Construction method and application of a modular gene editing vector

Abstract

The application discloses a construction method and application of a modular gene editing vector. Specifically, a modular vector assembly system is developed based on a type II restriction enzyme mediated Golden Gate molecular cloning technology. Since the type II restriction enzyme has the characteristics of different recognition sites and cutting sites, different cohesive ends can be generated by using the same type II restriction enzyme, and seamless cloning can be realized by using a T4 ligase. The whole operation of the system only needs two enzymes AarI and BsmBI , simplifies the experimental process, reduces the cost, improves the stability of the whole experimental process, and retains an assembly site of BsmBI on the final vector bone, so that multiple elements can be expanded without reengineering assembly, and more assembly requirements can be flexibly realized. Therefore, the modular gene editing vector has a good application prospect in plant gene editing or breeding.

Description

Technical Field

[0001] This invention relates to the field of gene editing vector technology, specifically to a method for constructing and applying a modular gene editing vector. Background Technology

[0002] In recent years, with the rapid development of genome engineering technology, significant progress has been made in constructing and modifying the genomes of organisms. These technologies have enabled efficient and scalable experimental procedures. Among them, the Golden Gate assembly technology based on Type IIS restriction endonucleases, due to its separate recognition and cleavage sites, can release different adapters under the cleavage of the same enzyme to achieve sequential ligation of multiple fragments. This has led to the development of modular assembly systems based on Golden Gate technology. The mainstream assembly systems suitable for plants include the GoldenBraid system, the Voytas system, and optimized derivative systems based on these two systems.

[0003] The Voytas system is a three-level assembly system for building modular CRISPR vectors specifically designed for plants. It includes a universal library of monocotyledonous and dicotyledonous elements and a fully compatible vector backbone adapted for Agrobacterium-mediated transformation. However, when adding elements or changing the order of elements, this system requires DNA fragments re-ligated to the corresponding restriction sites via PCR. Since the three levels of assembly cannot be skipped, the operation is more cumbersome. Furthermore, this system has certain limitations; for example, once the previous level of vector construction is completed, the next level of elements cannot be added, and adding new elements can only begin from the initial module construction. The GoldenBraid system overcomes the limitations of Golden Gate's single-round assembly by using iterative binary assembly logic to achieve multi-fragment assembly and ligation. It uses a universal α / Ω sticky end rule, making the assembly process unrestricted by hierarchy. The GoldenBraid system assembles single expression cassettes first, then splices multiple single expression cassettes together to form multiple expression cassettes. It also allows for optimized design of universal adapters to enable cross-vector reuse of expression cassettes. However, this system requires strict attention to the design of sticky ends, and the complex design process is prone to errors. In plants, CRISPR-Cas-based gene editing systems have seen rapid development. However, these systems require optimization for different species to improve their editing efficiency in specific contexts, such as changing promoters, nuclear localization signals, and optimizing vector structures. Furthermore, CRISPR-Cas gene editing systems offer numerous extension tools, such as base editing systems fused with nCas9 (D10A) and deaminase, and guided editing systems fused with nCas9 (H840A) and reverse transcriptase RT. Therefore, the development of efficient CRISPR-Cas-based gene editing tools in soybeans requires extensive vector modification and testing. Consequently, there is an urgent need to find a method for constructing an efficient, universal, and flexible vector construction system. Summary of the Invention

[0004] The purpose of this invention is to provide a method for constructing and applying modular gene editing vectors. To this end, this invention constructs a three-level modular vector construction system. This system comprises three levels of modular vectors, allowing for the insertion of arbitrary elements in the primary vector, the replacement and assembly of arbitrary elements in the secondary vector assembly, and the arbitrary combination of secondary vectors in the final vector assembly. Furthermore, assembly sites are retained in the complete final vector to allow for flexible addition of elements after the final expression vector construction is completed, thus achieving the system's high efficiency, versatility, and flexibility.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] In a first aspect, the present invention provides a method for constructing a modular gene editing vector, comprising the following steps:

[0007] (1) Construction of primary module vector pOFC: The target DNA fragment is standardized by PCR amplification technology. Type IIs restriction endonuclease recognition and cleavage sequences are introduced into the 5' end of the amplification primer of the target DNA fragment. The standardized DNA fragment is introduced into the pOFC empty vector by Golden Gate reaction mediated by Type IIs restriction endonuclease digestion to obtain the primary module vector pOFC.

[0008] (2) Assembly of secondary functional module pMOD: Multiple primary module vectors pOFC are digested with Type IIs restriction endonucleases to generate specific matching sticky ends. The empty pMOD vector is also digested with Type IIs restriction endonucleases. Then, the two modules are assembled in a directional and seamless manner through the Golden Gate reaction to obtain secondary functional module pMOD, which contains at least a Cas9 expression module, an sgRNA expression module, and a plant stable transformation screening resistance gene expression module.

[0009] (3) Construction of the final editing vector: Multiple secondary functional modules pMOD were digested with Type IIs restriction endonucleases, and the empty pTrans vector was also digested with Type IIs restriction endonucleases. Then, under the mediation of T4 DNA ligase, the digested secondary functional modules were directionally ligated with the pTrans vector in a Golden Gate reaction mediated by the same Type IIs restriction endonuclease to obtain the final gene editing expression vector pTrans-CRISPR.

[0010] Furthermore, the Type IIs restriction endonucleases include AarI and BsmBI enzymes.

[0011] Furthermore, after the target DNA fragment is standardized in step (1), it can be digested with AarI enzyme to produce the same sticky ends; the same sticky ends are CTCC at the 5' end and AGAG at the 3' end.

[0012] Furthermore, the sequence of the pOFC empty vector in step (1) is shown in SEQ ID NO: 1.

[0013] Furthermore, in step (1), at least three primary module carriers pOFC are constructed.

[0014] Furthermore, in step (2), during the assembly of the secondary functional module pMOD, the sticky end sequences generated by the adjacent primary module vector pOFC after BsmBI digestion are complementary and matched. The 3' sticky end sequence of the DNA fragment released by the previous primary module is consistent with the 5' sticky end sequence of the DNA fragment released by the next primary module, thereby achieving seamless assembly of multiple DNA fragments in a preset order.

[0015] Furthermore, in step (3), there are at least three secondary functional modules pMOD.

[0016] Furthermore, the pTrans-CRISPR gene editing expression vector obtained in step (3) can be expanded. Specifically, the pTrans-CRISPR vector is digested with BsmBI, and the primary module vector pOFC is digested with BsmBI to release the target DNA fragment. In the BsmBI-mediated Golden Gate reaction system, the target DNA fragment and the digested pTrans-CRISPR vector are sequentially and directionally assembled in a preset order under the action of T4 DNA ligase through complementary and matched sticky ends, thereby completing the final expansion of the pTrans-CRISPR vector.

[0017] Secondly, the present invention provides a modular gene editing vector prepared by the above-described construction method.

[0018] Thirdly, the present invention provides the application of the above-mentioned modular gene editing vector in plant gene editing or breeding.

[0019] The principle of this modular carrier assembly system is as follows:

[0020] The gene editing system is broken down into multiple primary modules, each of which can be assembled into larger secondary modules as needed. These secondary modules can then be further assembled into the final gene editing vector. Therefore, the system constructed in this invention can achieve systematic modification of any element or structure on the vector using only Golden Gate molecular cloning technology.

[0021] The advantages of this invention are:

[0022] Developing an efficient vector modification system is a prerequisite for developing efficient gene editing tools in soybeans. This invention first develops a modular vector assembly system based on Golden Gate molecular cloning technology mediated by type II restriction endonucleases. Because type II restriction enzymes have different recognition and cleavage sites, the same type II restriction endonuclease can generate different sticky ends, which can then be ligated using T4 ligase to achieve seamless cloning. This system requires only two enzymes, AarI and BsmBI, simplifying the experimental procedure, reducing costs, and improving the stability of the entire experimental process. This is a highly efficient one-step cloning technique. It utilizes type IIs restriction endonucleases to generate unique sticky ends after DNA cleavage. These ends act like keys and locks, pairing only with specific modules, ensuring that multiple fragments can be seamlessly spliced ​​in the correct order within a single reaction, greatly improving the efficiency and accuracy of vector construction. Furthermore, a BsmBI assembly site is retained on the final vector backbone, allowing for the expansion of multiple elements without de novo modification and assembly, flexibly meeting various assembly needs. Attached Figure Description

[0023] Figure 1 Design of a modular assembly system for soybean gene editing vectors.

[0024] Figure 2 This is a schematic diagram illustrating the design of the primary module and the standardization of DNA fragments.

[0025] Figure 3 This is a schematic diagram of the carrier structure of the secondary module.

[0026] Figure 4 A schematic diagram of assembling multiple primary carriers for a secondary carrier.

[0027] Figure 5 A schematic diagram of the assembly of the ultimate gene editing expression vector.

[0028] Figure 6 This is a schematic diagram illustrating the design of the component expansion module and the standardization of DNA fragments.

[0029] Figure 7 A schematic diagram of assembling multiple primary carriers for a component expansion module.

[0030] Figure 8 This is a schematic diagram of the assembly and construction of a multi-gene knockout vector.

[0031] Figure 9 The genotype of the homozygous multi-gene edited plant was determined. Detailed Implementation

[0032] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all reagents and materials used in the following examples are commercially available products.

[0034] CRISPR-Cas gene editing vectors in plants typically contain three core functional modules: a Cas9 expression module, an sgRNA expression module, and a resistance gene expression module used for stable transformation selection. To achieve efficient and flexible vector construction, this invention designs a modular gene editing vector assembly system (pTrans-CRISPR system) based on Golden Gate cloning technology, the overall design of which is as follows: Figure 1 As shown.

[0035] This invention uses two basic gene knockout vectors, dual-transcript and single-transcript, to divide the entire T-DNA region into nine primary modules (pOFC, plasmid of Original Fragment with Chloramphenicol). The functions of each module are defined as follows:

[0036] (1) pOFC-1: The promoter module that drives Cas9 expression. The promoters that can be selected include, but are not limited to, the constitutive promoters pM4, pYAO, pGmUBi3, pGmPRS5a, etc.

[0037] (2) pOFC-2: nuclear localization signaling module at the N-terminus of the Cas9 protein;

[0038] (3) pOFC-3: Cas9 expression module, which can be flexibly replaced according to the editing type.

[0039] a) Gene knockout: Cas9 coding sequence (such as SpCas9, SaCas9);

[0040] b) CBE base editing: a fusion protein of nCas9 (D10A) and deaminase;

[0041] c) Guided editing: a fusion protein of nCas9 (H840A) and reverse transcriptase RT;

[0042] (4) pOFC-4: In dual transcript vectors, it is the C-terminal nuclear localization signal and terminator module of Cas9 protein; in single transcript vectors, it is the C-terminal nuclear localization signal, poly-A and tRNA module of Cas9 protein.

[0043] (5) pOFC-5: in the dual transcript vector, it is the sgRNA promoter and ccdB expression module; in the single transcript, it is the ccdB expression module.

[0044] (6) pOFC-6: In dual transcripts, it consists of a scaffold structure and a terminator module; in single transcripts, it consists of a scaffold structure, a tRNA sequence, and a terminator module.

[0045] (7) pOFC-7: the promoter module of the resistance gene, which can be driven by a selectable promoter (such as p35S, pGmUBi3) to express the selection marker;

[0046] (8) pOFK-8: Resistance gene expression module, which can be herbicide resistance genes (such as Bar, Pat), antibiotic resistance genes (such as Hyg, Kan) or visual marker genes;

[0047] (9) pOFK-9: Terminate submodule.

[0048] The aforementioned nine primary modules can be further assembled into three secondary modules (pMODs, plasmid of modules) via the Golden Gate. pOFC-1 / 2 / 3 can be assembled into the Cas9 expression module pMOD-A; pOFC-4 / 5 / 6 can be assembled into the sgRNA expression module pMOD-B, which can be flexibly configured according to dual / single transcript requirements; and pOFC-7 / 8 / 9 can be assembled into the resistance gene expression module pMOD-C. Finally, the three secondary modules pMOD-A / B / C can be assembled into the final gene knockout vector pTrans-CRISPR (plasmid of transformation) through another round of Golden Gate reaction. This system, through standardized adapter design, achieves seamless, targeted splicing between modules, supports the rapid construction of various gene editing vectors, and can be extended to various editing types.

[0049] The modular gene editing vector assembly system described in this invention comprises components (including but not limited to various promoters, Cas9 and its derived editing enzymes, nuclear localization signals, terminators, sgRNA / pegRNA scaffolds, tRNA sequences, selection marker genes, etc.) that are all known functional elements in the art, and their nucleotide sequences and biological functions are well-known to those skilled in the art. This invention, for the first time, standardizes, modularizes, and recombines these known functional elements, establishing an efficient, flexible, and scalable vector assembly system through the design of a unified Golden Gate cloning adapter. Once those skilled in the art understand the modular design concept and assembly method of this invention, they can select suitable components known in the art according to actual needs, and rapidly construct gene editing vectors suitable for different plant species and different editing types (gene knockout, CBE / ABE base editing, guided editing, etc.) according to the Golden Gate reaction conditions and assembly process disclosed in this invention. The construction process of this system will be further elaborated in the following examples.

[0050] The sequences of the pOFC vector, pMOD-A vector, pMOD-B vector and pMOD-C vector involved in the following embodiments are shown in SEQ ID NO: 1~4.

[0051] Example 1: Design of the primary module pOFC and standardization of DNA fragments

[0052] The primary module was designed using AarI-mediated Golden Gate to construct the vector. First, DNA fragment normalization was performed via PCR amplification. Figure 2 a) This means that the primer design needs to add an AarI recognition sequence (red part in the figure) and a specific cutting sequence (black part in the figure) to its 5' end. Therefore, different DNA fragments can produce the same sticky ends ("CTCC" at the 5' end and "AGAG" at the 3' end) after PCR amplification and AarI digestion. Figure 2 As shown in b, the pOFC empty vector (sequence shown in SEQ ID NO: 1) has two AarI restriction sites (red parts in the figure) opposite to the DNA fragment cleavage direction, but also has the same cleavage site (black parts in the figure). Therefore, the substitution between the ccdB fragment and the target gene fragment on the pOFC empty vector can be completed through the AarI-mediated Golden Gate reaction. The constructed vector structure is shown in Figure 1. Figure 2As shown in c. The vector structure after replacement does not contain an AarI recognition site. Furthermore, because the pOFC empty vector has two BsmBI restriction sites (blue portion in the figure), the constructed vector can be cleaved by BsmBI and produce specific sticky ends (the 5' end is the first four bases of the DNA fragment, and the 3' end is the last four bases of the DNA fragment), as shown in Figure c. Figure 2 As shown in the black box in c.

[0053] Those skilled in the art can flexibly select appropriate target DNA fragments and corresponding amplification primers according to actual experimental needs. As long as the standardized design logic described above is followed, standardized processing of DNA fragments can be achieved. Through the above standardized design, DNA fragments from different sources and with different functions can be cloned into the pOFC empty vector using the same enzyme digestion and ligation conditions, achieving a unified and efficient fragment insertion process. Simultaneously, the design of amplification primers during DNA fragment standardization must ensure that two adjacent DNA fragments generate the same sticky ends in the BsmBI-mediated Golden Gate reaction, so that multiple fragments of the secondary module can be assembled in a preset order.

[0054] Example 2: Design of the secondary module pMOD and assembly of multiple primary modules

[0055] The design of the second-level module is as follows Figure 3 As shown, there are three vectors: pMOD-A (sequence shown in SEQ ID NO: 2), pMOD-B (sequence shown in SEQ ID NO: 3), and pMOD-C (sequence shown in SEQ ID NO: 4). All three vectors contain the ccdB suicide gene, two BsmBI restriction sites, two AarI restriction sites, and bleomycin resistance (Zeocin, Zeo + The adjacent BsmBI and AarI cleavage sequences are identical; for example, the 5' end of the ccdB sequence in the pMOD-A vector can produce a 5'-GATC-3' sticky end with both BsmBI and AarI digestion. Another feature of this vector design is that the second cleavage sequence in pMOD-A and one cleavage sequence in pMOD-B are both 5'-GATC-3' (red part in the figure), while the second cleavage sequence in pMOD-B and the first cleavage sequence in pMOD-C are both 5'-CCGG-3' (green part in the figure). The purpose of this design is to allow for the sequential splicing of the three vectors pMOD-A / B / C during the final expression vector assembly.

[0056] The construction of secondary modules enables the sequential assembly of multiple DNA fragments located on the primary vector. Taking the secondary vector pMOD-A as an example, the assembly of the two DNA fragments is performed as follows: Figure 4As shown in Figure a, under the action of BsmBI enzyme digestion, the two primary vectors pOFC release DNA-A and DNA-B fragments from the pOFC vector, and four sticky ends are generated at both ends of the DNA fragments. Figure 4 (As shown in the black box in section a). The sticky end sequence generated at the 5' end of DNA-A is 5'-GATC-3'; the sticky end sequence generated at the 3' end of DNA-A is the same as that generated at the 5' end of DNA-B, both being 5'-AGGC-3'; the sticky end sequence generated at the 3' end of DNA-B is 5'-GGAC-3'.

[0057] The secondary module pMOD-A, without any additional BsmBI enzymes, releases the ccdB fragment upon digestion with directions opposite to those of the primary module pOFC. This releases 5'-GATC-3' and 5'-GGAC-3' sticky ends, respectively, onto the vector backbone. Figure 4 (b) Therefore, under the BsmBI-mediated Golden Gate action, the DNA-A and DNA-B fragments released by the two primary modules can assemble with the pMOD-A vector backbone, and the assembled vector structure is as follows: Figure 4 As shown in c, DNA-A and DNA-B can be seamlessly assembled without introducing additional sequences. This vector no longer contains the BsmBI recognition site, while AarI can still cleave the same sequence for final expression vector assembly. The assembly process for secondary vectors pMOD-B and pMOD-C is the same as for pMOD-A.

[0058] The assembly of secondary modules is not limited to the assembly of two DNA fragments; multiple DNA fragments can also be assembled as needed without introducing additional sequences, achieving seamless assembly of multiple DNA fragments.

[0059] Example 3 Assembly of the final expression vector pTrans-CRISPR

[0060] The final step in the modular vector assembly system is the assembly of the final expression vector, which requires the simultaneous assembly of three secondary vectors: pMOD-A, B, and C. For example... Figure 5 As shown in a, pMOD-A / B / C completes the pairwise assembly of 6 DNA fragments, and can release 3 fragments under AarI restriction enzyme digestion. The tertiary module pTrans vector (sequence shown in SEQ ID NO: 5) carries two AarI restriction sites in the opposite direction to the secondary module pMOD. Figure 5(b) Similar to the principle of secondary vector construction, in the AarI-mediated Golden Gate reaction system, the three DNA fragments released by pMOD-A, pMOD-B, and pMOD-C can generate sticky ends with the pTrans vector backbone, and are assembled sequentially under the action of T4 ligase, thus completing the construction of the final gene editing expression vector (pTrans-CRISPR). Since the gene editing expression vector requires sgRNA construction, to improve its construction efficiency, the final assembled vector carries the ccdB sequence, and the tertiary module empty vector also carries ccdB. Therefore, to improve the modular assembly efficiency of the final vector, the ccdB gene carried in the DNA fragment is additionally fused with a chloramphenicol resistance expression gene, resulting in the final assembled expression vector exhibiting dual resistance to kanamycin and chloramphenicol. During the E. coli DB3.1 transformation process, dual-antibody selection is used to avoid false positives caused by the pTrans empty vector.

[0061] Example 4: Element expansion on the final expression vector pTrans-CRISPR

[0062] Element expansion in pTrans-CRISPR requires two steps: primary vector construction and final expression vector expansion. The first step, designing the primary module (using the primary module backbone), involves constructing the vector using AarI-mediated Golden Gate. First, DNA fragment normalization is performed via PCR amplification. Figure 6 a) Primer design requires the addition of an AarI recognition sequence (red portion in the diagram) and a specific cleavage sequence (black portion in the diagram) to the 5' end. Therefore, different DNA fragments can be amplified by PCR and then digested with AarI to produce the same sticky ends (5' end "CTCC", 3' end "AGAG"). Figure 6 As shown in b, the pOFC empty vector has two AarI restriction sites (red parts in the figure) opposite to the DNA fragment cleavage direction, but also has the same cleavage site (black parts in the figure). Therefore, the substitution between the ccdB fragment and the target gene fragment on the pOFC empty vector can be completed through the AarI-mediated Golden Gate reaction. The constructed vector structure is shown in Figure 1. Figure 6 As shown in c. The vector structure after replacement does not contain an AarI recognition site. Furthermore, because the pOFC empty vector has two BsmBI restriction sites (blue portion in the figure), the constructed vector can be cleaved by BsmBI and produce specific sticky ends (the 5' end is the first four bases of the DNA fragment, and the 3' end is the last four bases of the DNA fragment), as shown in Figure c. Figure 6 The box in c is shown.

[0063] The construction of the primary module can sequentially standardize all fragments of the gene editing system. Simultaneously, the design of amplification primers during DNA fragment standardization must ensure that two adjacent DNA fragments produce identical sticky ends in the BsmBI-mediated Golden Gate reaction, so that multiple fragments can be assembled in a predetermined order during the final expression vector expansion.

[0064] The second step involves element expansion in the final expression vector. The pTrans-CRISPR vector retains the BsmBI restriction site and is simultaneously assembled with the primary vector constructed in the first step. For example... Figure 7 As shown, the primary module releases two fragments upon BsmBI digestion, while the pTrans-CRISPR vector carries two BsmBI restriction sites in the opposite direction to the primary module's digestion. Similar to the principle of secondary vector construction, in a BsmBI-mediated Golden Gate reaction system, the two DNA fragments released from the primary module and the pTrans-CRISPR vector generate identical sticky ends and assemble sequentially under the action of T4 ligase, thus completing the final element expansion of the pTrans-CRISPR vector.

[0065] In summary, the modular assembly system designed in this invention is divided into three levels of module assembly ( Figure 1 The process begins with the standardization of DNA fragments and the construction of primary modules, which relies on an AarI-mediated Golden Gate reaction. The pOFC vector is chloramphenicol resistant. Next, multiple primary modules are sequentially assembled into secondary modules pMOD-A / B / C, relying on a BsmbI-mediated Golden Gate reaction. The pMOD-A / C vectors are bleomycin resistant, while pMOD-B, carrying ccdB, is bleomycin and chloramphenicol resistant. Finally, the three secondary modules pMOD-A, pMOD-B, and pMOD-C are assembled into the final expression vector after an AarI-mediated Golden Gate reaction. The empty pTrans vector is kanamycin resistant, and the assembled gene-editing vector is both kanamycin and chloramphenicol resistant. This modular assembly system can complete the construction of vectors of any structure or the replacement of any element on the vector within 8-10 days. Furthermore, to allow for more flexible expansion of additional components, we have reserved a BsmBI restriction site on the pTrans-CRISPR expression vector, which allows for the addition of more components through the primary module.

[0066] Example 5: Creating hypoallergenic soybeans by knocking out two soybean allergen targets based on this modular carrier assembly system.

[0067] In this embodiment, a total of nine primary vectors, three secondary vectors, and finally, pTrans-CRISPR expression vectors need to be constructed. For example... Figure 8 As shown in (a), normalized target fragments were amplified from the corresponding templates and ligated to the pOFC empty vector via an AarI-mediated Golden Gate reaction; the corresponding primary vectors were constructed onto the corresponding secondary empty vectors via a BsmBI-mediated Golden Gate reaction, namely pMOD-A1 / B1 / C1. Figure 8 As shown in (b), the elements from the three secondary vectors were assembled onto the pTrans empty vector via an AarI-mediated Golden Gate reaction, achieving the complete assembly of the pTrans-CRISPR expression vector, named pTrans-CRISPR-1. Figure 8 As shown in (c), the two target sgRNAs were finally linked to pTrans-CRISPR-1 to target the two sensitizing genes, soybean Gly mBd 30K and Gly mBd 28K (NCBI Locus: FJ616287.1, EU493461.1), respectively.

[0068] The specific experimental steps are as follows:

[0069] The template required for this embodiment is pGES501, and the primers were all synthesized by Beijing Qingke Biotechnology Co., Ltd. The primers corresponding to each vector are shown in Table 1.

[0070] Table 1 Primer list for Example 5

[0071]

[0072] 1. Primary Carrier Construction Process

[0073] 1.1 Fragment amplification reaction system and amplification system

[0074] Table 2 Fragment amplification reaction system

[0075]

[0076] Table 3 Fragment amplification reaction procedure

[0077]

[0078] After the amplification reaction was completed, the correct target band was identified by gel electrophoresis, and the concentration of the product was measured after gel extraction and recovery.

[0079] 1.2 Construction of the reaction system and procedure using the primary carrier Golden Gate

[0080] Nine primary vectors, namely pOFC-A1~3, pOFC-B1~3, and pOFC-C1~3, were constructed respectively. The system and procedure are as follows:

[0081] Table 4. Reaction system constructed using the primary support Golden Gate

[0082]

[0083] Table 5. Reaction Procedure for the Construction of the Primary Carrier Golden Gate

[0084]

[0085] All nine vectors were transformed into DH5α, and chloramphenicol was selected as the antibiotic. Finally, Sanger sequencing was used to confirm the correctness of the vectors.

[0086] 1.3 Construction of the reaction system and procedure using the secondary carrier Golden Gate

[0087] Three vectors, pMOD-A1, pMOD-B1, and pMOD-C1, were constructed respectively. The architecture and procedures are as follows:

[0088] Table 6. Reaction system for pMOD-A1Golden Gate construction

[0089]

[0090] Table 7. Reaction system for pMOD-B1 Golden Gate construction

[0091]

[0092] Table 8. Construction reaction system of pMOD-C1 Golden Gate

[0093]

[0094] Table 9 Reaction Procedure for the Construction of the Secondary Carrier Golden Gate

[0095]

[0096] Of the three vectors, pMOD-A1 and pMOD-C1 were transformed into DH5α, and bleomycin was used for antibiotic selection. pMOD-B1 was transformed into DB3.1, and bleomycin and chloramphenicol were used for antibiotic selection. Finally, Sanger sequencing was used to confirm the correctness of the vectors.

[0097] 1.4 Construction reaction system and procedure for the pTrans-CRISPR-1 final expression vector at the Golden Gate

[0098] Table 10. Construction reaction system of pTrans-CRISPR-1 Golden Gate

[0099]

[0100] Table 11. Construction reaction procedure for pTrans-CRISPR-1 Golden Gate

[0101]

[0102] pTrans-CRISPR-1 was transformed into DB3.1, and kanamycin and chloramphenicol were used for antibiotic selection. Finally, Sanger sequencing was used to confirm the correctness of the vector.

[0103] 1.5 Construction of Gly mBd 30K and Gly mBd 28K multi-knockout vectors

[0104] 1.5.1 Amplification of sgRNA Insert Fragment

[0105] Based on the primer table in Table 1 above, three sgRNA insert fragments were amplified and named as fragments 1-3.

[0106] The amplification reaction system and amplification procedure are as follows:

[0107] Table 12 Fragment amplification reaction system

[0108]

[0109] Table 13 Fragment Amplification Reaction Procedure

[0110]

[0111] After the amplification reaction was completed, the correct target band was identified by gel electrophoresis, and the concentration of the product was measured after gel extraction and recovery.

[0112] 1.5.2 Construction of Multiple Editing Carriers

[0113] The Golden Gate reaction system and amplification procedure are as follows:

[0114] Table 14. Construction reaction system of Golden Gate with multiple editing vectors

[0115]

[0116] Table 15. Construction Reaction Procedure for Golden Gate with Multiple Editable Vectors

[0117]

[0118] The vector was transformed into DH5α, and kanamycin was selected as the antibiotic. Sanger sequencing was then used to confirm the correctness of the vector. The correct vector was then transformed into Agrobacterium GV3101.

[0119] Subsequently, we obtained positive soybean plants with integrated core editing elements using stable soybean transformation technology. Sanger sequencing was used to identify the edited transformants, and after multiple generations of selection, we obtained homozygous Gly mBd 30K and Gly mBd 28K knockout soybean plants, as well as soybean plants without T-DNA insertion. Genotypes are as follows: Figure 9 As shown, this modular vector assembly system successfully knocked out two allergenic genes in soybean and created new low-allergenic soybean germplasm.

[0120] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a modular gene editing vector, characterized in that: Includes the following steps: (1) Construction of primary module vector pOFC: The target DNA fragment is standardized by PCR amplification technology. Type IIs restriction endonuclease recognition and cleavage sequences are introduced into the 5' end of the amplification primer of the target DNA fragment. The standardized DNA fragment is introduced into the pOFC empty vector by Golden Gate reaction mediated by Type IIs restriction endonuclease digestion to obtain the primary module vector pOFC. (2) Assembly of secondary functional module pMOD: Multiple primary module vectors pOFC are digested with Type IIs restriction endonucleases to generate specific matching sticky ends. The empty pMOD vector is also digested with the same Type IIs restriction endonuclease. Then, the two modules are assembled in a directional and seamless manner through the Golden Gate reaction to obtain secondary functional module pMOD, which contains at least a Cas9 expression module, an sgRNA expression module, and a plant stable transformation screening resistance gene expression module. (3) Construction of the final editing vector: Multiple secondary functional modules pMOD were digested with Type IIs restriction endonucleases, and at the same time, the... pTrans The empty vector was digested with a Type IIs restriction endonuclease, followed by a Golden Gate reaction mediated by the same Type IIs restriction endonuclease under the guidance of T4 DNA ligase, to connect the digested secondary functional modules with... pTrans The vector is directionally ligated and assembled to obtain the final gene editing expression vector pTrans-CRISPR.

2. The construction method according to claim 1, characterized in that: The Type IIs restriction endonucleases include AarI enzymes and BsmBI Enzymes.

3. The construction method according to claim 1, characterized in that: After standardization of the target DNA fragment in step (1), it is then passed through... AarI Enzymatic digestion produces identical sticky ends; the identical sticky ends are CTCC at the 5' end and AGAG at the 3' end.

4. The construction method according to claim 1, characterized in that: The sequence of the pOFC empty vector in step (1) is shown in SEQ ID NO:

1.

5. The construction method according to claim 1, characterized in that: In step (1), at least three primary module carriers pOFC are constructed.

6. The construction method according to claim 1, characterized in that: In step (2), during the assembly of the secondary functional module pMOD, the adjacent primary module carrier pOFC undergoes... BsmBI The sticky end sequences generated after enzyme digestion are complementary and matched. The 3' sticky end sequence of the DNA fragment released by the first primary module is consistent with the 5' sticky end sequence of the DNA fragment released by the second primary module, thereby achieving seamless assembly of multiple DNA fragments in a preset order.

7. The construction method according to claim 1, characterized in that: In step (3), there are at least 3 secondary functional modules pMOD.

8. The construction method according to claim 1, characterized in that: Element expansion can also be performed on the gene editing expression vector pTrans-CRISPR obtained in step (3), specifically by utilizing... BsmBI The pTrans-CRISPR vector was digested with enzymes, and the primary module vector pOFC was simultaneously... BsmBI Enzyme digestion releases the target DNA fragment, in BsmBI In the Golden Gate mediated reaction system, the target DNA fragment and the enzyme-digested pTrans-CRISPR vector are sequentially and directionally assembled in a preset order under the action of T4 DNA ligase through complementary and matched sticky ends, thereby completing the element expansion of the final pTrans-CRISPR vector.

9. The modular gene editing vector prepared by the construction method according to any one of claims 1 to 8.

10. The application of the modular gene editing vector as described in claim 9 in plant gene editing or breeding.

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