Method for site-directed mutagenesis of pig mitochondrial genome and application
The precise point mutation of MT-ND1 and MT-ND5 in the pig mitochondrial genome was achieved through the DdCBE base editor, solving the problem of difficulty in editing the pig mitochondrial genome in the existing technology, and providing an efficient mitochondrial disease research model.
Patent Information
- Application Number
- CN202411860810.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-17
- Publication Date
- 2025-07-18
AI Technical Summary
The prior art is difficult to achieve site-directed mutations in the pig mitochondrial genome, which makes it difficult to build animal models of mitochondrial gene-related disease models. Especially because there are significant differences in the special structure of the mitochondrial genome and the DNA damage repair mechanism from the nuclear genome, the commonly used CRISPR/Cas system is difficult to deliver to the mitochondria.
DdCBE base editor, which combines mitochondrial targeting signals, transcription activator-like effectors (TALE) and cytosine deaminase (DddA) to achieve precise point mutations of MT-ND1 and MT-ND5 in the pig mitochondrial genome, and edits them in pig cells and embryos through specific DdCBE vectors.
The efficient and precise base substitution of MT-ND1 and MT-ND5 of the pig mitochondrial genome was achieved, and a stable mutant cell and embryo model was obtained, providing an ideal animal model for human mitochondrial disease research.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of gene editing, and particularly relates to a method and application for site-directed mutagenesis of porcine mitochondrial genomes. Background Art
[0002] In addition to nuclear genomes, the genetic material of human and animal cells also contains a relatively independent mitochondrial genome (mtDNA) in mitochondria, showing typical maternal inheritance characteristics. The mitochondrial genome is a circular DNA about 16 kb in length, containing 37 genes, which are responsible for encoding 13 subunit proteins of the respiratory chain complex in the inner mitochondrial membrane and are crucial for maintaining the normal functions of mitochondria and cells.
[0003] Mitochondrial genome variations are closely related to many human diseases. For example, more than 90 pathogenic mtDNA mutations have been identified clinically, covering all 37 mitochondrial genes. These mtDNA mutation-related diseases have complex phenotypes, high heterogeneity, unclear etiologies, and no effective treatment methods. Constructing suitable animal models is essential for the research of related diseases.
[0004] Commonly used mouse models have been proven to have great limitations, and most clinical translations have not been successful. The high similarity between pigs and humans in terms of body size, anatomical structure, physiology, metabolism, etc. makes pigs an ideal model animal for studying human diseases. Site-directed mutagenesis of the porcine mitochondrial genome to simulate pathogenic mutations in clinical patients can provide an ideal animal model for the mechanism research and treatment method development of diseases.
[0005] In recent years, the rapid development of gene editing technologies has made the genetic modification of nuclear genomes efficient. Nuclease tools represented by CRISPR / Cas can perform site-directed gene knockout or knock-in; base editors derived from CRISPR / Cas can achieve site-directed precise base mutations. These gene editing tools have been widely used in the editing of porcine nuclear genomes to construct gene-modified pig models. However, due to the special structure of mitochondria where the mitochondrial genome is located and the significant differences in the DNA damage repair mechanism of the mitochondrial genome from that of the nuclear genome, the tools widely used for nuclear genome editing are difficult to be used for mitochondrial genome editing, and there are huge challenges in site-directed editing of the porcine mitochondrial genome. Due to the special structure of mitochondria affecting the entry of guide RNA (gRNA), these gene modification tools based on the CRISRP / Cas system are difficult to enter mitochondria.
[0006] Therefore, for the research of mtDNA mutation-related diseases, there is an urgent need to provide new mitochondrial gene-modified pig models. Summary of the Invention
[0007] The present invention provides a DdCBE vector targeting porcine mitochondrial genes MT-ND1 and MT-ND5, and a method for obtaining cells and embryos with precise point mutations MT-ND1 (m.G4505A) and MT-ND5 (m.G14111A) of porcine mitochondrial genes using the DdCBE vector.
[0008] In a first aspect of the present invention, there is provided a base editor for site-directed mutagenesis of porcine MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A), which is a combination of base editor 1 and base editor 2. Base editor 1 has a structure of formula I, and base editor 2 has a structure of formula II:
[0009] TALE Left-Z1-Z2-Z3-Z4-Z5(I)
[0010] TALE Right-Z1’-Z2’-Z3’-Z4’-Z5’(II)
[0011] In the formula,
[0012] Z1 and Z1’ are mitochondrial targeting signals MTS;
[0013] Z2 and Z2’ are a pair of TALE recognition sequences for porcine MT-ND1 and / or MT-ND5;
[0014] Z3 and Z3’ are a pair of split cytosine deaminases DddA;
[0015] Z4 and Z4’ are uracil glycosylase inhibitor UGI;
[0016] Z5 and Z5’ are fluorescent reporter proteins;
[0017] The "-" is none or a linker sequence.
[0018] In another preferred example, Z1 is SOD2 MTS, and the sequence of SOD2 MTS is as shown in SEQ ID NO:1.
[0019] In another preferred example, Z1’ is COX8A MTS, and the sequence of COX8A MTS is as shown in SEQ ID NO:2.
[0020] In another preferred example, the TALE recognition sequence is as shown in SEQ ID NO:3.
[0021] In another preferred example, Z2 and Z2’ are a pair of TALE recognition sequences for MT-ND1.
[0022] In another preferred example, the target site specific sequence recognized by Z2 is as shown in SEQ ID NO:12, and the target site specific sequence recognized by Z2’ is as shown in SEQ ID NO:13.
[0023] In another preferred example, Z2 and Z2’ are a pair of TALE recognition sequences of MT-ND5.
[0024] In another preferred example, the target site specific sequence recognized by Z2 is as shown in SEQ ID NO:14, and the target site specific sequence recognized by Z2’ is as shown in SEQ ID NO:15.
[0025] In another preferred example, the sequence of cytosine deaminase DddA is as shown in SEQ ID NO:4
[0026] In another preferred example, the split cytosine deaminase DddA includes: DddA split at the G1397 breakpoint and DddA split at the G1333 breakpoint.
[0027] In another preferred example, Z3 is G1333-N and Z3’ is G1333-C.
[0028] In another preferred example, the G1333-N sequence is as shown in SEQ ID NO:24.
[0029] In another preferred example, the G1333-C sequence is as shown in SEQ ID NO:25.
[0030] In another preferred example, the sequence of UGI is as shown in SEQ ID NO:5.
[0031] In another preferred example, Z5 and Z5’ are non-fusion expressed fluorescent reporter proteins linked by a 2A cleavage peptide.
[0032] In another preferred example, the sequence of the 2A cleavage peptide is as shown in SEQ ID NO:6.
[0033] In another preferred example, Z5 is an EGFP sequence linked by a 2A cleavage peptide.
[0034] In another preferred example, the sequence of EGFP is as shown in SEQ ID NO:7.
[0035] In another preferred example, Z5’ is an mCherry sequence linked by a 2A cleavage peptide.
[0036] In another preferred example, the sequence of mCherry is as shown in SEQ ID NO:8.
[0037] In another preferred example, a base editor for site-directed mutagenesis of porcine MT-ND1 (m.G4505A) is provided. The base editor is a combination of base editor 1 and base editor 2. The sequence of base editor 1 is shown in SEQ ID NO:20, and the sequence of base editor 2 is shown in SEQ ID NO:21.
[0038] In another preferred example, a base editor for site-directed mutagenesis of porcine MT-ND5 (m.G14111A) is provided. The base editor is a combination of base editor 1 and base editor 2. The sequence of base editor 1 is shown in SEQ ID NO:22, and the sequence of base editor 2 is shown in SEQ ID NO:23.
[0039] In a second aspect of the present invention, a polynucleotide is provided, which encodes the base editor described in the first aspect of the present invention.
[0040] In another preferred example, the polynucleotide is DNA, mRNA, cDNA, or a combination thereof.
[0041] In another preferred example, the polynucleotide is mRNA.
[0042] In another preferred example, the RNA is subjected to capping and tailing treatments.
[0043] In another preferred example, the polynucleotide includes a first polynucleotide molecule and / or a second polynucleotide molecule. The first polynucleotide molecule encodes the base editor for site-directed mutagenesis of porcine MT-ND1 (m.G4505A) described in the first aspect of the present invention, and the second polynucleotide molecule encodes the base editor for site-directed mutagenesis of MT-ND5 (m.G14111A) described in the first aspect of the present invention.
[0044] In a third aspect of the present invention, a vector is provided, which contains the polynucleotide described in the second aspect of the present invention.
[0045] In another preferred example, the vector includes a first vector component and / or a second vector component. The first vector component contains the first polynucleotide molecule in the polynucleotide described in the second aspect of the present invention, and the second vector component includes the second polynucleotide molecule in the polynucleotide described in the second aspect of the present invention.
[0046] In a fourth aspect of the present invention, a porcine cell is provided, which contains the vector described in the third aspect of the present invention.
[0047] In another preferred example, the porcine cell is selected from: porcine embryonic kidney cell line PK-15 or porcine fetal fibroblasts.
[0048] In another preferred example, the porcine cells are porcine fetal fibroblasts.
[0049] In the fifth aspect of the present invention, a method for constructing MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A) site-directed mutant porcine cells is provided, comprising the steps of:
[0050] (S1) Transfecting the vector described in the third aspect of the present invention into porcine cells; and
[0051] (S2) Picking cell monoclonal colonies and identifying the site-directed mutation effect, thereby obtaining MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A) site-directed mutant porcine cells.
[0052] In another preferred example, the transfection time is ≥48 h, preferably ≥96 h.
[0053] In another preferred example, the transfection time is 96 h.
[0054] In another preferred example, the identification refers to identification by Sanger sequencing.
[0055] In the sixth aspect of the present invention, the use of the base editor described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, the vector described in the third aspect of the present invention, or the porcine cells described in the fourth aspect of the present invention is provided for preparing experimental reagents for generating porcine embryos and / or porcine models with site-directed mutations in mitochondrial genes.
[0056] In another preferred example, the mitochondrial site-directed mutation is MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A) site-directed mutation.
[0057] In the seventh aspect of the present invention, a method for preparing MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A) site-directed mutant porcine embryos is provided, comprising the following steps:
[0058] (M1) Providing mRNA encoding the base editor described in the first aspect of the present invention;
[0059] (M2) Introducing the mRNA into porcine embryos, thereby obtaining MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A) site-directed mutant porcine embryos.
[0060] In another preferred example, before step (M1), there is also step (M0): fusing donor cells and enucleated mature porcine oocytes to obtain porcine embryos.
[0061] In another preferred example, the donor cells are porcine fetal fibroblasts.
[0062] In another preferred example, the porcine embryo is an activated porcine embryo.
[0063] In another preferred example, the introduction method is microinjection.
[0064] In another preferred example, the method is carried out ex vivo.
[0065] In another preferred example, the method includes step (M3): transplanting the porcine embryo obtained in step (M2) into a surrogate sow, thereby obtaining an MT-ND1 C10T and / or MT-ND5 C9T site-directed mutagenesis porcine model.
[0066] In another preferred example, the method is a method for non-diagnostic and non-therapeutic purposes.
[0067] In another preferred example, the method is a method for scientific research purposes.
[0068] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings
[0069] Figure 1 Shows the technical process of site-directed mutagenesis of the porcine mitochondrial genome.
[0070] Figure 2 Shows the schematic diagram of the components of the DdCBE base editor. Figure 2 A shows the schematic diagram of the structure of the mitochondrial base editor DdCBE; Figure 2 B shows the schematic diagram of the structures of four pairs of DdCBE respectively; Figure 2 C shows the schematic diagram of four pairs of DdCBE combined into a complete and active deaminase.
[0071] Figure 3 Shows the schematic diagram of DdCBE specifically targeting porcine MT-ND1 and MT-ND5. Figure 3 A shows the human pathogenic mtDNA mutation MT-ND1 and the corresponding mutation of the porcine mitochondrial gene MT-ND1; Figure 3 B shows the human pathogenic mtDNA mutation MT-ND5 and the corresponding mutation of the porcine mitochondrial gene MT-ND5; Figure 3 C shows the design of the target-site specific DdCBE vector for MT-ND1; Figure 3 D shows the design of the target-site specific DdCBE vector for MT-ND5.
[0072] Figure 4Shows the efficiency of DdCBE editing of MT-ND1 and MT-ND5 sites at the porcine cell level. Figure 4 A shows the editing efficiency of different DdCBE combinations for the base C at the target site of the MT-ND1 site; Figure 4 B shows the editing efficiency of different DdCBE combinations for the base C at the target site of the MT-ND5 site.
[0073] Figure 5 Shows the mutation levels of the MT-ND1 and MT-ND5 target sites in porcine cell lines carrying mutations. Figure 5 A shows the mutation ratio of G4505A (C10) in 13 mutant cell lines obtained for the MT-ND1 site; Figure 5 B shows the mutation ratio of G14111A (C9) in 18 mutant cell lines obtained for the MT-ND5 site; Figure 5 C shows that the ratio of the target mutation can be stably maintained after long-term culture of the mutant cell line.
[0074] Figure 6 Shows the flow chart of constructing a porcine model by mitochondrial base editing of porcine embryos.
[0075] Figure 7 Shows the gene editing efficiency of porcine embryo mitochondria, where the target base is framed by a dotted line. Figure 7 A shows the average mutation frequency of the target base (C10) at the MT-ND1 site; Figure 7 B shows the average mutation frequency of the target base (C9) at the MT-ND5 site. Detailed implementation mode
[0076] Through extensive and in-depth research, the present inventors unexpectedly discovered for the first time a base editor DdCBE that can achieve precise point mutations MT-ND1 (m.G4505A) and MT-ND5 (m.G14111A) in porcine mitochondrial genes by selecting appropriate TALE sequence target sites, optimizing the DddA splitting strategy, and the orientation of the DddA half-body and TALE fusion, and provided a method and its application for introducing MT-ND1 (m.G4505A) and MT-ND5 (m.G14111A) into the mitochondrial genomes of porcine cells and embryos, providing a strategic approach guarantee for constructing mtDNA mutant porcine models in the future. The present invention was completed on this basis.
[0077] Terms
[0078] To make the present disclosure easier to understand, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.
[0079] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the related listed items.
[0080] As used herein, "porcine MT-ND1 (m.G4505A)" and "porcine mitochondrial gene MT-ND1 (m.G4505A)" are used interchangeably and both refer to Figure 3 the site-directed mutation of porcine mitochondrial gene MT-ND1 shown in C, i.e., the G base at position 4505 of porcine mitochondrial gene MT-ND1 is mutated to an A base. In a specific embodiment, porcine mitochondrial gene MT-ND1 (m.G4505A) refers to the C Figure 4 base mutated to a T base as shown in A 10 base mutation.
[0081] As used herein, "porcine MT-ND5 (m.G14111A)" and "porcine mitochondrial gene MT-ND5 (m.G14111A)" are used interchangeably and both refer to Figure 3 the site-directed mutation of porcine mitochondrial gene MT-ND5 shown in D, i.e., the G base at position 14111 of porcine mitochondrial gene MT-ND5 is mutated to an A base. In a specific embodiment, porcine mitochondrial gene MT-ND5 (m.G14111A) refers to the C9 Figure 4 base mutated to a T base as shown in B.
[0082] As used herein, "split cytosine deaminase DddA", "split DddA", "DddA half" are used interchangeably and all include the two DddA halves resulting from the split of DddA at the G1397 breakpoint and the two DddA halves resulting from the split at the G1333 breakpoint.
[0083] As used herein, from the amino acid sequence of DddA, GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGG / PTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEG\AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQ ID NO:4, where the " / " marks the G1333 cleavage point and the "\" marks the G1397 cleavage point), the sequences of G1333-N, G1333-C, G1397-N, and G1397-C can be obtained. For example, the amino acid sequence corresponding to G1333-N, DddA(G1333-N): GSYALGPYQISAPQLPAYNGQTVGTFYYVN DAGGLESKVFSSGG (SEQ ID NO:24). Another example, the amino acid sequence corresponding to G1333C, DddA(G1333-C): PTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCV NMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQ ID NO:25).
[0084] In the present invention, the "TALE Left" refers to the TALE sequence of the strand where the target base C of the target sequence is located for recognition.
[0085] In the present invention, the "TALE Right" refers to the TALE sequence of the strand where the target base G of the target sequence is located for recognition.
[0086] Mitochondrial genome
[0087] Mitochondrial DNA (mtDNA), a multi-copy circular DNA present in the mitochondrial matrix, is the genetic material outside the cell nucleus.
[0088] Nuclease systems such as gene editing tools ZFN, TALEN, and CRISPR / Cas9, and CRISPR / Cas-derived base editors have been widely used for pig genome modification, but are only applicable to the editing of nuclear genomes. The CRISPR / Cas system requires gRNA, but it is difficult to deliver gRNA into mitochondria, making it difficult to use this system for pig mitochondrial genome editing. Although ZFN and TALEN do not require RNA and can be delivered into mitochondria by adding mitochondrial targeting signals, they can only cleave circular mitochondrial genomes and cannot achieve site-directed mutagenesis.
[0089] DdCBE base editor
[0090] Due to the mitochondrial genome being located within the special structure of mitochondria and the significant differences in the DNA damage repair mechanism of the mitochondrial genome compared to the nuclear genome, tools that are widely used for nuclear genome editing are difficult to apply to mitochondrial genome editing, presenting a huge challenge for site-directed editing of the porcine mitochondrial genome.
[0091] RNA-free zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), by adding an additional mitochondrial targeting signal, can be used for the cleavage of the mitochondrial genome but cannot be used to generate site-directed mutations, thus having relatively limited applications in porcine mitochondrial genome editing.
[0092] DdCBE does not contain an RNA component and is a cytosine editor that can be delivered into mitochondria to achieve the conversion of base C to T at the target site of mitochondrial genes.
[0093] The base editor of the present invention
[0094] The base editor of the present invention refers to the mitochondrial base editor (DddA-derived cytosine base editor, DdCBE) constructed by fusing a mitochondrial targeting signal, a transcription activator-like effector (TALE), and a deaminase. The DdCBE can solve the problem of delivering editing tools into porcine mitochondria, can be targeted and delivered into porcine mitochondria, and uses a cytosine deaminase for efficient and precise base substitution at MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A) of the porcine mitochondrial genome, enabling site-directed mutations of porcine mitochondrial genes.
[0095] Specifically, the base editor of the present invention refers to the base editor for site-directed mutation of MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A) of the porcine mitochondrial genome, and the base editor is the base editor formed by combining the base editor components as shown in Figure 2 Figure B in the manner shown in Figure 2 Figure C.
[0096] In another preferred example, the structure of the base editor is as shown in Figure 2 (c) in Figure C.
[0097] Therefore, in the present invention, based on the DdCBE, a method for site-directed editing of the porcine mitochondrial genome can be established, laying a foundation for constructing a porcine model of human mitochondrial diseases for disease research.
[0098] The method of the present invention
[0099] The method of the present invention refers to the present invention's strategy for the current lack of pig mitochondrial gene site-directed mutagenesis technology. By means of the mitochondrial base editor DdCBE, it simulates human pathogenic mtDNA mutations in the pig mitochondrial genome and establishes a technical strategy for pig mitochondrial gene site-directed mutagenesis. The technical strategy includes editing at the levels of pig somatic cells and embryos. Specifically, the method of the present invention is as Figure 1 shown, to open up a technical path for finally constructing an mtDNA mutant pig model.
[0100] The main advantages of the present invention include:
[0101] (a) The present invention provides DdCBE vectors for achieving precise point mutations MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A) in pig mitochondrial genes. The editing efficiency of the vectors for the target C base reaches about 25%, and there is almost no editing of non-target C bases in the target sequence region.
[0102] (b) The present invention provides pig cells with MT-ND1 (m.G4505A) and MT-ND5 (m.G14111A) mutations. The proportion of target base mutations in the pig cells is high, and they can remain stable after 23 days of subculture.
[0103] (c) The present invention provides methods for generating pig cells with MT-ND1 (m.G4505A) and MT-ND5 (m.G14111A) mutations and pig embryos with MT-ND1 (m.G4505A) and MT-ND5 (m.G14111A) mutations. The methods have a high mutation rate for target bases and a low off-target rate, so as to achieve efficient and precise pig mitochondrial gene base editing, and provide a model for the research on corresponding human pathogenic mtDNA mutations MT-ND1 (m.G3890A) and MT-ND5 (m.G13513A).
[0104] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions, such as the conditions described by Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight percentage and weight parts.
[0105] Example 1: Design of specific DdCBE vectors targeting pig MT-ND1 and MT-ND5 gene loci
[0106] As Figure 2As shown in A, the mitochondrial base editor DdCBE mainly consists of a mitochondrial targeting signal (MTS), a transcription activator-like effector (TALE) recognition module, a cytosine deaminase (DddA), and a uracil glycosylase inhibitor (UGI). In addition, a non-fused expressed fluorescent reporter protein (EGFP or mCherry) linked by a 2A cleavage peptide is added at the end, which can be used as a screening tag.
[0107] Among them, the complete DddA is split into two inactive halves, which are respectively fused with a TALE module. Only when this pair of TALEs binds to adjacent target sites, the DddA halves (DddAhalf) combine into a complete active deaminase, which mutates the target base C at the target site to T.
[0108] Since DddA has two effective splitting strategies (the G1397 breakpoint and the G1333 breakpoint), and there are two directions for the fusion of the split DddA halves with TALE (for example, for the G1397 breakpoint, G1397-N is fused with the left TALE, and G1397-C is fused with the right TALE module; or G1397-N is fused with the right TALE, and G1397-C is fused with the left TALE module). As Figure 2 shown in B, the components of the four pairs of DdCBE are respectively:
[0109] Left-G1397-N: SOD2 MTS-TALE-linker1-DddA(G1397-N)-linker2-UGI-linker3-2A-EGFP;
[0110] Right-G1397-C: COX8A MTS-TALE-linker1-DddA(G1397-C)-linker2-UGI-linker3-2A-EGFP;
[0111] Left-G1397-C: SOD2 MTS-TALE-linker1-DddA(G1397-C)-linker2-UGI-linker3-2A-EGFP;
[0112] Right-G1397-N: COX8A MTS-TALE-linker1-DddA(G1397-N)-linker2-UGI-linker3-2A-EGFP;
[0113] Left-G1333-N: SOD2 MTS-TALE-linker1-DddA(G1333-N)-linker2-UGI-linker3-2A-EGFP;
[0114] Right-G1333-C: COX8A MTS-TALE-linker1-DddA(G1333-C)-linker2-UGI-linker3-2A-EGFP;
[0115] Left-G1333-C: SOD2 MTS-TALE-linker1-DddA(G1333-C)-linker2-UGI-linker3-2A-EGFP;
[0116] Right-G1333-N: COX8A MTS-TALE-linker1-DddA(G1333-N)-linker2-UGI-linker3-2A-EGFP.
[0117] Among them,
[0118] The sequence of SOD2 MTS is as follows:
[0119] LSRAVCGTSRQLAPVLGYLGSRQKHSLPD (SEQ ID NO:1).
[0120] The sequence of COX8A MTS is as follows:
[0121] SVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO:2).
[0122] The sequence of TALE is as follows:
[0123] GTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAA LGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDT GQLLKIAKRGGVTAVEAVHAWRNALTGAPLN-SIVAQLSRPDPALAALTNDHLVAL ACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVA (SEQ ID NO:3, where the target site-specific sequence is inserted at the position marked by "-").
[0124] The complete protein sequence of DddA is as follows:
[0125] GSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGG / PTPYPNYA NAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPE G\AIPVKRGATGETKVFTGNSNSPKSPTKGGC (SEQ ID NO:4, where the breakpoint of G1333 is marked by " / ", and the breakpoint of G1397 is marked by "\").
[0126] The sequence of UGI is as follows:
[0127] TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML (SEQ ID NO:5).
[0128] The sequence of 2A is: EGRGSLLTCGDVEENPGP (SEQ ID NO:6).
[0129] The sequence of EGFP is as follows:
[0130] VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO:7).
[0131] The sequence of mCherry is as follows:
[0132] VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK (SEQ ID NO:8).
[0133] The sequence of Linker1 is: GS (SEQ ID NO:9).
[0134] The sequence of Linker2 is: SGGS (SEQ ID NO:10).
[0135] The sequence of Linker3 is: DICG (SEQ ID NO:11).
[0136] Therefore, for one target site, four pairs of DdCBE efficiencies need to be constructed and tested. The schematic diagram of the four pairs of DdCBEs assembled into a complete active deaminase is shown in Figure 2 Figure C.
[0137] Specifically, as shown in Figure 3 Figure A and Figure 3 Figure B, the human pathogenic mtDNA mutations MT-ND1 (m.G3890A) and MT-ND5 (m.G13513A) correspond to the MT-ND1 (m.G4505A) and MT-ND5 (m.G14111A) mutations of the porcine mitochondrial genome, respectively. The target site sequence information of the mitochondrial genome was obtained from the porcine genome (Sscrofa11.1) in NCBI. As shown in Figure 3 Figure C and Figure 3D, target-site specific DdCBE vectors were designed. Capital letters represent the recognition and binding sequences of TALE at the target site, and the bold letters represent the target bases for site-directed mutagenesis.
[0138] The target site sequences recognized by TALE in porcine mitochondrial MT-ND1 were designed as: 5’-ccttctgtaaggtcgaac-3’ (SEQ ID NO:12, ND1-TALE-Left) and 5’-ttatctcaaccctagcag-3’ (SEQ ID NO:13, ND1-TALE-Right); the target site sequences recognized by TALE in MT-ND5 were designed as: 5’-gctgcttcaatgat-3’ (SEQID NO:14, ND5-TALE-Left) and 5’-atctcacaggattct-3’ (SEQ ID NO:15, ND5-TALE-Right).
[0139] For these two groups of target sites, four pairs of DdCBE vector combinations were designed and constructed respectively: Left-G1397-N / Right-G1397-C, Left-G1397-C / Right-G1397-N, Left-G1333-N / Right-G1333-C, and Left-G1333-C / Right-G1333-N.
[0140] Using the TALE module library as a template, each repeat unit containing Esp3I recognition sites on both sides was amplified by PCR, and then each TALE repeat unit was assembled into the DdCBE basic expression plasmid by the "Golden Gate" cloning method to obtain a target site-specific DdCBE vector.
[0141] Example 2: Testing the editing efficiency and specificity of different DdCBE combinations
[0142] The target base editing efficiency of the designed different DdCBE combinations mediated on the target sites of porcine mitochondrial genes MT-ND1 and MT-ND5 was tested by transfecting cells.
[0143] First, porcine fetal fibroblasts (PFF) were cultured in PFF medium (DMEM medium, a high-glucose modified Eagle's medium supplemented with 15% v / v fetal bovine serum) in an incubator at 38.5 °C and 5% CO2. When the confluence reached 80%, the PFF cells were digested and collected.
[0144] Secondly, the corresponding DdCBE expression plasmid combinations were transfected into PFF cells using the Invitrogen TM Neon TM transfection system. Each group of cells was 2.5×10 5Each group received 5 μg of each of the two expression plasmids (Left and Right) of DdCBE. The electroporation parameters were: 1350 V, 30 ms, and 1 pulse. The transfected cells were seeded in a 24-well plate, fresh PFF medium was added, and the cells were cultured in an incubator at 38.5 °C and 5% CO2 for 48 h or 96 h to obtain transfected PFF cells. The transfected PFF cells were collected, and the total genome was extracted. Using the extracted total genome as a template, PCR was performed to amplify the fragment within the target site range (PCR product).
[0145] PCR reaction was carried out using 2×Rapid Taq Master Mix. The PCR reaction system was: 15 μL of 2×Rapid Taq premix, 0.3 μL of the forward primer with a concentration of 10 μM, 0.3 μL of the reverse primer with a concentration of 10 μM, 1 μL of the extracted genomic template, and 13.4 μL of ultrapure water, with a total volume of 30 μL. The PCR reaction conditions were: 95 °C for 3 min; [95 °C for 15 s, 55 °C for 15 s, 72 °C for 10 s] for 38 cycles; 72 °C for 5 min; hold at 12 °C. The PCR primers were as follows:
[0146] MT-ND1 locus:
[0147] Forward primer: gaccaggcacatcctcaatct (SEQ ID NO:16);
[0148] Reverse primer: ggtcgtatcggaatcgtggg (SEQ ID NO:17).
[0149] MT-ND5 locus:
[0150] Forward primer: agtgacaatcggcatcaacc (SEQ ID NO:18);
[0151] Reverse primer: aagataattcgagtgctgtaggc (SEQ ID NO:19).
[0152] The obtained PCR products were subjected to Sanger sequencing (first-generation gene sequencing). According to the sequencing peak map, the EditR tool was used to evaluate the efficiency of the conversion of the target base C to T at the target site, and the DdCBE combination that effectively edits the target site was determined. For a specific target site, different DdCBE combinations need to be tested to screen for the optimal design.
[0153] The results are as Figure 4 shown. Different DdCBE combinations have different editing efficiencies and specificities for multiple bases C at the target site. Moreover, the editing efficiency at 96 h after plasmid transfection is slightly higher than that at 48 h, indicating that appropriately extending the DdCBE editing time can improve the efficiency to a certain extent.
[0154] For the MT-ND1 locus, the editing efficiencies of different DdCBE combinations on multiple bases C at the target site are as Figure 4 shown in A. The results show that the DdCBE combination Left-G1333-N+Right-G1333-C has the highest editing efficiency (about 25%) for the target base (C10), and hardly edits other non-target bases (C8 and C9), with high specificity, which is the optimal DdCBE design for this locus.
[0155] For the MT-ND5 locus, the editing efficiencies of different DdCBE combinations on multiple bases C at the target site are as Figure 4 shown in B. The results show that the DdCBE combination Left-G1333-N+Right-G1333-C has the highest editing efficiency (about 25%) for the target base (C9), and hardly edits non-target bases (C11 and C12), which is the optimal DdCBE design for this locus.
[0156] Therefore, the DdCBE combinations ND1-Left-G1333-N+ND1-Right-G1333-C and ND5-Left-G1333-N+ND5-Right-G1333-C are the optimal combinations for site-directed mutagenesis of porcine mitochondrial genes MT-ND1 and MT-ND5, respectively.
[0157] The sequence of ND1-Left-G1333-N is as follows:
[0158] LSRAVCGTSRQLAPVLGYLGSRQKHSLPDGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN LTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQAL ETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLP VLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGL TPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQDHGLTPDQVVAI ASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQ ALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRL LPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAH GLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVV AIASHDGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLDICGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO:20, the underlined sequence is the target site-specific sequence).
[0159] The sequence of ND1-Right-G1333-C is shown as follows:
[0160] SVLTPLLLRGLTGSARRLPVPRAKIHSLGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAP LNLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALE TVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPV LCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLT PDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQA LETVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLL PVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHG LTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVA IASNNGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLDICGEGRGSLLTCGDVEENPGPVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK (SEQ ID NO:21, the underlined sequence is the target site specific sequence).
[0161] The sequence of ND5-Left-G1333-N is shown as follows:
[0162] LSRAVCGTSRQLAPVLGYLGSRQKHSLPDGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAPLN LTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQAL ETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLP VLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGL TPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAI ASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQ ALETVQRLLPVLCQDHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRL LPVLCQAHGLTPDQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSGSYALGPYQISAPQLPAYNGQTVGTFYYVNDAGGLESKVFSSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLDICGEGRGSLLTCGDVEENPGPVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO:22, the underlined sequence is the target site specific sequence).
[0163] The sequence of ND5-Right-G1333-C is shown below:
[0164] SVLTPLLLRGLTGSARRLPVPRAKIHSLGTVDLRTLGYSQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHPAALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEALLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRNALTGAP LNLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALE TVQRLLPVLCQDHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLLPV LCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQAHGLT PDQVVAIASHDGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQALETVQRLLPVLCQDHGLTPDQVVAIA SNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNNGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNIGGKQA LETVQRLLPVLCQDHGLTPDQVVAIASNGGGKQALETVQRLLPVLCQAHGLTPDQVVAIASNGGGKQALETVQRLL PVLCQAHGLTPDQVVAIASHDGGKQALETVQRLLPVLCQDHGLTPDQVVAIASNGGGRPALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDAVKKGLPHAPALIKRTNRRIPERTSHRVAGSPTPYPNYANAGHVEGQSALFMRDNGISEGLVFHNNPEGTCGFCVNMTETLLPENAKMTVVPPEGAIPVKRGATGETKVFTGNSNSPKSPTKGGCSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLDICGEGRGSLLTCGDVEENPGPVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK (SEQ ID NO:23, the underlined sequence is the target site specific sequence).
[0165] Example 3: Editing porcine somatic cell mitochondrial genes using DdCBE to obtain cell lines carrying mutations
[0166] In this example, Left-G1333-N + Right-G1333-C was used to edit porcine mitochondrial genes MT-ND1 and MT-ND5 to obtain porcine cells carrying MT-ND1 or MT-ND5 mutations.
[0167] Take 5 μg each of the ND1-Left-G1333-N and ND1-Right-G1333-C expression vectors and mix them to obtain the MT-ND1 site mixed plasmid; take 5 μg each of the ND5-Left-G1333-N and ND5-Right-G1333-C expression vectors and mix them to obtain the MT-ND5 site mixed plasmid. Use Invitrogen TM Neon TM transfection system to transfect the MT-ND1 site mixed plasmid or the MT-ND5 site mixed plasmid into porcine somatic cells PK-15 (2.5×10 5 cells), and the electroporation parameters are: 1500 V, 30 ms and 1 pulse to obtain the electroporated cells.
[0168] The electroporated cells were plated in a 12-well plate using a high-glucose DMEM medium supplemented with 15% (v / v) fetal bovine serum, and cultured in an incubator at 38.5°C and 5% CO2 (if not otherwise specified, the culture conditions remain unchanged below) for 2 days. Then, cells positive for the fluorescent reporter protein were sorted using a BD Aria IIU flow cytometer. Single cells were picked up under a microscope and continued to be cultured in a 96-well plate until they grew into clones, obtaining clones derived from single cells. The resulting clones were picked up and continued to be cultured in a 48-well plate until the cells filled the culture wells.
[0169] Three quarters of the cells were plated on a 24-well plate and cultured until the wells were full. The remaining quarter of the cells were lysed, and their genome was extracted as a template. The sequence fragments containing the target sites were amplified by PCR, and Sanger sequencing was used to identify whether the target bases were successfully edited at the MT-ND1 and MT-ND5 target sites. The operation was the same as that for the DdCBE combination test. The mutant cell lines identified as successfully edited were frozen.
[0170] Cell lines with different MT-ND1 mutation ratios and cell lines with different MT-ND5 mutation ratios are shown in Figure 2. Figure 5 A and Figure 5 As shown in B.
[0171] like Figure 5 As shown in A, for the MT-ND1 site, the G4505A (C10) mutation ratios of the 13 mutant cell lines obtained ranged from 23% to 90%, among which more than 84% of the mutant strains had a G4505A (C10) mutation ratio greater than 40%.
[0172] like Figure 5 As shown in B, for the MT-ND5 site, the G14111A (C9) mutation ratio of the 18 mutant cell lines obtained ranged from 15% to 90%, among which more than 72% of the mutant strains had a G14111A (C9) mutation ratio greater than 40%.
[0173] And, if Figure 5 As shown in C, after long-term culture (23 days), the proportion of target mutations in these mutant cell lines can still be stably maintained. Therefore, these mutant cell lines can be used as cell models for studying mtDNA mutations and for the study of related pathogenic mtDNA mutations.
[0174] Example 4: Editing pig embryo mitochondrial genes using DdCBE
[0175] The maternal inheritance characteristics of the mitochondrial genome make it impossible to use the conventional strategy of first editing somatic cells and then constructing a gene-edited pig model through somatic cell nuclear transfer to construct a mitochondrial gene-edited pig model. Therefore, in this example, a new strategy is established. As Figure 6 shown, recombinant embryos can be obtained by somatic cell nuclear transfer using wild-type somatic cells, or fertilized eggs can be used to microinject the mRNA of DdCBE at the 1-cell stage to obtain mitochondria gene-edited embryos, which can then be transplanted into surrogate sows to obtain a mitochondrial gene-edited pig model.
[0176] First, the mRNAs of MT-ND1 and MT-ND5 site-specific DdCBE are obtained by in vitro transcription. The ND1-Left-G1333-N, ND1-Right-G1333-C, ND5-Left-G1333-N, and ND5-Right-G1333-C are digested and linearized with the restriction enzyme KspAI, and the digested fragments are recovered and used as DNA templates for in vitro transcription. In vitro transcription uses the HiScribe TM SP6 RNA Synthesis Kit from NEB and is operated according to the capping RNA synthesis steps in the reagent instructions. The in vitro transcription system is as follows: SP6 Reaction Buffer (10X) 2.5 μL, ATP (50 mM) 2.5 μL, UTP (50 mM) 2.5 μL, CTP (50 mM) 2.5 μL, GTP (10 mM) 2.5 μL, ARCA cap analog (40 mM) 2.5 μL, linearized plasmid template 1 μg, SP6 RNAPolymerase Mix 2.5 μL, and nuclease-free water is added to make up to 25 μL. The transcription conditions are: 37 °C for 0.5 - 2 h. After transcription is completed, 2 μL of DNase I is added and reacted at 37 °C for 15 min to remove the template DNA. Then, the poly(A) tailing operation is carried out. The system is: 25 μL of the transcription reaction product, 5 μL of 10×Poly(A)Polymerase Reaction Buffer, 5 μL of Poly(A)Polymerase, and nuclease-free water is added to make up to 50 μL, and it is incubated at 37 °C for 30 min for poly(A) tailing. The product is then purified using the Cleanup Kit from QIAGEN and is operated according to the reagent instructions. The purified in vitro transcribed mRNA sample is used to determine the RNA quality by gel electrophoresis, and the concentration is measured. It is aliquoted into small portions and stored in a -80 °C refrigerator for later use.
[0177] Secondly, the mRNA of DdCBE was microinjected into porcine embryos for mitochondrial gene editing. Wild-type porcine fetal fibroblasts were used as donor cells for somatic cell nuclear transfer. Under the micromanipulator, the donor cells were injected into the perivitelline space of enucleated porcine mature oocytes, and fusion and activation were performed using an ECM2001 (BTX) cell fusion electroporator by electroactivation (parameters: 120 V / mm, 30 μsec, 2 pulses) to obtain reconstructed embryos. The embryos were placed in PZM-3 embryo medium and cultured briefly in an incubator at 38.5°C and 5% CO2. Six hours after activation, the in vitro transcribed mRNA of DdCBE was injected into the cytoplasm of the embryos using a microinjector (each of the two mRNAs of DdCBE was diluted to a final concentration of 150 ng / μL for use), and about 10 μL was injected into each embryo. The embryos after mRNA injection were continued to be cultured in PZM-3 embryo medium and cultured in an incubator at 38.5°C and 5% CO2 for 6 days until the blastocyst stage.
[0178] Finally, the efficiency of porcine embryo mitochondrial gene editing was detected. Individual porcine blastocysts were collected and lysed to extract genomic DNA. The target site fragment was amplified by PCR, and the mitochondrial gene mutation frequency of each embryo was detected by Sanger sequencing. The PCR conditions were the same as those for testing DdCBE at the cell level described above.
[0179] The results are as Figure 7 shown. The results showed that precise mutations of the target site bases occurred successfully in all the detected blastocysts.
[0180] Specifically, as Figure 7 shown in Figure A and Table 1, for the MT-ND1 locus, 6 blastocysts were detected, and the average mutation frequency of the target base (C10) was 43.8%;
[0181] For the MT-ND5 locus, as Figure 7 shown in Figure B and Table 2, 5 blastocysts were detected, and the average mutation frequency of the target base (C9) was 24.8%.
[0182] Table 1 Average Mutation Frequency of MT-ND1 Locus in Blastocysts
[0183] Locus ND1 C8 C9 C10 C-to-T conversion rate (%) 3.2 2.5 43.8
[0184] Table 2 Average Mutation Frequency of MT-ND5 Locus in Blastocysts
[0185] Locus ND5 C9 C11 C12 C-to-T conversion rate (%) 24.8 2.6 0.8
[0186] These embryos were transplanted into surrogate sows, and a porcine model with site-directed mitochondrial gene mutations could be obtained.
[0187] All documents mentioned in this invention are cited herein by reference as if each individual document was cited as reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.
Claims
1. A base editor with site-directed mutations in porcine MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A), characterized in that, The base editor is a combination of base editor 1 and base editor 2. Base editor 1 has a structure of formula I, and base editor 2 has a structure of formula II: TALE Left-Z1-Z2-Z3-Z4-Z5(I) TALE Right-Z1’-Z2’-Z3’-Z4’-Z5’(II) In the formulae, Z1 and Z1’ are mitochondrial targeting signals MTS; Z2 and Z2’ are a pair of TALE recognition sequences of porcine MT-ND1 and / or MT-ND5; Z3 and Z3’ are a pair of split cytosine deaminases DddA; Z4 and Z4’ are uracil glycosylase inhibitor UGI; Z5 and Z5’ are fluorescent reporter proteins; The "-" is none or a linker sequence.
2. The base editor according to claim 1, characterized in that, In the base editor for site-directed mutagenesis of porcine MT-ND1 (m.G4505A), the sequence of base editor 1 is as shown in SEQ ID NO:20, and the sequence of base editor 2 is as shown in SEQ ID NO:
21.
3. The base editor according to claim 1, characterized in that, In the base editor for site-directed mutagenesis of porcine MT-ND5 (m.G14111A), the sequence of base editor 1 is as shown in SEQ ID NO:22, and the sequence of base editor 2 is as shown in SEQ ID NO:
23.
4. A polynucleotide, characterized in that, The polynucleotide encodes the base editor described in claim 1.
5. A carrier, characterized in that, The vector comprises the polynucleotide described in claim 4.
6. A porcine cell, characterized in that, The porcine cell contains the vector described in claim 5.
7. A method for constructing MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A) site-directed mutant porcine cells, characterized in that, Comprising the steps of: (S1) Transfecting the vector described in claim 5 into a porcine cell; and (S2) Picking cell clones and identifying the site-directed mutagenesis effect, thereby obtaining porcine cells with site-directed mutagenesis of MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A).
8. Use of the base editor according to claim 1, the polynucleotide according to claim 4, the vector according to claim 5, or the porcine cell according to claim 6, characterized in that, An experimental reagent for preparing porcine embryos and / or porcine models with site-directed mitochondrial gene mutations.
9. A method for preparing MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A) site-directed mutagenesis porcine embryos, characterized in that, Comprising the following steps: (M1) Providing mRNA encoding the base editor described in claim 1; (M2) Introducing the mRNA into a porcine embryo, thereby obtaining a porcine embryo with site-directed mutagenesis of MT-ND1 (m.G4505A) and / or MT-ND5 (m.G14111A).
10. The method according to claim 9, wherein The method is a method for non-diagnostic and non-therapeutic purposes.