Method for improving growth rate of pigs

Abstract

The present invention belongs to the technical field of animal genetic engineering, and specifically relates to a method for improving the growth rate of pigs. The method comprises the following steps: fusing, in vivo, a coding region of a GH1 gene with a strong promoter of another endogenous high-expression gene without introducing an exogenous gene to generate a GH1 high-expression gene, wherein the strong promoter is a PSMC5 gene promoter, an ACTA1 gene promoter, an ACTB gene promoter, an NME2 gene promoter or an RPL23 gene promoter. According to the method, pig breeds with a rapid growth rate can be created.

Description

A method to improve the growth rate of pigs Technical Field

[0001] This invention belongs to the field of animal genetic engineering technology, and specifically relates to a method for improving the growth rate of pigs. Background Technology

[0002] In pigs, the growth hormone (GH) gene controls the rate of growth and development. The GH1 gene is the growth hormone gene in pigs. The GH1 gene is located on chromosome 12 of pigs, and its expression is regulated by the hypothalamus-pituitary-hepatic axis. By binding to the growth hormone receptor, it initiates a series of signal transduction pathways, thereby affecting growth, metabolism, immune function, and reproductive capacity.

[0003] GH1 gene expression affects pig growth and meat quality. On the one hand, it can accelerate growth rate; GH1 gene overexpression increases growth hormone secretion, promotes chondrocyte proliferation and differentiation, and accelerates bone and muscle growth. This allows pigs to reach slaughter weight faster under the same feeding conditions, shortening the breeding cycle. On the other hand, it can improve meat quality; appropriate regulation of GH1 gene expression can increase the lean meat ratio, reduce fat deposition, and improve the taste and nutritional value of the meat.

[0004] Developing fast-growing, feed-efficient pig breeds with high feed conversion rates is a major national need and has always been a key focus and challenge in genetic breeding. Summary of the Invention

[0005] This invention creates a fast-growing pig breed through gene editing, as detailed below:

[0006] This invention provides a method for improving the growth rate of pigs, comprising the following steps: without introducing exogenous genes, fusing the coding region of the GH1 gene with a strong promoter of another endogenous highly expressed gene to generate a GH1 highly expressed gene, wherein the strong promoter is the PSMC5 gene promoter, ACTA1 gene promoter, ACTB gene promoter, NME2 gene promoter or RPL23 gene promoter.

[0007] The present invention also provides an editing method for knocking up endogenous GH1 gene expression in pigs, comprising the following steps: without introducing exogenous genes, fusing the coding region of the GH1 gene with a strong promoter of another endogenous high-expression gene to generate a GH1 high-expression gene, wherein the strong promoter is the PSMC5 gene promoter, ACTA1 gene promoter, ACTB gene promoter, NME2 gene promoter or RPL23 gene promoter.

[0008] In one specific embodiment, the method includes the following steps:

[0009] DNA breaks can be simultaneously generated at two different specific locations in the genome of an organism. These locations are the genomic locus between the coding region of the GH1 gene and its promoter, and the genomic locus between the coding region of the endogenous highly expressed gene and its promoter. The DNA breaks are then interconnected through intracellular repair pathways, causing the GH1 gene coding region and the endogenous highly expressed gene promoter to fuse in vivo, ultimately producing the GH1 highly expressed gene. Alternatively, DNA breaks can be simultaneously generated at three different specific locations in the genome of an organism. These locations are two genomic loci capable of cutting out fragments of the endogenous highly expressed gene promoter, and the genomic locus between the coding region of the GH1 gene and its promoter. The DNA breaks are then interconnected through intracellular repair pathways, resulting in a translocation editing event where the endogenous highly expressed gene promoter fragment is inserted upstream of the coding region of the GH1 gene, ultimately producing the GH1 highly expressed gene.

[0010] Alternatively, DNA breaks may occur simultaneously at three different specific locations in the organism's genome. These specific locations are two genomic sites capable of cutting out the GH1 gene coding region fragment and a genomic site between the promoter of the endogenous highly expressed gene and its coding region. These sites are then interconnected through intracellular repair pathways to generate a translocation editing event in which the GH1 coding region fragment is inserted downstream of the promoter of the endogenous highly expressed gene, ultimately resulting in the GH1 highly expressed gene.

[0011] In one specific embodiment, the method further includes the following steps: designing primer pairs that can specifically distinguish the new combinations of gene elements for the specific positions mentioned above; screening cells or tissues containing the GH1 highly expressed gene by PCR; and sequencing to determine the characteristic sequence of the new combinations of gene elements.

[0012] In another specific embodiment, the primer pair includes at least one set of the following sequences: as shown in SEQ ID NO:14 and SEQ ID NO:15, as shown in SEQ ID NO:16 and SEQ ID NO:17, as shown in SEQ ID NO:67 and SEQ ID NO:68, as shown in SEQ ID NO:69 and SEQ ID NO:70, as shown in SEQ ID NO:71 and SEQ ID NO:72, as shown in SEQ ID NO:73 and SEQ ID NO:74, as shown in SEQ ID NO:75 and SEQ ID NO:76, as shown in SEQ ID NO:77 and SEQ ID NO:78, as shown in SEQ ID NO:79 and SEQ ID NO:80, and as shown in SEQ ID NO:81 and SEQ ID NO:82. In one specific embodiment, the method further includes: designing primer pairs for detection to screen cells or tissues that have no large fragment deletions at the promoters of the original PSMC5, ACTA1, ACTB, NME2, or RPL23 genes and have not been targeted at the upstream and downstream target sites of the promoters of the PSMC5, ACTA1, ACTB, NME2, or RPL23 genes.

[0013] In another specific embodiment, the primer pairs are as shown in SEQ ID NO:18 and SEQ ID NO:19, as shown in SEQ ID NO:18 and SEQ ID NO:30, and as shown in SEQ ID NO:31 and SEQ ID NO:19.

[0014] In one specific implementation, the DNA breakage is performed via a Meganuclease, Zinc finger nuclease, TALEN, or CRISPR / Cas system.

[0015] In another specific embodiment, the system is a CRISPR / Cas9 or CRISPR / KingCas12 system.

[0016] In one specific embodiment, the nucleic acid sequence of the strong promoter is shown in SEQ ID NO:3, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 or SEQ ID NO:52.

[0017] In one specific embodiment, the GH1 gene is a nucleotide encoding the amino acid sequence of SEQ ID NO:2.

[0018] In another specific embodiment, the GH1 gene nucleotide sequence is shown in SEQ ID NO:1.

[0019] In one specific embodiment, the GH1 highly expressed gene sequence is shown in SEQ ID NO:36, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 or SEQ ID NO:64.

[0020] In one specific embodiment, the pig growth traits are characterized by at least one of the following: increased daily weight gain throughout the entire growth stage, faster growth rate, lower feed conversion ratio, increased intramuscular fat percentage, reduced backfat thickness, and increased lean meat percentage.

[0021] The present invention also provides a GH1 high-expression gene, the nucleotide sequence of which is shown in SEQ ID NO:36, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 or SEQ ID NO:64.

[0022] The present invention also provides a pig having the above-mentioned GH1 high expression gene.

[0023] This invention also provides a target sequence designed to improve the growth rate of pigs or to knock up the expression of the endogenous GH1 gene in pigs, wherein the target sequence is selected from any one of the following groups:

[0024] (1) The sequence shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8 or their reverse complementary sequence;

[0025] (2) The sequences shown in SEQ ID NO:6, SEQ ID NO:53 and SEQ ID NO:54 or their reverse complementary sequences;

[0026] (3) The sequences shown in SEQ ID NO:6, SEQ ID NO:55 and SEQ ID NO:56 or their reverse complementary sequences;

[0027] (4) The sequences shown in SEQ ID NO:6, SEQ ID NO:57 and SEQ ID NO:58 or their reverse complementary sequences;

[0028] (5) The sequences shown in SEQ ID NO:6, SEQ ID NO:59 and SEQ ID NO:60 or their reverse complementary sequences.

[0029] Some of the terms used in this specification are defined as follows.

[0030] The term "pigs" in this invention refers to the family Suidae, a type of vertebrate. In one specific embodiment, the pigs include Yorkshire, Landrace, Duroc, and Pietrain.

[0031] The "Yorkshire pig" described in this invention is a mammal belonging to the genus *Sus* in the family Suidae of the order Artiodactyla. It is large, rectangular in shape, with white fur all over its body, occasionally with a few dark spots. It has a relatively long head, a slightly concave face, medium-sized ears that tilt forward, a long body, broad shoulders, and a slightly arched back. The large Yorkshire pig, also known as the Large White pig, is a cured meat type and is found worldwide due to its high reproductive rate, thin back fat, high lean meat content, and good meat quality.

[0032] The terms "wild type" and "mutation" as used in this invention are relative and refer to the phenotype with the highest frequency in a particular population, or the system, organism, or gene that has this phenotype.

[0033] The terms "protein," "polypeptide," and "peptide" are used interchangeably in this invention to refer to polymers of amino acid residues, including polymers in which one or more amino acid residues are chemical analogs of natural amino acid residues. The proteins and polypeptides of this invention can be generated through recombinant synthesis or through chemical synthesis.

[0034] As used in this article, “genome” refers to all the genetic material (genes and non-coding sequences) present in every cell, virus, or organelle of an organism, and / or the complete set of chromosomes inherited as a unit (haploid) from a parent.

[0035] The “gene” as described in this invention includes nucleic acid fragments that express functional molecules (such as, but not limited to, specific proteins), including regulatory sequences before (5' non-coding sequence) and after (3' non-coding sequence).

[0036] The DNA sequence that “encodes” a specific RNA is the DNA nucleic acid sequence that is transcribed into RNA. DNA polynucleotides can encode RNA (mRNA) that is translated into proteins, or DNA polynucleotides can encode RNA that is not translated into proteins (such as tRNA, rRNA, or RNA that targets DNA; also known as “non-coding” RNA or “ncRNA”).

[0037] The terms "nucleotide," "polynucleotide," "nucleic acid," "nucleic acid molecule," or "nucleic acid sequence" used in this invention are used interchangeably and refer to oligonucleotides, nucleotides, or polynucleotides and fragments or portions thereof, which may be single-stranded or double-stranded, and indicate sense or antisense strands. Nucleic acids include DNA, RNA, or hybrids thereof, and may have natural or synthetic origins. For example, nucleic acids may include mRNA or cDNA. Nucleic acids may include nucleic acids that have been amplified (e.g., using polymerase chain reaction). The nucleotide name "R" indicates a purine, such as guanine or adenine; "Y" indicates a pyrimidine, such as cytosine or thymine (or uracil if it is RNA); "M" indicates adenine or cytosine; "K" indicates guanine or thymine; and "W" indicates adenine or thymine.

[0038] Those skilled in the art will readily understand that, due to the degeneracy of the genetic code, a variety of different nucleic acid sequences can encode the amino acid sequences disclosed herein. Generating other nucleic acid sequences encoding the same protein is within the capabilities of those skilled in the art; therefore, this invention covers nucleic acid sequences encoding the same amino acid sequence due to the degeneracy of the genetic code. For example, to achieve high expression of a heterologous gene in a target host organism such as a plant, the gene can be optimized using codons preferred by the host organism to improve its expression.

[0039] The term "gene editing" refers to strategies and techniques for targeted and specific modifications to any genetic information or genome of a living organism. Therefore, the term includes editing of gene-coding regions, but also editing of regions other than the gene-coding regions of the genome. It also includes editing or modifying the nucleus (if present) and other genetic information within the cell.

[0040] The term “CRISPR / Cas” can refer to a CRISPR-based nuclease or the nucleic acid sequence encoding it, including but not limited to: 1) Cas9, including SpCas9, ScCas9, SaCas9, xCas9, VRER-Cas9, EQR-Cas9, SpG-Cas9, SpRY-Cas9, SpCas9-NG, NG-Cas9, NGA-Cas9(VQR), etc.; 2) Cas12, including LbCpf1, FnCpf1, AsCpf1, MAD7, KingCas12, etc.; or any variant or derivative of the aforementioned CRISPR-based nucleases. Preferably, the at least one of the CRISPR-based nucleases contains a mutation compared to the corresponding wild-type sequence, such that the obtained CRISPR-based nuclease recognizes a different PAM sequence.

[0041] The term "CRISPR" refers to a sequence-specific genetic manipulation technique that relies on clustered, regularly spaced short palindromic repeats, unlike RNA interference which regulates gene expression at the transcriptional level.

[0042] The terms "Cas9 nuclease" and "Cas9" are used interchangeably in this article, referring to RNA-guided nucleases that include the Cas9 protein or fragments thereof (e.g., proteins containing the active DNA-cutting domain of Cas9 and / or the gRNA-binding domain of Cas9). Cas9 is a component of the CRISPR / Cas (clustered regularly spaced short palindromic repeats and related systems) genome editing system, capable of targeting and cleaving DNA target sequences to form DNA double-strand breaks (DSBs) under the guidance of guide RNA.

[0043] In this document, "gRNA" refers to a guide RNA used for targeting specific genes and thus correcting them using CRISPR technology. It typically consists of partially complementary crRNA and tracrRNA molecules forming a complex. The crRNA contains a sequence sufficiently complementary to the target sequence to hybridize with it and guide the CRISPR complex (Cas9+crRNA+tracrRNA) to specifically bind to the target sequence. However, it is known in the art that a single guide RNA (sgRNA) can be designed that simultaneously incorporates features of both crRNA and tracrRNA.

[0044] The term "crRNA" or "CRISPR RNA" refers to a guide RNA suitable for the CRISPR system, which includes a recognition sequence (or spacer sequence) and a backbone region. The backbone region can interact with a CRISPR protein (or Cas protein), thereby causing the Cas protein and crRNA to form a complex, which guides the complex to bind to a target sequence. As described in this invention, crRNA is a short RNA that guides the KingCas12 protein / enzyme (SEQ ID NO: 13) to bind to a target genomic DNA sequence, and its recognition sequence is complementary to the target sequence.

[0045] The terms “complementary,” “complementarity,” “reverse complementarity,” or “reverse complementarity” are used interchangeably to refer to a nucleotide sequence that is complementary to a given nucleotide sequence, but in reverse order. For example, the sequence “5’-CAGT-3’” is complementary to the sequence “3’-GTCA-5’.” The term “complementary sequence” refers to a nucleic acid sequence that can form hydrogen bonds with another nucleic acid sequence through conventional Watson-Crick or other non-traditional types.

[0046] In some embodiments, guide RNA(one or more) and Cas9 or KingCas12 protein can be delivered to cells as a ribonucleoprotein (RNP) complex. The RNP consists of purified Cas9 or KingCas12 protein complexed with the guide RNA, and it is well known in the art that RNPs can be efficiently delivered to a variety of cell types, including but not limited to stem cells and immune cells (Addgene, Cambridge, MA; Mirus Bio LLC, Madison, WI).

[0047] The terms "translocation" or "chromosomal translocation" can be used interchangeably in this article. A change in the position of a chromosome segment is called a "translocation," which is accompanied by a change in the position of a gene. When a translocation occurs within a single chromosome, it is called a translocation or intrachromosomal translocation; when a translocation occurs between two homologous or non-homologous chromosomes, it is called an interchromosomal translocation.

[0048] Altering the expression of endogenous genes in organisms involves both intensity and spatiotemporal characteristics. Intensity includes upregulation (i.e., knock-up, high gene expression), downregulation (i.e., knock-down), and zeroing (i.e., knockout); spatiotemporal specificity includes time (reproductive period) specificity and space (tissue) specificity, as well as inducibility. Furthermore, it involves altering protein targeting, for example, changing a protein located in the cytoplasm to one located in chloroplasts or the nucleus. In this invention, a "highly expressed gene" refers to a gene whose expression level is higher than that of a normal gene in a specific tissue.

[0049] The terms "regulatory sequence" and "regulatory element" are used interchangeably. Both refer to nucleotide sequences located upstream (5' non-coding sequence), midway, or downstream (3' non-coding sequence) of a coding sequence that influence the transcription, RNA processing, stability, or translation of the relevant coding sequence. Plant expression regulatory elements are nucleotide sequences that can control the transcription, RNA processing, stability, or translation of a nucleotide sequence of interest in plants.

[0050] Regulatory sequences may include, but are not limited to, promoters, translational leader sequences, introns, and polyadenylation recognition sequences.

[0051] A promoter is a nucleic acid fragment that controls the transcription of another nucleic acid segment. A promoter is a specific DNA regulatory region located upstream of the coding sequence of a gene, whose function is to mediate and regulate the transcription initiation and expression level of a target gene. This region contains core promoter elements (such as the TATA box, initiator Inr, etc.) and adjacent regulatory sequences (such as the GC box, CAAT box, etc.), and can form a pre-initiation complex (PIC) by binding to RNA polymerase, universal transcription factors (GTFs), and specific transcription factors (TFs), thereby precisely controlling the spatiotemporally specific expression of the gene. In genetic engineering, promoters are operatively linked to the open reading frame (ORF) of the target gene to achieve the controlled expression of the foreign gene in the host cell. In some embodiments of this invention, the promoter is a promoter capable of controlling gene transcription in plant cells, regardless of whether it originates from plant cells. The promoter can be a constitutive promoter, a tissue-specific promoter, a developmental regulatory promoter, or an inducible promoter.

[0052] As used herein, the term "promoter" generally refers to a DNA molecule involved in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription. A promoter can initially be isolated from a DNA sequence within a 1-4000 bp range upstream of the 5' untranslated region (5'UTR) of a gene's genomic copy. Alternatively, a promoter can be a synthetically generated or manipulated DNA molecule. Promoters can also be chimeric, i.e., promoters generated by the fusion of two or more heterologous DNA molecules. Useful promoters in embodiments of the invention include SEQ ID NO:3, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52, or fragments or variants thereof. In specific embodiments of the invention, these promoters described herein, and any variants or derivatives thereof, are further defined as possessing promoter activity, i.e., the ability to function as a promoter in a host cell (e.g., in a transgenic plant). In a further specific embodiment, a fragment may be defined as exhibiting the promoter activity of the initiator from which it originates, or the fragment may contain a “minimal promoter” (which provides the basic level of transcription and consists of an equivalent sequence of a TATA box or a sequence for recognition and binding of the RNA polymerase II complex to enable transcription initiation).

[0053] In one embodiment, a fragment of the promoter sequence disclosed herein is provided. The promoter fragment may contain the promoter activity described above and may be used alone or in combination with other promoters and promoter fragments, as in a constructed chimeric promoter. In a particular embodiment, a promoter fragment comprising at least about 50, 95, 150, 250, 500, 750, 1000, 1500, 2000, 3000, or at least about 4000 consecutive nucleotides or longer of a polynucleotide molecule having the promoter activity disclosed herein is provided.

[0054] The promoter may be an isolated nucleic acid, wherein the sequence of said isolated nucleic acid comprises a sequence having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a sequence selected from SEQ ID NO:3, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52, or having at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with a fragment thereof.

[0055] The term "strong promoter" is well-known and widely used in the art, and many strong promoters are known in the art or can be identified by routine experiments. The promoter activity is higher than that of a promoter effectively linked to the nucleic acid molecule to be overexpressed in a wild-type organism, for example, a promoter with higher activity than the promoter of an endogenous gene. Preferably, the strong promoter activity is about 2%, 5%, 8%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000%, or more than 1000% higher than that of a promoter effectively linked to the nucleic acid molecule to be overexpressed in a wild-type organism. Those skilled in the art know how to determine promoter activity and compare the activities of different promoters.

[0056] In this invention, "comprising" or "including" means "including but not limited to".

[0057] The terminology used herein is for the purpose of describing particular embodiments and is not intended to be limiting. Unless otherwise expressly stated herein, the terms “a,” “an,” and “the” as used in the foregoing include their plural forms. The terms “comprises” and / or “comprising,” or “includes” and / or “including” as used herein specifically refer to the presence of the features, factors, and / or ingredients described herein, without excluding the presence and addition of one or more other features, factors, and ingredients. The term “and / or” as used above includes all or one of the items in the list of combinations.

[0058] This invention has been described in detail through a series of embodiments, but the invention is not limited to the disclosed embodiments. Any variations, substitutions, or replacements that fall within the scope of this invention, not described herein, may be modified according to public needs. Attached Figure Description

[0059] Figure 1. PCR identification results of PSMC5 promoter translocation in F1 generation. The left side connection represents the front-end interface of PSMC5 promoter initiating GH1 gene translocation event, and the right side connection represents the back-end interface.

[0060] Figure 2. PCR identification results of DDX5 promoter translocation. The left link represents the front-end interface of the DDX5 promoter initiating the GH1 gene translocation event, and the right link represents the back-end interface.

[0061] Figure 3. PCR identification results of RPS15A promoter translocation. The left link represents the front-end interface of the RPS15A promoter initiating the GH1 gene translocation event, and the right link represents the back-end interface.

[0062] Figure 4. Standard curve of ELISA detection of porcine GH1 high expression gene expression level;

[0063] Figure 5. Comparison of expression levels of the porcine GH1 overexpression gene by qPCR;

[0064] Figure 6-1. Comparison of average daily weight gain in pigs with GH1 gene knockout;

[0065] Figure 6-2. Comparison of pig GH1 gene knockout-in increase in body weight;

[0066] Figure 7. PCR identification results of PSMC5 promoter translocation in F2 generation. The left side connection represents the front-end interface of PSMC5 promoter initiating GH1 gene translocation event, and the right side connection represents the back-end interface.

[0067] Figure 8. PCR identification results of F2 generation ACTA1 promoter translocation. The left link represents the front-end interface of the ACTA1 promoter initiating the GH1 gene translocation event, and the right link represents the back-end interface.

[0068] Figure 9. F2 generation ACTB promoter translocation PCR identification results. The left link represents the front-end interface of the ACTB promoter initiating the GH1 gene translocation event, and the right link represents the back-end interface.

[0069] Figure 10. PCR identification results of NME2 promoter translocation in F2 generation. The left link represents the front-end interface of the NME2 promoter initiating the GH1 gene translocation event, and the right link represents the back-end interface.

[0070] Figure 11. PCR identification results of RPL23 promoter translocation in F2 generation. The left link represents the front-end interface of the RPL23 promoter initiating the GH1 gene translocation event, and the right link represents the back-end interface.

[0071] Sequence Description Detailed Implementation

[0072] The present invention will be further illustrated below with reference to examples. The following description is by way of example, but the scope of protection of the present invention should not be limited thereto.

[0073] Example 1: Design and preparation of GH1 overexpression gene RNPs

[0074] Strong promoters were selected to achieve high expression of GH1 (nucleotide sequence SEQ ID NO: 1, amino acid sequence SEQ ID NO: 2), namely PSMC5 (Proteasome 26S Subunit, ATPase 5, located on chromosome 12 of pigs, also known as SUG1 or TRIP1, SEQ ID NO: 3), DDX5 (SEQ ID NO: 4), and RPS15A (SEQ ID NO: 5). The goal is to obtain GH1 high-expression genes PSMC5-GH1, DDX5-GH1, and RPS15A-GH1 with promoter translocations of PSMC5, DDX5, and RPS15A. The specific steps are as follows:

[0075] Target sites were designed for the upstream sequence of the start codon of the Yorkshire pig GH1 gene and the upstream and downstream of the target strong promoter, as shown in Table 1.

[0076] Table 1. Target design for GH1 gene hyperexpression

[0077] Off-target prediction was performed using the Cas-OFFinder tool. PAM:YTTV was selected, species: Sus Scrofa was chosen, and seven crRNA target sequences (SEQ ID NO: 6-12) were entered. The results showed that none of the seven corresponding crRNAs had off-target sites. Genscript Biotech was commissioned to synthesize the crRNAs corresponding to the above targets. The synthesized crRNAs were mixed with purified KingCas12 (SEQ ID NO: 13) protein in a specific ratio, and RNase-free ultrapure water was added to make up the volume, resulting in a final crRNA concentration of 0.25 μM and a KingCas12 protein concentration of 0.125 μM. The mixture was incubated at room temperature for 10 minutes to form KingCas12 RNP complexes. Three groups of RNP samples were prepared by gently mixing equal volumes of the three KingCas12 RNP complexes. Example 2: Creation of a GH1 High-Expression Gene in Pigs

[0078] The three RNP samples were transfected into fetal porcine fibroblasts using electroporation. PCR detection was performed using the corresponding primers. Cells with promoter fragments inserted at the target site of crRNA1 and without deletions at the promoters of PSMC5, DDX5, and RPS15A genes were selected for single-cloning. The nuclei of these cells were then injected into enucleated oocytes using somatic cell cloning techniques. Recombinant embryos were fused, activated, and then transplanted into recipient sows. Representative primers are shown in Table 2.

[0079] Table 2 Primers for RNP-edited porcine DNA identification

[0080] Sows gave birth naturally between days 114 and 117. Molecular identification of F0 generation skin tissue revealed that the expected target bands (no bands were observed in wild-type WT) were amplified at both the anterior and posterior interfaces connecting the PSMC5 promoter and the GH1 gene in the F0 generation, with single-peak sequencing results at both interfaces, indicating PSMC5 promoter sequence insertion at the target site of crRNA1 in the F0 generation. The absence of large deletions in the F0 generation suggests that the PSMC5 promoter sequence at the target site of crRNA1 originates from the sister chromatid during replication and did not cause deletions at the target sites of crRNA2 and crRNA3 in the F0 generation. The amplification of expected target bands at the target sites of crRNA1, crRNA2, and crRNA3 in the F0 generation (with band sizes consistent with those amplified in wild-type) indicates heterozygous insertion of the PSMC5 promoter sequence at the target site of crRNA1 in the F0 generation. Sequencing results showed that the bands amplified at the target sites of crRNA1, crRNA2, and crRNA3 were all single peaks. Targeting was performed at the crRNA1 target site. An 8-base pair deletion was found in the 5'UTR region of the GH1 gene without causing a frameshift mutation. Targeting was not performed at the crRNA2 and crRNA3 target sites, and the sequences were consistent with the wild-type sequences, meeting expectations. F0 generation piglets were bred, and after farrowing, genotyping was performed in the F1 generation to obtain the target mutants for further breeding.

[0081] The molecular identification results of the GH1 gene initiated by the PSMC5 promoter in the F1 generation are shown in Figure 1. The F1 generation inherited the genotype of the F0 generation. On one chromosome 12, the expected target bands were amplified at both the anterior and posterior interfaces connecting the PSMC5 promoter and the GH1 gene (no band was observed in wild-type WT), and the sequencing results showed a single peak without large deletion fragments, indicating the insertion of the PSMC5 promoter fragment at the target site of crRNA1. The F1 generation amplified the expected target bands at the target sites of crRNA1, crRNA2, and crRNA3 (consistent in size with the bands amplified in wild-type). According to the sequencing results, the sequences at the target sites of crRNA1, crRNA2, and crRNA3 showed a single peak, and all were consistent with the wild-type WT sequence, indicating that the target sites of crRNA1, crRNA2, and crRNA3 on the other chromosome 12 of the F1 generation were all wild-type. The GH1 highly expressed gene PSMC5-GH1 (SEQ ID NO: 36) with heterozygous PSMC5 promoter translocation insertion at the target site of crRNA1 was obtained.

[0082] The molecular identification results of the F1 generation DDX5 and RPS15A promoters are shown in Figures 2 and 3. The GH1 highly expressed genes DDX5-GH1 (SEQ ID NO: 37) and RPS15A-GH1 (SEQ ID NO: 38) with heterozygous DDX5 and RPS15A promoter translocations at the crRNA1 target site were obtained.

[0083] The molecular identification results of the F2 generation PSMC5 promoter-driven GH1 gene are shown in Figure 7. Bands were amplified at both the front and back interfaces (no band was observed in wild-type WT), with no large deletions. No band was observed at the crRNA1 target site. Sequencing results at both the front and back interfaces showed a single peak, indicating homozygous PSMC5 promoter insertion at the crRNA1 target site on both chromosomes 12. Bands were amplified at the crRNA2 and crRNA3 target sites (same as wild-type WT). Sequencing results showed that the sequences at the crRNA2 and crRNA3 target sites were identical to the wild-type WT sequence, indicating that the crRNA2 and crRNA3 target sites on both chromosomes 12 in the F2 generation were wild-type. The GH1 high-expression gene PSMC5-GH1 (SEQ ID NO: 36) with homozygous PSMC5 promoter translocation insertion at the crRNA1 target site was obtained.

[0084] Example 3: Analysis of GH1 overexpression gene expression levels

[0085] 1. ELISA test

[0086] Three groups of F1 generation PSMC5-GH1, DDX5-GH1, and RPS15A-GH1 positive and wild-type Yorkshire pigs were respectively selected and grouped at the same time. They were weaned at 21 days of age and fed with the same nutritional levels and methods. Slaughter was carried out when they reached the required weight. Feed was withheld for 24 hours prior to slaughter, but water was provided freely. Blood was collected from the ear vein. The collected whole blood was allowed to coagulate naturally overnight at 4°C, centrifuged at 1000g for 20 minutes, and the supernatant was carefully collected.

[0087] Serum GH1 protein concentration in porcines was determined using the Hengyuan Biotechnology Porcine Growth Hormone Enzyme-Linked Immunosorbent Assay Kit (product number: HB131-Pg). A standard curve was established with the concentration of the standard as the x-axis and the OD value as the y-axis, and the linear regression equation of the standard curve was calculated, as shown in Figure 4. The actual concentration of the sample was calculated by substituting the OD value of the sample into the equation. The GH1 contents of the groups using RPS15A, DDX5, and PSMC5 as promoters and the wild type were 501.41 μg / L, 49.71 μg / L, 215.96 μg / L, and 10.12 μg / L, respectively. Compared with the wild type, RPS15A-GH1 content was the highest, approximately 50 times that of the wild type; PSMC5-GH1 content was the second highest, approximately 21 times that of the wild type; while DDX5-GH1 content was the lowest, approximately 5 times that of the wild type.

[0088] 2. qPCR detection

[0089] Total RNA was extracted from F1 generation PSMC5-GH1, DDX5-GH1, and RPS15A-GH1 positive and wild-type Yorkshire pig cells. After reverse transcription, qPCR was performed, and statistical analysis was conducted using GraphPad Prism 8.0 software (* represents P<0.05, ** represents P<0.01, ***** represents P<0.001). The qPCR primers are shown in Table 3. Primers were also designed in the CDS region of the GAPDH gene as an internal reference gene, and these primers were synthesized by Sangon Biotech Co., Ltd.

[0090] Table 3 qPCR detection primers

[0091] As shown in Figure 5, the mRNA levels of PSMC5-GH1, DDX5-GH1, and RPS15A-GH1 genes were significantly increased. Among them, the expression level of RPS15A-GH1 increased the most significantly, by 11,192 times compared with wild type; the expression level of PSMC5-GH1 increased the second most, by 7,646 times; and the expression level of DDX5-GH1 increased by 2,812 times.

[0092] Example 4: Phenotypic Identification of GH1 High-Expression Gene-Edited Pigs

[0093] Two groups of F1 generation positive and wild-type Yorkshire pigs were selected, and their average daily weight gain and body weight were measured, as shown in Figures 6-1 and 6-2. Compared with their wild-type littermates, PSMC5-GH1 knockout pigs had an average daily weight gain 1.7 times that of wild-type pigs after birth and a weaning weight 1.55 times that of wild-type pigs. They exhibited a faster growth rate after weaning, with significantly faster increases in body weight, body length, and body height. The corrected age at 100 kg was 105 days, far lower than the 160 days of the wild-type control. They had a higher lean meat percentage and no impact on reproduction or health, but no other phenotypic differences were observed. In addition, although the transcriptional strength of the RPS15A promoter was stronger than that of the PSMC5 promoter, the growth rate of the pigs was not significantly higher than that of the PSMC5-GH1 knockout pigs, and the pigs exhibited health problems, such as difficulty walking, rapid breathing, and decreased reproductive capacity. Although the transcriptional strength of the DDX5 promoter was weaker than that of the PSMC5 promoter, the pigs did not exhibit health problems, but their growth rate was significantly lower than that of the PSMC5-GH1 knockout pigs.

[0094] The results of testing and statistics on homozygous F2 generation gene-edited pigs were similar to those of F1 generation. PSMC5-GH1 knockout pigs showed significantly improved growth rate and no health problems.

[0095] Example 5: Obtaining Other Types of Pigs with High GH1 Gene Expression

[0096] Using the same method, more strong promoters were selected to achieve high expression of GH1, namely ACTA1 (actin alpha 1, located on pig chromosome 14, SEQ ID NO: 49), ACTB (β-actin, located on pig chromosome 3, SEQ ID NO: 50), NME2 (nucleoside diphosphate kinase 2, located on pig chromosome 12, also known as NME or NM23, SEQ ID NO: 51), and RPL23 (ribosomal protein L23, located on pig chromosome 12, SEQ ID NO: 52). This resulted in GH1-high expression genes ACTA1-GH1, ACTB-GH1, NME2-GH1, and RPL23-GH1 with promoter translocations of ACTA1, ACTB, NME2, and RPL23. Target design is shown in Table 4.

[0097] Table 4. Design of other types of GH1 gene high expression targets

[0098] Off-target prediction results showed that none of the eight corresponding crRNAs had off-target sites. RNP samples were prepared after crRNA synthesis.

[0099] RNP samples were transfected into fetal porcine fibroblasts using electroporation. PCR detection was performed with corresponding primers to amplify fragments containing the target gene, the anterior and posterior interface fragments connecting GH1 and the target gene, and large deletions at the target promoter. Cells with promoter fragments inserted at the target region of crRNA1 and without deletions at the promoters of ACTA1, ACTB, NME2, and RPL23 genes were selected for single-cloning. The nuclei were extracted using somatic cell cloning techniques and injected into enucleated oocytes. Recombinant embryos were then fused, activated, and transplanted into recipient sows. Primers for identifying the anterior and posterior interfaces are shown in Table 5.

[0100] Table 5 Primers for identifying other types of GH1-overexpressing pigs

[0101] After farrowing, molecular identification was performed on the skin tissue of the F0 generation. The F0 generation amplified the expected target band at both the front and back interfaces of the target promoter and GH1 gene (no band was observed in wild-type WT), and both the front and back interfaces showed a single peak sequencing result, indicating that the target promoter sequence was inserted at the target site of crRNA1 in the F0 generation. There was no large deletion in the F0 generation, indicating that the target promoter sequence at the target site of crRNA1 originated from the sister chromatid during replication. The F0 generation amplified the expected target band at the target site (the band size was consistent with that amplified in wild-type), indicating that the target promoter sequence at the target site of crRNA1 in the F0 generation was a heterozygous insertion. Sequencing results revealed that all amplified bands at the target sites were single-peaked, and targeting occurred at the crRNA1 target site. The 5'UTR region of the ACTA1 promoter translocation GH1 showed a 12-base deletion, the ACTB promoter translocation GH1 showed an 18-base deletion, and the NME2 promoter translocation GH1 showed a 4-base deletion. The RPL23 promoter translocation GH1 showed an insertion of a T at position +49 and a G at position +53, followed by an RPL23 promoter fragment, all without causing frameshift mutations. The target sites at crRNA8-15 at the promoter were not targeted, and the sequences were identical to the wild-type sequences, meeting expectations. F0 generation piglets were bred, and the F1 generation was genotype-selected after farrowing to obtain the target mutants for further breeding.

[0102] The F1 generation inherited the genotype of the F0 generation. The expected target bands were amplified at both the anterior and posterior interfaces connecting the target promoter and the GH1 gene (wild-type WT showed no bands), and the sequencing results showed a single peak with no large deletions, indicating insertion of the target promoter fragment at the crRNA1 target site. The F1 generation amplified the expected target bands at the target sites (consistent in size with the bands amplified by the wild type). Based on the single peak sequence at the target sites and the consistency with the wild-type WT sequence, it is clear that the target sites in the F1 generation were all wild-type. This yielded the GH1-overexpressing genes ACTA1-GH1, ACTB-GH1, NME2-GH1, and RPL23-GH1, which exhibit heterozygous target promoter translocation insertion at the crRNA1 target site.

[0103] The molecular identification results of the F2 generation ACTA1, ACTB, NME2, and RPL23 promoters for the GH1 gene are shown in Figures 8-11. Bands were amplified at both the anterior and posterior interfaces connecting the target promoter and the GH1 gene (no band was observed in wild-type WT). No large deletions were found, and no band was observed at the crRNA1 target site. The sequencing results at both the anterior and posterior interfaces showed a single peak, indicating homozygous insertion of the target promoter at the crRNA1 target site on both chromosomes 12. Bands were amplified at the target sites of promoter crRNAs 8-15 (same as wild-type WT). Based on the sequencing results, the promoter target crRNAs were identified. The sequences at the target sites of crRNA8-15 are all identical to the wild-type WT sequences, indicating that the two chromosomes at the target sites of the F2 generation promoter crRNA8-15 are all wild-type. The GH1 highly expressed genes ACTA1-GH1, ACTB-GH1, NME2-GH1 and RPL23-GH1 with homozygous target promoter translocation insertion at the target site of crRNA1 were obtained, and their sequences are shown in SEQ ID NO: 61-64.

[0104] Example 6: Phenotypic Identification of Other Types of GH1 High-Expression Gene-Edited Pigs

[0105] Two groups of F1 and F2 generation positive and wild-type Yorkshire pigs were selected respectively, and the average daily weight gain and body weight were measured. The results showed that, compared with their wild-type littermates, ACTA1-GH1 knockout pigs had an average daily weight gain of 1.73 times, a weaning weight of 1.6 times, and a corrected age of 100 kg per 100 days; ACTB-GH1 knockout pigs had an average daily weight gain of 1.78 times, a weaning weight of 1.64 times, and a corrected age of 95 days per 100 kg; NME2-GH1 knockout pigs had an average daily weight gain of 1.72 times, a weaning weight of 1.58 times, and a corrected age of 102 days per 100 kg; and RPL23-GH1 knockout pigs had an average daily weight gain of 1.76 times, a weaning weight of 1.62 times, and a corrected age of 98 days per 100 kg.

[0106] The above data indicate that GH1 knockout pigs exhibit a faster growth rate after weaning, with significantly faster increases in weight, body length, and body height compared to wild-type controls. Their corrected age at 100 kg is much lower than the control's 160 days, and they have a higher lean meat percentage without affecting reproduction or health, and no other phenotypic differences are observed. Therefore, the GH1 high-expression gene in pigs obtained in this invention can achieve ideal results, enabling pigs to grow rapidly and have high feed conversion rates without affecting reproduction and health, thus possessing significant industrial value.

[0107] All publications and patent applications mentioned in the specification are incorporated herein by reference as if each publication or patent application were individually and specifically incorporated herein by reference.

[0108] Although the invention has been described in considerable detail by way of example and embodiments for clarity, it will be apparent that certain changes and modifications may be made within the scope of the appended claims, and all such changes and modifications are within the scope of the invention.

Claims

1. A method of increasing the growth rate of a swine, comprising, The method comprises the following steps: The coding region of the GH1 gene is fused with a strong promoter of another endogenous high-expression gene in vivo to generate a GH1 high-expression gene without introducing an exogenous gene, and the strong promoter is a PSMC5 gene promoter, an ACTA1 gene promoter, an ACTB gene promoter, an NME2 gene promoter, or an RPL23 gene promoter.

2. An editing method for knocking up expression of an endogenous GH1 gene in a porcine, characterized by, The method comprises the following steps: The coding region of the GH1 gene is fused with a strong promoter of another endogenous high-expression gene in vivo to generate a GH1 high-expression gene without introducing an exogenous gene, and the strong promoter is a PSMC5 gene promoter, an ACTA1 gene promoter, an ACTB gene promoter, an NME2 gene promoter, or an RPL23 gene promoter.

3. The method according to claim 1 or 2, characterized in that, The method comprises the following steps: DNA is simultaneously cleaved at two different specific positions in the genome of the organism, which are a genomic site between the coding region of the GH1 gene and its promoter and a genomic site between the coding region of the endogenous high-expression gene and its promoter, and then connected to each other through an intracellular repair pathway to fuse the coding region of the GH1 gene with the promoter of the endogenous high-expression gene in vivo, thereby generating a GH1 high-expression gene; Or, DNA is simultaneously cleaved at three different specific positions in the genome of the organism, which are two genomic sites capable of cleaving a fragment of the promoter of the endogenous high-expression gene and a genomic site between the coding region of the GH1 gene and its promoter, and then connected to each other through an intracellular repair pathway to generate a translocation editing event of the fragment of the promoter of the endogenous high-expression gene inserted upstream of the coding region of the GH1 gene, thereby generating a GH1 high-expression gene; Or, DNA is simultaneously cleaved at three different specific positions in the genome of the organism, which are two genomic sites capable of cleaving a fragment of the coding region of the GH1 gene and a genomic site between the promoter of the endogenous high-expression gene and its coding region, and then connected to each other through an intracellular repair pathway to generate a translocation editing event of the fragment of the coding region of the GH1 gene inserted downstream of the promoter of the endogenous high-expression gene, thereby generating a GH1 high-expression gene.

4. The method of claim 3, wherein, The method further comprises the following steps: designing a primer pair that can specifically distinguish the new combination of genetic elements for the above-mentioned specific position combination, screening cells or tissues containing the GH1 high-expression gene by PCR, and determining the characteristic sequence of the new combination of genetic elements by sequencing; preferably, the primer pair comprises at least one of the following sequences: SEQ ID NO: 14 and SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, SEQ ID NO: 67 and SEQ ID NO: 68, SEQ ID NO: 69 and SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74, SEQ ID NO: 75 and SEQ ID NO: 76, SEQ ID NO: 77 and SEQ ID NO: 78, SEQ ID NO: 79 and SEQ ID NO: 80, and SEQ ID NO: 81 and SEQ ID NO:

82.

5. The method of claim 4, further comprising: A primer pair is designed for detection to screen cells or tissues without large fragment deletion at the promoter of the original PSMC5, ACTA1, ACTB, NME2 or RPL23 gene and without targeting at the target site upstream and downstream of the PSMC5, ACTA1, ACTB, NME2 or RPL23 gene promoter; preferably, the primer pair is as shown in SEQ ID NO: 18 and SEQ ID NO: 19, SEQ ID NO: 18 and SEQ ID NO: 30, and SEQ ID NO: 31 and SEQ ID NO:

19.

6. The method of claim 3, wherein: The DNA break is performed by a Meganuclease, a Zinc finger nuclease, a TALEN or a CRISPR / Cas system; preferably, the system is a CRISPR / Cas9 or CRISPR / KingCas12 system.

7. The method according to any one of claims 1 to 6, characterized in that, The nucleic acid sequence of the strong promoter is as shown in SEQ ID NO: 3, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO:

52.

8. The method according to any one of claims 1 to 7, characterized in that, The GH1 gene is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, preferably, the nucleotide sequence is as shown in SEQ ID NO:

1.

9. The method according to any one of claims 1 to 8, characterized in that, The GH1 high-expression gene sequence is as shown in SEQ ID NO: 36, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO:

64. The DNA break is performed by a Meganuclease, a Zinc finger nuclease, a TALEN or a CRISPR / Cas system; preferably, the system is a CRISPR / Cas9 or CRISPR / KingCas12 system. The nucleic acid sequence of the strong promoter is as shown in SEQ ID NO: 3, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 or SEQ ID NO:

52. The GH1 gene is a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2, preferably, the nucleotide sequence is as shown in SEQ ID NO:

1. The GH1 high-expression gene sequence is as shown in SEQ ID NO: 36, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63 or SEQ ID NO:

64.

10. The method according to any one of claims 1 to 9, characterized in that: The pig growth traits are at least one of the following characteristics: whole stage daily weight gain improvement, growth speed acceleration, feed conversion ratio reduction, intermuscular fat ratio increase, back fat thickness reduction, and lean meat rate improvement; preferably, the pigs include Yorkshire pigs, Landrace, Duroc, and Pietrain. 11.A pig with a GH1 high expression gene or having the gene, the nucleotide sequence of which is shown in SEQ ID NO: 36, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, or SEQ ID NO:

64.

12. A target sequence designed to improve growth rate in pigs or to knock up endogenous GH1 gene expression in pigs, characterized in that, The target sequence is selected from any one of the following groups: (1) SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, or reverse complements thereof; (2) SEQ ID NO: 6, SEQ ID NO: 53, and SEQ ID NO: 54, or reverse complements thereof; (3) SEQ ID NO: 6, SEQ ID NO: 55, and SEQ ID NO: 56, or reverse complements thereof; (4) SEQ ID NO: 6, SEQ ID NO: 57, and SEQ ID NO: 58, or reverse complements thereof; (5) SEQ ID NO: 6, SEQ ID NO: 59, and SEQ ID NO: 60, or reverse complements thereof.

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