A pilot editing system suitable for site-directed editing of an igf1r gene of a pig-derived cell and application thereof

Abstract

This invention discloses a pilot editing system and its application for site-specific editing of the IGF1R gene in porcine cells. The system includes a pilot editor expression component, an epigRNA expression component, and a nicking sgRNA expression component. The pilot editor integrates Cas9 protein, a functional domain of porcine La protein, and Tf1 reverse transcriptase, and co-expresses MLH1-SB protein. A selection marker is added to the epigRNA expression vector, namely RTT and PBS carrying targets to the F731A or Y517A / R518E sites of the porcine IGF1R gene. This system improves epigRNA stability and target mutation writing efficiency, reduces the adverse effects of mismatch repair on the retention of edited products, and is more suitable for editing the IGF1R gene in porcine fetal fibroblasts. It can efficiently achieve site-specific mutations, providing a tool for porcine cell editing, IGF1R research, and the construction of related cell models.

Description

Technical Field

[0001] This invention belongs to the field of gene editing technology, and relates to the adaptation and improvement of pilot editing tools in porcine cells, the design of targeted guide RNAs, and site-directed mutagenesis of the IGF1R gene. It relates to a technical strategy to improve the species adaptability of functional proteins and reverse transcriptase elements in pilot editing tools to enhance the efficiency of IGF1R gene editing in porcine cells. Specifically, it is a pilot editing system and its application suitable for site-directed editing of the IGF1R gene in porcine cells. Background Technology

[0002] Prime editing (PE) is a gene editing technology that uses a fusion protein of Cas nickase and reverse transcriptase as its core, and a prime editing guide RNA (pegRNA) to provide the target recognition sequence and editing template. Compared with the traditional CRISPR / Cas9 editing method that relies on DNA double-strand breaks, PE can achieve various types of site-specific rewriting, such as base substitution, small fragment insertion, and small fragment deletion, without introducing donor DNA templates or forming DNA double-strand breaks. Therefore, it has important application value in cell model construction and animal genetic trait analysis.

[0003] Existing lead editing systems typically include Cas9 nicking enzyme, reverse transcriptase, pegRNA, and in some systems, helper nicking guide RNA or repair regulatory elements to improve the retention rate of edited products. PegRNA usually consists of a target recognition sequence, an sgRNA backbone, a primer binding site (PBS), and a reverse transcription template (RTT). PBS affects the annealing stability between the broken DNA single strand and the pegRNA, while the RTT determines the target editing information to be written. Therefore, target sequence selection, PBS length, RTT length, helper nicking site location, and editor protein structure all significantly affect editing efficiency, editing purity, and cell adaptability.

[0004] However, the cell lines involved in existing PE systems are mostly human cell lines or immortalized cell lines. Compared with the above cell systems, porcine fetal fibroblasts (PFFs) and other porcine primary cells have differences in transfection efficiency, cell proliferation status, DNA damage response, RNA stability, and mismatch repair (MMR) activity. This leads to problems such as low editing efficiency of general-purpose PE tools in porcine cells, difficulty in enriching positive cells, decreased cell activity, and large differences in editing efficiency between sites. In particular, when carrying out gene-targeted editing, it is usually difficult to obtain a stable high proportion of target genotype cell populations by simply transplanting existing PE tools into porcine cells.

[0005] Insulin-like growth factor 1 receptor (IGF1R) is a key membrane receptor downstream of the IGF1 gene, belonging to the receptor tyrosine kinase family. It primarily initiates the expression of genes related to cell cycle and proliferation through the PI3K-AKT and Ras-MAPK pathways, thereby driving cells into the division cycle. Pigs share high similarities with humans in anatomical structure, physiological function, and genomic characteristics. Site-directed editing of the porcine IGF1R gene is of great significance for studying the function of body size-related genes, constructing porcine cell models, and investigating related disease mechanisms. However, as a target gene with a relatively complex sequence structure, site-directed mutation of IGF1R requires consideration of target specificity, editing efficiency, cell viability, and control of editing byproducts, placing higher demands on the adaptability of the PE system in porcine cells.

[0006] In summary, existing PE tools still suffer from problems such as insufficient species adaptability, limited targeted editing efficiency, insufficient enrichment of positive cells, and insufficient stability of editing results evaluation in porcine cells. Therefore, how to adapt the key functional components of the PE system to the physiological characteristics of porcine primary cells and improve the site-specific editing efficiency of the IGF1R gene while ensuring cell viability is an urgent technical problem to be solved. Summary of the Invention

[0007] (a) Purpose of the invention To address the shortcomings of existing PE tools in porcine primary cells, such as insufficient editing efficiency, weak species compatibility between functional proteins and reverse transcriptase elements, limited pegRNA stability, difficulty in enriching positive cells, and unstable site-directed mutagenesis efficiency at specific IGF1R sites, this invention aims to provide a pilot editing system and its application suitable for site-directed editing of the IGF1R gene in porcine cells. This system achieves highly efficient site-directed editing of the IGF1R gene at target sites in porcine cells by constructing a pilot editor expression component adapted to porcine cells, improving pegRNA stability with the functional domain of porcine La protein, introducing yeast Tf1 reverse transcriptase to enhance reverse transcription writing efficiency, and combining a pegRNA vector with selection markers and nicking sgRNA-assisted editing components. This improves the efficiency of obtaining target mutant cell populations and provides a stable technical tool for porcine IGF1R functional research and the construction of related cell models.

[0008] (II) Technical Solution To achieve the objective of this invention and solve its technical problems, the present invention adopts the following technical solution: The first objective of this invention is to provide a pilot editing fusion protein suitable for porcine cell pilot editing, comprising Cas9 protein, Tf1 reverse transcriptase, and a functional domain of porcine La protein: The Cas9 protein contains the H840A mutation and possesses single-stranded cleavage enzyme activity, the amino acid sequence of which is shown in SEQ ID NO.1. The single-stranded cleavage enzyme activity is the enzyme activity that forms a cleavage on one strand of double-stranded DNA. The Tf1 reverse transcriptase is derived from the PE6c leader editing system and is directly or indirectly linked to the Cas9 protein. It is used to write target mutation information based on the reverse transcription template (RTT) sequence in the engineered prime editing guide RNA (epegRNA), the amino acid sequence of which is shown in SEQ ID NO.2. The functional domain of the porcine La protein is directly or indirectly linked to Tf1 reverse transcriptase and is a fragment consisting of amino acids 1 to 194 in the amino acid sequence of the porcine La protein. The fragment contains the La motif and two RNA recognition motifs (RRMs) for binding to the polyuracil sequence at the 3′ end of epigRNA and inhibiting the degradation of epigRNA by nucleases. Its amino acid sequence is shown in SEQ ID NO.4.

[0009] The second objective of this invention is to provide a co-expression cassette for porcine cell lead editing, comprising a promoter, a first coding sequence of the lead editing fusion protein described above, a second coding sequence encoding a mismatch repair inhibitor protein, and a self-cleaving polypeptide coding sequence disposed between the first and second coding sequences. The mismatch repair inhibitor protein is an MLH1-SB protein, used to inhibit the reversion of the lead editing product by the DNA mismatch repair (MMR) pathway, and its amino acid sequence is shown in SEQ ID NO. 6. The self-cleaving polypeptide is a P2A self-cleaving peptide, and its amino acid sequence is shown in SEQ ID NO. 8.

[0010] The third objective of this invention is to provide a nucleic acid molecule encoding the aforementioned lead editing fusion protein, or the aforementioned co-expression cassette, wherein: When encoding a leader-edited fusion protein, the nucleic acid molecule contains a nucleotide sequence encoding a Cas9 protein, a nucleotide sequence encoding a Tf1 reverse transcriptase, and a nucleotide sequence encoding a functional domain of a porcine La protein; when containing a co-expression cassette, it also contains a nucleotide sequence encoding an MLH1-SB protein and a nucleotide sequence encoding a P2A self-cleaving peptide. Furthermore, the nucleotide sequence encoding the Cas9 protein is shown in SEQ ID NO.23, the nucleotide sequence encoding the Tf1 reverse transcriptase is shown in SEQ ID NO.3, the nucleotide sequence encoding the functional domain of the porcine La protein is shown in SEQ ID NO.5, the nucleotide sequence encoding the MLH1-SB protein is shown in SEQ ID NO.7, and the nucleotide sequence encoding P2A is shown in SEQ ID NO.9.

[0011] The fourth objective of this invention is to provide a recombinant expression vector, characterized in that the recombinant expression vector contains the aforementioned nucleic acid molecules.

[0012] The fifth objective of this invention is to provide an epigRNA expression vector for lead editing, comprising a U6 promoter, an epigRNA insertion site, RNA stabilizing structural elements, and a selection marker expression cassette. The U6 promoter is used to drive epigRNA transcription, and its nucleotide sequence is shown in SEQ ID NO.22; the epigRNA insertion site is used to insert a target recognition sequence, a single guide RNA scaffold sequence (sgRNA scaffold sequence), a reverse transcription template sequence (RTT), and a primer binding site (PBS); The RNA stabilizing structural element is the tevopreq1 RNA motif, used to improve the stability of epigRNA, and its nucleotide sequence is shown in SEQ ID NO.20. The selection marker expression cassette includes an enhanced green fluorescent protein (EGFP) coding sequence, a puromycin resistance gene (PuroR) coding sequence, a cytomegalovirus enhancer (CMV enhancer), a simian virus 40 enhancer (SV40 enhancer), and a polyA termination signal. The selection marker expression cassette is located in the epigRNA vector backbone linker region formed after linearization by Pci1 single enzyme digestion and is linked to the epigRNA vector backbone through a homologous recombination linker sequence.

[0013] Preferably, the epigRNA expression vector is an epigRNA expression vector with selection markers, comprising a U6 promoter for driving epigRNA transcription, a tevopreq1 RNA motif for improving epigRNA stability, epigRNA insertion sites for inserting target recognition sequences, sgRNA backbone sequences, RTT and PBS, and a selection marker expression cassette; the selection marker expression cassette includes an EGFP coding sequence, a PuroR coding sequence, a CMV enhancer, an SV40 enhancer, and a polyA termination signal. By combining EGFP labeling with puromycin resistance selection, porcine cells successfully incorporating the epigRNA expression component are enriched, thereby improving the efficiency of obtaining subsequent target edited cell populations.

[0014] The sixth objective of this invention is to provide a pilot editing system suitable for site-specific editing of the IGF1R gene in porcine cells, comprising a pilot editor expression component, an epigRNA expression component, and a nicking single guide RNA (nicking sgRNA) expression component: The lead editor expression component includes the recombinant expression vector of claim 4, or includes an expression component for expressing the lead editing fusion protein and MLH1-SB protein of claim 1, wherein the functional domain of the porcine La protein is used to improve the stability of the epigRNA in porcine cells, the Tf1 reverse transcriptase is used to write the target mutation information according to the RTT sequence, and the MLH1-SB protein is used to reduce the adverse effects of mismatch repair on the retention of the target editing product. The epigRNA expression component is used to express epigRNA targeting the porcine IGF1R gene editing site, comprising a target recognition sequence, an sgRNA backbone sequence, an RTT sequence, and a PBS sequence. The target editing site includes the F731A editing site for causing a mutation of phenylalanine at position 731 of the porcine IGF1R gene to alanine, and / or the Y517A / R518E editing site for causing a mutation of tyrosine at position 517 of the porcine IGF1R gene to alanine and arginine at position 518 to glutamic acid. The nicking sgRNA expression component is used to express nicking sgRNA that works in conjunction with epigRNA. The nicking sgRNA guides the Cas9 protein, which has single-stranded nicking enzyme activity, to form an auxiliary nick on the other DNA strand complementary to the epigRNA recognition strand in the DNA double strand where the target editing site of the porcine IGF1R gene is located.

[0015] Preferably, the leader editor expression component is a pPE-pro expression component, which sequentially includes a CMV initiation regulatory element, a nuclear localization signal, an nCas9 coding region, a nuclear localization signal, a PE6c-derived Tf1 reverse transcriptase coding region, a Linker coding region, a functional domain coding region of amino acids 1-194 of porcine La protein, a P2A self-cleaving peptide coding region, an MLH1-SB protein coding region, a nuclear localization signal, and a bGH poly(A) termination signal along the expression direction. Specifically, nCas9 contains R221K, N394K, and H840A mutations, and the functional domain of the porcine La protein is located downstream of the Tf1 reverse transcriptase and upstream of the MLH1-SB protein, enabling the leader editor to simultaneously enhance epegRNA stability, improve reverse transcription writing efficiency, and inhibit mismatch repair.

[0016] Preferably, epigRNA and nicking sgRNA combinations are constructed for the F731A and Y517A / R518E editing sites of the porcine IGF1R gene, respectively. For each target editing site, multiple combinations of PBS and RTT lengths are set, ensuring that the RTT sequence covers the target codon substitution information and that the PBS sequence anneals to the cleaved target DNA single strand. Simultaneously, a nicking sgRNA is set to act on the complementary strand of the epigRNA recognition strand, guiding the Cas9 protein to form an auxiliary nick on the other strand of the DNA double helix containing the target editing site. By comparing the proportion of target edited reads in porcine fetal fibroblasts with different epigRNA and nicking sgRNA combinations, the epigRNA combinations suitable for the F731A editing site and the epigRNA combinations suitable for the Y517A / R518E editing site are determined.

[0017] Furthermore, the F731A editing site uses a combination of the epigRNA shown in SEQ ID NO.10 and the nicking sgRNA shown in SEQ ID NO.14, wherein the target recognition sequence, RTT sequence, and PBS sequence in the epigRNA are shown in SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13, respectively; the Y517A / R518E editing site uses a combination of the epigRNA shown in SEQ ID NO.15 and the nicking sgRNA shown in SEQ ID NO.19, wherein the target recognition sequence, RTT sequence, and PBS sequence in the epigRNA are shown in SEQ ID NO.16, SEQ ID NO.17, and SEQ ID NO.18, respectively.

[0018] The seventh objective of this invention is to provide a gene editing kit for site-specific editing of the IGF1R gene in porcine cells, comprising the aforementioned lead editing system, and further comprising puromycin for screening positive cells, electroporation buffer for electroporation of porcine cells, and polymerase chain reaction (PCR) primers for amplifying the target editing site of the porcine IGF1R gene. Specifically, puromycin is used in conjunction with the PuroR coding sequence in the epigRNA expression vector to screen and enrich positive cells that have successfully incorporated the epigRNA expression component after transfection of porcine cells; the EGFP coding sequence is used for fluorescent labeling or auxiliary observation of the epigRNA expression vector incorporation.

[0019] The eighth objective of this invention is to provide a method for constructing the aforementioned pilot editing system, comprising at least the following steps: SS1. Obtaining the coding fragment of the functional domain of porcine La protein: Using porcine complementary deoxyribonucleic acid (cDNA) as a template, the nucleic acid fragment encoding amino acids 1 to 194 of porcine La protein was amplified; SS2. pPE7 vector construction: The nucleic acid fragment was ligated to the PEmax vector backbone to obtain the pPE7 vector expressing the lead editing fusion protein containing the functional domain of the porcine La protein; SS3. The nucleic acid fragment encoding the MLH1-SB protein was linked to the P2A self-cleaving peptide coding sequence and the leader editing fusion protein coding sequence in the pPE7 vector to obtain the pPE7-SB vector; SS4. Tf1 reverse transcriptase replacement and pPE-pro recombinant expression vector construction: Based on the PE6c vector, the Tf1 reverse transcriptase coding fragment and a fragment derived from the pPE7-SB vector containing the functional domain of the porcine La protein and the coding sequence of the MLH1-SB protein were ligated to the PE6c vector backbone to obtain the pPE-pro recombinant expression vector for expressing the leader editing fusion protein and co-expressing the MLH1-SB protein; SS5. Construction of epigRNA expression vector with selection marker: An epigRNA expression vector containing the U6 promoter, RNA stabilizing structural elements, epigRNA insertion site, enhanced green fluorescent protein (EGFP) coding sequence, and puromycin resistance gene (PuroR) coding sequence was constructed; SS6. Construction of IGF1R-targeting epigRNA and nicking sgRNA expression vectors: The target recognition sequence, reverse transcription template sequence (RTT), primer binding sequence (PBS), and single guide RNA (sgRNA) backbone sequence targeting the porcine IGF1R gene editing site were inserted into the epigRNA expression vector, and a nicking sgRNA expression vector was constructed to work in conjunction with the epigRNA.

[0020] The ninth objective of this invention is to provide a method for site-specific editing of the IGF1R gene in porcine cells, comprising the following steps: S1. The above-described preliminary editing system of the present invention is provided; S2. The pilot editor expression component, epigRNA expression component, and nickingsgRNA expression component from the pilot editing system are jointly introduced into porcine cells; S3. Positive cell selection and enrichment were performed on porcine cells after the expression component was imported; S4. Extract genomic nucleic acids from enriched porcine cells, amplify and sequence the target editing sites of the porcine IGF1R gene to obtain a porcine cell population with site-directed mutations.

[0021] Preferably, after the leader editor expression component, epigRNA expression component, and nicking sgRNA expression component are co-electropoized into porcine cells, the medium is replaced with fresh antibiotic-free medium 12 h after electroporation. Positive cells are screened using 2 μg / mL puromycin 24 h after electroporation, and the screening time is 3 days to obtain an enriched porcine cell population. The enriched porcine cell population is used for amplification of the porcine IGF1R target editing site, sequencing analysis, and obtaining the target mutant cell population.

[0022] The tenth objective of this invention is to provide a porcine cell or porcine cell population obtained by in vitro editing, wherein the porcine cell or porcine cell population is obtained by the above method, and its IGF1R gene contains Y517A mutation and / or R518E mutation and F731A mutation.

[0023] The eleventh objective of this invention is to provide the application of the above-mentioned pilot editing system, gene editing kit, and gene-targeted editing method in the preparation of porcine IGF1R gene mutant cell research materials, or in the construction of porcine IGF1R functional cell models.

[0024] (III) Technical Effects Compared with existing technologies, the pilot editing system and its application for site-specific editing of the IGF1R gene in porcine cells provided by this invention have the following beneficial and significant technical effects: (1) This invention introduces the functional domain of porcine La protein, Tf1 reverse transcriptase and MLH1-SB protein into the lead editing system, so that the editor can simultaneously improve the stability of epigRNA, enhance the reverse transcription writing efficiency and mismatch repair interference in porcine cells, which can improve the problem of insufficient adaptability of existing lead editing tools in porcine fetal fibroblasts.

[0025] (2) This invention constructs and screens combinations of epigRNA and nicking sgRNA targeting the F731A and Y517A / R518E editing sites in the porcine IGF1R gene, enabling the target codons to undergo site-specific substitution in a predetermined manner. Experimental results show that the pPE-pro system achieves a maximum target editing efficiency of approximately 50% at the Y517A / R518E editing site, with an average of approximately 45.07% across three biological replicates; and a maximum target editing efficiency of 16.80% at the F731A editing site, with an average of approximately 13.60% across three biological replicates, representing an efficiency improvement of nearly 5 times compared to the basic PE system.

[0026] (3) The present invention improves the enrichment effect of positive transfected cells by setting EGFP and Puro selection markers in the epigRNA expression vector; the porcine La protein has less impact on the activity of porcine fetal fibroblasts than the human La protein, which is conducive to obtaining a stable IGF1R site-directed mutant porcine cell population.

[0027] (iv) Description of the sequence list The SEQ ID NOs involved in this invention are all used to define or describe the PE fusion protein, encoding nucleic acid molecule, epigRNA expression element, target editing site, and helper nick guide RNA used in this invention. To facilitate understanding of the correspondence between each sequence and the technical solution, the sequences involved are described as follows: SEQ ID NO.1 is the amino acid sequence of the Cas9 protein; SEQ ID NO.23 is the nucleotide sequence encoding the Cas9 protein shown in SEQ ID NO.1. SEQ ID NO.2 is the amino acid sequence of the Tf1 reverse transcriptase; SEQ ID NO.3 is the nucleotide sequence encoded by the Tf1 reverse transcriptase. SEQ ID NO.4 is the amino acid fragment from position 1 to 194 of the porcine La protein; SEQ ID NO.5 is the nucleotide sequence encoded by the amino acid fragment from position 1 to 194 of the porcine La protein. SEQ ID NO.6 is the amino acid sequence of the MLH1-SB protein; SEQ ID NO.7 is the nucleotide sequence encoded by the MLH1-SB protein. SEQ ID NO.8 is the amino acid sequence of the P2A self-cleaving peptide; SEQ ID NO.9 is the nucleotide sequence encoded by the P2A self-cleaving peptide.

[0028] SEQ ID NO.10 is the complete nucleotide sequence of the epigRNA corresponding to the F731A editing site, SEQ ID NO.11 is the target recognition sequence corresponding to the F731A editing site, SEQ ID NO.12 is the RTT sequence corresponding to the F731A editing site, SEQ ID NO.13 is the PBS sequence corresponding to the F731A editing site, and SEQ ID NO.14 is the nicking sgRNA target recognition sequence used in conjunction with the F731A editing site.

[0029] SEQ ID NO.15 is the complete nucleotide sequence of the epigRNA corresponding to the Y517A / R518E editing site; SEQ ID NO.16 is the target recognition sequence corresponding to the Y517A / R518E editing site; SEQ ID NO.17 is the RTT sequence corresponding to the Y517A / R518E editing site; SEQ ID NO.18 is the PBS sequence corresponding to the Y517A / R518E editing site; and SEQ ID NO.19 is the nicking sgRNA target recognition sequence used in conjunction with the epigRNA at the Y517A / R518E editing site.

[0030] SEQ ID NO. 20 is the tevopreq1 RNA motif nucleotide sequence, used to improve epigRNA stability; SEQ ID NO. 21 is the sgRNA backbone sequence; SEQ ID NO. 22 is the U6 promoter nucleotide sequence, used to drive epigRNA transcription. SEQ ID NO. 24 is the porcine IGF1R coding sequence (CDS), and SEQ ID NO. 25 is the porcine IGF1R protein amino acid sequence. Attached Figure Description

[0031] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. Hereinafter, embodiments of the invention will be described in detail with reference to the accompanying drawings, wherein: Figure 1 This is a map of epigRNA expression vectors with selection markers, specifically the epigRNA-pU6-tevopreq1-GG-acceptor-EGFP-Puro plasmid map. This vector includes a U6 promoter, a tevopreq1 RNA motif, an epigRNA insertion site, an EGFP coding sequence, and a PuroR coding sequence, which are used to achieve epigRNA expression, enhanced RNA stability, and enrichment of positively transfected cells. Figure 2 The results of screening for combinations of epigRNA and nicking sgRNA targeting different sites of the porcine IGF1R gene are shown in Figure 1. (a) Comparison of editing efficiency of epigRNA and nicking sgRNA with different PBS and RTT lengths at the F731A editing site. (b) Comparison of editing efficiency of epigRNA and nicking sgRNA with different PBS and RTT lengths at the Y517A / R518E editing sites. Figure 3 This is a Sanger sequencing peak diagram of the functional domain coding fragment of a porcine La protein; Figure 4 This is a schematic diagram of the pPE7 plasmid structure, which includes CMV initiation regulatory elements, nCas9, MMLVRT, Linker, porcine La protein functional domain, nuclear localization signal, and bGH poly(A) termination signal. Figure 5 This is a comparison of the cellular activity of human and porcine La proteins in porcine fetal fibroblasts; Figure 6 This is a schematic diagram of the pPE7-SB plasmid structure, in which the MLH1-SB protein forms a co-expression structure with the pPE7 system through the P2A self-cleaving peptide; Figure 7 This is a schematic diagram of the pPE-pro plasmid structure. The pPE-pro plasmid contains nCas9, PE6c-derived Tf1 reverse transcriptase, a functional domain of porcine La protein, P2A self-cleaving peptide, and MLH1-SB protein. Figure 8This is a comparison of the editing efficiency of different lead editing systems for the target editing sites of the porcine IGF1R gene. Among them, (a) is a comparison of the editing efficiency of the F731A editing site under the conditions of PEmax, PE7, pPE7, pPE7-SB and pPE-pro, and (b) is a comparison of the editing efficiency of the Y517A / R518E editing site under the conditions of PEmax, PE7, pPE7, pPE7-SB and pPE-pro. Detailed Implementation

[0032] The materials involved in this invention include, but are not limited to, the materials in the following embodiments, and the preparation methods involved include, but are not limited to, the preparation methods used in the following examples. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those skilled in the art. The reagents or raw materials used in this invention can be obtained through conventional means. Unless otherwise specified, the reagents or raw materials used in this invention are used in accordance with conventional methods in the art or according to the product instructions. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. The preferred embodiments and materials described in this invention are for illustrative purposes only. The invention will now be further described with reference to the accompanying drawings and specific embodiments.

[0033] In this invention, epigRNA refers to an RNA molecule formed by introducing RNA stabilizing structural elements into ordinary pegRNA. It includes a target recognition sequence, an sgRNA backbone sequence, an RTT sequence, a PBS sequence, and the RNA stabilizing structural elements. The epigRNA guides the lead editing fusion protein to the target editing site and carries the target mutation information via the RTT. Furthermore, in this invention, nicking sgRNA refers to a guide RNA used in conjunction with epigRNA to guide the Cas9 protein, which has single-stranded nicking enzyme activity, to form an auxiliary nick on the other DNA strand complementary to the epigRNA recognition strand in the DNA double helix containing the target editing site, thereby increasing the retention rate of the target editing product.

[0034] Example 1: Construction of an epeRNA vector with a selection tag This embodiment illustrates the construction process of an epigRNA expression vector for lead editing. The epigRNA expression vector includes a U6 promoter for driving epigRNA transcription, an epigRNA insertion site for inserting a target recognition sequence, an sgRNA backbone sequence, an RTT sequence, and a PBS sequence, a tevopreq1 RNA motif as a stable structural element of RNA, and a selection marker expression cassette containing an EGFP coding sequence, a PuroR coding sequence, a CMV enhancer, an SV40 enhancer, and a polyA termination signal.

[0035] (1) Obtain the fragment with filter tags Using pX458 and pX459 plasmids as templates, the coding sequences of enhanced green fluorescent protein (EGFP) and puromycin resistance gene (PuroR) were amplified by PCR, and the fragments related to the selection markers were obtained by gel recovery after separation by agarose gel electrophoresis.

[0036] (2) Obtain the start and stop segments Using plasmids such as pX458 and pX459 as templates, PCR was used to amplify the three parts: cytomegalovirus enhancer, simian virus 40 enhancer, and polyA termination signal. The regulatory element fragments used for screening marker expression cassette construction were then recovered by gel extraction.

[0037] (3) Enzyme digestion of the epe RNA vector backbone The epigRNA plasmid was linearized by single-restriction digestion with Pci1 to obtain the epigRNA vector backbone for homologous recombination ligation. The specific enzyme digestion system is as follows: Table 1. Linearization and Enzyme Digestion System for epegRNA Vector Backbone (4) Construct the pU6-tevopreq1-GG-acceptor-EGFP-Puro plasmid In steps (1) and (2) above, a 15-29 nt homologous ligation sequence was added to the 5′ end of the PCR amplification primers. The EGFP coding sequence, PuroR coding sequence, CMV enhancer, SV40 enhancer, and polyA signal were ligated to the Pci1 linearized epigRNA vector backbone using homologous recombination. The homologous recombination reaction system is shown in Table 2 below. The reaction was carried out at 50°C for 30-60 min. After transformation and antibiotic selection, the positive clones were selected for PCR identification and sequencing. Positive clone vectors with correct insertion direction and sequence were obtained. The plasmid was extracted to obtain the vector with the selection tag, namely the epigRNA-pU6-tevopreq1-GG-acceptor-EGFP-Puro plasmid vector. The vector map is shown below. Figure 1As shown, this vector is used for subsequent experiments. It drives epigRNA transcription via the U6 promoter, enhances epigRNA stability through the tevopreq1 RNA motif, and improves the enrichment efficiency of positively transfected cells through EGFP and PuroR selection markers, thus providing a vector basis for subsequent site-specific IGF1R editing in porcine cells. Specifically, the EGFP coding sequence is used to form an observable fluorescent marker to help determine the epigRNA expression vector delivery status; the PuroR coding sequence is used to confer puromycin resistance to porcine cells with the delivered vector, allowing cells that failed to deliver the epigRNA expression component to be removed under puromycin treatment. The combination of EGFP labeling and PuroR drug screening can increase the proportion of positive cells for subsequent IGF1R target site detection and reduce the impact of fluctuations in porcine fetal fibroblast transfection efficiency on the evaluation of editing results.

[0038] Table 2 Homologous recombination linkage reaction system Example 2: Construction of epigRNA and nicking sgRNA vectors targeting the porcine IGF1R gene This embodiment illustrates the construction and screening process of the epigRNA expression component and the nicking sgRNA expression component. In this embodiment, the target editing sites of the porcine IGF1R gene are determined based on the nucleotide sequence of the coding region of porcine IGF1R shown in SEQ ID NO.24 and the amino acid sequence of the porcine IGF1R protein shown in SEQ ID NO.25. Specifically, the F731A editing site corresponds to phenylalanine at position 731 of the porcine IGF1R protein shown in SEQ ID NO.25, and the Y517A / R518E editing sites correspond to tyrosine at position 517 and arginine at position 518 of the porcine IGF1R protein shown in SEQ ID NO.25. By setting the coding codons corresponding to the above amino acid sites as the target editing regions, and combining the PAM position, spacer sequence, RTT sequence, and PBS sequence to design epigRNA and nicking sgRNA, site-specific replacement of preset codons can be achieved.

[0039] In this embodiment, the design of epigRNA and nicking sgRNA is not based solely on a single target recognition sequence. Instead, it comprehensively considers the target codon substitution relationship, the coverage of the target mutation information by the RTT, the annealing stability between PBS and the cleaved target DNA single strand, and the auxiliary nicking sgRNA's position on the complementary strand of the epigRNA recognition strand. Through the synergistic setting of these parameters, the epigRNA is responsible for carrying the target mutation information and guiding the leader editor to its location, allowing the nicking sgRNA to form an auxiliary nick on the other DNA strand, thereby increasing the retention rate of the target editing product in porcine cells.

[0040] Specifically, in this embodiment, the Primer3web website was used to design sgRNAs targeting the Y517A (TAC>GCC) / R518E (AGG>GAA) and F731A (TTT>GCC) mutation sites of the Bama pig IGF1R gene. The Y517A / R518E editing site replaces the codon TAC encoding tyrosine at position 517 with the codon GCC encoding alanine, and replaces the codon AGG encoding arginine at position 518 with the codon GAA encoding glutamate. The F731A editing site replaces the codon TTT encoding phenylalanine at position 731 with the codon GCC encoding alanine. A nicking sgRNA was also designed for each of these two sites to work in conjunction with the corresponding epigRNA.

[0041] To obtain epigRNAs with higher editing efficiency, three different lengths of PBS and RTT were designed for each target editing site to compare their editing efficiency. Single-stranded spacers, RTTs, PBS elements, and sgRNA scaffold sequences were obtained through primer synthesis, followed by annealing to obtain double-stranded fragments with sticky ends. The annealed double-stranded fragments were then ligated with BsaI-digested pU6-tevopreq1-GG-acceptor-EGFP-Puro (epegRNA vector backbone) and pU6-pegRNA-GG-acceptor (nicking sgRNA vector backbone) using T4 DNA ligase. After transformation and preliminary PCR identification, the samples were sent to a sequencing company for Sanger sequencing. Ultimately, six epigRNA candidate vectors for the two target editing sites and nicking sgRNA vectors for use with the corresponding epigRNAs for the two target editing sites were obtained.

[0042] For the F731A editing site, three candidate epigRNA vectors with different PBS and RTT lengths were constructed and used in combination with corresponding nicking sgRNA vectors. Similarly, for the Y517A / R518E editing site, three candidate epigRNA vectors with different PBS and RTT lengths were constructed and used in combination with corresponding nicking sgRNA vectors. After transfection of PFFs with each candidate combination, the percentage of sequencing reads containing the target-specific edit was used as the screening criterion to compare the editing efficiency of different combinations, thus identifying epigRNA combinations suitable for the F731A editing site and those suitable for the Y517A / R518E editing site.

[0043] To further clarify the correspondence between PBS, RTT structural parameters and target editing efficiency screening, this embodiment specifically sets up the epigRNA candidate combinations used for two porcine IGF1R target editing sites. For the same target editing site, each epigRNA candidate combination uses the same spacer and PAM. Different combinations are formed by changing the length and sequence of PBS and RTT, and are used in conjunction with the nicking sgRNA corresponding to the target editing site. The epigRNA candidate combinations used for the Y517A / R518E editing sites are shown in Table 3.

[0044] Table 3. Candidate combinations of epegRNAs and nicking sgRNAs for the Y517A / R518E editing site. As shown in Table 3, the three epigRNA candidate combinations for the Y517A / R518E editing site used the same spacer and PAM. EpigRNA1 and epigRNA2 had the same RTT but different PBS lengths, while epigRNA1 and epigRNA3 had the same PBS but different RTT lengths and sequences. This resulted in candidate combinations with different PBS and RTT configurations, which were used to compare the effects of different primer binding and reverse transcription template configurations on the target mutation writing efficiency of the Y517A / R518E co-editing site.

[0045] The candidate epigRNA combinations used for the F731A editing site are shown in Table 4. As shown in Table 4, the three candidate epigRNA combinations for the F731A editing site use the same spacer and PAM. EpigRNA1 and epigRNA2 use the same 24nt RTT with PBS lengths of 13nt and 15nt, respectively, which can be used to compare the effect of PBS length variation on target editing efficiency. EpigRNA2 and epigRNA3 use the same 15nt PBS with RTT lengths of 24nt and 22nt, respectively, which can be used to compare the effect of different RTT configurations on the target mutation writing efficiency at the F731A site.

[0046] Table 4. Candidate combinations of epigRNAs and nicking sgRNAs for the F731A editing site. To clarify the core editing sequence combination used in this invention, in this embodiment, the epigRNA and nicking sgRNA at the F731A and Y517A / R518E editing sites are mapped as follows. The F731A editing site is used to replace the codon TTT encoding phenylalanine at position 731 in the porcine IGF1R gene with the codon GCC encoding alanine. The complete nucleotide sequence of the epigRNA obtained by screening at this site is shown in SEQ ID NO.10, the target recognition sequence is shown in SEQ ID NO.11, the RTT sequence is shown in SEQ ID NO.12, the PBS sequence is shown in SEQ ID NO.13, and the nicking sgRNA target recognition sequence used in conjunction with this epigRNA is shown in SEQ ID NO.14.

[0047] The Y517A / R518E editing site is used to replace the codon TAC encoding tyrosine at position 517 in the porcine IGF1R gene with the codon GCC encoding alanine, and to replace the codon AGG encoding arginine at position 518 with the codon GAA encoding glutamate. The complete nucleotide sequence of the epigRNA obtained by screening at this site is shown in SEQ ID NO.15, the target recognition sequence is shown in SEQ ID NO.16, the RTT sequence is shown in SEQ ID NO.17, the PBS sequence is shown in SEQ ID NO.18, and the nicking sgRNA target recognition sequence used in conjunction with this epigRNA is shown in SEQ ID NO.19.

[0048] It should be noted that the lengths of PBS and RTT affect the annealing stability between the broken DNA single strand and the epigRNA, as well as the efficiency of writing the target mutation information. This embodiment screened out highly efficient epigRNA combinations suitable for the F731A and Y517A / R518E editing sites by setting three different PBS and RTT length combinations for each target editing site and comparing their editing efficiencies. This provides a guide RNA basis for subsequent site-specific editing experiments using the lead editing system.

[0049] Subsequently, these six epigRNA plasmids and their corresponding nicking sgRNA plasmids, along with PEmax, were electroporated into Bama pig fetal fibroblasts (PFFs) using the U-012 electroporator program. After 24 h of electroporation, positive cells were screened with 2 μg / mL puromycin for 3 days. After enrichment, the genomes of the mixed cells were extracted, and high-throughput comparative editing efficiency was measured. The editing efficiency was characterized by the percentage of target edited reads to the total number of sequencing reads, and the target editing efficiency of different epigRNA candidate combinations was compared. Figure 2 As shown, for the Y517A / R518E editing site, the editing efficiencies of three biological repeats of epigRNA1 were 14.10%, 14.80%, and 21.40%, respectively; the editing efficiencies of three biological repeats of epigRNA2 were 17.00%, 17.90%, and 23.60%, respectively; and the editing efficiencies of three biological repeats of epigRNA3 were 27.10%, 24.70%, and 26.40%, respectively. Therefore, the combination of epigRNA3 and its nickingsgRNA was selected for the Y517A / R518E editing site. For the F731A editing site, the editing efficiencies of epigRNA1 in three biological replicates were 3.40%, 3.70%, and 3.20%, respectively; those of epigRNA2 were 1.20%, 1.40%, and 1.40%, respectively; and those of epigRNA3 were 2.10%, 2.20%, and 2.20%, respectively. EpigRNA1 showed the highest overall editing efficiency. Therefore, epigRNA1 and its corresponding nicking sgRNA were selected for subsequent F731A site editing experiments. All data were biological replicates and analyzed using the PE-analyzer website, with statistical analysis performed using Student's t-test.

[0050] Specifically, this refers to the combination of epigRNA1 and its nicking sgRNA at the IGF1R F731A site, and the combination of epigRNA3 and its nicking sgRNA at the Y517A / R518E site. Subsequent formal experiments will utilize these two sets of epigRNAs and nicking sgRNAs. Specifically, the complete nucleotide sequence of the epigRNA obtained through screening at the F731A editing site is shown in SEQ ID NO. 10, and its target recognition sequence, RTT sequence, and PBS sequence are shown in SEQ ID NO. 11, SEQ ID NO. 12, and SEQ ID NO. 13, respectively. The nucleotide sequence of the nicking sgRNA used in conjunction with this epigRNA is shown in SEQ ID NO. 14. The complete nucleotide sequence of the epigRNA obtained by screening the Y517A / R518E editing site is shown in SEQ ID NO.15. Its target recognition sequence, RTT sequence and PBS sequence are shown in SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, respectively. The nucleotide sequence of the nicking sgRNA used in conjunction with this epigRNA is shown in SEQ ID NO.19.

[0051] Example 3: Construction of pPE7 vector plasmid containing the functional domain of porcine La protein This embodiment illustrates the acquisition and connection of the functional domain of the porcine La protein in the lead-edited fusion protein and its adaptation effect in porcine cells.

[0052] (1) Obtain the coding fragment of the functional domain of porcine La protein (pLa). La proteins are a class of RNA-binding proteins. Their complete structure includes a highly conserved La motif, RNA recognition motif 1 (RRM1), RNA recognition motif 2 (RRM2), and a C-terminal flexible regulatory region. The N-terminal functional domain La(1-194), composed of amino acids 1 to 194, mainly contains the La motif and RRM1, and can bind to the 3' polyuracil sequence of RNA with high affinity. Studies have shown that La proteins, through their N-terminus binding to the 3' polyU sequence of pegRNA, promote the stability and integrity of pegRNAs and epigRNAs transcribed by Pol III. Therefore, in this embodiment, the N-terminal functional domain composed of amino acids 1 to 194 of porcine La proteins, rather than the complete porcine La protein, is selected as the RNA protection functional domain of the leader editor to control the structural size of the editor fusion protein while retaining its ability to bind to the 3' polyU sequence.

[0053] To apply the above strategy to Bama pig cells and reduce the editor size while maintaining efficiency, this embodiment incorporates the protective amino acid domains from amino acid position 1 to 194 at the N-terminus of the porcine La protein into the PE editing system to construct the pPE7 system (porcine-PE7). First, total RNA was extracted from porcine fetal fibroblasts (PFFs), and complementary deoxyribonucleic acid (cDNA) was obtained through reverse transcription. Using this cDNA as a template, PCR amplification was performed on the N-terminal 194 amino acid residues of the pLa protein coding region. The resulting fragment was then subjected to Sanger sequencing. Comparison of the sequencing results with the retrieved pLa protein sequence revealed consistency. A representative Sanger sequencing peak diagram is shown below. Figure 3 As shown, a functional domain encoding fragment of the porcine La protein was obtained for constructing the pPE7 vector. This fragment encodes the N-terminal functional domain consisting of amino acids 1-194 of the porcine La protein, which will be subsequently ligated downstream of the reverse transcriptase of the leader editor.

[0054] (2) Enzyme digestion of the PEmax plasmid backbone The PEmax plasmid was linearized by double digestion with EcoRI and AgeI. The specific digestion system is as follows: Table 5. PEmax vector backbone double enzyme digestion system (3) Construction of pPE7 vector plasmid A 15-29 nt homologous linker sequence was added to the 5′ end of the porcine La protein functional domain amplification primers from step (1) above, and the sequence was ligated to the PEmax vector backbone via homologous recombination. Specifically, the PEmax vector backbone, linearized by double digestion with EcoRI and AgeI, was reacted with the porcine La protein functional domain coding fragment according to the homologous recombination ligation system. The reaction conditions were 50 °C for 30-60 min, and the homologous recombination ligation reaction system is shown in Table 6 below.

[0055] Table 6. Homologous recombination ligation reaction system constructed using pPE7 vector. After the ligation reaction was completed, the ligation product was transformed into competent cells. After antibiotic selection, positive clones were selected for PCR identification and sequencing. Positive clone vectors with the target insertion fragment orientation and sequence matching the expectations were selected. The plasmid was extracted to obtain the pPE7 vector plasmid for subsequent experiments. Figure 4The pPE7 vector shown includes a CMV initiation regulatory element, a nuclear localization signal, an nCas9 containing R221K, N394K, and H840A mutations, an MMLV RT, a linker, a porcine La protein functional domain, a nuclear localization signal, and a bGH poly(A) termination signal. The porcine La protein functional domain, consisting of amino acids 1 to 194, is located downstream of the MMLV RT and linked to the upstream editor via the linker. This structure allows the porcine La protein functional domain to be co-expressed with the leader editor and enhances epigRNA stability by binding to the 3′ polyU sequence of epigRNA.

[0056] Compared to directly introducing the complete La protein, this embodiment uses the La(1-194) functional domain, which enables it to perform RNA protection functions related to binding to the polyU sequence at the 3′ end of the epigRNA, while reducing the length of the additional fusion protein fragment. This provides a structural basis for balancing editor expression, epigRNA stability, and cell tolerance in porcine primary cells.

[0057] (4) Detection of the effects of porcine La protein and human La protein on PFF cell viability To assess the impact of La protein source differences on the viability of Bama pig fetal fibroblasts (PFFs), the same epigRNA targeting the IGF1R gene was co-electrotransfected into PFFs with plasmids fused with either porcine La protein (pLa) or human La protein (hLa) using the optimal transfection program U012. This ensured that the main treatment conditions, except for the source of the La protein functional domains, were consistent between the two groups to evaluate the cell adaptation of porcine La protein functional domains to human La protein functional domains in PFFs. Cell viability was assessed 2 and 3 days post-electrotransfection using a CCK-8 assay kit. Absorbance (OD450) was measured at 450 nm using a microplate reader, and the OD450 value reflected the relative metabolic activity of PFFs under the same detection conditions.

[0058] like Figure 5 The results showed that, compared with human LA protein, porcine LA protein caused less damage to PFF cell viability. Furthermore, the difference in cell viability increased with plasmid expression. Therefore, expressing homologous proteins in PFFs can reduce damage, and using the functional domains of homologous porcine LA protein in porcine cells is beneficial in reducing the impact of the editing system on host cell viability.

[0059] Example 4: Construction of pPE7-SB vector plasmid This embodiment illustrates the configuration of the MLH1-SB protein and P2A self-cleaving peptide in a co-expression cassette. Based on the pPE7 vector, the coding sequence of the MLH1-SB protein is ligated to the coding sequence of the P2A self-cleaving peptide and the coding sequence of the lead editing fusion protein in pPE7, resulting in the pPE7-SB vector plasmid. The MLH1-SB protein is used to inhibit the reversion of the lead editing product by the DNA mismatch repair (MMR) pathway, and the P2A self-cleaving peptide is used to enable the co-expression of the lead editing fusion protein and the MLH1-SB protein within the same expression cassette. A schematic diagram of the pPE7-SB vector structure is shown below. Figure 6 As shown. By Figure 6 It is evident that pPE7-SB further incorporates the P2A self-cleaving peptide and MLH1-SB protein coding modules on the basis of the pPE7 structure, thereby forming an expression system capable of simultaneously expressing the lead editing fusion protein and the MLH1-SB protein.

[0060] Example 5: Construction of pPE-pro vector plasmid This example illustrates the process of introducing Tf1 reverse transcriptase and constructing the pPE-pro recombinant expression vector.

[0061] (1) Obtaining yeast Tf1 reverse transcriptase fragment Using the PE6c plasmid as a template, the coding fragment of yeast Tf1 reverse transcriptase (Tf1 RT) derived from PE6c was amplified by PCR, and the Tf1 RT coding fragment was obtained by gel extraction. The Tf1 RT was used to replace the original MMLV reverse transcriptase in the pPE7-SB system to construct a leader editor expression component using PE6c-derived Tf1 RT.

[0062] (2) Obtain the pLa-SB fragment Using pPE7-SB plasmid as a template, the pLa-SB portion containing the functional domain of porcine La protein and the coding sequence of MLH1-SB protein was amplified by PCR, and the corresponding fragments were obtained by gel extraction.

[0063] Specifically, the pLa-SB fragment contains the coding sequence for the functional domain of the porcine La protein, the coding sequence for the P2A self-cleaving peptide, and the coding sequence for the MLH1-SB protein, which is used to preserve the expression structure of the porcine La protein functional domain and the MLH1-SB protein in pPE-pro. By using the already constructed pPE7-SB as an amplification template, the linkage relationship between the porcine La protein functional domain, the P2A self-cleaving peptide, and the MLH1-SB protein can be preserved in the subsequent pPE-pro expression vector.

[0064] (3) Enzyme digestion of the PE6c vector backbone The PE6c plasmid was linearized by single-restriction digestion with EcoRI. The specific digestion system is shown in Table 7 below. Table 7 PE6c vector backbone single enzyme digestion system (4) Constructing the pPE-pro vector plasmid In steps (1) and (2) above, a 15-29 nt homologous sequence was added to the 5' end of the primers for amplifying the Tf1 RT coding fragment and the pLa-SB fragment. Homologous recombination was then used to ligate these fragments to the PE6c vector backbone. Specifically, the PE6c vector backbone linearized by single digestion with EcoRI, the PE6c-derived Tf1 RT coding fragment, and the pLa-SB fragment derived from the pPE7-SB vector were added to the homologous recombination ligation system for a multi-fragment reaction at 50°C for 30-60 min. The homologous recombination ligation reaction system is shown in Table 8 below. Table 8. Homologous recombination ligation reaction system constructed using pPE-pro vector After transformation and screening, the ligation products were subjected to PCR identification and sequencing of the clones. Positive clones with correct insertion orientation and sequence of both the Tf1 RT coding fragment and the pLa-SB fragment were selected. Plasmids were extracted from the correctly sequenced positive clones to obtain the pPE-pro vector plasmid for subsequent experiments. A schematic diagram of the pPE-pro vector structure is shown below. Figure 7 As shown, the pPE-pro vector includes a CMV initiation regulatory element, nCas9, a PE6c-derived Tf1 RT, a linker, a porcine La protein functional domain, a P2A self-cleaving peptide, MLH1-SB protein, a nuclear localization signal, and a bGH poly(A) termination signal. This structure enables the lead editing system to simultaneously possess porcine cell adaptation, enhanced epigRNA stability, improved reverse transcription writing efficiency, and mismatch repair interference functions.

[0065] Specifically, such as Figure 7As shown, the lead editor expression box in the pPE-pro vector is sequentially set along the expression direction as follows: CMV initiation regulatory element, nuclear localization signal upstream of nCas9, nCas9 coding region, nuclear localization signal between nCas9 and Tf1 RT, PE6c-derived Tf1 RT coding region, Linker coding region, functional domain coding region of amino acids 1-194 of porcine La protein, P2A self-cleaving peptide coding region, MLH1-SB protein coding region, nuclear localization signal downstream of MLH1-SB protein, and bGH poly(A) termination signal. The nCas9 coding region encodes a Cas9 nickase containing R221K, N394K, and H840A mutations; the Tf1 RT coding region enhances RTT-mediated reverse transcription writing; the porcine La protein functional domain coding region expresses a porcine RNA-binding domain capable of binding to the 3′ polyU sequence of epigRNA; the P2A self-cleaving peptide coding region enables co-expression of the lead editing fusion protein and the MLH1-SB protein; and the MLH1-SB protein coding region expresses a repair regulatory protein that inhibits mismatch repair pathway reversion. Through this modular configuration, the pPE-pro vector can coordinate target site recognition, single-strand nick formation, RTT information writing, epigRNA stability maintenance, and editing product retention within the same expression system.

[0066] Therefore, pPE-pro does not simply replace a single leader editing element. Instead, it replaces the original MMLV reverse transcriptase with PE6c-derived Tf1RT on the basis of the co-expression structure of the porcine La protein functional domain formed by pPE7-SB and MLH1-SB, forming the pPE-pro leader editor expression system optimized based on pPE7-SB. This allows functions such as target site recognition and single-strand cut formation, RTT-mediated mutation information writing, epigRNA stability maintenance, and mismatch repair and reversion inhibition to be configured within the same leader editor expression system. This specific module configuration is used for subsequent comparisons of porcine IGF1R target editing efficiency with PEmax, PE7, pPE7, and pPE7-SB.

[0067] Example 6: Detection of IGF1R gene site-specific editing efficiency in porcine cell transfection This embodiment illustrates a method for site-specific editing of the IGF1R gene in porcine cells.

[0068] Observe the PFF cell status and perform transfection when the cell count reaches approximately 80%. Resuspend the cells in an appropriate electroporation buffer to form a homogeneous cell suspension. Add the epigRNA plasmid and the corresponding nicking sgRNA plasmid, as well as PEmax / PE7 / pPE7 / pPE7-SB / pPE-pro, to the cell suspension and gently mix to avoid air bubbles. Then, place the mixture containing cells and DNA in an electroporation cuvette, set the optimal transfection program U012, and immediately after electroporation, transfer the cells to a culture dish containing preheated medium and return them to the incubator for recovery. Replace with fresh antibiotic-free medium 12 h after electroporation. After culturing for 24 h, select positive cells with 2 μg / mL puromycin for 3 days. After enrichment, collect the mixed cells for editing efficiency testing.

[0069] In this screening process, puromycin screening, in conjunction with the PuroR coding sequence in the epigRNA expression vector, is used to remove negative cells that have not acquired puromycin resistance; the remaining cell population is enriched with positive cells that have successfully incorporated the epigRNA expression component. Since porcine fetal fibroblasts are primary cells, their transfection efficiency and cell state are easily affected by culture and electroporation conditions. The aforementioned drug screening and enrichment steps can increase the detection rate of the target editing event in subsequent sequencing samples and improve the stability of editing efficiency comparisons between different editing systems.

[0070] Enriched cellular genomic DNA was extracted, and primers capable of amplifying the edited sequence were designed based on the sequencing requirements of the target editing site of the porcine IGF1R gene. Adapter sequences were added to the 5′ end of the primers according to sequencing requirements. Using the edited mixed cellular genomic DNA as a template, PCR amplification was performed using primers with adapters, and the target band was then recovered for high-throughput sequencing.

[0071] This embodiment uses PE-Analyzer to analyze high-throughput sequencing results. Editing efficiency is calculated as the percentage of target edited reads to the total number of sequenced reads, i.e.: Editing efficiency = Number of target edited reads / Total number of sequenced reads × 100%. Specifically, for the F731A editing site, the replacement of the codon encoding phenylalanine at position 731 in the porcine IGF1R gene with TTT to GCC was used as the criterion for target editing; for the Y517A / R518E editing site, the replacement of the codon encoding tyrosine at position 517 with TAC to GCC and the replacement of the codon encoding arginine at position 518 with AGG to GAA were used as the criteria for target editing. In addition to target edited reads, other substitution reads and insertion and deletion (Indel) reads were also counted, and the sum of the number of other substitution reads and the number of Indel reads was used as the byproduct read count to help evaluate the generation of non-target edited products by different lead editing systems. To evaluate the effects of sequential introduction of the functional domain of porcine La protein, MLH1-SB protein, and PE6c-derived Tf1 reverse transcriptase on the editing efficiency of porcine IGF1R target sites, five lead editing systems (PEmax, PE7, pPE7, pPE7-SB, and pPE-pro) were compared in parallel under the same target site and corresponding epigRNA and nicking sgRNA combinations. All experimental results were derived from three biological replicates.

[0072] (1) Comparison of editing efficiency of Y517A / R518E edit sites The high-throughput sequencing results of five lead editing systems for the Y517A / R518E editing site are shown in Table 9.

[0073] Table 9 Editing efficiency of different lead editing systems at the Y517A / R518E edit site Based on three biological replicates, the average target editing efficiencies of PEmax, PE7, pPE7, pPE7-SB, and pPE-pro at the Y517A / R518E edit site were approximately 27.93%, 23.67%, 32.87%, 38.23%, and 45.07%, respectively. Among them, pPE-pro achieved target editing efficiencies of 41.60%, 45.60%, and 48.00% in the three replicates, respectively, demonstrating the highest overall editing efficiency among the five lead editing systems.

[0074] The results of the hierarchical system comparison show that pPE7 has a higher average editing efficiency than PE7 containing the human La protein functional domain. This indicates that under the conditions of this embodiment, replacing the human La protein functional domain with the porcine La protein functional domain improves the target editing efficiency of the Y517A / R518E site in porcine cells. After further introducing MLH1-SB to obtain pPE7-SB, the average editing efficiency increased from approximately 32.87% to approximately 38.23%. Based on this, after constructing pPE-pro using PE6c-derived Tf1 RT, the average editing efficiency further increased to approximately 45.07%.

[0075] (2) Comparison of editing efficiency of F731A editing sites The high-throughput sequencing results of five lead editing systems for the F731A editing site are shown in Table 10.

[0076] Table 10 Editing efficiency of different lead editing systems for F731A edit sites Based on three biological replicates, the average target editing efficiencies of PEmax, PE7, pPE7, pPE7-SB, and pPE-pro at the F731A editing site were approximately 2.63%, 2.80%, 3.73%, 6.23%, and 13.60%, respectively. With the gradual introduction of porcine La protein functional domains, MLH1-SB, and Tf1 RT-related modifications, the target editing efficiency at the F731A site generally showed a gradual increasing trend, with pPE-pro achieving editing efficiencies of 11.50%, 12.50%, and 16.80% in the three replicates, respectively.

[0077] Comparing the arithmetic mean of three biological replicates, pPE-pro's average editing efficiency at the F731A editing site was approximately 5.17 times that of the basic PEmax system and approximately 2.18 times that of the pPE7-SB system. Therefore, it can be more accurately stated that the nearly 5-fold efficiency improvement described in this invention refers to an average target editing efficiency increase of approximately 5.17 times for pPE-pro relative to the basic PEmax system at the F731A site, rather than a ratio between the maximum values ​​of a single experiment.

[0078] (3) Statistical analysis and comparison of the effects of different pilot editing systems In this embodiment, editing efficiency is expressed as the percentage of sequencing reads containing the target-specific edit out of the total sequencing reads. For each experimental group, the total number of sequencing reads, the number of unedited reads, the number of targeted edited reads, the number of insertion / deletion reads, and the number of reads containing non-target substitutions or partial write events are counted. Targeted edited reads refer to sequencing reads that simultaneously meet the preset codon substitution results at the target editing site of porcine IGF1R; insertion / deletion reads and non-target substitution reads are used to evaluate the editing byproducts. When analyzing the F731A editing site, TTT replaced with GCC is used as the criterion for target editing; when analyzing the Y517A / R518E editing site, TAC replaced with GCC and AGG replaced with GAA are used as the criterion for target editing. By comparing the proportion of target edited reads in different systems such as PEmax, PE7, pPE7, pPE7-SB, and pPE-pro, the effects of the functional domain of porcine La protein, MLH1-SB protein, and Tf1 reverse transcriptase on the site-specific editing efficiency of porcine IGF1R can be evaluated.

[0079] The experimental results for the two porcine IGF1R target editing sites mentioned above are biologically replicate data. Sequencing results were analyzed using PE-Analyzer, and statistical analysis was performed using Student's t test. Figure 8 middle This indicates that P < 0.05. This indicates that P < 0.01. P < 0.001 indicates that the difference is not statistically significant, and ns indicates that the specific comparison relationship between groups is as follows. Figure 8 The square brackets should be used as the standard.

[0080] like Figure 8 The results showed that in the precise editing of IGF1R at the F731A and Y517A / R518E sites: after replacing the human La protein with the porcine La protein, the editing efficiency of pPE7 was improved compared to PE7, indicating that homologous proteins can perform better in cells, and the porcine La protein is more adapted to PFF cell editing, thus improving the species adaptability of the lead editing system in PFFs; pPE-pro further improved the editing efficiency compared to pPE7-SB, indicating that after replacing the original RT enzyme with the yeast Tf1 reverse transcriptase of PE6c based on the functional domain of porcine La protein and the introduction of MLH1-SB, the editing efficiency of IGF1R target sites in porcine cells was further improved. Among them, the average target editing efficiency of pPE-pro at the IGF1R F731A editing site in three biological replicates was about 5.17 times higher than that of the basic PEmax system, indicating that the pPE-pro system can significantly improve the editing efficiency in PFF cells.

[0081] Further analysis of Tables 9 and 10 shows that the stepwise modification employed in this invention does not only produce effects at a single target site. At the Y517A / R518E site, the average target editing efficiencies of the pPE7, pPE7-SB, and pPE-pro systems are approximately 32.87%, 38.23%, and 45.07%, respectively; at the F731A site, the average target editing efficiencies of the three systems are approximately 3.73%, 6.23%, and 13.60%, respectively. Both IGF1R target sites with different basic editing difficulties show that pPE-pro has higher target editing efficiency, experimentally supporting the combined application of porcine La protein functional domains, mismatch repair regulatory modules, and Tf1 RT in the lead editing system of this invention. Among them, the functional domain of the porcine La protein is mainly used to improve the adaptability between the leader editor and the PFFs cellular environment and maintain the stability of epigRNA; MLH1-SB is used to reduce the adverse effects of the mismatch repair pathway on the retention of the target editing product; and Tf1 RT is used to complete the writing of target mutation information based on RTT. The above technical means, together with the site-adaptive epigRNA and nicking sgRNA obtained by screening in Example 2, constitute the technical system of this invention in the site-specific editing of IGF1R in porcine cells.

[0082] To further evaluate the editing specificity of the lead editing system, candidate off-target sites can be predicted within the porcine genome based on the target recognition sequences of the F731A and Y517A / R518E editing sites. High-scoring candidate off-target sites are then selected for PCR amplification and sequencing analysis. The selection of candidate off-target sites considers the number of spacer sequence mismatches, mismatch location, PAM sequence type, and the genomic region where the candidate site is located. Statistical analysis of sequencing reads from candidate off-target sites evaluates the likelihood of the lead editing system generating unexpected edits outside the target site.

[0083] Example 7: Obtaining gene editing kits and porcine IGF1R mutant cell research materials This embodiment illustrates the gene editing kit, in vitro editing of porcine cells, and related applications. The gene editing kit for site-specific editing of the IGF1R gene in porcine cells includes the aforementioned lead editing system, puromycin for positive cell selection, electroporation buffer for porcine cell electroporation, and PCR primers for amplifying the target editing site of the porcine IGF1R gene. The lead editing system includes a lead editor expression component, an epigRNA expression component, and a nicking sgRNA expression component; the epigRNA expression component can be constructed using the epigRNA expression vector obtained in Example 1; the lead editor expression component can be constructed using the pPE-pro vector obtained in Example 5. Electroporation buffer was used to improve cell tolerance when the lead editor expression component, epigRNA expression component, and nicking sgRNA expression component were co-introduced into porcine cells; puromycin was used in conjunction with the PuroR selection marker in the epigRNA expression vector to enrich positive cells that had been successfully introduced into the epigRNA expression component; PCR primers were used to amplify the target regions containing the F731A and Y517A / R518E editing sites to detect the editing results in the enriched cell population.

[0084] Using the method described in Example 6, in vitro edited porcine cells or porcine cell populations can be obtained. The IGF1R gene of the porcine cells or porcine cell populations contains at least one of the following mutations: Y517A, R518E, and F731A. These cells or cell populations can be used to prepare porcine IGF1R gene mutant cell research materials or to construct porcine IGF1R functional research cell models.

[0085] It should be noted that the porcine cells or porcine cell populations described in this invention are research materials at the in vitro cell level and do not directly limit or protect the individual gene-edited pigs or animal breeds themselves.

[0086] Furthermore, this invention does not simply apply existing lead editing systems directly to porcine cells. Instead, it addresses the limitations of lead editing efficiency, insufficient adaptation to heterologous functional proteins, low epigRNA stability, and susceptibility of target editing products to mismatch repair in porcine fetal fibroblasts by systematically modifying the lead editing tool for the porcine cell environment. This modification is manifested in at least four aspects: replacement of the functional domain of porcine La protein, introduction of a mismatch repair regulatory module, replacement of Tf1 reverse transcriptase, and screening of IGF1R-specific site epigRNA combined with nicking sgRNA. These aspects are not isolated but collectively serve the technical goal of improving the efficiency of IGF1R gene-specific editing and reducing cell viability damage in porcine cells.

[0087] On one hand, this invention replaces the functional domain of the human La protein in the PE7 system with the functional domain of the porcine La protein. Due to differences in expression adaptability, RNA binding stability, and cell tolerance among RNA-binding proteins from different species in porcine cells, directly using the human La protein cannot fully meet the editing requirements in PFFs. To address this situation and considering the research subjects, this invention replaces the human La protein in the original PE7 system with a porcine La protein. Addressing the issues of insufficient species adaptability, limited epigRNA stability, and susceptibility to cell activity in the pilot editing system in porcine fetal fibroblasts, this invention introduces the functional domain composed of amino acids 1 to 194 of the porcine La protein into the pilot editing system. This functional domain mainly contains the La motif and RNA recognition motif 1 (RRM1), which can bind to the polyuracil sequence at the 3′ end of epigRNA, thereby inhibiting epigRNA degradation by nucleases and improving the stability and integrity of epigRNA in porcine cells. Based on specific experiments on porcine cells, pPE7 showed better porcine cell adaptability than PE7 containing the human La protein functional domain, and exhibited higher target editing efficiency at relevant IGF1R target sites. Meanwhile, the porcine La protein functional domain had little effect on the activity of PFFs cells.

[0088] On the other hand, this invention further replaces the RT enzyme in pPE7-SB with the yeast Tf1 RT enzyme from PE6c, forming the pPE-pro lead editing tool. During the lead editing process, the reverse transcriptase needs to use the RTT in the epigRNA as a template to synthesize a new DNA strand carrying the target mutation information, and its catalytic performance directly affects the target mutation writing efficiency. This invention combines the epigRNA stability enhancement provided by the functional domain of porcine La protein, the inhibition of DNA mismatch repair pathway reversal by MLH1-SB protein, and the enhancement of RTT-mediated reverse transcription writing by Tf1 reverse transcriptase, enabling pPE-pro to exhibit high editing efficiency at both the porcine IGF1R F731A site and the Y517A / R518E site, especially at the F731A site, where it achieves an efficiency improvement of nearly 5 times compared to the basic editing system.

[0089] Furthermore, this invention designs and screens epigRNAs with different PBS and RTT length combinations targeting the F731A and Y517A / R518E sites of the porcine IGF1R gene, respectively, and simultaneously configures corresponding nicking sgRNAs. These are then combined with epigRNA expression vectors carrying EGFP and Puro selection markers to achieve positive cell enrichment. Thus, this invention forms a synergistic technical system encompassing editor structure modification, guide RNA structural stabilization, auxiliary nick site configuration, positive cell enrichment, and sequencing evaluation, enabling the stable acquisition of porcine cells or porcine cell populations with site-directed mutations in the IGF1R gene. This system provides a reproducible technical foundation for porcine IGF1R functional studies, in vitro cell model construction, and optimization of precise gene editing tools for large animal cells.

[0090] In summary, this invention addresses the issues of insufficient species adaptability and limited editing efficiency in porcine cell lead editing applications. It proposes a lead editing system centered on the synergistic configuration of porcine La protein functional domains, Tf1 reverse transcriptase, MLH1-SB protein, epigRNA stable structural elements, and nicking sgRNA. This system balances epigRNA stability, reverse transcription writing efficiency, retention of target editing products, and maintenance of cell viability in porcine cells, effectively improving the site-specific editing efficiency of the porcine IGF1R gene and providing a reliable tool for subsequent research on porcine IGF1R gene mutant cells.

[0091] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pilot editing fusion protein suitable for pilot editing of porcine cells, characterized in that, This includes the functional domains of Cas9 protein, Tf1 reverse transcriptase, and porcine La protein, among which: The Cas9 protein contains an H840A mutation and possesses single-strand nickase activity; its amino acid sequence is shown in SEQ ID NO.

1. The Tf1 reverse transcriptase is derived from the PE6c leader editing system and is linked to the Cas9 protein. It is used to write target mutation information based on the RTT sequence in the epigRNA; its amino acid sequence is shown in SEQ ID NO.

2. The functional domain of the porcine La protein is linked to the Tf1 reverse transcriptase and is a fragment composed of amino acids 1 to 194 in the porcine La protein amino acid sequence. This fragment contains a La motif and two RRMs, which are used to bind to the polyuracil sequence at the 3′ end of the epigRNA and inhibit the degradation of the epigRNA by nucleases; its amino acid sequence is shown in SEQ ID NO.

4.

2. A co-expression cassette for porcine cell lead editing, characterized in that, It includes a promoter, a first coding sequence encoding the lead editing fusion protein of claim 1, a second coding sequence encoding a mismatch repair inhibitor protein, and a self-cleaving polypeptide coding sequence disposed between the first and second coding sequences, wherein the mismatch repair inhibitor protein is an MLH1-SB protein, the amino acid sequence of which is shown in SEQ ID NO.6; and the self-cleaving polypeptide is a P2A self-cleaving peptide, the amino acid sequence of which is shown in SEQ ID NO.

8.

3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the lead editing fusion protein of claim 1, or comprises the co-expression cassette of claim 2, wherein: When the nucleic acid molecule encodes the lead editing fusion protein of claim 1, the nucleic acid molecule comprises a nucleotide sequence encoding the Cas9 protein, a nucleotide sequence encoding the Tf1 reverse transcriptase, and a nucleotide sequence encoding the functional domain of the porcine La protein; When the nucleic acid molecule contains the co-expression cassette of claim 2, it further contains a nucleotide sequence encoding the MLH1-SB protein and a nucleotide sequence encoding the P2A self-cleaving peptide; Furthermore, the nucleotide sequence encoding the Cas9 protein is shown in SEQ ID NO.23, the nucleotide sequence encoding the Tf1 reverse transcriptase is shown in SEQ ID NO.3, the nucleotide sequence encoding the functional domain of the porcine La protein is shown in SEQ ID NO.5, the nucleotide sequence encoding the MLH1-SB protein is shown in SEQ ID NO.7, and the nucleotide sequence encoding P2A is shown in SEQ ID NO.

9.

4. An epigRNA expression vector for lead editing, characterized in that, This includes the U6 promoter, epegRNA insertion site, RNA stability structural elements, and selection marker expression cassette, among which: The U6 promoter is used to drive epigRNA transcription, and its nucleotide sequence is shown in SEQ ID NO.22; the epigRNA insertion site is used to insert a target recognition sequence, an sgRNA backbone sequence, an RTT sequence, and a PBS sequence; the RNA stabilizing structural element is a tevopreq1 RNA motif, and its nucleotide sequence is shown in SEQ ID NO.20; the selection marker expression cassette includes an EGFP coding sequence, a PuroR coding sequence, a CMV enhancer, an SV40 enhancer, and a polyA termination signal.

5. A pilot editing system suitable for site-specific editing of the IGF1R gene in porcine cells, characterized in that, It includes a leader editor expression component, an epigRNA expression component, and a nicking sgRNA expression component, wherein the leader editor expression component includes an expression component for expressing the leader editing fusion protein and MLH1-SB protein of claim 1; The epigRNA expression component is used to express epigRNA targeting the porcine IGF1R gene editing site, comprising a target recognition sequence, an sgRNA backbone sequence, an RTT sequence, and a PBS sequence. The target editing site includes the F731A editing site for causing a mutation of phenylalanine at position 731 of the porcine IGF1R gene to alanine, and / or the Y517A / R518E editing site for causing a mutation of tyrosine at position 517 of the porcine IGF1R gene to alanine and arginine at position 518 to glutamic acid. The nicking sgRNA expression component is used to express nicking sgRNA that works in conjunction with epigRNA. The nicking sgRNA guides the Cas9 protein, which has single-stranded nicking enzyme activity, to form an auxiliary nick on the other DNA strand complementary to the epigRNA recognition strand in the DNA double strand where the target editing site of the porcine IGF1R gene is located.

6. The pilot editing system according to claim 5, characterized in that, The epigRNA is used to replace the codon TTT encoding phenylalanine at position 731 with the codon GCC encoding alanine. Its nucleotide sequence is shown in SEQ ID NO.10, its target recognition sequence is shown in SEQ ID NO.11, its RTT sequence is shown in SEQ ID NO.12, and its PBS sequence is shown in SEQ ID NO.

13. The target recognition sequence of the nicking sgRNA used in conjunction with the epigRNA is shown in SEQ ID NO.

14.

7. The pilot editing system according to claim 5, characterized in that, The epigRNA is used to replace the codon TAC encoding tyrosine at position 517 with the codon GCC encoding alanine, and to replace the codon AGG encoding arginine at position 518 with the codon GAA encoding glutamic acid. Its nucleotide sequence is shown in SEQ ID NO.15, its target recognition sequence is shown in SEQ ID NO.16, its RTT sequence is shown in SEQ ID NO.17, its PBS sequence is shown in SEQ ID NO.18, and the target recognition sequence of the nicking sgRNA used in conjunction with the epigRNA is shown in SEQ ID NO.

19.

8. A gene editing kit for site-specific editing of the IGF1R gene in porcine cells, characterized in that, The system includes the lead editing system according to any one of claims 5 to 7, and further includes puromycin for positive cell screening, electroporation buffer for porcine cell electroporation, and PCR primers for amplifying the target editing site of the porcine IGF1R gene.

9. A method for constructing the pilot editing system according to any one of claims 5 to 7, characterized in that, Includes the following steps: SS1. Obtaining the coding fragment of the functional domain of porcine La protein: Using porcine cell cDNA as a template, the nucleic acid fragment encoding amino acids 1 to 194 of porcine La protein was amplified; SS2. pPE7 vector construction: The nucleic acid fragment was ligated to the PEmax vector backbone to obtain the pPE7 vector expressing the lead editing fusion protein containing the functional domain of the porcine La protein; SS3. The nucleic acid fragment encoding the MLH1-SB protein was linked to the P2A self-cleaving peptide coding sequence and the leader editing fusion protein coding sequence in the pPE7 vector to obtain the pPE7-SB vector; SS4. Tf1 reverse transcriptase replacement and construction of pPE-pro recombinant expression vector: Based on the PE6c vector, the Tf1 reverse transcriptase coding fragment and a fragment from the pPE7-SB vector containing the functional domain of the porcine La protein and the coding sequence of the MLH1-SB protein were ligated to the PE6c vector backbone to obtain the pPE-pro recombinant expression vector for expressing the leader editing fusion protein and co-expressing the MLH1-SB protein; SS5. Construct an epigRNA expression vector containing a U6 promoter, RNA stabilizing structural elements, an epigRNA insertion site, an EGFP coding sequence, and a PuroR coding sequence; SS6. The target recognition sequence, RTT, PBS, and sgRNA backbone sequence targeting the porcine IGF1R gene editing site were inserted into the epegRNA expression vector to construct the nicking sgRNA expression vector.

10. A method for site-specific editing of the IGF1R gene in porcine cells, characterized in that, include: S1. To provide the pilot editing system according to any one of claims 5 to 7; S2. The pilot editor expression component, epigRNA expression component, and nicking sgRNA expression component from the pilot editing system were jointly introduced into porcine cells; S3. Positive cell selection and enrichment were performed on porcine cells after the expression component was imported; S4. Extract genomic nucleic acids from enriched porcine cells, amplify and sequence the target editing sites of the porcine IGF1R gene to obtain a porcine cell population with site-directed mutations.

11. A porcine cell or porcine cell population obtained through in vitro editing, characterized in that, The porcine cells or porcine cell population are obtained by the method of claim 10, and their IGF1R gene contains at least one of the Y517A mutation, R518E mutation, and F731A mutation.

12. The pilot editing system according to any one of claims 5 to 7, or the gene editing system according to claim 8. The kit and the method of claim 10 are used in the preparation of porcine IGF1R gene mutant cell research materials, or in the construction of porcine IGF1R functional cell models.

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