Method for realizing large-fragment DNA (Deoxyribose Nucleic Acid) insertion by utilizing IVC (Intravariant Vitamin C) donor system with shortened homologous arm in Amplipleta parula

Abstract

The invention discloses a method for realizing large-fragment DNA (Deoxyribose Nucleic Acid) insertion by utilizing an IVC (Intravariant Vitamin C) donor system with a shortened homologous arm in Amplistrum canescens. Aiming at the problem of high cloning complexity caused by dependence of an existing IVC donor system on a long homologous arm (about 1kb), the invention initiatively proposes that the homologous arm is shortened to 250-510bp, and the insertion efficiency of an IVC ring donor and a linearized donor after the homologous arm is shortened is compared and evaluated for the first time, and the result shows that the insertion efficiency of the IVC ring donor is obviously higher than that of the linearized donor, and the insertion efficiency of the linearized donor is obviously higher than that of the linearized donor. And it is proved that the IVC ring donor with the shortened homologous arm can still keep about 50% of insertion efficiency. According to the method, the cloning difficulty is remarkably reduced while the relatively high insertion efficiency is maintained, the technical bottleneck that long homologous arms are needed for large-fragment DNA insertion is successfully broken through, and an efficient tool is provided for plant synthetic biology and crop improvement.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for inserting large DNA fragments into *Sclerotium spp.* using an IVC donor system with shortened homologous arms. Background Technology

[0002] CRISPR / Cas9 technology has been widely used in genome editing of various plants, enabling precise insertion of target genes. However, as the length of the DNA fragment to be inserted increases, the insertion efficiency decreases significantly. This phenomenon is particularly prominent in plant genome modification, severely limiting the application of inserting large DNA fragments.

[0003] Due to the ease of protoplast isolation and the high biological activity and regeneration capacity of its protoplasts, *Sphaerocarpus spp.* is widely considered an important model organism in plant biology, particularly in basic plant science and evolutionary biology research. Currently, the transformation system for *Sphaerocarpus spp.* is relatively mature, typically employing a polyethylene glycol (PEG)-mediated method to introduce target DNA into protoplasts, followed by culture and resistance selection to obtain modified plants. In this system, by providing sgRNA, Cas9, and a donor template containing homologous arms, the homology-directed repair (HDR) mechanism can be used to achieve precise insertion of the target sequence.

[0004] However, in *Moss simulans*, the insertion efficiency of large fragments decreases significantly with increasing fragment length. Existing in vivo IVC donor technology has improved insertion efficiency (Reference: Gu X, Lang J, Chang Y, Zhang M. (2025) Cleavable donor-assisted CRISPR / Cas9 system significantly improves the efficiency of large DNA insertion in...). Physcomitrium patens (The Plant Journal, 121(4):e70020.), but it relies on relatively long homologous arms (usually about 1kb). Such long homologous arms not only increase the workload and difficulty of molecular cloning during vector construction, but also easily introduce point mutations during PCR amplification, affecting the accuracy and stability of subsequent applications. Therefore, how to effectively shorten the length of homologous arms while maintaining high insertion efficiency has become a key problem that urgently needs to be solved in this field, and it is also the core bottleneck for promoting the further development of IVC donor technology.

[0005] Furthermore, the efficiency of directly using linearized donor templates for CRISPR / Cas9 targeted insertion is unknown, and the in vitro preparation of linearized donor templates presents numerous inconveniences and uncertainties. Currently, common in vitro DNA template linearization methods mainly fall into two categories: one is donor linearization via enzymatic digestion, relying on specific restriction endonucleases. While this method can yield linearized donors with high integrity, it places high demands on enzyme types, reaction systems, and purification steps, resulting in high economic costs. The other method is obtaining linearized donors through PCR amplification. However, in large-fragment PCR reactions, high-fidelity polymerases are easily affected by factors such as template structure and GC content, readily introducing point mutations or small fragment misassemblies, thus affecting the accuracy and stability of subsequent homologous recombination repair events. In summary, traditional in vitro linearized donors have shortcomings in both efficiency and reliability, suggesting the need to develop more efficient, stable, and lower-cost donor preparation methods to improve the overall efficiency of homologous recombination editing in the *Sphaerocera minor* genome. Summary of the Invention

[0006] The technical problem this invention aims to solve is how to develop more efficient, stable, and lower-cost donor preparation methods to improve the overall efficiency of homologous recombination editing of the genotype of *Sphagnum moss*.

[0007] To address the aforementioned technical problems, this invention first provides a method for inserting large DNA fragments into *Moss styracifolium*.

[0008] The method for achieving large DNA fragment insertion in *Stylos spp.* provided by this invention includes the following steps: introducing a CRISPR / Cas9 editing vector and an IVC donor plasmid into *Stylos spp.*; The CRISPR / Cas9 editing vector expresses Cas9 nuclease and sgRNA; The IVC donor plasmid contains a donor fragment, which sequentially includes the sgRNA target sequence, a left homologous arm sequence, a large DNA fragment to be inserted, a right homologous arm sequence, and the reverse complementary sequence of the sgRNA target sequence; the sgRNA targets the sgRNA target sequence in the donor fragment. The size of the left homologous arm sequence and the right homologous arm sequence does not exceed 510 bp.

[0009] In the above method, the size of the left homologous arm sequence and the right homologous arm sequence can be 250-510 bp.

[0010] In some implementations, the left homologous arm sequence and the right homologous arm sequence are 306 bp and 250 bp in size, respectively.

[0011] In some implementations, the left homologous arm sequence and the right homologous arm sequence are 507 bp and 500 bp in size, respectively.

[0012] In the above method, the size of the large DNA fragment to be inserted can be 7000-11000 bp.

[0013] In some implementations, the size of the large DNA fragment to be inserted is 7338 bp.

[0014] In the above method, the large DNA fragment to be inserted can be any sequence.

[0015] In some embodiments, the large DNA fragment to be inserted contains an FtsZ1-TurboID-YFP expression cassette.

[0016] The FtsZ1-TurboID-YFP expression cassette includes, in sequence, the following: LexAop: m35S The promoter, FtsZ1 encoded sequence, TurboID encoded sequence, YFP encoded sequence, and Tpea3A terminator.

[0017] The LexAop: m35S The promoter sequence is shown in positions 2499-2849 of sequence 1.

[0018] The FtsZ1 encoding sequence is shown as bits 2860-4188 of sequence 1.

[0019] The TurboID encoding sequence is shown as bits 4219-5175 of sequence 1.

[0020] The YFP encoding sequence is shown as bits 5194-5910 of sequence 1.

[0021] The Tpea3A termination subsequence is shown in positions 5953-6424 of sequence 1.

[0022] In some implementations, the large DNA fragment to be inserted also contains an XVE expression cassette.

[0023] The XVE expression box includes, in sequence, a ProGX8 promoter, an XVE coding sequence, and a TrbcS terminator.

[0024] The ProGX8 starter sequence is shown as positions 6425-7941 of sequence 1.

[0025] The XVE encoding sequence is shown as bits 7951-9402 of sequence 1.

[0026] The TrbcS termination subsequence is as shown in positions 9542-9836 of sequence 1.

[0027] In the above method, the molar ratio of the CRISPR / Cas9 editing vector to the IVC donor plasmid is 1:1.

[0028] In some implementations, the CRISPR / Cas9 editing vector is the pHyg-Cas9-sgRNA2 vector (see reference: Gu X, Lang J, Chang Y, Zhang M. (2025) Cleavable donor-assisted CRISPR / Cas9 system significantly improves the efficiency of large DNA insertion). Physcomitrium patens. The Plant Journal, 121(4):e70020.). The target sequence of sgRNA2 expressed by the pHyg-Cas9-sgRNA2 vector is as follows: 5'-GAGGACGGTTCGGCCGACATGGG-3'.

[0029] In some embodiments, the IVC donor plasmid is the p2xsgRNA2-500AR-EI-FtsZ1-TurboID-YFP-HA vector. The nucleotide sequence of the p2xsgRNA2-500AR-EI-FtsZ1-TurboID-YFP-HA vector is obtained by inserting the sgRNA2 target sequence (5'-GAGGACGGTTCGGCCGACATGGG-3') between positions 1991 and 1992 of sequence 1, and inserting the inverse complementary sequence of the sgRNA2 target sequence (5'-CCCATGTCGGCCGAACCGTCCTC-3') between positions 10358 and 10359 of sequence 1, and deleting positions 1547-1991 and 10359-10920 of sequence 1.

[0030] In some embodiments, the IVC donor plasmid is the p2xsgRNA2-300AR-EI-FtsZ1-TurboID-YFP-HA vector. The p2xsgRNA2-300AR-EI-FtsZ1-TurboID-YFP-HA vector is obtained by inserting the sgRNA2 target sequence (5'-GAGGACGGTTCGGCCGACATGGG-3') between positions 2192 and 2193 of sequence 1, and inserting the inverse complementary sequence of the sgRNA2 target sequence (5'-CCCATGTCGGCCGAACCGTCCTC-3') between positions 10108 and 10109 of sequence 1, and deleting positions 1547-2192 and 10109-10920 of sequence 1.

[0031] The above method also includes the following steps: introducing the CRISPR / Cas9 editing vector and IVC donor plasmid into the protoplasts of *Stylosporium simulans*, and obtaining positive transformants by screening with antibiotics (such as hygromycin) and genotyping.

[0032] In some embodiments, the method of introducing CRISPR / Cas9 editing vectors and IVC donor plasmids into *Phyllostachys pubescens* protoplasts, followed by screening with antibiotics (such as hygromycin) and genotyping to obtain positive transformants includes the following steps: preparing a solution of *Phyllostachys pubescens* protoplasts (e.g., at a concentration of 2 × 10⁻⁶) 6 The solution of *Phyllostachys edulis* protoplasts (cells / mL), CRISPR / Cas9 editing vector and IVC donor plasmid were mixed (e.g., 300 μL of *Phyllostachys edulis* protoplast solution was mixed with 30 μg of total plasmid (CRISPR / Cas9 editing vector and IVC donor plasmid)). PEG (e.g., 40% PEG) was added, and the mixture was thoroughly mixed and allowed to stand at room temperature (e.g., 5 min at room temperature). After heat shock (e.g., 45℃ heat shock for 5 min), it was allowed to stand at room temperature (e.g., 10 min at room temperature). Then it was placed in mannitol (e.g., 8.5% mannitol) and incubated in the dark (e.g., 2 h incubation). After incubation, it was centrifuged (e.g., 170 g centrifuged for 5 min), the supernatant was discarded, and the precipitate was resuspended in PRML medium and transferred to PRMB medium for culture. After 5 days of culture, it was transferred to BCDAT medium containing antibiotics for further culture. After 7 days of culture, the green clones were transferred to BCDAT medium for further culture. After 7 days of culture, the DNA of the positive green clones was randomly selected for genotyping.

[0033] In some embodiments, the primer sequences for genotype identification are F1: 5'-CACCTTGGAGAGTCTGCCAG-3' and R1: 5'-TGATTTAAGAGTGGGATTAGAGATTTGA-3'.

[0034] To address the aforementioned technical problems, the present invention also provides a complete set of carriers.

[0035] The complete set of vectors provided by the present invention includes the above-mentioned CRISPR / Cas9 editing vector and the above-mentioned IVC donor plasmid.

[0036] The application of the above-mentioned complete vector in the editing of the genome sequence of *Sphagnum moss* or in the preparation of products with edited genome sequences of *Sphagnum moss* is also within the scope of protection of this invention; the editing of the genome sequence includes the insertion of large DNA fragments.

[0037] To address the aforementioned technical problems, the present invention ultimately provides a product for achieving large-fragment DNA insertion in *Moss sclerotium*.

[0038] The product provided by this invention for achieving large-fragment DNA insertion in *Sclerotium pulmonale* contains the aforementioned complete set of vectors.

[0039] In some embodiments, the product includes reagents required to introduce the complete vector into the protoplasts of *Styrax spp.*, such as MMM solution, PEG (e.g., 40% PEG), and mannitol (e.g., 8.5% mannitol).

[0040] In some implementations, the product includes reagents required for culturing and screening protoplasts of *Styrax spp.* introduced into a complete vector, such as PRMB medium and BCDAT medium.

[0041] Any of the above-mentioned small-bowl mosses can be the Gransden strain.

[0042] This invention addresses the problem of high cloning complexity caused by the reliance on long homologous arms (approximately 1 kb) in existing IVC donors. It innovatively proposes IVC donor plasmids with homologous arms shortened to approximately 250-510 bp (such as the p2xsgRNA2-300AR / 500AR-EI-FtsZ1-TurboID-YFP-HA vector). Simultaneously, it reveals for the first time the key advantages of the circular donor form: when inserting a 7.3 kb fragment, the insertion efficiency of the short-arm IVC donor is approximately 50%, slightly lower than that of the long-arm IVC donor (61.3%), but significantly higher than that of the standard circular donor (17.7%). Under the same short-arm conditions, the efficiency of directly using in vitro linearized donors is only about 20%. This invention achieves a significant reduction in homologous arms while maintaining high-efficiency site-specific insertion of large fragments, significantly simplifying the donor preparation process while maintaining high insertion efficiency, providing a highly efficient gene editing tool for plant synthetic biology and crop improvement.

[0043] The beneficial effects of this invention are as follows: In early studies of homologous recombination in *Phyllostachys nigra*, donor DNA was mostly introduced into protoplasts in a linearized form, and it has been proven that high homologous recombination efficiency can be obtained. Simultaneously, linearized donors can transform "in vivo cleavage" into "in vitro cleavage," thereby skipping the Cas9 cleavage effect on the donor, theoretically helping to reduce system complexity and uncertainty. However, this invention, through comparative evaluation of the insertion efficiency of IVC circular donors with shortened homologous arms and in vitro linearized donors, found that the insertion efficiency of IVC circular donors is significantly higher than that of linearized donors, and confirmed that IVC circular donors with shortened homologous arms can still maintain an insertion efficiency of approximately 50%. Attached Figure Description

[0044] Figure 1 This image shows the genotype identification results for different donor patterns.

[0045] Figure 2 A comparison chart of large fragment insertion efficiency for different donor modes. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0048] The wild-type Gransden strain in the following examples is described in the literature "Ashton, NW & Cove, DJ (1977) The isolation and preliminary characterisation of auxotrophic and analogue-resistant mutants of the moss, Physcomitrella patens In Molecular and General Genetics: MGG, 154, 87–95,” the growth conditions were: 23°C, 16 hours of light / 8 hours of darkness.

[0049] The pHyg-Cas9-sgRNA2, pEI-FtsZ1-TurboID-YFP-HA, and p2xsgRNA2-EI-FtsZ1-TurboID-YFP-HA vectors used in the following examples are all described in the literature "Gu X, Lang J, Chang Y, Zhang M. (2025) Cleavable donor-assisted CRISPR / Cas9 system significantly improves the efficiency of large DNA insertion in Physcomitrium patens . The Plant Journal, 121(4):e70020."

[0050] The formulations of the solutions and culture media involved in the following examples are shown in Tables 1-4.

[0051] Table 1. Commonly Used Mother Solution Formulas for Pseudomonas aeruginosa

[0052] Table 2. Preparation of BCDAT medium for *Moss simulans*

[0053] Table 3. Preparation of PRML culture medium for *Moss simulans*

[0054] Table 4. Preparation of PRMB culture medium for *Moss simulans*

[0055] Example 1: A method for efficiently inserting large DNA fragments into *Moss simulans* using an IVC donor system with shortened homologous arms. I. Carrier Construction 1. CRISPR / Cas9 editing vector The sgRNA targeting gene locus of the CRISPR / Cas9 editing vector used was *Moss sclerotium*. Pp6c18_3160 (See reference: Gu X, Lang J, Chang Y, Zhang M. (2025) Cleavable donor-assisted CRISPR / Cas9 system significantly improves the efficiency of large DNA insertion in) Physcomitrium patens . The Plant Journal, 121(4):e70020.), that is, the vector in the literature: pHyg-Cas9-sgRNA2.

[0056] 2. Construction of IVC donor plasmids with shortened homologous arms Using the p2xsgRNA2-EI-FtsZ1-TurboID-YFP-HA vector (nucleotide sequence shown in Sequence 1) as a template, PCR amplification was performed using primers 500AR-F / 500AR-R and 300AR-F / 300AR-R, respectively. The amplified target fragments were then added to the T vector (Clone Smarter, catalog number C5851) via TOPO cloning, resulting in the p2xsgRNA2-500AR-EI-FtsZ1-TurboID-YFP-HA and p2xsgRNA2-300AR-EI-FtsZ1-TurboID-YFP-HA vectors, respectively. The primer sequences are as follows: 500AR-F: 5'-GAGGACGGTTCGGCCGACATGGGCCCAAAGAAGAAAGAATGAGGT-3' 500AR-R: 5'-GAGGACGGTTCGGCCGACATGGGTCCATGGCCATTCGAATACC-3'.

[0057] 300AR-F: 5'-GAGGACCGTTTCGGCCGACATGGGCTAGAAAAAAAATTGGTCCATTTTATGACC-3' 300AR-F: 5'-GAGGACGTTCGGCCGACATGGGCCTCCACCACAGCTCGAATTCA-3'.

[0058] The p2xsgRNA2-500AR-EI-FtsZ1-TurboID-YFP-HA vector is a vector containing p2xsgRNA2-EI-FtsZ1-TurboID-YFP-HA. Pp6c18_3160 Left homologous arm and Pp6c18_3160 Add sgRNA2 target sequences to both ends of the right homologous arm and Pp6c18_3160 Left homologous arm and Pp6c18_3160 The vector obtained after shortening the size of the right homologous arm to approximately 500 bp ( Pp6c18_3160 The left homologous arm was shortened to 507 bp. Pp6c18_3160 The right homologous arm was shortened to 500 bp.

[0059] The nucleotide sequence of the p2xsgRNA2-500AR-EI-FtsZ1-TurboID-YFP-HA vector is obtained by inserting the sgRNA2 target sequence (5'-GAGGACGGTTCGGCCGACATGGG-3') between positions 1991 and 1992 of sequence 1, and inserting the reverse complementary sequence of the sgRNA2 target sequence (5'-CCCATGTCGGCCGAACCGTCCTC-3') between positions 10358 and 10359 of sequence 1, and deleting positions 1547-1991 and 10359-10920 of sequence 1.

[0060] The p2xsgRNA2-300AR-EI-FtsZ1-TurboID-YFP-HA vector is a vector containing p2xsgRNA2-EI-FtsZ1-TurboID-YFP-HA. Pp6c18_3160 Left homologous arm and Pp6c18_3160 Add sgRNA2 target sequences to both ends of the right homologous arm and Pp6c18_3160 Left homologous arm and Pp6c18_3160 The vector obtained after shortening the size of the right homologous arm to approximately 300 bp ( Pp6c18_3160 The left homologous arm was shortened to 306 bp. Pp6c18_3160The right homologous arm was shortened to 250 bp.

[0061] The nucleotide sequence of the p2xsgRNA2-300AR-EI-FtsZ1-TurboID-YFP-HA vector is obtained by inserting the sgRNA2 target sequence (5'-GAGGACGGTTCGGCCGACATGGG-3') between positions 2192 and 2193 of sequence 1, and inserting the reverse complementary sequence of the sgRNA2 target sequence (5'-CCCATGTCGGCCGAACCGTCCTC-3') between positions 10108 and 10109 of sequence 1, and deleting positions 1547-2192 and 10109-10920 of sequence 1.

[0062] 3. Obtaining linearized donor fragments Using the p2xsgRNA2-500AR-EI-FtsZ1-TurboID-YFP-HA vector as a template, PCR amplification was performed using primers 500ARN-F and 500ARN-R to obtain the linearized 500AR-EI-FtsZ1-TurboID-YFP-HA fragment.

[0063] Using the p2xsgRNA2-300AR-EI-FtsZ1-TurboID-YFP-HA vector as a template, PCR amplification was performed using primers 300ARN-F and 300ARN-R to obtain the linearized 300AR-EI-FtsZ1-TurboID-YFP-HA fragment.

[0064] The primer sequences are as follows: 500ARN-F: 5'-GGGCCCAAAGAAGAAAGAATGAGG-3'.

[0065] 500ARN-R: 5'-TCCATGGCCATTCGAATACCCG-3'.

[0066] 300ARN-F: 5'-GGGCTAGAAAAAAAATTGGTCCATTTTATGACC-3'.

[0067] 300ARN-R: 5'-CCTCCACCACAGCTCGAATTCA-3'.

[0068] II. Small bowl moss steadily turns The CRISPR / Cas9 editing vector and donor were transformed into the protoplasts of *Sclerotium spp.*, and were divided into 6 groups according to the different transfectants. The CRISPR / Cas9 editing vector and donor in each group are shown in Table 5.

[0069] Table 5

[0070] The specific transfer method includes the following steps: After extracting protoplasts from *Gransden* (a strain of *Moss spp.*), the protoplasts are diluted with MMM solution (9.1% mannitol, 15mM MgCl2, 0.1% [w / v] MES-KOH, pH 5.6) to a concentration of 2×10⁻⁶. 6 Cells / mL, then add 30 μg total plasmid (CRISPR / Cas9 editing vector: donor molar ratio of 1:1) and 300 μL protoplasts to a 2.0 mL EP tube. Finally, add an equal volume of 40% PEG to the protoplasts and plasmids, mix thoroughly, let stand at room temperature for 5 min, heat shock at 45℃ for 5 min, then let stand at room temperature for 10 min, and then incubate in 8.5% mannitol in the dark for at least 2 h. After incubation, centrifuge at 170g for 5 min, discard the supernatant, resuspend the precipitate with 2 mL of PRML medium, and then spread 500 μL each on PRMB plates (culture plates containing PRMB medium). After culturing for 5 days, transfer to BCDAT plates (culture plates containing BCDAT medium) containing Hyg (25 μg / mL) resistance for 7 days of continued growth. After 7 days, transfer the green clones to BCDAT plates (culture plates containing BCDAT medium) for 7 days of continued growth.

[0071] III. Genotyping 1. Randomly select 30-46 positive green clones. Take an appropriate amount of material from each clone into a 1.5 mL EP tube, add 20 μL of crude DNA extraction and grinding solution of *Sphagnum moss* (solution formula: 670 mM Tris-HCl pH 8.8, 160 mM (NH4)2SO4, 0.1% Tween 20), and grind the material with a glass rod.

[0072] 2. Using the extracted DNA as a template, PCR amplification was performed using primers F1 and R1: The PCR reaction system was 20 μL, and the specific composition was as follows: 10 μL of 2 × Rapid Taq Plus Master Mix (Vazyme), 1 μL each of primers F1 and R1, 1 μL of DNA template and 7 μL of ddH2O.

[0073] The PCR reaction program is as follows: pre-denaturation 95℃, 2 min; denaturation 98℃, 10 s; annealing 58℃, 30 s; extension 68℃, 2 min 30 s; a total of 35 cycles; and final extension 68℃, 5 min.

[0074] The primer sequences are as follows: F1: 5'-CACCTTGGAGAGTCTGCCAG-3'.

[0075] R1: 5'-TGATTTAAGAGTGGGATTAGAGATTTGA-3'.

[0076] Run the PCR product on an agarose gel, stain, develop, and check if the band size meets expectations (a single band of 9588 bp obtained from PCR product amplification is the clone with the inserted large fragment), and calculate the insertion efficiency. Insertion efficiency = (number of clones with the inserted large fragment / total number of clones) × 100%.

[0077] Genotype identification and insertion efficiency statistics are as follows: Figure 1 and Figure 2 As shown, the results of two repeated experiments indicated that the insertion efficiency of the donor pEI-FtsZ1-TurboID-YFP-HA (standard circular group) was 17.7%; the insertion efficiency of the IVC donor p2xsgRNA2-EI-FtsZ1-TurboID-YFP-HA (IVC-1000AR group) was 61.3%; and the insertion efficiency of the IVC donor p2xsgRNA2-500AR-EI-FtsZ1-TurboID-YFP-HA with homologous arms shortened to approximately 500 bp was 47% (IVC-500AR group); the insertion efficiency of the homologous arm shortened to approximately 300 bp was... The insertion efficiency of the IVC donor p2xsgRNA2-300AR-EI-FtsZ1-TurboID-YFP-HA was 46.5% (IVC-300AR group); the insertion efficiencies of linearized 500AR-EI-FtsZ1-TurboID-YFP-HA (linearized 500AR group) and linearized 300AR-EI-FtsZ1-TurboID-YFP-HA (linearized 300AR group) were 20.9% and 17.7%, respectively.

[0078] The results above demonstrate that even with shortened homologous arm lengths, the IVC circular donor can still maintain approximately 50% insertion efficiency for large fragments, achieving high insertion efficiency while simplifying vector construction. Further comparison revealed that the insertion efficiency of the IVC circular donor was significantly higher than that of the in vitro linearized donor. This may be because linearized DNA is more susceptible to nuclease degradation in protoplasts, and the linearized donor is partially or completely destroyed before participating in homologous recombination. Especially under conditions of large fragment insertion, the integrity of the donor is more critical, thus limiting the effective insertion efficiency of linearized DNA. In contrast, the IVC circular donor structure is more stable and more conducive to efficient site-specific insertion of large fragments.

[0079] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for achieving large-fragment DNA insertion in *Stylos spp.*, comprising the following steps: introducing a CRISPR / Cas9 editing vector and an IVC donor plasmid into *Stylos spp.*; The CRISPR / Cas9 editing vector expresses Cas9 nuclease and sgRNA; The IVC donor plasmid contains a donor fragment, which sequentially includes the sgRNA target sequence, the left homologous arm sequence, the large DNA fragment to be inserted, the right homologous arm sequence, and the reverse complementary sequence of the sgRNA target sequence. The size of the left homologous arm sequence and the right homologous arm sequence does not exceed 510 bp.

2. The method according to claim 1, characterized in that: The size of the left homologous arm sequence and the right homologous arm sequence is 250-510 bp.

3. The method according to claim 1 or 2, characterized in that: The sizes of the left homologous arm sequence and the right homologous arm sequence are 306 bp and 250 bp, respectively; Alternatively, the sizes of the left homologous arm sequence and the right homologous arm sequence are 507 bp and 500 bp, respectively.

4. The method according to any one of claims 1-3, characterized in that: The large DNA fragment to be inserted contains the FtsZ1-TurboID-YFP expression cassette.

5. The method according to any one of claims 1-4, characterized in that: The large DNA fragment to be inserted contains an XVE expression cassette.

6. The method according to any one of claims 1-5, characterized in that: The molar ratio of the CRISPR / Cas9 editing vector to the IVC donor plasmid is 1:

1.

7. The method according to any one of claims 1-6, characterized in that: The method includes the following steps: introducing the CRISPR / Cas9 editing vector and IVC donor plasmid into the protoplasts of *Moss styracifolium*, and obtaining positive transformants through antibiotic screening and genotyping.

8. A complete set of vectors, the complete set of vectors comprising the CRISPR / Cas9 editing vector as described in any one of claims 1-7 and the IVC donor plasmid as described in any one of claims 1-7.

9. The use of the complete vector kit of claim 8 in the editing of the genome sequence of *Sphaerocera minor* or in the preparation of products containing the edited genome sequence of *Sphaerocera minor*; wherein the editing of the genome sequence includes the insertion of large DNA fragments.

10. A product for enabling large-fragment DNA insertion in *Sclerotium spp.*, comprising the kit vector as described in claim 8.

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