6b). Considering that the maternal parent, Col-0, can only provide one WT allele, the KO allele is transmitted from the paternal parent through pollen. This means that a small number of pollen carrying the NPG1KO allele and lacking the drive element can still germinate, indicating that the non-germination phenotype of the NPG1KO allele is not 100%, that is, there is incomplete penetrance.

[0158] To understand the potential population-level effects of DNA cleavage failure and incomplete penetrance on CAIN-driven transmission, computational simulations based on the Wright-Fisher model were performed. This individual-based stochastic model assumes a finite, randomly mating population that reproduces in discrete, non-overlapping generations. Starting with 9900 wild-type individuals and 100 TPD-CAIN / + individuals, the population size was maintained, mating pairs were randomly selected, and DNA cleavage and segregation bias were restricted to the male parent. Based on the estimated male germline cleavage efficiency and penetrance (98.4% and 96.0%, respectively; Figure 13a), simulations indicated that population transmission of TPD-CAIN from 1% to 99% would require approximately 17 generations, only one generation more than under optimal conditions (i.e., 100% DNA cleavage efficiency and full penetrance; Figure 7a). These results indicate that despite these effects, the efficiency of TPD-CAIN transmission remains relatively robust.

[0159] Example 5: Common NPG1 allele conversion in reproductive cells of plants carrying TPD-CAIN

[0160] Observed that TPD-CAIN; NPG1 - Pollen in wild-type plants (NPG1 + / + ) pollinated FAST+F1 plants, NPG1 was not detected by Sanger sequencing. + Genotype (Figure 3b; n = 16). This suggests that the maternal NPG1 + The allele may have undergone CRISPR-mediated DSB. To further test this hypothesis, leaf and inflorescence samples of three FAST+F1 plants from paternal parent T18 were analyzed for detailed NPG1 genotypes at the gRNA11 target site using Illumina sequencing technology. In addition to the major (average 52.9%) genotype "NPG1" that may have been inherited from T18, the -8 ” (Figure 3b), several other genotypes were observed, but the uncut NPG1 + The allele was relatively rare (average 0.8%, Figure 3c). + The allele does undergo CRISPR-mediated DSBs, and it is speculated that Cas9 activity arises after zygote formation, with DSBs being repaired primarily through an end-joining mechanism.

[0161] The Cas9 activity responsible for generating these DSBs could originate from one of two possible scenarios: Cas9 expressed from the embryonic genome after fertilization (i.e., zygote), or paternal inheritance of the Cas9 protein. The latter scenario seems unlikely due to the limited protein / RNA content of sperm cells. To investigate these two hypotheses, the NPG1 genotype of F2 plants previously generated by F1 reciprocal crosses was examined using Sanger sequencing (Figure 6c). Regardless of whether TPD-CAIN was inherited from the paternal or maternal parent, NPG1 + None of the genotypes were found in the FAST+F2 progeny. In contrast, NPG1 was detected in all FAST-F2 progeny, even if the father carried TPD-CAIN. + These observations are consistent with postzygotic expression of Cas9 and contradict the interpretation of paternal inheritance.

[0162] Analysis of the NPG1 genotypes transmitted from FAST+F1 to F2 plants (Fig. 6c) revealed that the frequency of a certain NPG1 genotype would be dominant (i.e., the frequency was much greater than 50%), even though the genotypes of these F1 parents at the time of initial zygote formation were heterozygous for NPG1. + / - For example, although at the gRNA11 target site, FAST+F1 father T18-2-1 (NPG1-8 ) were transmitted to the three genotypes in F2 plants, but NPG1 -8 Appeared in 13 / 16 F2 plants, the remaining two genotypes NPG1 -C and NPG1 -30 These findings suggest that DSBs in germ cells undergo HDR repair compared to somatic tissues, with paternal NPG1 - The allele serves as a template and produces allelic conversion.

[0163] Example 6: Cleavage of NPG1 in female reproductive cells promotes CAIN transmission

[0164] The cleavage of NPG1 in female germ cells and its subsequent repair have the potential to enhance the spread of CAIN by complementing the NPG1 knockout achieved in male germ cells. To accurately estimate the efficiency of NPG1 cleavage in female germ cells, the genotypes of FAST-F2 plants were analyzed, because no additional NPG1 cleavage occurs after F2 zygotes are formed, providing information on the original genotype inherited from their parents. Among the 34 FAST-F2 progeny produced by FAST+F1 plants (i.e., TPD-CAIN / +) and wild-type pollen, 33 showed heterozygous NPG1 at the gRNA11 target site. + / - genotype, based on which the NPG1 cleavage efficiency in female germ cells was estimated to be 94.1% ( FIG13 b ).

[0165] To assess the extent to which NPG1 cleavage in female germ cells promotes CAIN spread, this parameter was incorporated into simulations, with the initial frequency of CAIN carrier introduction set to 1% (Figure 7b). The results revealed a context-dependent effect: when male germ cell cleavage efficiency was 50%, an additional 50% female germ cell cleavage accelerated CAIN propagation by approximately three generations. However, the observed male germ cell cleavage efficiency in TPD-CAIN plants was 98.4%, while the actual female germ cell cleavage efficiency was 94.1%, resulting in only about one generation of acceleration (Figure 7b). Simulations also showed that CAIN spread was delayed by only a few generations compared to the homing form. However, this was still significantly faster than the frequency-dependent TARE-driven approach, which requires a sufficiently high initial introduction rate to spread rapidly (Figure 7b).

[0166] Compared with TARE, which has a high initial launch ratio, CAIN / TADS also spreads significantly faster. 18 , which also shows its potential as a population suppression tool when integrated into haplosufficient male fertility genes 25To measure its suppressive effect, the population dynamics of CAIN were simulated with an initial release ratio of 1% (Figure 7c). These simulations showed a rapid increase in the number and frequency of CAIN carriers (primarily heterozygotes in the early stages and primarily homozygotes in the later stages). Simultaneously, the total population size gradually decreased until extinction at the 26th generation (Figure 7c), likely due to a surge in the number of male-sterile CAIN homozygotes. Overall, the results of the computer simulations suggest that the rapid spread of CAIN gives it the potential to achieve population suppression.

[0167] In this study, the inventors developed CAIN, a CRISPR-based poison-antidote gene drive system targeting a key gene essential for male gametophyte function in Arabidopsis thaliana, achieving super-Mendelian inheritance and significantly reducing the generation of resistant alleles compared to homing-based drives. The inventors not only provide key insights into the design and application of synthetic TA gene drive systems in plants, but also propose an innovative solution to address pressing ecological and agricultural challenges.

[0168] Homing-based drives have been successfully applied to mosquitoes 3-7 , often produces resistance alleles in species with low HDR repair rates, hindering their spread. Artificially synthesized TA gene drive systems that mimic nature can overcome this problem. For example, the synthetic Medea 20,21 The TARE system (maternal effect dominant embryonic arrest), inspired by the natural Medea gene drive in flour beetles, exploits a complex balance between maternal miRNAs (as poisons) and antidotes expressed in the zygote and was implemented in Drosophila. However, its broader application is limited because it relies on a detailed understanding of Drosophila embryonic development. 10 (also known as ClvR 9 The design of Medea and TARE / ClvR avoids this by targeting essential genes during individual development, although it relies on the carryover of Cas9 activity from the egg to the zygote. Therefore, the design of Medea and TARE / ClvR is biased towards female germ cells, which may compromise fertility. 22 In contrast, our design targets male reproductive cells. Given that pollen grains far outnumber ovules, this is likely to minimize the fitness cost.

[0169] The inventors' design focuses on the key genes for the normal function of male gametophytes. The inventors' unique strategy utilizes the common male gametophyte formation process in plants, that is, after meiosis, there are two rounds of mitosis cycles, which eventually form mature pollen grains. 23 The pollen grains continue to germinate and elongate, passing two sperm cells through the stigma to achieve final double fertilization, and then they can be truly passed on to the offspring. The design and efficacy of the inventors' gene drive system depends on the selection of a target gene that is critical for pollen grain germination (NPG1 in this case), coupled with the selection of an efficient promoter for high Cas9 activity in the reproductive system. This method, tailored for plants, may also be applied in animals if the key essential genes for sperm formation can be identified.

[0170] By adding FAST 17 As a cargo, the inventors have determined that CAIN can achieve efficient partial segregation in plants. Cargo can also be replaced and used to solve various ecological problems, depending on the specific situation and goals (Figure 14). For example, the CAIN system can be used to control invasive plants by selecting specific genes - such as those that affect megaspore mother cells or embryo development to achieve population control. Or CAIN can be used to spread beneficial traits - such as drought or disease resistance genes - thereby enhancing the survival rate of endangered species in the wild. Similarly, specific herbicide susceptibility genes can be introduced to more effectively manage weeds, etc. If this strategy is widely used, it may herald a new era of ecological management and sustainable agriculture.

[0171] Due to the heated debate and regulatory scrutiny surrounding gene drive technology, the inventors took specific measures to ensure ecological safety when designing CAIN. The choice of Arabidopsis thaliana, a self-reproducing model plant, eliminates the possibility of accidental spread of this system, as gene drives require hybridization to effectively spread within a population. Furthermore, CAIN's design incorporates a degree of specificity, enabling the specific targeting of certain genotypes or ecotypes through the selection of gRNAs. Like homing-based drives, CAIN has a zero-threshold mechanism, meaning that theoretically, the release of a single individual containing a drive element can gradually spread throughout a population. The speed of its spread can be controlled by utilizing weaker gRNAs or promoters to control Cas9 expression, as the effectiveness of the drive is closely related to its cutting efficiency. This additional flexibility adds a layer of control to the spread of gene drives, enhancing their safe application in diverse ecological environments.

[0172] gRNAs may have certain off-target effects, which is a problem worthy of attention. Although off-target phenomena are unlikely to hinder the spread of CAIN, they may introduce unexpected genomic mutations, thereby increasing the genetic load. It is worth mentioning that although it was not emphasized in the results section, the inventors conducted a small-scale test and used CRISPR-P 2.0 24 , predicted 16 potential off-target sites for the four gRNAs, and determined the genotypes of these sites by Sanger sequencing in 16 F1 plants generated by four T1 lines. These potential off-target sites were not edited in the 16 plants tested, which also supported that the four gRNAs selected in CAIN specifically targeted NPG1.

[0173] The flexibility to update existing gene drive elements in a population is a key safety issue. The inventors designed CAIN with the ability to be functionally replaced. There are three prerequisites to achieve this process (Figure 12).

[0174] First, the new CAIN driver, CAIN n+1 , must be compatible with CAIN n Integration into the same genomic location. Although the probability of targeted integration by homology repair (HDR) in plants is relatively low, screening of a sufficient number of transformants can compensate for this limitation. Homologous locations can force CAIN n+1 and CAIN n Direct competition ensures that only one drive component remains.

[0175] Secondly, CAIN n+1 Different gRNA targets are needed to disrupt essential genes, thus acting as new poisons. This change makes CAIN n Both the recoded NPG1 and the NPG1 in the genome become the targets of cleavage.

[0176] Finally, you will not be CAIN n and CAIN n+1 The new recoded NPG1 targeted by the gRNA in the gene serves as a new antidote.

[0177] With these three prerequisites, the original CAIN n Can be CAIN n+1 This approach eliminates the need to select new target genes and preserves the overall size of the gene drive element, thereby improving compatibility and integration efficiency.

[0178] In summary, the inventors have designed CAIN, a CRISPR-based gene drive system specifically tailored for plants. By targeting the extended male gamete stage of the plant life cycle, the inventors have successfully demonstrated the efficacy of CAIN in Arabidopsis thaliana, setting a benchmark for its application in other species. Given that the key component, the NPG1 gene, exhibits sequence conservation across various plants, CAIN's species-wide applicability is promising. Looking ahead, further refinement of this gene drive system is needed, including research into its reversibility and adaptability, as well as the mechanisms underlying its controllability. CAIN and similar gene drive systems have the potential to reshape ecological management, agriculture, and species conservation in substantial and transformative ways.

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Claims

1. An artificial gene drive system for a plant, comprising: A first nucleic acid comprising a coding sequence of a gene editing system component, wherein the gene editing system can target a gene such as a coding sequence of a protein essential for pollen tube development in the plant and cause the endogenous protein essential for pollen tube development to lose function, wherein the coding sequence of the gene editing system component is operably linked to a promoter that mediates specific expression during pollen formation; A second nucleic acid comprising a recoded coding sequence of the pollen tube development essential protein, wherein the recoded sequence encodes the wild-type endogenous pollen tube development essential protein and cannot be targeted by the gene editing system, and is operably linked to a natural promoter of the pollen tube development essential gene; and A third nucleic acid comprising a coding sequence for a cargo to be propagated among a population of said plant.

2. The artificial gene drive system of claim 1, wherein the first nucleic acid, the second nucleic acid and the third nucleic acid are located on the same expression construct.

3. The artificial gene drive system of claim 1 or 2, wherein the protein essential for pollen tube development is No Pollen Germination 1 (NPG1).

4. The artificial gene drive system of claim 3, wherein the NPG1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO:

1.

5. The artificial gene drive system of claim 3, wherein the coding sequence of endogenous NPG1 in the plant comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO:

2.

6. The artificial gene drive system of claim 3, wherein the coding sequence of the recoded NPG1 comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity with SEQ ID NO:3, and the coding sequence of the recoded NPG1 cannot be targeted by the gene editing system, thereby not losing function due to the expression of the gene editing system.

7. The artificial gene drive system of claim 3, wherein the native promoter of NPG1 comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO:

4.

8. The artificial gene drive system according to any one of claims 1 to 7, wherein the promoter that mediates specific expression during pollen formation is the promoter of the DMC1 (Disruption of Meiotic Control 1) gene.

9. The artificial gene drive system of claim 8, wherein the DMC1 promoter comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO:

5.

10. The artificial gene drive system according to any one of claims 1 to 7, wherein the promoter that mediates specific expression during pollen formation is the promoter of the TPD1 (Tapetum Determinant 1) gene.

11. The artificial gene drive system of claim 10, wherein the TPD1 promoter comprises a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or even 100% sequence identity to SEQ ID NO:

6.

12. The artificial gene drive system of any one of claims 1 to 11, wherein the gene editing system is selected from a gene editing system based on CRISPR, ZFN or TALEN, preferably, the gene editing system is a gene editing system based on CRISPR.

13. The artificial gene drive system of claim 12, wherein the CRISPR gene editing system comprises a CRISPR nuclease and at least one guide RNA, preferably, the CRISPR nuclease is a Cas9 nuclease.

14. The artificial gene drive system of claim 13, wherein the coding sequence of the CRISPR nuclease is operably linked to the promoter that mediates specific expression during pollen formation, preferably, is operably linked to the TPD1 promoter.

15. The artificial gene drive system of claim 13, wherein the gene editing system comprises a Cas9 nuclease and at least one gRNA targeting endogenous NPG1.

16. The artificial gene drive system of claim 15, wherein the at least one gRNA targeting endogenous NPG1 targets a nucleotide sequence selected from any one of SEQ ID NOs: 7-10.

17. The artificial gene drive system of any one of claims 1 to 16, wherein the expression of the cargo is harmful to the plant when the plant is exposed to a specific compound or condition, for example, the cargo is a herbicide-sensitive gene, a gene that destroys herbicide resistance, a gene that improves environmental adaptability, or a gene that improves disease resistance.

18. A method for producing modified plants for gene drive modification of plant populations, the method comprising introducing the artificial gene drive system of any one of claims 1 to 17 into at least one plant, thereby obtaining at least one modified plant whose genome is integrated with the first nucleic acid, the second nucleic acid and the third nucleic acid.

19. The method of claim 18, wherein the first nucleic acid, the second nucleic acid and the third nucleic acid integrated into the genome of the modified plant are closely linked, such as located at the same locus.

20. Use of modified plants for gene drive modification of plant populations, wherein the modified plants are obtained by the method of claim 18 or 19 or the modified plants are introduced with the artificial gene drive system for plants according to any one of claims 1 to 17, so that the first nucleic acid, the second nucleic acid and the third nucleic acid are integrated into their genome.

21. A modified plant for gene drive modification of a plant population, wherein the modified plant is obtained by the method of claim 18 or 19 or the modified plant is introduced with the artificial gene drive system for plants according to any one of claims 1 to 17, so that the first nucleic acid, the second nucleic acid and the third nucleic acid are integrated into its genome.

22. A method for modifying a plant population by gene drive, the method comprising placing at least one modified plant of claim 21 into the plant population and allowing the at least one modified plant to hybridize with other plants in the plant population.

23. The method of claim 22, wherein said method allows progeny of a cross between said at least one modified plant and other plants in said plant population to cross with other plants and / or progeny in said population.

24. The method of claim 22 or 23, which results in said modified plant population comprising an increased proportion of plants carrying said cargo compared to a non-modified plant population.

Images