A method for cultivating a good variety of crops based on genetic engineering

Abstract

The application discloses a kind of crop elite cultivation methods based on genetic engineering, it is related to crop elite cultivation technical field, method includes constructing multifunctional expression vector: Cas9 protein is fused with single-stranded DNA binding domain expression, and the recognition sequence specifically paired with the domain is set at the end of donor recombination template, so that donor template is anchored in the instant breakage site at double-stranded break, improve homology-mediated repair efficiency, adopt early specific promoter to drive the time sequence misplacement expression of development regulation gene, late specific promoter drives recombination enzyme, constitute automatic excision type self-cleaning loop, while realizing regeneration breakthrough, automatically excise exogenous gene element in bud point differentiation stage, obtain isogenic line replacement plant without transgenic residue, can be used for stubborn commercial crop variety Precision elite cultivation.

Description

Technical Field

[0001] This invention relates to the field of crop breeding technology, specifically a method for breeding improved crop varieties based on genetic engineering. Background Technology

[0002] Gene editing technologies, exemplified by CRISPR-Cas9, use sgRNA to guide the Cas9 protein to induce DNA double-strand breaks at specific sites in the genome. Cells then repair these breaks through two pathways: non-homologous end joining or homologous-mediated repair. Homologous-mediated repair relies on a homologous donor DNA template and can achieve precise replacement of superior alleles, making it highly valuable for breeding. However, this pathway has an extremely low natural occurrence rate in plant somatic cells, typically below one percent, which is one of the core bottlenecks limiting the application of gene editing technology in precision breeding. The closest existing technology to this invention involves constructing a gene editing vector containing both a Cas9 protein expression cassette and an sgRNA expression cassette, co-transforming it with a donor DNA template carrying superior allele sequences, and then transferring it into recipient cells via Agrobacterium-mediated transformation, relying on the cell's own repair mechanisms to complete random repair. To improve the transformation and regeneration efficiency of recalcitrant commercial crop varieties, existing methods typically combine the donor DNA template with the genome... In contrast to the conventional view that the expression cassette of developmental regulatory genes and genome editing components (such as single-stranded DNA-binding domain fusion proteins) need to be independently regulated to balance regeneration efficiency and controllable excision timing, this invention breaks through this conventional understanding by placing both under the same early-specific promoter for synchronous and equal expression. This achieves precise overlap between the genome editing window and the cell regeneration window without introducing additional regulatory elements, resulting in a self-clearance timing reliability improvement that cannot be expected by using any existing technology alone. In the above schemes, the donor template does not form a physical association with the break site, leading to low repair efficiency. Continuous expression of developmental regulatory genes can easily cause pleiotropic toxicity in regenerated plants, such as dwarfing, leaf curling, and sterility. Chemical or heat-induced clearance methods are cumbersome to operate and have the risk of missed expression and transgene residue. Therefore, we propose a method for breeding improved crop varieties based on genetic engineering. Summary of the Invention

[0003] The purpose of this invention is to provide a method for breeding superior crop varieties based on genetic engineering.

[0004] The technical problem this invention aims to solve is that: existing genetic engineering breeding technologies have extremely low homology-mediated repair efficiency, making it difficult to achieve precise replacement of superior alleles in crop cells; at the same time, existing methods that rely on developmental regulatory genes to improve transformation efficiency have the problem of gene toxicity; and existing chemical or heat shock-induced gene removal methods are cumbersome to operate and have the risk of missed expression and transgenic residues, making it difficult for gene editing technology to achieve precise, safe, and scarless breeding of superior varieties in stubborn commercial crop varieties.

[0005] To address the aforementioned technical problems, this invention provides a method for breeding improved crop varieties based on genetic engineering, comprising: constructing a multifunctional expression vector, wherein the multifunctional expression vector includes a fusion protein expression cassette formed by fusing Cas9 protein with a single-stranded DNA binding domain via a flexible linker peptide, an sgRNA expression cassette linked thereto, a donor recombinant template expression cassette carrying homologous flanking sequences at both ends and a recognition sequence specifically paired with the single-stranded DNA binding domain, a developmental regulatory gene expression cassette driven by an early-specific promoter, a recombinase expression cassette driven by a late-specific promoter, and an resistance marker gene expression cassette, wherein the developmental regulatory gene expression cassette, the fusion protein expression cassette, and the resistance marker gene expression cassette are linked by a pair of site-specific recombination recognition sites aligned in the same direction. The vector is wrapped around the donor recombinant template outside the wrapped area. The vector is transformed into embryogenic callus of crop recipient material. During the callus induction culture stage, the early specific promoter is activated, so that the expression cassette of developmental regulatory genes and the expression cassette of fusion protein are expressed synchronously. On the one hand, it enhances the regeneration capacity of recipient cells. On the other hand, it enables the single-stranded DNA binding domain in the fusion protein to bind to the recognition sequence at the end of the donor recombinant template, anchoring the donor template near the double-strand break site. Allele replacement is completed through homology-mediated repair. During the shoot tip meristem formation stage, the late specific promoter is activated, driving recombinase expression and automatically excising the wrapped foreign gene element. Finally, plants with target allele replacement and no foreign transgenic elements are obtained by molecular detection screening.

[0006] Compared with the prior art, the beneficial effects of the present invention by adopting the above technical solution are as follows:

[0007] 1. This invention utilizes the physical anchoring effect mediated by fusion proteins to locally enrich the donor recombinant template at the break site during the instantaneous repair window caused by double-strand breaks. Compared with existing schemes where the donor template is distributed in a free state, this invention can significantly enhance the competitive advantage of homologous repair pathways over non-homologous end joining pathways, thereby improving the efficiency of precise allele replacement.

[0008] 2. This invention achieves temporal and spatial misalignment of the expression of developmental regulatory genes by having the expression cassette driven by an early embryonic cell-specific promoter and the recombinase expression cassette driven by a late shoot apical meristem-specific promoter. This forms a self-contained, self-removing loop that can be automatically triggered without exogenous chemical or physical induction. Developmental regulatory genes are expressed only during the window period required for cell dedifferentiation and regeneration to fulfill their functional mission. Once the shoot apical meristem is formed, they are automatically removed. This avoids the pleiotropic toxicity caused by continuous expression of developmental regulatory genes, such as plant dwarfing, leaf curling, and sterility. Furthermore, since the activation of the recombinase depends entirely on the developmental physiological signals of the recipient cell itself, there is no need to artificially control the timing and intensity of induction, thus avoiding the problems of missed expression and transgene residues commonly found in chemical or heat shock induction methods.

[0009] 3. In the regenerated plants after the excision treatment of this invention, only the superior allele sequence integrated by homology-mediated repair is retained at the original genome target site. There are no exogenous transgenic elements such as developmental regulatory genes, fusion protein coding genes and resistance marker genes. This achieves the effect of genotype-independent and transgenic-free precision breeding, which can be used for the breeding of superior varieties of stubborn commercial crops. Attached Figure Description

[0010] Figure 1 This is a schematic diagram of the overall process of breeding improved crop varieties based on genetic engineering in an embodiment of the present invention;

[0011] Figure 2 This is a schematic diagram of the timing workflow of the carrier module in an embodiment of the present invention;

[0012] Figure 3 This is a schematic diagram of the self-clearing loop logic flow in an embodiment of the present invention. Detailed Implementation

[0013] The following is in conjunction with the appendix Figure 1-3 The specific embodiments of the present invention will be further described below. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0014] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0015] I. Overall Structure of a Multifunctional Expression Carrier:

[0016] The multifunctional expression vector used in this embodiment is functionally divided into four expression units within the T-DNA region, arranged sequentially from left to right boundary. The first expression unit is a fusion protein expression cassette, whose coding sequence is formed by fusing the Cas9 protein coding region and the single-stranded DNA binding domain coding region through a flexible linker peptide sequence. Downstream, it is linked to an sgRNA expression cassette targeting the allele site to be replaced. The fusion protein expression cassette is driven by a constitutive promoter or an early specific promoter. The second expression unit is a donor recombinant template expression cassette, which carries flanking sequences homologous to the genomic target site and sequences specifically paired with the single-stranded DNA binding domain at both ends. The recognition sequence consists of a superior allele fragment to be replaced in the middle, a third expression unit that is a developmental regulatory gene expression cassette driven by an early specific promoter, and a fourth expression unit that is a recombinase expression cassette and an resistance marker gene expression cassette driven by a late specific promoter. The third expression unit, the fusion protein expression cassette in the first expression unit, and the resistance marker gene expression cassette are all wrapped by a pair of site-specific recombination recognition sites aligned in the same direction, forming a self-cleaning box to be excised. The donor recombination template of the second expression unit and the superior allele sequence that is finally integrated into the genome are located outside this wrapping region, so that they can still be retained in the genome after the self-cleaning box is excised.

[0017] II. Working principle of the donor spatiotemporal anchoring module:

[0018] The fusion protein, guided by sgRNA, recognizes and binds to the genomic target sequence. While the Cas9 domain cleaves the double-stranded DNA to produce a break, the single-stranded DNA binding domain fused with it simultaneously recognizes and binds to the donor recombination template, which has been exposed after single-stranding treatment. Since the two functional domains are on the same polypeptide chain, the donor recombination template is forcibly physically pulled to the vicinity of the break site, forming a local high-concentration donor enrichment region. Thus, during the window period when the cell selects repair pathways, homologous-mediated repair gains a competitive advantage over non-homologous end joining. Finally, homologous recombination repair is completed using the donor template as a template, integrating the superior allele sequence into the genomic target site.

[0019] To illustrate the impact of local enrichment on the choice of remediation pathway, the following simplified model can be used to describe the relationship between the probability of homology-mediated remediation and the local donor concentration:

[0020] ;

[0021] in, This indicates the probability that the double-strand break site will eventually be repaired via the homology-mediated repair pathway within the repair window. The rate constant, which reflects the efficiency with which the donor recombination template binds to the break site and participates in the repair reaction, is determined by the binding affinity between the single-stranded DNA binding domain and the terminal recognition sequence of the donor recombination template. This indicates the effective concentration of donor recombination template within the local region of the double-strand break site. This invention utilizes the physical anchoring effect mediated by the fusion protein to achieve... This is significantly improved compared to the case where the donor template is distributed in a free state. This represents the effective repair window duration that exists before cells choose between non-homologous end joining or homologous-mediated repair pathways. As can be seen from the model above, in... and Under relatively constant conditions, increasing the local donor concentration It can significantly improve This is precisely the core effect achieved by the donor spatiotemporal anchoring module of the present invention. The above model is used to illustrate the working mechanism of the technical solution of the present invention. In the specific embodiment of the present invention, PCR molecular detection has confirmed that the allele replacement at the target site was successful and the exogenous transgenic element was completely excised, which verifies the actual role of the enrichment effect described by the model in improving the repair efficiency.

[0022] III. The temporal logic of autophagy-based developmental regulatory circuits:

[0023] In the early dedifferentiation stage after the completion of exogenous DNA transformation, the early specific promoter is activated, driving the efficient expression of developmental regulatory genes and fusion proteins. The developmental regulatory genes enable recipient cells to overcome the regeneration barrier of the stubborn genotype. At the same time, the fusion protein completes genome editing and donor template anchoring, realizing homology-mediated repair. As the callus tissue further differentiates into microbuds and enters the organogenesis stage, the activity of the early specific promoter gradually shuts down. The late specific promoter is specifically activated in the newly formed shoot apical meristem, driving recombinase expression. The recombinase recognizes and binds to paired site-specific recombination recognition sites, mediating the precise excision and degradation of the self-cleaning cassette between the two sites (including the expression cassette of developmental regulatory genes, the expression cassette of fusion proteins, and the expression cassette of resistance marker genes) from the genome. Finally, the shoot apical meristem of the regenerated plant and all the tissues that develop thereafter no longer contain any exogenous transgenic elements, and only retains the excellent allele sequence integrated through homology-mediated repair at the original genome target site.

[0024] IV. Method Implementation Steps:

[0025] The first step involves constructing a multifunctional expression vector. The Cas9 protein-coding sequence and the single-stranded DNA binding domain-coding sequence are fused using a flexible linker peptide sequence to construct a fusion protein expression cassette. Downstream of this fusion protein expression cassette, an sgRNA expression cassette targeting the allele site to be replaced is linked to construct a donor recombination template carrying a superior allele sequence. Flanking sequences homologous to the genomic target site and recognition sequences specifically paired with the single-stranded DNA binding domain are introduced at both ends of this template. An early-specific promoter is linked to the coding sequence of a developmental regulatory gene to construct an early developmental regulatory gene expression cassette. A late-specific promoter is linked to the coding sequence of a recombinase to construct a late recombinase expression cassette. The developmental regulatory gene expression cassette, the fusion protein expression cassette, and the resistance marker gene expression cassette are placed between a pair of co-aligned site-specific recombination recognition sites. These functional units are then sequentially linked into a T-DNA backbone vector to obtain the multifunctional expression vector.

[0026] The second step is to transform the above-mentioned vector into embryogenic callus or immature embryos of crop recipient materials using Agrobacterium-mediated transformation.

[0027] In the third step, during the callus induction culture stage, the early specific promoter in the recipient cells is specifically activated by the early embryonic cells, driving the synchronous and efficient expression of developmental regulatory genes and fusion proteins. The developmental regulatory genes and proteins enable the originally difficult-to-regenerate stubborn genotype cells to break through the regeneration barrier and enter an active dedifferentiation and regeneration process.

[0028] In the fourth step, the fusion protein recognizes and binds to the genomic target site under the guidance of sgRNA. The Cas9 domain cuts the double-stranded DNA to produce a break. At the same time, the single-stranded DNA binding domain recognizes and binds to the recognition sequence at the end of the donor recombination template, physically anchoring the donor template near the break site, forming a local donor enrichment effect.

[0029] Fifth, during the repair window, the cells use the donor recombination template, which is physically anchored to the break site, as a template to complete homologous recombination repair through the homologous-mediated repair pathway. The superior allele sequence carried in the donor template is integrated into the genomic target site, replacing the original allele.

[0030] In the sixth step, as the callus tissue further differentiates to form microbuds, the recipient cells enter the shoot tip meristem formation stage. The activity of the early specific promoter gradually shuts down, while the late specific promoter is specifically activated in the newly formed shoot tip meristem, driving the expression of recombinase.

[0031] In the seventh step, the recombinase recognizes and binds to paired site-specific recombination recognition sites that encapsulate developmental regulatory gene expression frames, fusion protein expression frames, and resistance marker gene expression frames, mediating the precise excision of DNA fragments in this region from the genome, thereby achieving the self-clearance of foreign gene elements.

[0032] The eighth step involves molecular testing of the regenerated plants obtained from the seventh step, including PCR testing of allele replacement at the target site and whether the exogenous transgenic element has been completely removed. Plants containing only superior allele replacements and no exogenous transgenic residues are selected, and these plants are then subjected to routine field trait identification and progeny propagation. Specific Implementation

[0033] Taking the replacement of superior alleles related to agronomic traits in a recalcitrant maize inbred line as an example, the specific implementation of this invention is illustrated. When constructing the multifunctional expression vector, the single-stranded DNA-binding domain in the fusion protein expression cassette is selected from the HUH endonuclease domain, which is fused to the Cas9 protein coding region via a flexible linker peptide sequence. Downstream, an sgRNA expression cassette targeting the site to be replaced is linked. Flanking sequences homologous to the genomic target site and single-stranded DNA recognition sequences specifically binding to the HUH endonuclease domain are introduced at both ends of the donor recombinant template. The middle section carries the superior allele fragment to be replaced. The early-stage specific promoter is the maize early embryogenic callus-specific promoter ZmLBD1, which drives the developmental regulatory gene expression cassette formed by the fusion of growth regulator GRF4 and transcription co-activator GIF1. The late-stage specific promoter is the shoot apex meristem-specific promoter STM, which drives Cre recombinase expression. The resistance marker gene was selected from herbicide resistance screening marker genes. The expression frames of the developmental regulatory gene, fusion protein, and resistance marker gene were all wrapped by a pair of LoxP sites aligned in the same direction. The donor recombination template was located outside this wrapped region. The constructed vector was transformed into immature maize embryos by Agrobacterium-mediated transformation and cultured on callus induction medium. The ZmLBD1 promoter drove the synchronous expression of the developmental regulatory gene and fusion protein, which prompted the stubborn callus to break through the regeneration barrier and simultaneously complete the editing of the genome target site and homology-mediated repair. After the callus further differentiated into buds, the STM promoter was specifically activated and drove Cre recombinase expression. The self-clearing box was removed from the genome. The obtained regenerated plants were tested by PCR to confirm that the target site allele replacement was successful and that there were no transgenic residues of the developmental regulatory gene, fusion protein encoding gene, and resistance marker gene. After field phenotypic identification, it can be used for the selection of subsequent breeding materials.

[0034] VI. Alternative Implementation Methods:

[0035] In the donor spatiotemporal anchoring module, the HUH endonuclease domain can be replaced with either a phage single-stranded DNA binding protein-derived domain or a Diphtheria virus Rep protein-derived domain. By introducing the corresponding specific recognition sequence at the end of the donor template, the physical enrichment of the donor template to the break site can also be achieved, thereby improving the technical effect of homology-mediated repair efficiency.

[0036] In the autophagy-type developmental regulatory circuit, the combination of Cre and LoxP sites can be replaced with the combination of Flp recombinase and FRT sites. The late-stage specific promoter can still be the STM promoter or other shoot apical meristem-specific promoters, which can also achieve the technical effect of automatically activating recombinase and completing the self-clearance of exogenous gene elements during the bud differentiation stage.

[0037] The method described in this invention is applicable to various crops that require precise replacement of superior alleles, especially to stubborn commercial crop varieties with low tissue culture regeneration capacity. Those skilled in the art can make corresponding adjustments to the promoter, developmental regulatory genes, recombinase system and donor template sequence according to the specific crop type and target trait gene, all of which fall within the scope of protection claimed by this invention.

[0038] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make possible changes and modifications without departing from the spirit and scope of the present invention. Therefore, any modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for breeding superior crop varieties based on genetic engineering, characterized in that, Includes the following steps: A multifunctional expression vector is constructed, comprising: a fusion protein expression cassette formed by fusing a Cas9 protein-coding sequence and a single-stranded DNA-binding domain-coding sequence through a flexible linker peptide sequence; an sgRNA expression cassette linked to the fusion protein expression cassette and targeting the allele site to be replaced in the crop genome; the fusion protein expression cassette being driven by an early-specific promoter; a donor recombination template expression cassette, wherein the donor recombination template carries flanking sequences homologous to the allele site to be replaced in the genome and recognition sequences specifically paired with the single-stranded DNA-binding domain at both ends; a superior allele fragment to be replaced in the middle of the donor recombination template; a developmental regulatory gene expression cassette driven by an early-specific promoter; a recombinase expression cassette driven by a late-specific promoter; and an resistance marker gene expression cassette. The developmental regulatory gene expression cassette, the fusion protein expression cassette, and the resistance marker gene expression cassette are surrounded by a pair of site-specific recombination recognition sites aligned in the same direction, and the donor recombination template is located outside the region surrounded by the site-specific recombination recognition sites. The multifunctional expression vector was transformed into embryogenic callus of crop receptor material using Agrobacterium-mediated transformation. During the callus induction and culture stage, the early specific promoter is activated, driving the expression of the developmental regulatory gene expression cassette and the fusion protein expression cassette, thereby enhancing the regeneration capacity of the recipient cells. Since the fusion protein expression cassette and the developmental regulatory gene expression cassette are synchronously driven by the same early specific promoter, their expression intensity and timing are completely consistent, enabling the three functions of genome editing, donor template anchoring, and enhanced cell regeneration to occur synergistically within the same developmental window. The single-stranded DNA binding domain in the fusion protein binds to the recognition sequence at the end of the donor recombination template, anchoring the donor recombination template near the double-strand break site generated by the Cas9 protein in the fusion protein cleaving the genome. Through homology-mediated repair, the superior allele fragment is integrated into the genome at the allele site to be replaced, replacing the original allele. During the shoot apical meristem formation stage, the early-specific promoter activity is turned off, and the late-specific promoter is activated, driving the expression of the recombinase expression cassette. The recombinase recognizes and binds to the site-specific recombination recognition site, and removes the DNA fragment within the region enclosed by the site-specific recombination recognition site from the genome. Molecular testing was performed on the regenerated plants after excision to screen for plants containing the superior allele replacements and without exogenous transgenic elements.

2. The method for breeding improved crop varieties based on genetic engineering according to claim 1, characterized in that: The single-stranded DNA binding domain is the HUH endonuclease domain, and the recognition sequence at the end of the donor recombination template is a single-stranded DNA recognition sequence that specifically binds to the HUH endonuclease domain.

3. The method for breeding improved crop varieties based on genetic engineering according to claim 1, characterized in that: The single-stranded DNA binding domain is one of the following: a phage single-stranded DNA binding protein-derived domain or a Geminivirus Rep protein-derived domain.

4. The method for breeding improved crop varieties based on genetic engineering according to claim 1, characterized in that: The early-specific promoter is the ZmLBD1 promoter, a maize early embryogenic callus-specific promoter; the developmental regulatory gene is the GRF4-GIF1 developmental regulatory gene, formed by the fusion of growth regulator GRF4 and transcription coactivator GIF1; the late-specific promoter is the STM promoter, a shoot apex meristem-specific promoter; the recombinase is Cre recombinase; and the site-specific recombination recognition site is the LoxP site.

5. The method for breeding improved crop varieties based on genetic engineering according to claim 1, characterized in that: The recombinase is an Flp recombinase, and the site-specific recombination recognition site is the FRT site.

6. The method for breeding improved crop varieties based on genetic engineering according to claim 1, characterized in that: The molecular detection includes: detecting the allele replacement status of the genomic allele loci to be replaced by PCR, and detecting whether the developmental regulatory gene expression cassette, the fusion protein expression cassette, and the resistance marker gene expression cassette have been excised.

7. The method for breeding improved crop varieties based on genetic engineering according to claim 1, characterized in that: The crop recipient material is a recalcitrant genotype crop material.

8. A multifunctional expression vector for breeding improved crop varieties, characterized in that, This includes a T-DNA region, within which four expression units are arranged sequentially from left to right boundary: The first expression unit is a fusion protein expression box formed by fusing the Cas9 protein coding sequence and the single-stranded DNA binding domain coding sequence through a flexible linker peptide sequence, and its downstream is connected to an sgRNA expression box that targets the allele site to be replaced in the crop genome. The second expression unit is a donor recombination template expression frame. The donor recombination template carries flanking sequences homologous to the allele sites to be replaced in the genome at both ends, as well as recognition sequences specifically paired with the single-stranded DNA binding domain. The donor recombination template carries a superior allele fragment to be replaced in the middle. The third expression unit is the expression cassette of developmental regulatory genes driven by early-specific promoters; The fourth expression unit consists of a late-stage promoter-driven recombinase expression cassette and an antibiotic resistance marker gene expression cassette. The third expression unit, the first expression unit, and the resistance marker gene expression frame are enclosed by a pair of co-located site-specific recombination recognition sites, and the second expression unit is located outside the enclosed region of the site-specific recombination recognition sites.

9. A multifunctional expression vector for breeding improved crop varieties according to claim 8, characterized in that: The single-stranded DNA binding domain is the HUH endonuclease domain; the early-specific promoter is the maize early embryogenic callus-specific promoter ZmLBD1; the developmental regulatory gene is the GRF4-GIF1 developmental regulatory gene formed by the fusion of growth regulator GRF4 and transcription coactivator GIF1; the late-specific promoter is the shoot apex meristem-specific promoter STM; the recombinase is Cre recombinase; and the site-specific recombination recognition site is the LoxP site.

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