A method for creating uORF for regulating expression of MSTN protein and application thereof

Abstract

The present application relates to a method for creating uORF for regulating MSTN protein expression and application thereof. The present application provides a pig MSTN gene containing a newly created upstream open reading frame (uORF) in a non-coding region (5'UTR). The present application also provides an sgRNA and a gene editing system for creating an upstream open reading frame in the 5'UTR of a pig MSTN gene. The present application also provides a method for regulating a pig MSTN gene, a pig fetal fibroblast with reduced MSTN protein expression level, and a method for producing a lean pig. The system and method provided by the present application can achieve low expression of MSTN protein, thereby creating a lean pig model for livestock breeding improvement, which is efficient and safe.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and animal genetics and breeding. Specifically, this invention relates to a method for creating a uORF for regulating MSTN protein expression and its application. More specifically, this invention relates to a porcine MSTN gene with a created uORF in its 5'UTR, as well as sgRNA, gene editing system, method, and application for creating the uORF. Background Technology

[0002] Myosin (MSTN) is a major negative regulator of skeletal muscle growth and development, inhibiting the proliferation and differentiation of myoblasts and suppressing muscle growth. Therefore, MSTN is an important candidate gene for animal breed improvement, particularly showing significant potential in promoting muscle mass increase and improving muscle growth traits.

[0003] In pigs, the MSTN gene is located on chromosome 15 and is primarily expressed in skeletal muscle tissue. This gene is highly conserved across species, structurally consisting of three exons and two introns, encoding a protein of 375 amino acids. As an extracellular signaling molecule, MSTN binds to receptors on the myoblast membrane, triggering receptor phosphorylation and activating a series of intracellular signaling pathways. These signals act on the regulatory region of myogenic determinant factor (MyoD), inhibiting muscle cell proliferation and differentiation, thereby limiting skeletal muscle growth. This mechanism makes MSTN a crucial molecular switch regulating muscle growth. Studies have found that mutations in this gene can significantly improve meat production efficiency in livestock; the famous double-muscle trait of the Belgian Blue cattle is attributed to mutations in the MSTN gene. To accelerate pig genetic breeding improvement, researchers used the CRISPR / Cas9 gene editing system to knock out the MSTN gene in pigs using a biallelic knockout method, followed by somatic cell nuclear transfer. This successfully produced homozygous MSTN knockout pigs from Bama, China, which exhibited significantly increased growth rate and muscle fiber number (Zhu et al. 2020). However, the survival rate of "muscle pigs" created by knocking out the MSTN gene is low. Kim Jin-soo and colleagues at Seoul National University in South Korea obtained 32 "muscle pigs" by knocking out the MSTN gene; after eight months, only 13 piglets survived, and only one was subsequently rated as "healthy." Therefore, healthy "muscle pigs" can be obtained by using non-coding sequences to regulate MSTN gene expression.

[0004] uORF stands for "upstream open reading frame," which is widely present in eukaryotes. Typically, uORF regulates gene expression by inhibiting the translation of coding regions (CDS). Traditionally, it is believed that proteins in cells are produced by ribosomes translating the coding regions of mRNA. However, recent high-throughput sequencing of RNA fragments being translated by ribosomes has revealed that ribosomes can also translate untranslated regions (UTRs) upstream or downstream of the CDS in mRNA, as well as some non-coding RNAs. These non-classical translational sequences, composed of UTRs and non-coding RNAs, play important roles in developmental regulation, stress responses, immune responses, and human diseases such as tumors. Research on the mechanism of action of uORF has found that it regulates gene expression through three mechanisms: 1) uORF inhibits CDS translation, thus regulating gene expression; 2) uORF affects CDS transcription, thereby influencing gene expression; and 3) uORF regulates CDS stability, thus affecting gene expression.

[0005] The creation of uORF sequences to regulate gene expression using exogenous gene expression systems has been applied in plants, but its application in animal models has not yet been reported. Therefore, creating uORF sequences to reduce MSTN gene expression and ultimately obtain healthy lean-type pigs remains a pressing technical problem that needs to be solved. Summary of the Invention

[0006] Therefore, the purpose of this invention is to address the shortcomings of existing technologies by providing a method for regulating MSTN protein expression through the creation of uORF by introducing mutations into the non-coding region. This involves a porcine MSTN gene with a created uORF in its 5'UTR, as well as sgRNA for creating the uORF, a gene editing system, and a method. This invention also provides porcine fetal fibroblasts with reduced MSTN protein expression levels, and a method for producing lean-type pigs using these porcine fetal fibroblasts.

[0007] The technical solution of this invention is proposed based on the following findings and research:

[0008] The MSTN gene in animals is a key gene controlling muscle growth; mutations or knockouts of this gene are beneficial for muscle growth in livestock. The inventors of this invention created a uORF sequence by constructing an exogenous gene expression system to regulate MSTN gene expression. uORF is an open reading frame with the start codon located in the 5'UTR. During uORF creation, the inventors studied the 5'UTR DNA sequence of the MSTN gene, analyzing its potential start codon AUG and the mutation sites to be introduced. Compared to knocking out the MSTN gene, this invention uses sgRNA to edit the 5'UTR of the porcine MSTN gene, employing a non-coding method to regulate MSTN gene expression, which is more efficient and safer.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] On the one hand, the present invention provides a porcine MSTN gene with an innovative upstream open reading frame in the 5'UTR, which has an A to G mutation at position chr15:94628476 compared with the wild-type porcine MSTN gene.

[0011] In this invention, the nucleotide sequence of the 5'UTR of the wild-type pig MSTN gene is shown in SEQ ID NO. 1.

[0012] Specifically, the nucleotide sequence shown in SEQ ID NO. 1 is as follows:

[0013] tctctcagacagtgcaggcattaaaattttgcttggcgttactcaaaagcaaaagtaaaaggaagaaataagaacaaggagaaagattgtattgattttaaaatc.

[0014] The inventors of this invention studied and discovered that the editing site on the 5'UTR of the wild-type pig MSTN gene is located at chr15: 94628545~94628441. Therefore, the inventors used an sgRNA designed based on the 5'UTR region of the wild-type pig MSTN gene (as shown in SEQ ID NO. 1) to introduce a mutation site through site editing, thereby creating a uORF sequence in the regulatory region of the MSTN gene.

[0015] When designing uORF modification creation sites, mutation sites in the 5'UTR of the wild-type pig MSTN gene were determined based on uORF characteristics. Based on the sequence characteristics of the MSTN gene 5'UTR, mutations were introduced at chr15:94628476 (A to G), chr15:94628458 (T to G), and chr15:94628459 (T to A), respectively, thereby creating uORF-mut1, uORF-mut2, and uORF-mut3 in the 5'UTR region of the pig MSTN gene, with lengths of 84 bp, 66 bp, and 9 bp, respectively. After introducing mutations at positions chr15:94628476, chr15:94628458, and chr15:94628459 in the porcine MSTN gene, the corresponding nucleotide sequences of the MSTN gene 5'UTR are shown in SEQ ID NO. 2, SEQ ID NO. 21, and SEQ ID NO. 22, respectively. Based on the results of dual-luciferase reporter assays, it was found that uORF-mut1 significantly inhibited MSTN translation.

[0016] Specifically, the nucleotide sequence shown in SEQ ID NO. 2 is as follows:

[0017] tctctcagacagtgcaggcattaaaattttgcttggcgttatactcaaaagcaaaagtaaaaggaagaaat G agaacaaggagaaagattgtattgattttaaaatc (The underlined bases are the mutated bases).

[0018] The nucleotide sequence shown in SEQ ID NO. 21 is as follows:

[0019] tctctcagacagtgcaggcattaaaattttgcttggcgttactcaaaagcaaaagtaaaaggaagaaataagaacaaggagaaagat G gtattgattttaaaatc (The underlined bases are the mutated bases).

[0020] The nucleotide sequence shown in SEQ ID NO. 22 is as follows:

[0021] tctctcagacagtgcaggcattaaaattttgcttggcgttactcaaaagcaaaagtaaaaggaagaaataagaacaaggagaaaga Atgtattgattttaaaatc (The underlined bases are the mutated bases).

[0022] On the other hand, the present invention provides an sgRNA for creating an upstream open reading frame in the 5'UTR of the porcine MSTN gene.

[0023] As described above, the inventors designed an sgRNA based on the 5'UTR of the wild-type pig MSTN gene (as shown in SEQ ID NO. 1). Specifically, the sgRNA is complementary to a DNA sequence fragment of 18-22 bp (preferably 20 bp) in length located in the 5'UTR of the MSTN gene and immediately adjacent to the 5'-NG-3' protospacer adjacent motif (PAM sequence).

[0024] Therefore, according to some embodiments of the present invention, uORF-mut1 is created based on the chr15:94628476 (A mutated to G) position, wherein the nucleotide sequence of the sgRNA comprises the nucleotide sequence shown in SEQ ID NO. 3.

[0025] Preferably, the nucleotide sequence of the sgRNA is shown in SEQ ID NO. 3.

[0026] Specifically, the nucleotide sequence shown in SEQ ID NO. 3 is as follows:

[0027] ataagaacaaggagaaagat.

[0028] On the other hand, the present invention provides a gene editing system for creating an upstream open reading frame in the 5'UTR of the porcine MSTN gene, comprising the sgRNA and CRISPR protein described in this invention. This gene editing system can mutate an A to a G mutation (i.e., A->G) at position 94628476 on chromosome 15 of the porcine MSTN gene (i.e., chr15: 94628476 position).

[0029] According to some embodiments of the present invention, the CRISPR protein is selected from one of dCas9, nCas9 and Cas12; more preferably, the CRISPR protein is dCas9 or nCas9.

[0030] According to some embodiments of the present invention, the system is a base editor (BE).

[0031] Preferably, the base editor is an adenine base editor (ABE), which is a genome editing tool that uses the CRISPR-Cas system to achieve the conversion of adenine (A) to guanine (G) at a specific position without causing DNA double-strand breaks.

[0032] On the other hand, the present invention provides a method for regulating the porcine MSTN gene.

[0033] In one embodiment of the present invention, the method for regulating the porcine MSTN gene includes: introducing an expression vector containing the coding region of the sgRNA described in the present invention and a vector expressing CRISPR protein into porcine fetal fibroblasts to edit the non-coding region of the MSTN gene, thereby creating a uORF in the 5'UTR of the porcine MSTN gene.

[0034] The expression vector containing the sgRNA coding region is a plasmid containing the sgRNA coding region, preferably the pGL3-U6 plasmid containing the sgRNA coding region. The pGL3-U6 plasmid backbone can be obtained commercially.

[0035] Preferably, the pGL3-U6 plasmid may also contain the coding region of the tdTomato red fluorescent marker protein.

[0036] It should be noted that the construction method of the plasmid containing the coding region of the sgRNA and the coding region of the tdTomato red fluorescent marker protein can refer to the relevant schemes in the prior art, and will not be elaborated here.

[0037] The vector for expressing the CRISPR protein is a plasmid expressing the CRISPR protein, preferably the NG-ABE8e plasmid. The NG-ABE8e plasmid can be obtained commercially.

[0038] In another embodiment of the present invention, the method for regulating the porcine MSTN gene of the present invention includes: introducing the gene editing system of the present invention into porcine fetal fibroblasts, thereby reducing the expression of porcine MSTN protein.

[0039] On the other hand, the present invention provides porcine fetal fibroblasts with reduced MSTN protein expression levels, which are obtained by the method of regulating the porcine MSTN gene described in the present invention.

[0040] On the other hand, the present invention provides a method for producing lean-type pigs, comprising:

[0041] The nucleus of the porcine fetal fibroblast described in this invention is injected into a porcine enucleated oocyte in vitro via somatic cell nuclear transfer, thereby fusing the two cells to construct a recombinant embryo.

[0042] It should be noted that the specific methods of somatic cell nuclear transfer technology can refer to relevant solutions in the existing technology, and will not be elaborated upon in this invention.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The inventors of this invention discovered a site in which modifying the 5'UTR region of the porcine MSTN gene can reduce MSTN protein expression. Based on this, they proposed a system and method for creating uORF sgRNA in the 5'UTR of the MSTN gene, which can effectively reduce MSTN protein expression in porcine fetal fibroblasts.

[0045] The method of this invention involves regulating the expression of porcine MSTN protein through non-coding sequences. Since uORFs can regulate gene expression by inhibiting CDS translation, a new uORF is created by introducing a point mutation at the 5'UTR position of the MSTN gene using CRISPR / Cas9 base editing technology to reduce MSTN gene translation, thereby promoting muscle cell proliferation and differentiation. The porcine primary cell lines obtained using this gene regulation method can be used for nuclear transfer to obtain lean-type pigs. This method has significant value for livestock genetics and breeding. Attached Figure Description

[0046] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0047] Figure 1 This is a map of the MSTN-LUC-WT recombinant vector constructed in Example 1 of this invention.

[0048] Figure 2 This is a map of the MSTN-LUC-mut1 recombinant vector constructed in Example 1 of this invention.

[0049] Figure 3 This is a map of the MSTN-LUC-mut2 recombinant vector constructed in Example 1 of this invention.

[0050] Figure 4 This is a map of the MSTN-LUC-mut3 recombinant vector constructed in Example 1 of this invention.

[0051] Figure 5Figure 1 shows the results of the uORF dual luciferase activity study in Example 1 of this invention; where a is a schematic diagram of the 5'UTR structure of MSTN, the black line is the 5'UTR, the light gray square is the coding region, and the dark gray square is the created uORF; b is the result of dual luciferase activity detection after electroporation of MSTN-LUC-WT, MSTN-LUC-mut1, MSTN-LUC-mut2, and MSTN-LUC-mut3, respectively.

[0052] Figure 6 This is a map of the MSTN-FLAG-WT recombinant vector constructed in Embodiment 1 of the present invention.

[0053] Figure 7 This is a map of the MSTN-FLAG-mut1 recombinant vector constructed in Example 1 of this invention.

[0054] Figure 8 Figure 1 shows the results of the study on the effect of uORF on MSTN expression in Example 1 of this invention. Among them, a is a quantitative data graph of RT-qPCR detection after electroporation of MSTN-FLAG-WT and uORF-FALG-mut1, respectively; b is a Western blot detection graph after electroporation of MSTN-FLAG-WT and uORF-FALG-mut1, respectively; c is a quantitative data graph of Western blot detection after electroporation of MSTN-FLAG-WT and uORF-FALG-mut1, respectively.

[0055] Figure 9 This is a graph showing the results of sgRNA editing efficiency detection in Example 1 of this invention; where a is the editing efficiency of NGABE8e / sgRNA1 output after analysis by the EditR online website, with solid boxes representing editing efficiency and dashed boxes representing parasite editing efficiency; b is the editing efficiency of NGABE8e / sgRNA2 output after analysis by the EditR online website, with solid boxes representing editing efficiency and dashed boxes representing parasite editing efficiency; c is a quantitative data graph of editing efficiency; and d is a quantitative data graph of off-target efficiency.

[0056] Figure 10 This is a sequencing result diagram of monoclonal positive cells in Example 2 of this invention. The dashed line represents the 5'UTR region of the MSTN, the solid line represents the CDS region of the MSTN, and the red boxes indicate the sites of post-editing mutations.

[0057] In each figure, MSTN-WT is also MSTN-LUC-WT, uORF-mut1 is also MSTN-LUC-mut1, uORF-mut2 is also MSTN-LUC-mut2, and uORF-mut3 is also MSTN-LUC-mut3. Detailed Implementation

[0058] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0059] Example 1

[0060] 1. uORF modification creation site design

[0061] (1) Gene information and mutation sites

[0062] For the target gene editing, the coding region of the porcine MSTN sequence was located. This gene has two transcripts, and this embodiment designs the sequence encoding the ENSSCG00000016047 transcript. This transcript is 8276 bp long, has three exons, and encodes the MSTN protein, which is 375 amino acids long, as shown in SEQ ID NO. 4. uORFs were designed to be created at three positions in the 5'UTR region of the MSTN gene (chr15: 94628545~94628441, 105 bp in length).

[0063] The amino acid sequence shown in SEQ ID NO. 4 is as follows:

[0064] MQKLQIYVYIYLFMLIVAGPVDLNENSEQKENVEKEGLCNACMWRQNTKSSRLEAIKIQILSKLRLETAPNISKDAIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDLLMQVEGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPMKDGTRYTG IRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTPFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS.

[0065] (2) Target design

[0066] The A position at chr15:94628476 of the porcine MSTN gene was mutated to G, the T position at chr15:94628458 to G, and the T position at chr15:94628459 to A.

[0067] MSTN identification primers are as follows:

[0068] Forward primer MSTN-5UTR-GT-F1: 5'-tgaatcagctcacccttgact-3' (SEQ ID NO. 6);

[0069] Reverse primer MSTN-5UTR-GT-R1: 5'-acatgcattacacagcccctc-3' (SEQ ID NO. 7).

[0070] 2. Dual-luciferase reporter gene assay

[0071] (1) To verify whether the modified 5'UTR in the MSTN gene inhibits the translation of the downstream coding region, a dual luciferase reporter vector was constructed. SEQ ID NO. 1 (nucleotide sequence encoded by the 5'UTR region of the wild-type MSTN gene), SEQ ID NO. 2 (nucleotide sequence encoded by the 5'UTR region of the MSTN gene with A mutation to G), SEQ ID NO. 21 (nucleotide sequence encoded by the 5'UTR region of the MSTN gene with T mutation to G), and SEQ ID NO. 22 (nucleotide sequence encoded by the 5'UTR region of the MSTN gene with T mutation to A) were cloned into the dual luciferase reporter vector and named MSTN-LUC-WT, MSTN-LUC-mut1, MSTN-LUC-mut2, and MSTN-LUC-mut3, respectively.

[0072] (1.1) RNA was extracted from pig muscle cells and reverse transcribed into cDNA. The cDNA was used as a template for PCR amplification to obtain the 5'UTR of MSTN with homologous arms at both ends.

[0073] The primers used for PCR amplification are as follows:

[0074] Forward primer MSTN-IFWT-F1:

[0075] 5'-tatataagcagagcttctctcagacagtgcaggcat-3' (SEQ ID NO. 8);

[0076] Reverse primer MSTN-IFWT-R1:

[0077] 5'-CTTAGCATCGGCCATgattttaaaatcaatacaatctttctccttgttcttatttct-3' (SEQ ID NO. 9).

[0078] The PCR reaction system is shown in Table 1:

[0079]

[0080] The PCR reaction procedure is shown in Table 2:

[0081]

[0082] (1.2) The mutation was introduced by PCR amplification, and uORF was created in the 5' UTR of MSTN. The PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2. The primers used for PCR amplification are as follows:

[0083] Forward primer MSTN-IFWT-F1:

[0084] 5'-tatataagcagagcttctctcagacagtgcaggcat-3' (SEQ ID NO. 8);

[0085] Reverse primer MSTN-IFMUT1-R2:

[0086] 5'-CTTAGCATCGGCCATgattttaaaatcaatacaatctttctccttgttctCatttct-3' (SEQ ID NO. 10);

[0087] Reverse primer MSTN-IFMUT2-R2:

[0088] 5'-CTTAGCATCGGCCATgattttaaaatcaatacaTtctttctccttgttcttatttct-3' (SEQ ID NO. 23);

[0089] Reverse primer MSTN-IFMUT3-R2:

[0090] 5'-CTTAGCATCGGCCATgattttaaaatcaatacCatctttctccttgttcttatttct-3' (SEQ ID NO. 24).

[0091] (1.3) The In-Fusion Seamless Cloning Kit was used. ®Snap Assembly Master Mix (Takara, catalog number 638947) ligated the 5'UTRs shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 21, and SEQ ID NO. 22 to a dual luciferase reporter plasmid (vector backbone: CMV-5'UTR-Firefly luciferase-SV40-Renillaluciferase) to obtain recombinant vectors MSTN-LUC-WT, MSTN-LUC-mut1, MSTN-LUC-mut2, and MSTN-LUC-mut3, as shown in the diagrams below. Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown.

[0092] (2) The recombinant vectors MSTN-LUC-WT, MSTN-LUC-mut1, MSTN-LUC-mut2, and MSTN-LUC-mut3 were transiently expressed in porcine fetal fibroblasts via electroporation, and cells were collected after 48 h. LUC / REN activity was measured using a dual luciferase reporter gene assay kit (Beyotime, RG027), and the results are as follows: Figure 5 As shown. With the coding region start site set to 0, the created uORF-mut1 is 129 bp long, overlapping the coding region by 66 bp, starting at -38 bp, with the mutation site located at -36 bp; the created uORF-mut2 is 111 bp long, overlapping the coding region by 91 bp, starting at -20 bp, with the mutation site located at -18 bp; the created uORF-mut3 is 9 bp long, not overlapping the coding region, starting at -19 bp, with the mutation site located at -19 bp. From... Figure 5 As can be seen, compared with cells electroporated with the MSTN-LUC-WT recombinant vector (WT group), the luciferase activity detected in cells electroporated with the MSTN-LUC-mut1, MSTN-LUC-mut2, and MSTN-LUC-mut3 recombinant vectors was significantly reduced. Furthermore, the MSTN-LUC-mut1 activity was approximately 0.1 times that of the WT group, indicating that the created uORF-mut1 can significantly inhibit MSTN translation.

[0093] 3. Measurement of MSTN gene expression

[0094] (1) Construction of overexpression vector

[0095] (1.1) RNA was extracted from porcine muscle cells and reverse transcribed into cDNA. The cDNA was then used as a template for PCR amplification to obtain the CDS of MSTN. The PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2. The primers used for PCR amplification are as follows:

[0096] Forward primer MSTN-CDS-F2:

[0097] 5'-atgcaaaaactgcaaatctatgtttatatttacctg-3' (SEQ ID NO. 11);

[0098] Reverse primer MSTN-CDS-R1:

[0099] 5'-agcacccacagcgatctact-3' (SEQ ID NO. 12).

[0100] (1.2) A FLAG tag was introduced to the 3' end of the CDS via PCR amplification, and homologous arms were introduced at both ends. Specifically, using MSTN CDS as a template, the PCR reaction system is shown in Table 1, and the PCR reaction procedure is shown in Table 2. The primers used for PCR amplification are as follows:

[0101] Forward primer IF-MSTN-FLAG-F2:

[0102] 5'-tgattttaaaatcatgcaaaaactgcaaatctatgtttatatttacctg-3' (SEQ ID NO. 13);

[0103] Reverse primer MSTN-FLAG-R2:

[0104] 5'-TAGACTCGAGCGGCCtcacttgtcatcgtcatccttgtaatctgagcacccacagcgatcta-3' (SEQ ID NO. 14).

[0105] (1.3) Using MSTN-LUC-WT and MSTN-LUC-mut1 recombinant vectors as templates, the 5'UTR containing the CMV promoter was amplified by PCR, and a 15 bp sequence homologous to the CDS of the MSTN gene was introduced. The PCR reaction system is shown in Table 1, and the PCR reaction program is shown in Table 2. The primers used for PCR amplification are as follows:

[0106] Forward primer IF-MSTN-FLAG-F1:

[0107] 5'-GGCCAGATATACGCGTgacattgattattgactagttattaatagtaatcaattacggg-3' (SEQ ID NO. 15);

[0108] Reverse primer IF-MSTN-FLAG-R1:

[0109] 5'-ttgcagtttttgcatgattttaaaatcaatacaatctttctccttgttcttatttct-3' (SEQ ID NO. 16).

[0110] (1.4) The two fragments obtained in step (1.3) were ligated to the 5' end of CDS-FLAG, respectively, and the In-Fusion seamless cloning kit was used. ® The fragments obtained using Snap Assembly Master Mix (Takara, catalog number 638947) were ligated into the pcDNA3.1 overexpression vector (Miaoling Biotechnology, P0157) digested with restriction endonucleases MluI and NotI, resulting in the MSTN-FLAG-WT and MSTN-FLAG-mut1 recombinant vectors, as shown in the diagrams below. Figure 6 and Figure 7 As shown.

[0111] (2) The recombinant vectors MSTN-FLAG-WT and MSTN-FLAG-mut1 were electroporated into porcine fetal fibroblasts, respectively. After 48 h, cells were collected and RNA and protein were extracted. The expression of the MSTN gene was measured by RT-qPCR (YEASEN) and Western Blot experiments (for specific methods, see: Ruigao Song, et al., One-step base editing in multiple genes by directembryo injection for pig trait improvement. Science China Life Sciences, 2022. 65: p. 739-752. Or see: https: / / doi.org / 10.1007 / s11427-021-2013-8). The results are as follows. Figure 8 As shown. From Figure 8It can be seen that, compared with cells electroporated with the MSTN-FLAG-WT recombinant vector (MSTN-WT group), there was no significant difference in MSTN mRNA levels in cells electroporated with the MSTN-FLAG-mut1 recombinant vector (uORF-mut1 group), while MSTN protein expression was significantly reduced. This result indicates that the created uORF blocks MSTN translation but does not affect MSTN transcription.

[0112] 4. Construction of sgRNA expression vector

[0113] (1) sgRNA design

[0114] In this embodiment, the sgRNA was designed after identifying the chr15:94628476 mutation site based on uORF characteristics. It was designed around the goal of mutating ATA to ATG, and based on the "NG" restriction in the PAM region, two sgRNAs were designed 18-20 bp upstream and downstream of it. sgRNA1 has a higher off-target rate, making it easier for ATA to mutate into GTG, while sgRNA2 can achieve the ATA to ATG mutation.

[0115] The specific research process is as follows:

[0116] Two 20bp sgRNAs were designed targeting the mutation site chr15:94628476 and the PAM structure (5'-NG-3'). Details are as follows:

[0117] sgRNA1: 5'-agaaataagaacaaggagaa-3' (SEQ ID NO. 5);

[0118] sgRNA2: 5'-ataagaacaaggagaaagat-3' (SEQ ID NO. 3).

[0119] The designed sequence was compared with the pig genome sequence using blast to avoid sgRNA binding specifically to non-target regions.

[0120] (2) Construction of sgRNA expression vector

[0121] Using the pGL3-U6 plasmid (Miaoling Biotechnology, P1139) as the vector backbone, the pGL3-U6-sgRNA-tdTomato vector was constructed. In addition to expressing the sgRNA backbone, it also expresses the tdTomato red fluorescent protein. Based on the sequence information of sgRNA1 and sgRNA2, two single-stranded oligonucleotides with special sequences added to their 5' ends (ACCG added to the forward primer and AAAAC added to the reverse primer) were first synthesized. Then, the paired single-stranded oligonucleotides were annealed according to the following system to form double-stranded DNA fragments with sticky ends. The annealing systems for sgRNA1 and sgRNA2 were the same, as shown in Table 3.

[0122]

[0123] The mixture was incubated at 95°C for 5–10 min, and then annealed at 25°C for 30 min. The annealing primers are as follows:

[0124] sgRNA1:

[0125] Forward primer sgRNA1-F: 5'-accgagaaataagaacaaggagaa-3' (SEQ ID NO. 17);

[0126] Reverse primer sgRNA1-R: 5'-AAACttctccttgttcttatttct-3' (SEQ ID NO. 18).

[0127] sgRNA2:

[0128] Forward primer sgRNA2-F: 5'-accgataagaacaaggagaaagat-3' (SEQ ID NO. 19);

[0129] Reverse primer sgRNA2-R: 5'-AAACatctttctccttgttcttat-3' (SEQ ID NO. 20).

[0130] In-Fusion Seamless Cloning Kit ® The Snap Assembly Master Mix (Takara, catalog number 638947) was used to ligate the obtained sgRNA1 and sgRNA2 products, respectively, into the pGL3-U6-sgRNA-tdTomato vector backbone digested with restriction endonuclease Bsa I-HF (NEB, R3733S). The ligation products were transformed into *E. coli* competent cells (TAKARA), seeded into LB agar plates containing ampicillin, and incubated overnight at 37°C. Single clones were picked the following day and sequenced.

[0131] Positive monoclonal antibodies were enriched in 30 mL of agarose medium containing ampicillin and cultured overnight at 37°C. Recombinant plasmids were then extracted using the Tiangen endotoxin-free plasmid mini-extraction kit (DP118-02).

[0132] 5. Detection of base editing efficiency in mixed cells

[0133] The sgRNA1 and sgRNA2 expression vectors constructed in step 4 were co-electropoized into porcine fetal fibroblasts with NG-ABE8e (MiaoLingBio, P36211). Cells were collected after 48 h, and the cell samples were resuspended in α-MEM medium containing 10% FBS. Using a BD FACSAria flow cytometer, cell samples expressing tdTomato red fluorescence were directly sorted into 1.5 mL centrifuge tubes, and cell pellets were obtained by centrifugation. The cell pellets were resuspended in 20 μL of cell lysis buffer (Vazyme) and transferred to 0.2 mL PCR tubes for lysis and PCR.

[0134] The pyrolysis procedure is shown in Table 4:

[0135]

[0136] The PCR system and procedure are shown in Tables 1 and 2, and the PCR primers are shown in SEQ ID NO. 6 and 7.

[0137] Finally, the PCR products were subjected to Sanger sequencing, and the base editing efficiency of the mixed cells was analyzed using the EditR online database. The results are as follows: Figure 9 As shown, although sgRNA1 has a slightly higher editing efficiency than sgRNA2, its paratopic editing efficiency (i.e., off-target rate) is also significantly higher than that of sgRNA2 (as indicated by the dashed box). Therefore, in order to achieve the goal of ATA mutation to ATG, sgRNA2 was selected as the sgRNA for subsequent examples in this experiment.

[0138] Example 2

[0139] Example 1 screened for a highly efficient sgRNA, sgRNA2, targeting an editing site capable of creating uORF in porcine MSTN. In this example, 5'UTR porcine primary cells with gene-edited MSTN were obtained by flow cytometry sorting of single-clonal positive cells.

[0140] NG-ABE8e and sgRNA expression vectors were electroporated into porcine fetal fibroblasts. Cells were collected after 48 h, and the cell samples were resuspended in culture medium containing 15% fetal bovine serum (HyClone), 1% non-essential amino acids (Gibco), and 2 mmol / L. −1 MEM-α (Gibco) from GlutaMAX (Gibco).

[0141] Using a BD FACSAria flow cytometer, following the software operation steps and adjusting the parameters, cell samples were loaded. Single cells expressing tdTomato red fluorescence were directly sorted into 15 96-well cell culture plates. Fresh culture medium was replaced on day 4 after cell sorting. On day 7, the wells containing single-cell clones were observed and labeled under a microscope, with the culture medium only replaced in the labeled wells. The culture medium was then replaced every 3 days until the cell clone confluence in the 96-well plates reached approximately 90%. The cells were then transferred to 24-well plates, and when the cell clone confluence in the 24-well plates reached approximately 90%, the cells were digested. 9 / 10 of the cells were cryopreserved; 1 / 10 of the cells were transferred to a 0.2 mL PCR tube, centrifuged at 5000 r / min for 5 min, and the supernatant was discarded to obtain the cell pellet. The cell pellet in the PCR tube was resuspended in 20 μL of cell lysis buffer and lysed. The specific lysis system and procedure were the same as step 5 in Example 1. Finally, the PCR products were subjected to Sanger sequencing to identify the genotype of the single-clone cells. Finally, the monoclonal cell with the sequencing sequence SEQ ID NO. 2 was retained, and the identification results are as follows: Figure 10 As shown. From Figure 10 The results show that homozygous mutant cells that mutate from ATA to ATG have been successfully screened out.

[0142] By identifying and screening 5'UTR porcine primary cells with MSTN modified through gene editing, a lean pig model with low MSTN protein expression can be constructed using nuclear transfer technology.

[0143] The above descriptions are merely several exemplary embodiments of the present invention and are not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any equivalent or related embodiments obtained by those skilled in the art through minor modifications or variations of the disclosed technical content without departing from the scope of the present invention fall within the scope of the present invention.

Claims

1. A gene editing system for creating an upstream open reading frame in the 5'UTR of the porcine MSTN gene, characterized in that, It contains sgRNA with nucleotide sequences as shown in SEQ ID NO. 3 and CRISPR protein; The gene editing system is an adenine base editor; Using this gene editing system, a mutation from A to G was induced in the MSTN gene of wild-type pigs at position chr15:94628476. The transcript of the pig MSTN gene is ENSSSCG00000016047. The nucleotide sequence of the 5'UTR of the porcine MSTN gene is shown in SEQ ID NO. 2; An 84bp upstream open reading frame was created in the 5'UTR region of the porcine MSTN gene. The open reading frame started 38bp upstream of the start codon in the CDS region of the MSTN gene, and the mutation site was located 36bp upstream of the start codon in the CDS region of the MSTN gene.

2. A porcine fetal fibroblast with reduced MSTN protein expression levels, characterized in that, Porcine fetal fibroblasts were obtained by introducing the gene editing system of claim 1 into porcine fetal fibroblasts to reduce the expression of MSTN protein.

3. A method for producing lean-type pigs, characterized in that, It includes: The nucleus of the porcine fetal fibroblast as described in claim 2 is injected into a porcine enucleated oocyte in vitro via somatic cell nuclear transfer, thereby fusing the two cells to construct a recombinant embryo.

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