Novel separated guide editor based on Csy4 system and related protein

Abstract

The invention discloses a novel separated guide editor based on a Csy4 system and related protein. The invention belongs to the technical field of biology, and particularly relates to a novel separated guide editor based on a Csy4 system and related protein. The fusion protein is a protein obtained by fusing Csy4 nuclease of which the amino acid sequence is SEQ ID No: 1 to one end of RT protein, the Csy4 nuclease is fused to the N end or C end of the RT protein, and the amino acid sequence of the fusion protein is SEQ ID No: 2 or SEQ ID No: 3. The fusion of Csy4 improves the editing efficiency of separated guided editing, and compared with separated PE3max, the editing efficiency of 12 important agronomic trait targets of 11 genes of rice is greatly improved. The research lays a foundation for developing a Csy4-based separated guide editor with higher editing efficiency in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a novel split guide editor based on the Csy4 system and related proteins. Background Art

[0002] Precision editing is an advanced CRISPR / Cas-derived technology aimed at achieving precise genetic modification at specific genomic loci, including base substitutions, insertions, and deletions. Different from traditional homologous recombination repair methods, precision editing does not require double-stranded DNA breaks and exogenous donor DNA templates, making it a safer and more efficient method, especially in the context of gene therapy.

[0003] Current research shows that Prime Editors (PEs) have broad application scenarios in fields such as biomedicine. However, current precision editors (PEs) are too large in size and exceed the packaging limit of the delivery vector adenovirus (AAVs) during in vivo gene therapy. Split PEs express the Cas9 protein and reverse transcriptase RT separately, both of which reach a size that can be accommodated by viral vectors, providing a more flexible and efficient method for therapeutic applications. However, the efficiency of split guide editors is relatively low and needs to be further optimized.

[0004] Csy4 (also known as Cas6f) is a key enzyme responsible for crRNA production in the CRISPR I-F subtype. It specifically cleaves the pre-crRNA substrate at the 3' end of the 20-nucleotide Csy4 recognition sequence (Csy4RS) to produce crRNA composed of a unique spacer sequence and Csy4RS. Existing research has shown that the Csy4 system can complete the precise processing of sgRNA and pegRNA, and the Csy4 protein has a high affinity for both its substrates and products.

[0005] An important reason for the low efficiency of split guide editors is the lack of a recruitment mechanism after the separate expression of reverse transcriptase RT and Cas9. Therefore, by fusing the Csy4 protein with reverse transcriptase RT, the high affinity of the Csy4 protein for the pegRNA product processed by it can recruit the reverse transcriptase to the vicinity of the Cas9 protein, thereby improving the editing efficiency of split guide editing. Summary of the Invention

[0006] The technical problems to be solved by the present invention are how to efficiently perform guide editing on organisms, and / or how to improve the gene editing efficiency of the guide editing system and / or how to optimize the guide editing system.

[0007] To solve the above problems, the present invention provides a fusion protein.

[0008] The fusion protein provided by the present invention is a protein obtained by fusing the Csy4 nuclease with an amino acid sequence of SEQ ID No: 1 to one end of the reverse transcriptase RT.

[0009] In the above-mentioned fusion protein, the Csy4 nuclease can be fused to the N-terminus or C-terminus of the RT enzyme.

[0010] In this article, the reverse transcriptase (Reverse transcriptase) RT is optimized, and its amino acid sequence is SEQ ID No: 7.

[0011] Furthermore, the Csy4 nuclease can be fused to the N-terminus of the RT enzyme through sequences such as linker, and the specific amino acid sequence of the fused protein is SEQ ID No: 2; the Csy4 nuclease can also be fused to the C-terminus of the RT enzyme through sequences such as linker, and the specific amino acid sequence of the fused protein is SEQ ID No: 3.

[0012] In this article, the RT enzyme is derived from the split guide editor 9s2V1.

[0013] The specific information of the split guide editor 9s2 is as follows: The split guide editor 9s2 contains two parts, the Cas9-KK840 protein and the reverse transcriptase RT. The amino acid sequence of the Cas9-KK840 protein of the split guide editor 9s2 is SEQ ID No: 9, and the amino acid sequences of the reverse transcriptase RT are SEQ ID No: 7.

[0014] In this article, the Cas9-KK840 protein and the reverse transcriptase RT in the split guide editor 9s2 can be codon-optimized. The nucleotide sequence of the codon-optimized Cas9-KK840 protein-encoding gene is SEQ ID No: 10, and the nucleotide sequence of the codon-optimized reverse transcriptase RT-encoding gene is SEQ ID No: 8.

[0015] The guide editor generated by fusing the Csy4 protein to the N-terminus of the RT enzyme in the split guide editor 9s2 is named guide editor 9s42; the amino acid sequence of the fusion protein of guide editor 9s42 is SEQ ID No: 2.

[0016] The guide editor generated by fusing the Csy4 protein to the C-terminus of the RT enzyme in the split guide editor 9s2 is named guide editor 9s24; the amino acid sequence of the fusion protein of guide editor 9s24 is SEQ ID No: 3.

[0017] The present invention also provides a nucleic acid molecule encoding the aforementioned fusion protein.

[0018] Wherein the nucleic acid molecule is formed by fusing the coding gene of the Csy4 nuclease and the coding gene of the RT enzyme through a linker;

[0019] The coding gene of the Csy4 nuclease is any of the following:

[0020] (a1) A DNA molecule whose nucleotide sequence of the coding strand is positions 1-561 of SEQ ID No: 5 or SEQ ID No: 4;

[0021] (a2) A DNA molecule that hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide;

[0022] (a3) A DNA molecule that has a homology of more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% with the DNA sequence defined in (a1) or (a2) and encodes the polypeptide;

[0023] The coding gene of the RT enzyme is any of the following:

[0024] (b1) A DNA molecule whose nucleotide sequence of the coding strand is positions 664-2694 of SEQ ID No: 5 or the DNA molecule shown in SEQ ID No: 8;

[0025] (b2) A DNA molecule that hybridizes with the DNA molecule defined in (b1) under stringent conditions and encodes the same protein;

[0026] (b3) A DNA molecule that has a homology of more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% with the DNA sequence defined in (b1) or (b2) and encodes the same protein.

[0027] The nucleotide sequence of the linker can be positions 562 to 663 of SEQ ID No: 5, or positions 2089 to 2184 of SEQ ID No: 6.

[0028] The present invention also provides a biological material related to the nucleic acid molecule described above, and the biological material is any of the following:

[0029] B1) An expression cassette containing the nucleic acid molecule described above;

[0030] B2) A recombinant vector containing the nucleic acid molecule described above, or a recombinant vector containing the expression cassette described in B1);

[0031] B3) A recombinant microorganism containing the nucleic acid molecule described above, or a recombinant microorganism containing the expression cassette described in B1), or a recombinant microorganism containing the recombinant vector described in B2);

[0032] B4) A transgenic plant cell line containing the nucleic acid molecule described above, or a transgenic plant cell line containing the expression cassette described in B1);

[0033] B6) A transgenic plant tissue containing the nucleic acid molecule described above, or a transgenic plant tissue containing the expression cassette described in B1);

[0034] B7) A transgenic plant organ containing the nucleic acid molecule described above, or a transgenic plant organ containing the expression cassette described in B1).

[0035] Furthermore, the recombinant vector described in B2) can be recombinant vectors 9s2V1, 9s42, 9s24.

[0036] The present invention also provides a multi-gene prime editing system, and the prime editing system contains the fusion protein described above.

[0037] The prime editing system also contains pegRNA. Compared with sgRNA, pegRNA has an additional reverse transcriptase template (rtT) containing new genetic information and a primer binding site (PBS) sequence at the 3' end (abbreviated as RT sequence and PBS sequence), that is, pegRNA is composed of a spacer sequence, a gRNA backbone sequence, an RT sequence, and a PBS sequence in sequence.

[0038] The spacer sequence is used to identify the target site on the target gene; the RT sequence is a reverse complementary sequence after the target site on the genome, and a target mutation is introduced therein as the reverse transcription template of reverse transcriptase to reverse transcribe a DNA sequence, and then as a repair template to repair the genomic DNA; the PBS sequence is a primer binding site, which can be complementary to the 3' end of the broken target DNA strand to initiate the reverse transcription process. The design method or principle of the RT sequence and the PBS sequence can refer to the design methods or principles related to the RT sequence and the PBS sequence of pegRNA in the reported prime editing technology (PE) in the prior art.

[0039] Furthermore, the prime editing system also contains epegRNA (engineered prime editing guideRNA), and the epegRNA has an evopreQ1 sequence added to the 3' end of the 3' end of the above-mentioned pegRNA to stabilize the pegRNA.

[0040] The epegRNA is designed according to the target site to be edited. In the present invention, the information of the pegRNA can be specifically as shown in Table 2.

[0041] In a specific embodiment, the target site of the epegRNA of the present invention may be located in the OsSPL14 and OsSLR1 genes of rice.

[0042] In this article, the gene editing may specifically be site-directed replacement, deletion or insertion of bases, including single-base replacement, multi-base replacement, single-base deletion, multi-base deletion, single-base insertion, and multi-base insertion. In addition, it should also include large-fragment insertion and deletion or chromosomal structure variations such as chromosomal doubling and inversion completed by prime editing.

[0043] In this article, the multi-gene may be two or more genes.

[0044] The present invention also provides the application of the fusion protein or its related biological material described above in any of the following:

[0045] 1) Application in the preparation of a gene prime editing system;

[0046] 2) Application in improving gene editing efficiency;

[0047] 3) Application in the preparation of disease treatment products;

[0048] 4) Application in plant genetic improvement.

[0049] The present invention also provides the application of the nucleic acid molecule and its related biological material described above in any of the following:

[0050] 1) Application in the preparation of a gene prime editing system;

[0051] 2) Application in improving gene editing efficiency;

[0052] 3) Application in the preparation of disease treatment products;

[0053] 4) Application in plant genetic improvement.

[0054] In this application, Csy4 was first fused with the reverse transcriptase RT protein to form a new fusion protein without destroying the original functions of the Csy4 protein and the reverse transcriptase. Utilizing the high affinity of the Csy4 protein for its reaction substrate, the separately expressed reverse transcriptase RT and Cas9 protein were recruited together, improving the assembly efficiency of split PE and thus further enhancing the split prime editing efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Schematic diagrams of the structures of three split prime editing PE proteins and prime editing vectors.

[0056] Figure 2Editing efficiency of three types of dual-target PE vectors at 12 important agronomic trait targets of 11 genes. Ho, He, and Chi are homozygous, heterozygous, and chimeric mutant lines, respectively; Re: by-products derived from DNA repair, that is, only some of the target bases are edited when editing multiple bases simultaneously; Sc: by-products derived from the pegRNA scaffold. TAP-IVS, T173I, A174V, and P177S; W548 and P171 represent two targets of OsALS.

[0057] Figure 3 Comparative analysis of the prime editing efficiency of three types of dual-target PE vectors in transgenic lines of 12 targets (11 genes) in rice.

[0058] Figure 4 Average fold change in the prime editing efficiency of the dual-target PE vector fused with Csy4 and the control vector 9s2V1 at the target sites of OsSPL14, OsSLR1, OsNR2, OsNRT1.1B, OsAK, OsDHDPS, OsCold1, OsTubA2, OsEPSPS, OsALS-W548, OsACC, and OsALS-P171 in rice. Detailed implementation manners

[0059] The present invention will be further described in detail below in conjunction with the detailed implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0060] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0061] Unless otherwise specified, the quantitative tests in the following embodiments are all set with three repeated experiments, and the results are averaged.

[0062] The Gateway TM LR Clonase TM II enzyme is provided by Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is 11791020.

[0063] The vector pUC57 in the following embodiments is provided by GenScript Biotech Corporation, and the product number is SD1176.

[0064] pG3R23-PE3max-35C.5 in the following examples has been described in: Jiang, Y., et al. (2022) Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS. Mol Plant 15, 1646-1649. The biological material can be obtained from the applicant and is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0065] pL2R4-OsMLH1dn in the following examples has been described in: Jiang, Y., et al. (2022) Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS. Mol Plant 15, 1646-1649. The biological material can be obtained from the applicant and is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0066] pL4L3-Hyg2 in the following examples has been described in: Jiang, Y., et al. (2022) Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS. Mol Plant 15, 1646-1649. The biological material can be obtained from the applicant and is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0067] pG3H-840KK2 in the following examples has been described in: Jiang, Y., et al. (2022) Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS. Mol Plant 15, 1646-1649. The biological material can be obtained from the applicant, and it is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0068] Agrobacterium tumefaciens LBA4404 / pVS1-VIR2 in the following examples has been described in: Zhang, Q., et al. (2019) A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR / Cas Delivery in Maize. Plant Physiol 181, 1441-1448. The biological material can be obtained from the applicant, and it is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0069] Zhonghua 11 rice in the following examples has been described in: Jiang, Y., et al. (2022) Optimized prime editing efficiently generates glyphosate-resistant rice plants carrying homozygous TAP-IVS mutation in EPSPS. Mol Plant 15, 1646-1649. The biological material can be obtained from the applicant, and it is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0070] The primer-related sequences used in the examples of the present invention are shown in Table 1.

[0071] Table 1. Primer sequences for deep sequencing

[0072]

[0073]

[0074]

[0075] In the following embodiments, epegRNA is obtained by adding a reverse transcriptase template (rtT) containing new genetic information and a primer binding site (PBS) sequence to the 3' end of a single-stranded guide RNA (sgRNA), and simultaneously adding the evopreQ1 sequence that stabilizes pegRNA to the 3' end using a linker sequence. The sgRNA in epegRNA consists of a target sequence and an sgRNA scaffold sequence. The final structure of epegRNA is target-sgRNA scaffold–rtT–PBS–linker-evopreQ1. Among them, the target and rtT / PBS / Linker sequences need to be designed according to the target site to be edited. Since the PE3 strategy is used for editing in this experiment, each target also includes an additional guide RNA that cleaves the non-edited strand, which consists of a nicking sequence and an sgRNA scaffold sequence, and the nicking sequence needs to be designed according to the target site to be edited. The specific relevant sequences are shown in Table 2 below.

[0076] Table 2. epegRNA-related sequence information

[0077]

[0078]

[0079]

[0080] Example 1. Construction of the separated guide editing control vector 9s2V1

[0081] 1. Construction of the vector backbone:

[0082] a. Replace the fragment between BsaI of pG3H-840KK2 with synthetic fragment 1 (SEQ ID No: 11), and name it pG3H-KK840.

[0083] The structural description of the pG3H-KK840 vector is as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No: 11 between the BsaI cleavage sites of the starting vector pG3H-840KK2, while keeping other sequences of the vector pG3H-840KK2 unchanged.

[0084] b. Replace the fragment between XbaI and SacI of pG3R23-PE3max-35C.5 with the fragment between XbaI and SacI of pG3H-KK840 (SEQ ID No: 12) to obtain pG3R23-KK840.

[0085] The structure of the pG3R23-KK840 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No:12 between the XbaI and SacI restriction enzyme cleavage sites of the starting vector pG3R23-PE3max-35C.5, while keeping other sequences of the vector pG3R23-PE3max-35C.5 unchanged.

[0086] c. Replace the fragment between HindIII and SpeI of pG3R23-KK840 with the synthetic fragment 2 (SEQ ID No:13) digested with HindIII and SpeI, and name it pG3R23-KK840-dV3.1.

[0087] The structure of the pG3R23-KK840-dV3.1 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No:13 between the HindIII and SpeI restriction enzyme cleavage sites of the starting vector pG3R23-KK840, while keeping other sequences of the vector pG3R23-KK840 unchanged.

[0088] d. Synthesize RT2, name it the synthetic fragment 3 (SEQ ID No:14), and replace the fragment between XbaI and SacI of pL2R4-OsMLH1dn to obtain pL2R4-RT2.

[0089] The structure of the pL2R4-RT2 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No:14 between the XbaI and SacI restriction enzyme cleavage sites of the starting vector pL2R4-OsMLH1dn, while keeping other sequences of the vector pL2R4-OsMLH1dn unchanged.

[0090] e. Through Gateway TM LR Clonase TM II enzyme assembly steps: Assemble pG3R23-KK840-dV3.1 obtained in step c, pL2R4-RT2 obtained in step d, and pL4L3-Hyg2 to generate pG3H-9s2-dV3.1.

[0091] The specific assembly method is as follows: attR2 on the pG3R23-KK840-dV3.1 vector is recombinantly ligated with attL2 on the pL2R4-RT2 vector, attR4 on the pL2R4-RT2 vector is recombinantly ligated with attL4 on the pL4L3-Hyg2 vector, and attL3 on the pL4L3-Hyg2 vector is recombinantly ligated with attR3 on the pG3R23-KK840-dV3.1 vector.

[0092] 2. Construct a guide editing vector for targeting OsSPL14 to achieve the L292I mutation, while deleting 84 bp of OsSLR1 and with the PE protein being a codon-optimized one.

[0093] The synthesized fragment 4 (SEQ ID No: 15) digested by BsaI was inserted between the two BsaI sites of pG3H-9s2-dV3.1 obtained in step 1, and the positive clone obtained by transforming Escherichia coli was named E.coli / 9s2V1-SPL14-SLR1.

[0094] Extract the recombinant vector 9s2V1-SPL14-SLR1 from the above positive clone using a plasmid kit. The structure of the 9s2V1-SPL14-SLR1 vector is described as follows: It is a recombinant vector obtained by inserting the DNA fragment of SEQ ID No: 15 (the sequence after BsaI digestion) between the two BsaI sites of the starting vector pG3H-9s2-dV3.1, while keeping the other sequences of the vector pG3H-9s2-dV3.1 unchanged.

[0095] 3. The method for constructing guide editing vectors for respectively targeting the other 5 groups of double-gene targets to simultaneously achieve corresponding point mutations, insertions or deletions is similar to the construction method of 9s2V1-SPL14-SLR1 above, and recombinant vectors 9s2V1-NR2-NRT1.1B, 9s2V1-AK-DHDPS, 9s2V1-TubA2-Cold1, 9s2V1-TAP-W548, and 9s2V1-ACC-P171 are obtained.

[0096] The structures of the recombinant vectors 9s2V1-NR2-NRT1.1B, 9s2V1-AK-DHDPS, 9s2V1-TubA2-Cold1, 9s2V1-TAP-W548, and 9s2V1-ACC-P171 are described as follows: They are recombinant vectors obtained by respectively inserting the synthesized fragments 5 to 9 digested by BsaI (the nucleotide sequences are SEQ ID No: 16 - SEQ ID No: 20) between the two BsaI sites of the starting vector pG3H-9s2-dV3.1, while keeping the other sequences of the vector pG3H-9s2-dV3.1 unchanged.

[0097] Example 2. Construction of the 9s42 guide editing vector

[0098] 1. Construct the vector backbone:

[0099] a. Synthesize the 2xPE3RS fragment, named synthetic fragment 10 (SEQ ID No: 21), and after digestion with HindIII and SpeI, replace the fragment between HindIII and SpeI of pG3R23-KK840 obtained in step b to obtain pG3R23-KK840-dV3RS.

[0100] The structure of the pG3R23-KK840-dV3RS vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No: 21 between the XbaI and SacI restriction enzyme sites of the starting vector pG3R23-KK840, while keeping other sequences of the vector pG3R23-KK840 unchanged.

[0101] b. Synthesize the Csy4-RT2 fragment, named synthetic fragment 11 (SEQ ID No: 22), and replace the fragment between XbaI and SacI of pL2R4-OsMLH1dn to obtain pL2R4-Csy4-RT2.

[0102] The structure of the pL2R4-Csy4-RT2 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No: 22 between the XbaI and SacI restriction enzyme sites of the starting vector pL2R4-OsMLH1dn, while keeping other sequences of the vector pL2R4-OsMLH1dn unchanged.

[0103] c. Through Gateway TM LR Clonase TM II enzyme assembles pG3R23-KK840-dV3RS, pL4L3-Hyg2 and pL2R4-Csy4-RT2 to generate pG3H-9s42-dV3RS.

[0104] The specific assembly method is as follows: attR2 on the pG3R23-KK840-dV3RS vector is recombinantly ligated with attL2 on the pL2R4-Csy4-RT2 vector, attR4 on the pL2R4-Csy4-RT2 vector is recombinantly ligated with attL4 on the pL4L3-Hyg2 vector, and attL3 on the pL4L3-Hyg2 vector is recombinantly ligated with attR3 on the pG3R23-KK840-dV3RS vector.

[0105] 2. Construct a guide editing vector for targeting OsSPL14 to achieve the L292I mutation, while deleting 84bp of OsSLR1 and the PE protein has been codon-optimized

[0106] The synthetic fragment 12 (SEQ ID No: 23) digested with BsaI was inserted between the two BsaI sites of pG3H-9s42-dV3RS obtained in step 1, and the positive clone obtained by transforming Escherichia coli was named E.coli / 9s42-SPL14-SLR1.

[0107] The recombinant vector 9s42-SPL14-SLR1 in the above positive clone was extracted using a plasmid kit. The structure of the 9s42-SPL14-SLR1 vector is described as follows: It is a recombinant vector obtained by inserting the DNA fragment with the sequence of SEQ ID No: 23 digested with BsaI between the two BsaI sites of the starting vector pG3H-9s42-dV3RS, while keeping the other sequences of the vector pG3H-9s42-dV3RS unchanged.

[0108] 3. The method for constructing guide editing vectors for separately targeting the other 5 groups of double-gene targets and simultaneously achieving corresponding point mutations, insertions or deletions is similar to the construction method of the above 9s42-SPL14-SLR1, and the recombinant vectors 9s42-NR2-NRT1.1B, 9s42-AK-DHDPS, 9s42-TubA2-Cold1, 9s42-TAP-W548, 9s42-ACC-P171 were obtained.

[0109] The structures of the recombinant vectors 9s42-NR2-NRT1.1B, 9s42-AK-DHDPS, 9s42-TubA2-Cold1, 9s42-TAP-W548, 9s42-ACC-P171 are described as follows: They are recombinant vectors obtained by respectively inserting the synthetic fragments 13 to 17 (nucleotide sequences are SEQ ID No: 24 - SEQ ID No: 28) digested with BsaI between the two BsaI sites of the starting vector pG3H-9s42-dV3RS, while keeping the other sequences of the vector pG3H-9s42-dV3RS unchanged.

[0110] Example 3. Construction of 9s24 guide editing vector

[0111] 1. Construction of vector backbone

[0112] a. Synthesize the RT2-Csy4 fragment, named synthetic fragment 18 (SEQ ID No: 29), and replace the fragment between XbaI and SacI of pL2R4-OsMLH1dn to obtain pL2R4-RT2-Csy4.

[0113] The structure of the pL2R4-RT2-Csy4 vector is described as follows: It is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No:29 between the XbaI and SacI restriction enzyme sites of the starting vector pL2R4-OsMLH1dn, while keeping other sequences of the vector pL2R4-OsMLH1dn unchanged.

[0114] b. Through Gateway TM LR Clonase TM II enzyme pG3R23-KK840-dV3RS, pL4L3-Hyg2 and pL2R4-RT2-Csy4 to generate pG3H-9s24-dV3RS.

[0115] The specific assembly method is as follows: attR2 on the pG3R23-KK840-dV3RS vector is recombinantly ligated with attL2 on the pL2R4-RT2-Csy4 vector, attR4 on the pL2R4-RT2-Csy4 vector is recombinantly ligated with attL4 on the pL4L3-Hyg2 vector, and attL3 on the pL4L3-Hyg2 vector is recombinantly ligated with attR3 on the pG3R23-KK840-dV3RS vector.

[0116] 2. The method for constructing a prime editing vector for targeting OsSPL14 to achieve the L292I mutation and simultaneously delete 84 bp of OsSLR1 is the same as the method in Example 2. The synthesized fragment 12 (SEQ ID No:23) digested with BsaI is inserted between the two BsaI sites of pG3H-9s24-dV3RS obtained in step 1, and the positive clone obtained by transforming Escherichia coli is named E.coli / 9s24-SPL14-SLR1.

[0117] 3. The method for constructing prime editing vectors for respectively targeting another 5 groups of dual-gene targets and simultaneously achieving corresponding point mutations, insertions or deletions is similar to the construction method of 9s24-SPL14-SLR1 above, and recombinant vectors 9s24-NR2-NRT1.1B, 9s24-AK-DHDPS, 92s4-TubA2-Cold1, 9s24-TAP-W548, 92s4-ACC-P171 are obtained.

[0118] Recombinant vectors 9s24-NR2-NRT1.1B, 9s24-AK-DHDPS, 92s4-TubA2-Cold1, 9s24-TAP-W548, 92s4-ACC-P171: are recombinant vectors obtained by inserting synthetic fragments 13 to 17 (SEQ ID No: 24 - SEQ ID No: 28) digested by BsaI between the two BsaI sites of the starting vector pG3H-9s24-dV3RS, while keeping other sequences of the vector pG3H-9s24-dV3RS unchanged.

[0119] Example 4. Stable transformation of rice and testing of editing efficiency

[0120] 1. Respectively introduce the above control vector 9s2V1 and the improved prime editing vector 9s42 and 9s24 fused with Csy4 ( Figure 1 ) into the engineered Agrobacterium tumefaciens LBA4404 / pVS1-VIR2 containing the helper vector pVS1-VIR2 to form strains containing a ternary vector system. Use the above Agrobacterium strains to transform the rice variety Zhonghua 11, and screen transgenic lines using 50 mg / L hygromycin.

[0121] 2. DNA extraction from stably transformed positive plants. After collecting the leaves of transgenic positive lines corresponding to each vector, extract leaf DNA (30 μL) using the Tiangen kit method, and measure its concentration (30 - 80 ng / μL) using a NanoDrop ultra-micro spectrophotometer, and store at -20 °C.

[0122] 3. Amplicons sequencing analysis (taking the OsSPL14 gene as an example)

[0123] (1) Using the rice transgenic line genome as a template, perform PCR amplification of the fragment containing the target site with the SPL14-IDF / SPL14-IDR primers, and identify the product by 1% agarose gel electrophoresis;

[0124] (2) Using the SPL14-F / SPL14-R primers and the sequencing primers containing Barcode, perform a second round of PCR amplification with the above amplified product diluted 100 times as a template. Separate the PCR products by 2% agarose gel electrophoresis, and use the TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 to recover the target fragment from the gel, and send it to the company for amplicons sequencing analysis.

[0125] (3) After the sequencing is completed, split the raw data, use WT as a control, and compare and analyze the editing types and editing efficiencies of the products at the gene target site.

[0126] The amplification analysis steps for the remaining 11 gene targets (see Table 2 for details) were similar to the identification of the above-mentioned OsSPL14 gene.

[0127] When 100% of the deep sequencing reads of a certain strain represent only one mutant type with the desired editing, it is rated as a homozygous (Ho) strain; when a non-homozygous strain contains more than one mutant type with the required editing, it is respectively rated as a heterozygous (He) strain (≥45% desired editing) or a chimeric (Chi) strain (<45% desired editing); when the main mutant type in a chimeric strain is the Re by-product, it is rated as a Re strain; when the main mutant type in a chimeric strain is the Indel by-product, it is rated as an Indel strain; when the main mutant type in a chimeric strain is the Sc by-product, it is rated as an Sc strain. The mutation efficiency is calculated based on the ratio of the number of mutant strains to the total number of positive transgenic plants and classified. The editing efficiencies of three different dual-gene editing vectors are shown in Table 3 and Figure 2 as follows.

[0128] Table 3. Editing efficiencies of novel prime editors and controls at 12 important agronomic trait targets of 11 genes

[0129]

[0130]

[0131]

[0132] Compare the editing efficiencies of 3 PEs ( Figure 1 ) at 12 targets from 11 genes in transgenic rice, and evaluate the performance of sPE (9s2V1) without RT binding at 12 targets from 11 genes ( Figure 2 ; Figure 3 ). The results show that the Csy4-based split PE significantly improved the overall editing efficiency compared with the non-Csy4 split PE control (9s2V1), with improvement multiples of 3.62 (9s42) and 4.37 (9s24) respectively ( Figure 4 ).

[0133] These results indicate that the Csy4-based sPE provides a better choice in various applications compared with the sPE without RT binding. Since the fusion of Csy4 overall improves the editing efficiency of prime editing, this study lays a foundation for the future development of split PEs with higher editing efficiency.

[0134] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses, or improvements to the present invention, including changes made with conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. A fusion protein, which is a protein obtained by fusing the Csy4 nuclease with an amino acid sequence of SEQ ID No: 1 at one end of the reverse transcriptase RT.

2. The fusion protein according to claim 1, wherein: The Csy4 nuclease is fused to the N-terminus or C-terminus of the reverse transcriptase RT.

3. The fusion protein according to claim 2, wherein: The amino acid sequence of the fusion protein is SEQIDNo:2 or SEQ ID No:

3.

4. A nucleic acid molecule encoding the fusion protein according to any one of claims 1-3.

5. The nucleic acid molecule according to claim 3, characterized in that: The nucleic acid molecule is formed by fusing the encoding gene of the Csy4 nuclease and the encoding gene of the reverse transcriptase RT through a linker; The encoding gene of the Csy4 nuclease is any one of the following: (a1) A DNA molecule with a nucleotide sequence of SEQ ID No: 4 in the coding strand; (a2) A DNA molecule that hybridizes with the DNA molecule defined in (a1) under stringent conditions and encodes the polypeptide; (a3) A DNA molecule having a homology of more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% with the DNA sequence defined in (a1) or (a2) and encoding the polypeptide; The encoding gene of the reverse transcriptase RT is any one of the following: (b1) A DNA molecule with a nucleotide sequence of positions 664-2694 of SEQ ID No: 5 or SEQ ID No: 8 in the coding strand; (b2) A DNA molecule that hybridizes with the DNA molecule defined in (b1) under stringent conditions and encodes the same protein; (b3) A DNA molecule having a homology of more than 99%, more than 95%, more than 90%, more than 85%, or more than 80% with the DNA sequence defined in (b1) or (b2) and encoding the same protein.

6. A biological material related to the nucleic acid molecule according to claim 4 or 5, and the biological material is any one of the following: B1) An expression cassette containing the nucleic acid molecule according to claim 4 or 5; B2) A recombinant vector containing the nucleic acid molecule according to claim 4 or 5, or a recombinant vector containing the expression cassette described in B1); B3) A recombinant microorganism containing the nucleic acid molecule according to claim 4 or 5, or a recombinant microorganism containing the expression cassette described in B1), or a recombinant microorganism containing the recombinant vector described in B2); B4) A transgenic plant cell line containing the nucleic acid molecule according to claim 4 or 5, or a transgenic plant cell line containing the expression cassette described in B1); B6) A transgenic plant tissue containing the nucleic acid molecule according to claim 4 or 5, or a transgenic plant tissue containing the expression cassette described in B1); B7) A transgenic plant organ containing the nucleic acid molecule according to claim 4 or 5, or a transgenic plant organ containing the expression cassette described in B1).

7. Multigene-guided editing system, characterized in that : The prime editing system comprises the fusion protein according to any one of claims 1-3.

8. Use of the fusion protein according to any one of claims 1-3 or its related biological material in any of the following: 1) Use in the preparation of a gene prime editing system; 2) Use in improving gene editing efficiency; 3) Use in the preparation of disease treatment products; 4) Use in plant genetic improvement.

9. Use of the nucleic acid molecule according to claim 4 or 5 and its related biological materials in any of the following: 1) Use in the preparation of a gene-guided editing system; 2) Use in improving gene editing efficiency; 3) Use in the preparation of disease treatment products; 4) Use in plant genetic improvement.

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