Creation method and application of uORF for regulating MSTN protein expression

Abstract

The invention relates to a creation method and application of uORF for regulating and controlling MSTN protein expression. The invention provides a pig MSTN gene containing a newly created upstream open reading frame (uORF) in a non-coding region (5 'UTR). The invention also provides the sgRNA for creating the upstream open reading frame at the 5 'UTR of the pig MSTN gene and a gene editing system. The invention also provides a method for regulating and controlling the pig MSTN gene, a pig fetus fibroblast with reduced MSTN protein expression level and a production method of a lean type pig. The system and the method provided by the invention can realize low expression of the MSTN protein, so that a lean type pig model can be created for livestock breeding improvement, and the system and the method are efficient and safe.

Description

Technical Field

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

[0002] Myostain (MSTN) is a major negative regulator in the growth and development of skeletal muscle, which can inhibit the proliferation and differentiation of myoblasts and inhibit muscle growth. Therefore, MSTN is an important candidate gene in animal breed improvement, especially in promoting muscle mass increase and improving muscle growth traits.

[0003] In pigs, the MSTN gene is located on chromosome 15 and is mainly expressed in skeletal muscle tissue. This gene is highly conserved among different species. It is structurally composed of 3 exons and 2 introns, encoding a protein composed of 375 amino acids. As an extracellular signaling molecule, MSTN binds to receptors on the myoblast membrane, triggers the phosphorylation of the receptor itself, and then activates a series of intracellular signal transduction pathways. These signals act on the regulatory region of the myogenic determining factor (MyoD), inhibiting the proliferation and differentiation of muscle cells, thereby limiting the growth of skeletal muscle. This mechanism makes MSTN an important molecular switch for regulating muscle growth. Studies have found that mutations in this gene can significantly improve the meat production efficiency of livestock. The double muscle trait of the famous Belgian Blue cattle in nature is caused by mutations in the MSTN gene. In order to accelerate the improvement of pig genetic breeding, researchers used the CRISPR / Cas9 gene editing system to perform biallelic gene knockout of pig MSTN, and then performed somatic cell nuclear transplantation. The Chinese Bama pig with homozygous knockout of the MSTN gene was successfully prepared, and its growth rate and muscle fiber number were significantly increased (Zhu et al. 2020). However, the survival rate of "muscle pigs" created by gene knockout of MSTN is low. Kim Jin-soo and his colleagues at Seoul National University in South Korea obtained 32 "muscle pigs" by knocking out the MSTN gene. After 8 months, only 13 piglets survived, and only one was subsequently rated as "healthy". Therefore, healthy "muscle pigs" can be prepared by using non-coding sequences to regulate the expression of the MSTN gene.

[0004] uORF refers to "upstream open reading frame", which is widely present in eukaryotes. Usually, uORF can regulate gene expression by inhibiting the translation of coding regions (CDS). The traditional view is that proteins in cells are produced by ribosomes translating the coding regions of mRNA. In recent years, by capturing RNA fragments being translated by ribosomes for high-throughput sequencing, people have found that ribosomes can also translate untranslated regions (UTRs) upstream or downstream of CDS in mRNA and some non-coding RNAs. These non-classical translation groups composed of UTR and non-coding RNA translation play an important role in the developmental regulation, stress response and immune response of organisms, as well as human diseases such as tumors. Studies on the mechanism of action of uORF have found that uORF can regulate gene expression through three mechanisms: 1) uORF inhibits the translation of CDS to regulate gene expression; 2) uORF affects the transcription of CDS, thereby affecting gene expression; 3) uORF regulates the stability of CDS, thereby affecting gene expression.

[0005] The creation of uORF sequences to regulate gene expression by constructing an exogenous gene expression system has been applied in plants, but has not been reported in the construction of animal models. Therefore, creating uORF sequences to reduce the expression of MSTN genes and ultimately obtain healthy lean pigs is still a technical problem that needs to be solved urgently. Summary of the invention

[0006] Therefore, the purpose of the present invention is to provide a method for regulating the expression of MSTN protein by introducing mutations in non-coding regions to create uORF, and to provide a porcine MSTN gene with a created uORF in the 5'UTR, as well as sgRNA, gene editing system and method for creating uORF. The present invention also provides porcine fetal fibroblasts with reduced expression levels of MSTN protein, and a method for producing lean pigs using the porcine fetal fibroblasts.

[0007] The technical solution of the present invention is proposed on the basis of the following discoveries and studies:

[0008] Animal MSTN gene is the main effect gene controlling muscle growth, and mutation or knockout of this gene is beneficial to the growth of livestock muscle. The inventors of the present invention created uORF sequence to regulate the expression of MSTN gene by constructing an exogenous gene expression system. Among them, uORF is an open reading frame with a start codon located in 5'UTR. When creating uORF, the inventors studied the DNA sequence at the 5'UTR end of MSTN gene, analyzed its potential start codon AUG and the mutation sites that need to be introduced. Compared with knocking out MSTN gene, the present invention performs gene editing on the 5'UTR of pig MSTN gene by designing sgRNA, that is, adopting non-coding regulation of MSTN gene expression method is more efficient and safe.

[0009] The objective of the present invention is achieved through the following technical solutions:

[0010] In one aspect, the present invention provides a porcine MSTN gene having a 5'UTR with a created upstream open reading frame, and compared with the wild-type porcine MSTN gene, the porcine MSTN gene has a mutation from A to G at position chr15:94628476.

[0011] In the present invention, the nucleotide sequence of the 5'UTR of the wild-type porcine 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 the present invention studied and found that the editing site on the 5'UTR of the wild-type porcine MSTN gene is located at chr15: 94628545-94628441. Therefore, the inventors used sgRNA designed according to the 5'UTR region of the wild-type porcine MSTN gene (as shown in SEQ ID NO. 1) to perform site editing to introduce a mutation site, thereby creating a uORF sequence in the regulatory region of the MSTN gene.

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

[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] In another aspect, the present invention provides a sgRNA for creating an upstream open reading frame in the 5'UTR of the porcine MSTN gene.

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

[0024] Thus, according to some embodiments of the present invention, uORF-mut1 was created based on the position of chr15:94628476 (A mutated to G), and the nucleotide sequence of the sgRNA comprised 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, which comprises the sgRNA and CRISPR protein of the present invention. The gene editing system can mutate the A at position 94628476 (i.e., chr15:94628476) of chromosome 15 of the porcine MSTN gene to G (i.e., A->G).

[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 double-strand breaks in DNA.

[0032] In another aspect, the present invention provides a method for regulating porcine MSTN gene.

[0033] In one embodiment of the present invention, the method for regulating the porcine MSTN gene of the present invention comprises: introducing an expression vector comprising the coding region of the sgRNA described in the present invention and a vector expressing the CRISPR protein into porcine fetal fibroblasts, editing 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 a pGL3-U6 plasmid containing the sgRNA coding region. The pGL3-U6 plasmid backbone can be purchased from the market.

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

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

[0037] Wherein, the vector expressing CRISPR protein is a plasmid expressing CRISPR protein, preferably NG-ABE8e plasmid. NG-ABE8e plasmid can be purchased from the market.

[0038] In another embodiment of the present invention, the method for regulating the porcine MSTN gene of the present invention comprises: 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 a porcine fetal fibroblast with reduced expression level of MSTN protein, which is obtained by the method for regulating porcine MSTN gene of the present invention.

[0040] In another aspect, the present invention provides a method for producing lean pigs, comprising:

[0041] The cell nuclei of the porcine fetal fibroblasts of the present invention are injected into the enucleated porcine oocytes in vitro through somatic cell nuclear transplantation, so that the two cells fuse and a recombinant embryo is constructed.

[0042] It should be noted here that the specific method of somatic cell nuclear transplantation technology can refer to the relevant schemes in the prior art, and the present invention will not elaborate on it.

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

[0044] The inventors of the present invention have discovered through research that modification in the 5'UTR region of the porcine MSTN gene can reduce the site of MSTN protein expression, and based on this, proposed sgRNA, system and method for creating uORF in the 5'UTR of the MSTN gene, thereby effectively reducing the expression of MSTN protein in porcine fetal fibroblasts.

[0045] The method of the present invention is to regulate the expression of porcine MSTN protein through non-coding sequences. Since uORF can regulate gene expression by inhibiting the translation of CDS, a new uORF is created by introducing a point mutation at the 5'UTR genomic position of the MSTN gene through CRISPR / Cas9 base editing technology to reduce the translation of the MSTN gene, which can promote the proliferation and differentiation of muscle cells. The primary porcine cell line obtained by this gene regulation method can be used for nuclear transplantation to obtain lean pigs, and this method has great value for livestock genetic breeding. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

[0051] Figure 51 is a graph showing the results of the dual luciferase activity study of uORF in Example 1 of the present invention; wherein, a is a schematic diagram of the structure of the 5'UTR of MSTN, the black straight 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 results of the dual luciferase activity detection after electroporation of MSTN-LUC-WT, MSTN-LUC-mut1, MSTN-LUC-mut2, and MSTN-LUC-mut3, respectively.

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

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

[0054] Figure 8 1 is a graph showing the research results of the effect of uORF on MSTN expression in Example 1 of the present invention; wherein, 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; and c is a quantitative data graph of Western blot detection after electroporation of MSTN-FLAG-WT and uORF-FALG-mut1, respectively.

[0055] Fig. 9 : is a graph showing the editing efficiency detection result of sgRNA in Example 1 of the present invention; wherein, a is the editing efficiency of NGABE8e / sgRNA1 output after analysis by the EditR online website, the solid line box represents the editing efficiency, and the dotted line box represents the by-site editing efficiency; b is the editing efficiency of NGABE8e / sgRNA2 output after analysis by the EditR online website, the solid line box represents the editing efficiency, and the dotted line box represents the by-site editing efficiency; c is a quantitative data graph of editing efficiency; d is a quantitative data graph of off-target efficiency.

[0056] Fig.10 : is a diagram of the sequencing results of monoclonal positive cells in Example 2 of the present invention. Among them, the dotted line is the 5'UTR region of MSTN, the solid line is the CDS region of MSTN, and the red box is the site of mutation after editing.

[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 DESCRIPTION

[0058] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting 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 object of gene editing, the coding region of the porcine MSTN sequence was searched. The gene has two transcripts. This embodiment designs the sequence encoding the ENSSSCG00000016047 transcript. The transcript is 8276 bp long, has 3 exons, and encodes a MSTN protein with a length of 375 amino acids, as shown in SEQ ID NO. 4. The design creates uORFs at three positions in the 5'UTR region of the MSTN gene (chr15: 94628545~94628441, 105 bp long).

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

[0064] MQKLQIYVYIYLFMLIVAGPVDLNENSEQKENVEKEGLCNACMWRQNTKSSRLEAIKIQILSKLRLETAPNISKDAIRQLLPKAPPLRELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDLLMQVEGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVKTPTTVFVQILRLIKPMKDGTRYTG IRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTPFPGPGEDGLNPFLEVKVTDTPKRSRRDFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTKMSPINMLYFNGKEQIIYGKIPAMVVDRCGCS.

[0065] (2) Target design

[0066] The A at position 94628476 of the porcine MSTN gene was mutated to G, the T at position 94628458 was mutated to G, and the T at position 94628459 was mutated 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 encoding the 5'UTR region of the wild-type MSTN gene), SEQ ID NO. 2 (nucleotide sequence encoding the 5'UTR region of the MSTN gene with A mutated to G), SEQ ID NO. 21 (nucleotide sequence encoding the 5'UTR region of the MSTN gene with T mutated to G), and SEQ ID NO. 22 (nucleotide sequence encoding the 5'UTR region of the MSTN gene with T mutated 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. PCR amplification was performed using cDNA as a template to obtain the 5'UTR of MSTN with homology 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 program is shown in Table 2:

[0081]

[0082] (1.2) Introduce mutations by PCR amplification to create uORF 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) Using the seamless cloning kit In-Fusion ®Snap Assembly Master Mix (Takara, Cat. No. 638947) was used to connect the 5'UTRs shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 21, and SEQ ID NO. 22 to the dual luciferase reporter plasmid (the vector backbone is CMV-5'UTR-Firefly luciferase-SV40-Renillaluciferase) to obtain MSTN-LUC-WT, MSTN-LUC-mut1, MSTN-LUC-mut2, and MSTN-LUC-mut3 recombinant vectors. The maps are shown in Figure 2. Figure 1 , Figure 2 , Figure 3 and Figure 4 shown.

[0092] (2) The recombinant vectors of MSTN-LUC-WT, MSTN-LUC-mut1, MSTN-LUC-mut2, and MSTN-LUC-mut3 were transiently expressed in porcine fetal fibroblasts by electroporation, and the cells were collected after 48 h. The LUC / REN activity was measured using a dual luciferase reporter gene assay kit (Biyuntian, RG027). The results are shown in Table 1. Figure 5 As shown. Taking the start site of the coding region as 0, the created uORF-mut1 is 129 bp long, overlaps with the coding region by 66 bp, starts at -38 bp, and the mutation site is located at -36 bp; the created uORF-mut2 is 111 bp long, overlaps with the coding region by 91 bp, starts at -20 bp, and the mutation site is located at -18 bp; the created uORF-mut3 is 9 bp long, does not overlap with the coding region, starts at -19 bp, and the mutation site is located at -19 bp. Figure 5 It can be seen that compared with cells electroporated with MSTN-LUC-WT recombinant vector (WT group), the luciferase activity detected in cells electroporated with MSTN-LUC-mut1, MSTN-LUC-mut2, and MSTN-LUC-mut3 recombinant vectors was significantly reduced. And MSTN-LUC-mut1 was about 0.1 times that of the WT group, which shows that the created uORF-mut1 can significantly inhibit the translation of MSTN.

[0093] 3. Determination of MSTN gene expression

[0094] (1) Construction of overexpression vector

[0095] (1.1) RNA from pig muscle cells was extracted and reverse transcribed into cDNA, and PCR amplification was performed using cDNA as a template to obtain 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) Introduce the FLAG tag at the 3' end of the CDS by PCR amplification, and introduce homology arms at both ends. Specifically, using the CDS of MSTN 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, PCR was used to amplify the 5'UTR containing the CMV promoter as an element, 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 procedure 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) Connect the two fragments amplified in step (1.3) to the 5' end of CDS-FLAG respectively, and use the seamless cloning kit In-Fusion ® Snap Assembly Master Mix (Takara, Cat. No. 638947) was used to connect the obtained fragments to the pcDNA3.1 overexpression vector (Miaoling Biotechnology, P0157) digested with restriction endonucleases MluI and NotI to obtain MSTN-FLAG-WT and MSTN-FLAG-mut1 recombinant vectors. The maps are shown in Figure 6 and Figure 7 shown.

[0111] (2) The MSTN-FLAG-WT and MSTN-FLAG-mut1 recombinant vectors were electroporated into porcine fetal fibroblasts, respectively. After 48 h, the 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 direct embryo 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 shown in the figure. Figure 8 As shown. Figure 8It can be seen that compared with cells electroporated with MSTN-FLAG-WT recombinant vector (MSTN-WT group), there was no significant difference in the mRNA level of MSTN in cells electroporated with MSTN-FLAG-mut1 recombinant vector (uORF-mut1 group), while the protein expression of MSTN was significantly reduced. This result shows that the created uORF will block the translation of MSTN, but will not affect the transcription of MSTN.

[0112] 4. Construction of sgRNA expression vector

[0113] (1) sgRNA design

[0114] In this example, sgRNA was designed after determining the mutation site of chr15:94628476 based on the uORF characteristics, with the purpose of mutating ATA to ATG, and based on the restriction of "NG" in the PAM region, two sgRNAs were designed 18 to 20 bp upstream and downstream. Among them, sgRNA1 has a higher off-target rate, and ATA is easily mutated to GTG, while sgRNA2 can achieve the mutation of ATA to ATG.

[0115] The specific research process is as follows:

[0116] Two sgRNAs with a length of 20 bp were designed for the target mutation site chr15: 94628476 and the PAM structure (5'-NG-3'). The 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 by blast to avoid specific binding of sgRNA in non-target regions.

[0120] (2) Construction of sgRNA expression vector

[0121] The pGL3-U6 plasmid (Miaoling Biotechnology, P1139) was used as the vector backbone to construct the pGL3-U6-sgRNA-tdTomato vector. In addition to expressing the sgRNA backbone, the tdTomato red fluorescent protein was also expressed. According to the sequence information of sgRNA1 and sgRNA2, two single-stranded oligonucleotides with special sequences added to the 5' end (ACCG added to the forward primer and AAAC added to the reverse primer) were first synthesized, and then the paired single-stranded oligonucleotides were annealed according to the following system to form double-stranded DNA fragments with sticky ends. The annealing system of sgRNA1 and sgRNA2 is the same, as shown in Table 3:

[0122]

[0123] Incubate the mixture at 95°C for 5-10 min, then anneal 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 ® Snap Assembly Master Mix (Takara, Cat. No. 638947) was used to connect the obtained sgRNA1 and sgRNA2 products to the pGL3-U6-sgRNA-tdTomato vector backbone after digestion with restriction endonuclease Bsa I-HF (NEB, R3733S). The ligation products were transformed into E. coli competent cells (TAKARA), inoculated into LB culture plates with ampicillin, and cultured at 37°C overnight. Single clones were picked and sequenced the next day.

[0131] The positive monoclones were enriched in 30 mL of agarose medium containing ampicillin, cultured at 37°C overnight, and then the recombinant plasmid was extracted using the Tiangen Endotoxin-Free Plasmid Miniprep Kit (DP118-02).

[0132] 5. Base editing efficiency detection in mixed cells

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

[0134] The cracking 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 NOs. 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 website. Fig. 9 As shown, although the editing efficiency of sgRNA1 is slightly higher than that of sgRNA2, the editing efficiency of sgRNA1 (i.e., off-target rate) is also significantly higher than that of sgRNA2 (as shown in the dotted box). Therefore, in order to achieve the purpose of mutating ATA to ATG, this experiment selected sgRNA2 as the sgRNA for subsequent examples.

[0138] Example 2

[0139] For the editing site that can create uORF in porcine MSTN, a high-efficiency sgRNA, namely sgRNA 2, was screened in Example 1. In this example, the 5'UTR porcine primary cells modified by gene editing of MSTN were obtained by flow sorting of monoclonal positive cells.

[0140] The NG-ABE8e and sgRNA expression vectors were electroporated into porcine fetal fibroblasts. After 48 h, the cells were collected and 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, follow the software operation steps, adjust the parameters, load the cell samples, and directly sort the single cells expressing tdTomato red fluorescence into 15 96-well cell culture plates. Replace fresh culture medium on the 4th day after cell sorting; observe and mark the cell wells that form single cell clones under a microscope on the 7th day, and only replace the culture medium for the marked cell wells; then replace the culture medium every 3 days until the cell clone confluence in the 96 wells is about 90%, transfer the cells to a 24-well plate, and digest the cells when the cell clone confluence in the 24 wells is about 90%. Take 9 / 10 cells and freeze them; take 1 / 10 cells and transfer them to a 0.2 mL PCR tube, centrifuge at 5000 r / min for 5 min, discard the supernatant, and obtain a cell pellet. Resuspend the cell pellet in the PCR tube with 20 μL of cell lysis buffer and lyse it. The specific lysis system and lysis procedure are the same as step 5 of Example 1. Finally, the PCR product was Sanger sequenced to identify the genotype of the monoclonal cell. Finally, the monoclonal cell with the sequencing sequence SEQID NO. 2 was retained, and the identification result was as follows Fig.10 As shown. Fig.10 The results show that homozygous mutant cells in which ATA mutated to ATG have been successfully screened out.

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

[0143] The above are only several exemplary embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention is disclosed as above with preferred embodiments, they are not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, can make some changes or modifications to obtain equivalent or equivalent embodiments using the above disclosed technical content, which fall within the scope of the present invention.

Claims

1. A porcine MSTN gene with a 5'UTR having a created upstream open reading frame, and compared with the wild-type porcine MSTN gene, the porcine MSTN gene has a mutation from A to G at position chr15:94628476.

2. A sgRNA for creating an upstream open reading frame in the 5'UTR of the porcine MSTN gene, characterized in that: The sgRNA is complementary to a DNA sequence fragment with a length of 18 to 22 bp located in the 5'UTR of the MSTN gene and adjacent to the 5'-NG-3' protospacer sequence adjacent motif.

3. The sgRNA according to claim 2, characterized in that The nucleotide sequence of the sgRNA comprises the nucleotide sequence shown in SEQ ID NO. 3, or the nucleotide sequence of the sgRNA is shown in SEQ ID NO.

3.

4. A gene editing system for creating an upstream open reading frame in the 5'UTR of the porcine MSTN gene, characterized in that: It comprises the sgRNA and CRISPR protein according to claim 2 or 3.

5. The gene editing system according to claim 4, characterized in that: The CRISPR protein is dCas9 or nCas9.

6. The gene editing system according to claim 4 or 5, characterized in that: The gene editing system is a base editor; Preferably, the base editor is an adenine base editor.

7. A method for regulating porcine MSTN gene, characterized in that: It includes: Introducing an expression vector comprising the coding region of the sgRNA according to claim 2 or 3 and a vector expressing the CRISPR protein into porcine fetal fibroblasts to edit the non-coding region of the MSTN gene; or The gene editing system according to any one of claims 4 to 6 is introduced into porcine fetal fibroblasts to reduce the expression of MSTN protein.

8. The method according to claim 7, characterized in that The expression vector comprising the sgRNA coding region is a plasmid comprising the sgRNA coding region; Preferably, the plasmid comprising the sgRNA coding region is a pGL3-U6 plasmid comprising the sgRNA coding region; Optionally, the vector expressing the CRISPR protein is a plasmid expressing the CRISPR protein; Preferably, the plasmid expressing the CRISPR protein is a NG-ABE8e plasmid.

9. A porcine fetal fibroblast with reduced MSTN protein expression level, characterized in that: It is obtained by the method according to claim 7 or 8.

10. A method for producing lean pigs, characterized in that: It includes: The cell nucleus of the porcine fetal fibroblast according to claim 9 is injected into the enucleated porcine oocyte in vitro by somatic cell nuclear transplantation, and the two cells are fused to construct a recombinant embryo.

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