Gene editing method capable of realizing fragment insertion or fragment replacement

Abstract

The invention relates to a gene editing method capable of realizing fragment insertion or fragment replacement, and provides a gene editing system which can insert an exogenous sequence donor into an expected position of a target genome. The gene editing system comprises a DNA donor expression cassette, an mRNA donor expression cassette, a gene editing tool expression cassette and a gRNA expression cassette. By improving the gene editing system, the efficiency of fragment insertion or fragment replacement is improved.

Description

[0001] This application claims priority to Chinese patent application CN202510303249.8, filed on March 14, 2025. The entire contents of the aforementioned Chinese patent application are incorporated herein by reference. Technical Field

[0002] This invention belongs to the field of biotechnology, specifically relating to a gene editing method that enables fragment insertion or fragment replacement. Background Technology

[0003] Gene editing technology, as a core tool of modern biotechnology, has evolved from early zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to the third-generation CRISPR gene editing system. These technologies achieve site-specific modifications to the genome by targeting and cutting the DNA double strand and relying on cellular repair mechanisms (such as non-homologous end joining or homologous recombination).

[0004] Since its development, the CRISPR-Cas9 system has been rapidly applied to fields such as agricultural breeding, disease treatment, and industrial microbial modification due to its advantages of high efficiency, programmability, and low cost. For example, in agriculture, editing crop genes can enhance insect resistance, stress resistance, and increase yield; in the medical field, CRISPR technology provides a new pathway for gene repair of genetic diseases such as β-thalassemia.

[0005] In recent years, the technology of precise insertion of large DNA fragments has become a research hotspot in the field of gene editing due to its potential in agricultural breeding (such as enhancing stress resistance), disease treatment (such as gene replacement therapy) and synthetic biology (such as metabolic pathway reconstruction).

[0006] In this application, the inventors improved the efficiency of using gene editing technology to insert or replace fragments by modifying the gene editing vector. Summary of the Invention

[0007] This invention improves the efficiency of fragment insertion by modifying the gene editing system to simultaneously design DNA donor expression cassettes and mRNA donor expression cassettes within the same vector backbone.

[0008] On one hand, the present invention provides a gene editing system capable of inserting a foreign sequence donor into a desired location in a target genome. The gene editing system includes a first expression frame, a second expression frame, a third expression frame, and a fourth expression frame. The first expression frame is used to express a gene editing tool enzyme; the second expression frame is used to express a guide RNA (gRNA), the gRNA including a region capable of binding to the gene editing tool enzyme and a region capable of binding to a target sequence in the target genome. The gRNA guides the gene editing tool enzyme to cleave in the region binding to the target sequence in the target genome to obtain the desired insertion location of the foreign donor sequence; the third expression frame is used to provide a DNA-based foreign sequence donor; and the fourth expression frame is used to express an mRNA-based foreign sequence donor.

[0009] In this invention, the gene editing tool enzyme is selected from Cas9, Cas12 protein (e.g., Cas12a, Cas12b, Cas12i, Cas12j, Cas12f, etc.), Cas13 protein, and Cas14 protein.

[0010] In one embodiment, the gene editing tool enzyme is selected from Cas12i3 or a variant thereof, preferably, the amino acid sequence of the gene editing tool enzyme is shown in SEQ ID No. 9.

[0011] In one embodiment, the first expression box further includes a promoter, a terminator, and a kernel localization sequence.

[0012] In one embodiment, the second expression box further includes a transcription promoter and a terminator.

[0013] In one embodiment, the transcription promoter is a class II promoter or a class III promoter.

[0014] In one embodiment, the third expression box includes a first unit and a second unit.

[0015] The first unit is provided with the DNA donor, which includes a foreign sequence donor and homologous arms. Specifically, the two ends of the foreign sequence donor are respectively provided with homologous arms that hybridize with the target genome.

[0016] The length of the homologous arm is 50bp-2000bp, for example, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1700bp, 1800bp or 1900bp.

[0017] Furthermore, the DNA donor is provided with target sequences at both ends that can be cleaved by the gene-editing enzyme to release the DNA donor. The target sequences that can be cleaved by the gene-editing enzyme are the regions of the gRNA that can bind to the target genome sequence.

[0018] The second unit is provided with virus replication-related proteins to perform rolling circle replication of the DNA donor. Furthermore, the virus replication-related proteins are connected to LIR (long intergenic region) and SIR (short intergenic region) at their two ends, respectively.

[0019] In one embodiment, the viral replication-related protein is a geminivirus replication-related protein; for example, the Rep / RepA protein.

[0020] In one embodiment, the Rep or RepA protein is a naturally occurring protein.

[0021] In one embodiment, the RepA protein is a mutant RepA protein, which, compared with the parental RepA protein, has a mutation at amino acid position 263 corresponding to the sequence shown in SEQ ID No. 4; preferably, the mutated amino acid position 263 is D.

[0022] In one embodiment, the amino acid sequence of the Rep protein is shown in SEQ ID No. 6, and the amino acid sequence of the RepA protein is shown in SEQ ID No. 4 or SEQ ID No. 7.

[0023] In one embodiment, the Rep / RepA protein is encoded by the sequence shown in SEQ ID No. 5 or SEQ ID No. 8.

[0024] In one embodiment, the sequences of the LIR and SIR are shown as SEQ ID No. 1 and SEQ ID No. 2, respectively.

[0025] The second unit is placed at the 5' or 3' end of the first unit.

[0026] In one embodiment, the mRNA donor comprises a foreign sequence donor and homologous arms. Specifically, the foreign sequence donor has homologous arms at both ends that hybridize with the target genome.

[0027] The length of the homologous arm is 50bp-2000bp, for example, 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1100bp, 1200bp, 1300bp, 1400bp, 1500bp, 1600bp, 1700bp, 1800bp or 1900bp.

[0028] Furthermore, the mRNA donor is provided with HH ribozyme sequences and / or HDV ribozyme sequences at both ends to release the mRNA donor.

[0029] Furthermore, the fourth expression box also includes a transcription promoter (class II promoter) and a terminator; the transcription promoter transcribes the mRNA donor.

[0030] In one embodiment, the gene editing system further includes the MCP-MS2 system.

[0031] In one implementation, the MCP is placed in the first expression box, and the MS2 is placed in the fourth expression box.

[0032] In one embodiment, the MCP is positioned at the 5' or 3' end of the gene editing tool enzyme;

[0033] In one embodiment, the MS2 is positioned at the 5' or 3' end of the mRNA donor.

[0034] In one embodiment, the first expression box, the second expression box, the third expression box, and the fourth expression box are placed on the same carrier.

[0035] In one embodiment, the gRNA may be one or more gRNAs (e.g., two, three or more).

[0036] If it is a single gRNA, the gene editing tool can cut the target sequence to obtain the intended insertion site of the exogenous donor sequence under the guidance of the gRNA. At this time, the target sequences at both ends of the DNA donor, which are cut by the gene editing tool enzyme, are the regions of the aforementioned gRNA that can bind to the target genome sequence.

[0037] If multiple gRNAs are used, for example, two gRNAs (gRNA1 and gRNA2), the gene editing tool can cleave the regions where gRNA1 and gRNA2 bind to the target sequence to obtain the desired insertion site of the exogenous donor sequence. In this case, the target sequences cleaved by the gene editing tool enzyme at both ends of the DNA donor can be regions within the same gRNA that bind to the target genome sequence, or regions within different gRNAs that bind to the target genome sequence.

[0038] In one embodiment, the exogenous sequence donor is selected from any sequence, which may be 100bp-10kb in length, or longer, for example, 200bp, 300bp, 500bp, 1kb, 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, or more than 10kb.

[0039] The exogenous sequence donor corresponds to an endogenous or natural chromosomal sequence. For example, the exogenous sequence donor may be substantially identical to a portion of the genome / chromosomal sequence at or near the target site, but it contains at least one nucleotide change. Thus, the exogenous sequence donor may be a modified form of the wild-type sequence at the target site such that, when integrated or exchanged with the natural sequence, the sequence at the target site contains at least one nucleotide change. For example, the change may be the insertion of one or more nucleotides, the deletion of one or more nucleotides, the substitution of one or more nucleotides, or a combination thereof.

[0040] On the other hand, the present invention also provides a carrier, the carrier including the first expression box, the second expression box, the third expression box and the fourth expression box described above.

[0041] In one embodiment, the carrier includes a third expression box, a second expression box, a fourth expression box, and a first expression box connected in sequence.

[0042] In one embodiment, the vector includes a cloning vector, an expression vector, a shuttle vector, and an integration vector.

[0043] In one embodiment, the carrier is an expression carrier.

[0044] In one embodiment, the sequence of elements of the vector is codon-optimized for expression in prokaryotic cells.

[0045] In one embodiment, the sequence of elements of the vector is codon-optimized for expression in eukaryotic cells (e.g., animal or plant cells).

[0046] On the other hand, the present invention also provides a host cell containing the above-mentioned carrier.

[0047] On the other hand, the present invention also provides the use of the above-described gene editing system, the above-described vector, or the above-described host cell in inserting a foreign sequence donor into the intended location of the target genome.

[0048] On the other hand, the present invention provides a method for integrating a foreign donor sequence into a target location in the genome of a cell, the method comprising the step of delivering the above-described gene editing system or the above-described vector into the cell, and further comprising culturing the cell so that the foreign sequence donor is inserted into the target location in the genome.

[0049] The gene editing system of the present invention can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipid transfection, nuclear transfection, microinjection, acoustic pore effect, gene gun, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendritic transfection, heat shock transfection, nuclear transfection, magnetic transfection, puncture transfection, optical transfection, reagent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, artificial viruses, Agrobacterium-mediated transformation, etc.

[0050] In one embodiment, the cells are selected from prokaryotic cells or eukaryotic cells.

[0051] In some implementations, the cell is a prokaryotic cell.

[0052] In some implementations, the cells are eukaryotic cells.

[0053] In some embodiments, the cell is an animal cell or a plant cell.

[0054] In some embodiments, the cells are human cells or non-human mammalian cells, such as cells of non-human primates, cattle, sheep, pigs, dogs, monkeys, rabbits, or rodents (such as rats or mice). In some embodiments, the cells are non-mammal eukaryotic cells, such as cells of poultry (such as chickens), fish, or crustaceans (such as clams or shrimp).

[0055] In one embodiment, the cell is a plant cell. The method further includes a method for obtaining plant cells into which a foreign sequence donor is inserted at a desired location in the target genome, and then cultivating the plant cells into a plant.

[0056] In one embodiment, the plant is a monocotyledonous or dicotyledonous plant; for example, rice, corn, soybeans, wheat, tomatoes, potatoes, etc.

[0057] In one embodiment, the cell is a plant cell. The exogenous sequence donor can be a sequence that induces plant resistance, such as disease resistance, herbicide resistance, stress resistance, drought resistance, cold resistance, or insect resistance. The exogenous sequence donor can also be a sequence that increases the content of a specific protein.

[0058] In one embodiment, the cells are either isolated or in vivo cells.

[0059] In one embodiment, the cell is an animal cell. The exogenous sequence donor may be a sequence capable of treating a disease, or a sequence capable of inducing an animal to contract a disease, thereby establishing an animal model.

[0060] In one embodiment, the nucleotide sequence is codon-optimized for expression in prokaryotic cells. In another embodiment, the nucleotide sequence is codon-optimized for expression in eukaryotic cells.

[0061] As used herein, the term "promoter" has the meaning known to those skilled in the art, referring to a non-coding nucleotide sequence located upstream of a gene that initiates the expression of a downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, results in the production of the gene product in the cell substantially only when the cell is a cell of the tissue type corresponding to that promoter.

[0062] A “nuclear localization signal” or “nuclear localization sequence” (NLS) is an amino acid sequence that “tags” a protein to allow it to be transported to the nucleus via nuclear transport; that is, a protein with an NLS is transported to the nucleus. Typically, an NLS contains positively charged Lys or Arg residues exposed on the protein surface. Exemplary nuclear localization sequences include, but are not limited to, NLS from the following: SV40 large T antigen, EGL-13, c-Myc, and TUS protein. In some embodiments, the NLS contains any of the following sequences: PKKKRKV, AVKRPAATKKAGQAKKKKLD, PAAKRVKLD, KLKIKRPVK, KRPAATKKAGQAKKKK, ASPKRPRDRHDGELGGRKRARG, or MSRRRKANPTKLSENAKKLAKEVEN. In some embodiments, the NLS contains the sequence. Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP A1, the KIPIK sequence in the yeast transcriptional repressor Matα2, and PY-NLS.

[0063] The term "vector" refers to a nucleic acid molecule capable of delivering another nucleic acid molecule linked to it. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules including one or more free ends, or without free ends (e.g., circular); nucleic acid molecules including DNA, RNA, or both; and a wide variety of other polynucleotides known in the art. A vector can be introduced into a host cell through transformation, transduction, or transfection, thereby enabling the expression of its carried genetic material elements in the host cell. A vector can be introduced into a host cell to produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, etc., as described herein (e.g., CRISPR transcripts, such as nucleic acid transcripts, proteins, or enzymes). A vector may contain a variety of elements controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site.

[0064] One type of vector is a "plasmid," which is a circular double-stranded DNA loop into which another DNA fragment can be inserted, for example, using standard molecular cloning techniques.

[0065] Another type of vector is the viral vector, in which a virus-derived DNA or RNA sequence is present in a vector used to package the virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). Viral vectors also contain polynucleotides carried by the virus used for transfection into a host cell. Some vectors (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) are capable of autonomous replication in the host cells into which they are introduced.

[0066] Other vectors (e.g., non-attachment mammalian vectors) integrate into the host cell's genome upon introduction and thereby replicate along with the host genome. Furthermore, some vectors are capable of directing the expression of genes they are operatively linked to. Such vectors are referred to herein as "expression vectors."

[0067] The term “plant cell” should be understood as any cell that is derived from or found in a plant and is capable of forming, for example: undifferentiated tissues such as callus, differentiated tissues such as embryos, components of a plant, or seeds.

[0068] In this invention, amino acid residues can be represented by a single letter or by three letters, for example: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamic acid (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), proline (Pro, P), isoleucine (Ile, I), histidine (His, H), and arginine (Arg, R).

[0069] The sequence involved in this invention is as follows:

[0070] Table 1. Nucleotide and amino acid sequences involved in this application

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] The main advantages of the present invention are:

[0079] This invention improves the efficiency of fragment insertion or fragment replacement by optimizing gene editing vectors. Attached Figure Description

[0080] Figure 1Fluorescence microscopy observations of rice protoplasts transformed with different vectors.

[0081] Figure 2 Enzyme digestion results of amplified products from rice seedlings transformed with vector P50.

[0082] Figure 3 Sequencing results of amplified products from rice seedlings with inserted fragments.

[0083] Figure 4 Electrophoresis results of amplification products from positive rice seedlings transformed with P5967 vector.

[0084] Figure 5 Sequencing results of amplified products from positive rice seedlings transformed with the P5967 vector. Detailed Implementation

[0085] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0086] Example 1: Optimization of viral replication-related proteins

[0087] In this embodiment, the RepA protein required for WDV rolling circle replication was optimized, further improving the rolling circle replication capability.

[0088] Construct an EGFP expression vector according to the following structure:

[0089] LIR-Rep / RepA-SIR-EGFP-LIR-OsU6-gRNA1-gRNA2-HDV-PolyT-ZmUBI-NLS-CasSF01-Nos.

[0090] The above-mentioned OsU6 and ZmUBI are promoters, Nos is a terminator, CasSF01 is the Cas enzyme used, and EGFP has a sequence recognized by gRNA1 and a sequence recognized by gRNA2 at both ends.

[0091] The nucleotide sequences of LIR and SIR are shown in Table 1.

[0092] Vectors were constructed using wild-type Rep / RepA proteins (wild-type Rep protein as shown in SEQ ID No. 3, wild-type RepA protein as shown in SEQ ID No. 4) and mutant Rep / RepA proteins (mutant Rep protein as shown in SEQ ID No. 6, mutant RepA protein as shown in SEQ ID No. 7). The Rep and RepA proteins are different proteins resulting from alternative splicing. The vectors were constructed using the wild-type nucleic acid sequence shown in SEQ ID No. 5 and the mutant nucleic acid sequence shown in SEQ ID No. 8. Due to the presence of alternative splicing, the Rep protein translated from SEQ ID No. 5 is shown in SEQ ID No. 3, and the RepA protein is shown in SEQ ID No. 4; the Rep protein translated from SEQ ID No. 8 is shown in SEQ ID No. 6, and the RepA protein is shown in SEQ ID No. 7. The amino acid sequence of the translated Rep protein remained unchanged, while the RepA protein underwent a mutation; the mutated RepA protein had a G-to-D mutation at position 263 relative to the wild-type RepA protein.

[0093] The vector constructed using the mutant Rep / RepA protein is designated P5557, and the vector constructed using the wild-type Rep / RepA protein is designated P5675. Both vectors contain a LIR-SIR rolling circle for EGFP expression, and the vectors contain a Cas protein that can cleave the single-stranded EGFP-expressing DNA following the rolling circle.

[0094] Vectors P5557 and P5675 were transformed into rice protoplasts, and the EGFP fluorescence expression level was detected. The specific method is as follows: Dehulled rice seeds were sterilized and sown in 1 / 2 MS medium boxes and cultured at 26-28℃ in the dark for 9-14 days. Rice seedling stems were taken and cut into 0.5 mm thin slices with a blade. The slices were transferred to a 90 mm culture dish containing 15 mL of 0.6 M D-Mannitol solution and incubated at 55 rpm for 30 min at room temperature in the dark. The liquid was discarded, and 15 mL of enzyme digest was added. The mixture was incubated at 55 rpm for 3-5 hours at room temperature in the dark. 15 mL of W5 solution was added, shaken well, and filtered through a 0.4 μm cell sieve. The filtered liquid was transferred to a 50 mL round-bottom centrifuge tube. The rice tissue on the cell sieve was rinsed several times with W5 solution and gently squeezed with the back of a spoon. The mixture was centrifuged (300-350 g for 3 min (acceleration and deceleration at 3)) and the supernatant was carefully removed. Add 6 mL of W5 solution, gently tap the bottom of the centrifuge tube to resuspend the protoplasts, and centrifuge (300-350g for 3 min, acceleration / deceleration rate 3). Carefully remove the supernatant, resuspend the protoplasts with 1 mL of W5 solution, place on ice for 30 min, and centrifuge (300-350g for 3 min, acceleration / deceleration rate 3). Carefully remove the supernatant, and resuspend the protoplasts with MMg solution to a protoplast concentration of 1-5 × 10⁻⁵. 6 Cells / mL. Add 20 μg plasmid and 200 μL protoplasts to a 2 mL round-bottom centrifuge tube and gently swirl to mix. Add 220 μL of 40% PEG solution, gently swirl to mix, and incubate at 28-30℃ for 15 min. Add 880 μL of W5 Solution, gently swirl to mix, and terminate the reaction. Centrifuge (300-350g for 3 min, acceleration / deceleration rate 3). Carefully aspirate the supernatant, resuspend the protoplasts in 1 mL of W5 Solution, transfer to a 6-well plate containing 1 mL of W5 Solution, seal with a breathable membrane, and incubate at 26-28℃ in the dark for 48 h. Observe under a fluorescence microscope to find EGFP-positive cells and photograph and record the results. (See attached image.) Figure 1 As shown, the EGFP brightness of protoplasts obtained from the P5557 vector was significantly higher than that from the P5675 vector. In other words, the EGFP brightness of the mutated Rep / RepA protein was significantly increased compared to the unmutated protein. The mutation of the Rep / RepA protein (the G263D mutation of the RepA protein) can enhance the rolling circle replication ability of the LIR-SIR sequence, thereby increasing the expression level of EGFP.

[0095] Example 2: Fragment insertion based on gene editing technology

[0096] In order to utilize gene editing technology to achieve fragment insertion or fragment replacement, this embodiment employs both a DNA donor and an mRNA donor, and places the donor, Cas protein, and gRNA in the same vector, thereby improving the efficiency of fragment insertion.

[0097] In this embodiment, the Cas protein is Cas-SF01, its promoter is ZmUBI, and its terminator is Nos. The expression cassette of Cas-SF01 is constructed in the following manner:

[0098] 5'-ZmUBI-NLS-Cas SF01-MCP-NLS-Nos ter-3'.

[0099] In this embodiment, the Cas protein used is Cas-SF01, which belongs to the Cas12i family of proteins. It is a Cas protein obtained by mutating amino acid sites on the basis of Cas12i3 (Cas12f.4 in CN111757889B, which is referred to as Cas12i3 in this embodiment).

[0100] In other implementations, other Cas proteins may also be used, such as Cas9, Cas12 proteins (e.g., Cas12a, Cas12b, Cas12i, Cas12j, Cas12f, etc.), Cas13 proteins, and Cas14 proteins.

[0101] In this embodiment, two gRNAs are used. The gRNAs include a region that binds to the Cas protein and a region that binds to the target sequence. The gRNA expression cassette is shown below:

[0102] 5'-OsU6-Gly / tRNA-gRNA1-Gly / tRNA-gRNA2-HDV-PolyT-3'.

[0103] In other implementations, different gRNAs can be selected depending on the specific insertion or replacement fragment location.

[0104] The DNA donor expression frame is shown below:

[0105] 5'-LIR-Rep / RepA-SIR-gRNA1 targeting sequence-homologous arm 1-insertion sequence-homologous arm 2-gRNA2 targeting sequence-LIR-3'; Rep / RepA is a mutant sequence, the nucleic acid sequence is shown in SEQ ID No. 8, the amino acid sequence of the translated Rep protein is shown in SEQ ID No. 6, and the amino acid sequence of the RepA protein is shown in SEQ ID No. 7.

[0106] Geminid replication-associated proteins Rep / RepA perform rolling circle replication on DNA donors, thereby increasing the DNA donor copy number. This embodiment employs a mutated Rep / RepA protein, which enhances rolling circle replication capability, further increasing the DNA donor copy number. Target sequences for gRNA are positioned at both ends of the DNA donor, allowing Cas enzyme cleavage of these target sequences to release the DNA donor (homologous arm 1 - insert sequence - homologous arm 2).

[0107] The mRNA donor expression cassette is shown below:

[0108] 5'-SbUBI-HH ribozyme sequence-homologous arm 1-insertion sequence-homologous arm 2-MS2-HDV ribozyme sequence-E9 ter-3'.

[0109] HH ribozyme and HDV ribozyme are used to release the mRNA donor (homologous arm 1 - insertion sequence - homologous arm 2), and the MS2 system is used to bind to the MCP linked to the Cas protein, recruiting the mRNA donor to the DSB and improving the efficiency of fragment insertion.

[0110] The sequence of elements used in each of the above expression boxes is shown in Table 1.

[0111] In other implementations, other alternative elements in the art can also be used. For example, other promoters can be selected, such as UBI, SlEF1α, LLDAV, EVCV, DMMV, BSV PRO, CYMV PRO FL, ZmUBI PRO, SI-UB3 PRO, SB-UBI PRO, USB1ZM PRO, ZM-GOS2 PRO, ZM-H1B PRO, IN2-2, NOS, etc.; other commonly used sequences can also be selected for the nuclear localization sequence.

[0112] In this embodiment, the Cas-SF01 expression cassette, gRNA expression cassette, DNA donor expression cassette, and mRNA donor expression cassette are mounted on the same vector backbone, and are sequentially linked to construct vectors P50 and P5967 in the order of DNA donor expression cassette, gRNA expression cassette, mRNA donor expression cassette, and Cas-SF01 expression cassette. In other embodiments, other linking orders can be used to link the expression cassettes sequentially, as long as each expression cassette can be successfully expressed; this is a conventional technique and operation in the art.

[0113] In this embodiment, the constructed vectors P50 and P5967 were transformed into Agrobacterium tumefaciens EHA105 and genetically transformed in rice variety Wanzhijing 006. Hygromycin was used for screening, and DNA was extracted from leaf samples of positive seedlings to identify whether sequence substitution had occurred.

[0114] In this embodiment, the inserted sequences in the DNA and mRNA donors of P50 are partial gene sequences of the rice ACCase protein. The 1879th amino acid of the rice ACCase protein is replaced with G, and a synonymous substitution without affecting the amino acid is performed at another location, creating a new KpnI restriction site. Amplification primers are designed on the right homologous arm and outside the left homologous arm to avoid amplifying to the T-DNA region inserted into the rice genome, ensuring that the amplified product is entirely a genomic sequence. The amplified product (1422 bp) is digested with KpnI; if cleaved bands (890 bp and 532 bp) are found, it confirms the successful sequence insertion on the donor. The inserted sequence in the DNA and mRNA donors of P5967 is P2A-EGFP, inserted at the CDS end of the rice UBQ2 gene. Amplification primers were designed separately for the left homologous arm and the inserted sequence, as well as for the right homologous arm and the inserted sequence. This avoids amplifying the T-DNA region inserted into the rice genome, ensuring that the amplified product is entirely a genomic sequence. Successful amplification of bands of 831 bp and 1454 bp on the left and right sides, respectively, confirms the successful amplification of the donor sequence.

[0115] The above-mentioned P50 vector was used to transform rice variety Wanzhijing 006. A total of 120 positive seedlings were detected. Among them, the amplification products of 10 positive seedlings showed cleaved bands (890bp and 532bp) after KpnI enzyme digestion. Partial enzyme digestion electrophoresis results are shown below. Figure 2 As shown in the figure. The amplification products of these 10 positive seedlings were sent for first-generation sequencing, which revealed that the sequence insertion was successfully completed at the expected position. The sequencing results are as follows. Figure 3 As shown in the figure. Using the above-mentioned P5967 vector to transform rice variety Wanzhijing 006, a total of 64 positive seedlings were detected. The amplification products of 10 of these positive seedlings showed bands of 831 bp and 1454 bp. Electrophoresis results are shown in the figure. Figure 4 As shown in the figure. The amplification products of these 10 positive seedlings were sent for first-generation sequencing, which revealed that the sequence insertion was successfully completed at the expected position. The sequencing results are as follows. Figure 5 As shown.

[0116] We also designed a control vector, CK1. Compared to the aforementioned vector P50, CK1 lacks the mRNA donor expression cassette, while other elements of CK1 are identical to those of P50. Using vector CK1, rice variety Wanzhijing 006 was transformed using the same procedures described above, yielding 100 positive seedlings. Upon identification, the amplified product showed no cleavage after KpnI restriction enzyme digestion; in other words, no sequence insertion at the expected position was observed in the 100 positive seedlings obtained using the control vector CK1.

[0117] Further, the control vector CK2 was designed. Compared with the above-mentioned vector P50, the DNA donor expression cassette of CK2 lacks the expression element of the geminivirus replication-related protein. That is, the DNA donor expression cassette of CK2 is as follows: 5' - gRNA1 target sequence - homologous arm 1 - insertion sequence - homologous arm 2 - gRNA2 target sequence - LIR-3'; other elements of CK2 are the same as those of P50. Using vector CK2, rice Wanzhijing 006 was transformed according to the same operation as above to obtain positive seedlings, which were then identified. The results showed that no cleavage was observed after KpnI digestion of the amplified product; that is, no sequence insertion at the expected position was observed in the 100 positive seedlings obtained using the control vector CK2.

[0118] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A gene editing system capable of inserting a foreign sequence donor into a desired location in a target genome, the gene editing system comprising a first expression frame, a second expression frame, a third expression frame, and a fourth expression frame; The first expression box is used to express gene editing tool enzymes; The second expression box is used to express guide RNA (gRNA), which includes a region that can bind to the gene editing tool enzyme and a region that can bind to the target genome sequence. The gRNA guides the gene editing tool enzyme to cut in the region that binds to the target genome sequence to obtain the expected location for insertion of the exogenous donor sequence. The third expression box is used to provide a DNA-based exogenous sequence donor, which is a DNA donor; the third expression box includes a first unit and a second unit; the first unit is provided with the DNA donor, which includes an exogenous sequence donor and a homologous arm; the two ends of the DNA donor are respectively provided with target sequences that can be cleaved by the gene editing tool enzyme to release the DNA donor; the second unit is provided with virus replication-related proteins to perform rolling circle replication on the DNA donor; The fourth expression box is used to express an mRNA-based exogenous sequence donor, which is an mRNA donor; the mRNA donor includes an exogenous sequence donor and a homologous arm.

2. The gene editing system according to claim 1, characterized in that, The second unit is provided with a geminivirus replication-related protein to perform rolling circle replication of the DNA donor; preferably, the geminivirus replication-related protein is connected to an LIR (long intergenic region) and a SIR (short intergenic region) at its two ends, respectively.

3. The gene editing system according to claim 1, characterized in that, The gene editing system also includes the MCP-MS2 system.

4. The gene editing system according to claim 1, characterized in that, The mRNA donor is further provided with HH ribozyme sequences and / or HDV ribozyme sequences at both ends to release the mRNA donor.

5. The gene editing system according to claim 1, characterized in that, The first expression box, the second expression box, the third expression box, and the fourth expression box are placed on the same carrier.

6. A carrier comprising a first expression box, a second expression box, a third expression box, and a fourth expression box as described in any one of claims 1-5.

7. A host cell comprising the vector of claim 6.

8. Use of the gene editing system of any one of claims 1-5, the vector of claim 6, or the host cell of claim 7 in inserting a foreign sequence donor into a desired location in the target genome.

9. A method for integrating a foreign donor sequence into a target genome of a cell at a desired location, the method comprising the step of delivering a gene editing system of any one of claims 1-5 or a vector of claim 6 into the cell; optionally, the method further comprising the step of culturing the cell to allow the foreign sequence donor to be inserted into the desired location of the target genome.

10. The method according to claim 9, characterized in that, The cells can be isolated or in vivo.

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