CRISPR / Cas12i-mediated crRNA and application thereof in knocking out bovine MSTN gene

Abstract

The present application relates to the technical field of genetic engineering, in particular to a CRISPR / Cas12i mediated crRNA and its application in knocking out bovine MSTN genes. The crRNA provided by the present application is a crRNA with high editing efficiency (up to 89.22%) designed and screened for 7 natural mutation sites (F94L, nt419, E226X, Q204X, nt821, E291X and C313Y) of the MSTN gene, and the different mutation sites of the MSTN gene of the Huaxi cattle can be genetically edited by the CRISPR / Cas12i system to obtain an MSTN gene knockout cell line. The present application not only improves the specificity and efficiency of gene editing, but also helps to accelerate the breeding process of excellent varieties in the field of agricultural biotechnology, provides new tools and strategies for genetic improvement of Huaxi cattle and other livestock, and has important scientific value and application prospect.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of genetic engineering, in particular to a CRISPR / Cas12i-mediated crRNA and application thereof in knocking out a bovine MSTN gene. BACKGROUND

[0002] With the rapid development of molecular biology and genetic engineering technology, gene editing technology plays an increasingly important role in life science research and biotechnology applications. In particular, in the field of livestock breeding, gene editing technology shows great application potential and provides a new technical means for rapidly and efficiently improving target traits. Among numerous gene editing tools, the CRISPR / Cas system, as the third generation of gene editing tools, has attracted much attention due to its simple operation, high efficiency and low cost.

[0003] The MSTN gene, also known as growth differentiation factor 8 (GDF8), is a negative regulator that mainly functions to inhibit muscle growth and development. Numerous studies have shown that natural mutations or artificial knockout of the MSTN gene can cause animals to exhibit a "double muscle" phenotype, i.e., significant muscle hypertrophy and increased muscle fiber number. This phenotype naturally exists in breeds such as Belgian Blue cattle, making these breeds have a higher lean meat rate and better meat quality. Therefore, the MSTN gene has become an important target for improving muscle growth in beef cattle breeding.

[0004] The CRISPR / Cas12i system exhibits higher average gene editing efficiency compared to currently widely used gene editing tools. Compared to Cas9 and Cas12a, the Cas12i has a smaller molecular weight and higher transfection efficiency, but there is currently a lack of crRNA with high editing efficiency for different mutation sites of the bovine MSTN gene. SUMMARY

[0005] To solve the above problems, the application provides a CRISPR / Cas12i-mediated crRNA and application thereof in knocking out a bovine MSTN gene. The crRNA provided by the application can perform gene editing on different mutation sites of the MSTN gene of the Huaxi cattle, and has high editing efficiency.

[0006] To achieve the above purpose, the application provides the following technical solutions:

[0007] The application provides a crRNA targeting a MSTN gene, including one or more of 94-1, 419-3, 226-1, 204-2, 821-3, 291-2 and 313-3; the nucleotide sequence of the 94-1 is shown as SEQ ID NO. 1; the nucleotide sequence of the 419-3 is shown as SEQ ID NO. 5; the nucleotide sequence of the 226-1 is shown as SEQ ID NO. 7; the nucleotide sequence of the 204-2 is shown as SEQ ID NO. 12; the nucleotide sequence of the 821-3 is shown as SEQ ID NO. 15; the nucleotide sequence of the 291-2 is shown as SEQ ID NO. 17; and the nucleotide sequence of the 313-3 is shown as SEQ ID NO. 21.

[0008] The application provides an expression vector, including a basic vector and a double-stranded nucleic acid molecule inserted into the basic vector; the double-stranded nucleic acid molecule is composed of a positive strand and a negative strand, and the positive strand and the negative strand are inversely complementary to each other; the positive strand includes a nucleic acid molecule encoding the crRNA in the above technical solution.

[0009] Preferably, the basic vector includes a Puc19 vector.

[0010] Preferably, the double-stranded nucleic acid molecule is located between the BsaI enzyme cutting sites of the Puc19 vector.

[0011] The application provides a kit for knocking out a MSTN gene, including the crRNA in the above technical solution and / or the expression vector in the above technical solution.

[0012] Preferably, the kit further includes a Cas12i nuclease and / or a Cas12i nuclease expression vector.

[0013] The application provides the application of the crRNA in the above technical solution, the expression vector in the above technical solution or the kit in the above technical solution in knocking out a bovine MSTN gene and / or constructing a bovine MSTN gene knockout cell line.

[0014] Preferably, the original cell of the bovine MSTN gene knockout cell line includes a fibroblast.

[0015] The application provides a method for constructing a bovine MSTN gene knockout cell line, including the following steps:

[0016] The expression vector in the above technical solution and the Cas12i nuclease expression vector are introduced into bovine fibroblasts to obtain positive knockout cells; the positive knockout cells are cultured and subcultured to obtain the bovine MSTN gene knockout cell line.

[0017] Preferably, the mass ratio of the expression vector and the Cas12i nuclease expression vector is 2:3.

[0018] Advantages:

[0019] The application provides a crRNA targeting an MSTN gene, which comprises one or more of 94-1, 419-3, 226-1, 204-2, 821-3, 291-2 and 313-3; the nucleotide sequence of the 94-1 is shown in SEQ ID NO. 1; the nucleotide sequence of the 419-3 is shown in SEQ ID NO. 5; the nucleotide sequence of the 226-1 is shown in SEQ ID NO. 7; the nucleotide sequence of the 204-2 is shown in SEQ ID NO. 12; the nucleotide sequence of the 821-3 is shown in SEQ ID NO. 15; the nucleotide sequence of the 291-2 is shown in SEQ ID NO. 17; and the nucleotide sequence of the 313-3 is shown in SEQ ID NO. 21. The crRNA provided by the application is a crRNA with high editing efficiency (up to 89.22 %) obtained by screening and designing for seven natural mutation sites (F94L, nt419, E226X, Q204X, nt821, E291X and C313Y) of the MSTN gene, and the MSTN gene knockout cell line can be obtained by editing different mutation sites of the MSTN gene of the West China cattle through the CRISPR / Cas12i system. The application not only improves the specificity and efficiency of gene editing, but also helps to accelerate the breeding process of excellent varieties in the field of agricultural biotechnology, provides new tools and strategies for genetic improvement of the West China cattle and other domestic animals, and has important scientific value and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the drawings needed in the embodiments will be briefly introduced below.

[0021] Figure 1 is a fluorescence graph of 7 crRNAs;

[0022] Figure 2 and Figure 3 is a crRNA T7E1 enzyme digestion result graph;

[0023] Figure 4 is a crRNA editing efficiency statistical graph;

[0024] Figure 5 is a different editing type mutation result graph. DETAILED DESCRIPTION

[0025] The application provides a crRNA targeting an MSTN gene, and the crRNA comprises one or more of 94-1, 419-3, 226-1, 204-2, 821-3, 291-2 and 313-3; the nucleotide sequence of the 94-1 is shown as SEQ ID NO. 1; the nucleotide sequence of the 419-3 is shown as SEQ ID NO. 5; the nucleotide sequence of the 226-1 is shown as SEQ ID NO. 7; the nucleotide sequence of the 204-2 is shown as SEQ ID NO. 12; the nucleotide sequence of the 821-3 is shown as SEQ ID NO. 15; the nucleotide sequence of the 291-2 is shown as SEQ ID NO. 17; and the nucleotide sequence of the 313-3 is shown as SEQ ID NO. 21.

[0026] The application designs multiple pairs of crRNAs in different exon regions of the MSTN gene, uses a CRISPR / Cas12i system for gene editing, and statistically analyzes the editing efficiency of different crRNAs, so that the most suitable and efficient crRNA (the editing efficiency is as high as 89.22 %) is screened out. The high-efficiency crRNA sequence screened out can be directly used for editing the MSTN gene, improves the editing efficiency, helps to accelerate the breeding process of excellent varieties in the field of agricultural biotechnology, provides a new tool and strategy for genetic improvement of Huaxi cattle and other domestic animals, and has important scientific value and application prospect.

[0027] Based on the above advantages, the application provides an expression vector, which comprises a basic vector and a double-stranded nucleic acid molecule inserted into the basic vector; the double-stranded nucleic acid molecule is composed of a positive strand and a negative strand, and the positive strand and the negative strand are reversely complementary to each other; the positive strand comprises a nucleic acid molecule encoding the crRNA in the above technical solution. The expression vector in the application is an expression vector for expressing the crRNA in the above technical solution.

[0028] As an implementation manner, the basic vector comprises a Puc19 vector.

[0029] As an implementation manner, the double-stranded nucleic acid molecule is located between BsaI enzyme cutting sites of the Puc19 vector.

[0030] The application provides a kit for knocking out an MSTN gene, which comprises the crRNA in the above technical solution and / or the expression vector in the above technical solution.

[0031] As an implementation manner, the kit further comprises a Cas12i nuclease and / or a Cas12i nuclease expression vector.

[0032] As an embodiment, the Cas12i nuclease expression vector of the present application is obtained from the Key Laboratory of Stem Cell and Reproductive Biology of the Institute of Animal Sciences of the Chinese Academy of Sciences, which is disclosed in the literature

Chen, Y., Y. Hu, X. Wang, S. Luo, N. Yang, Y. Chen, Z. Li, Q. Zhou, and W. Li. 2022. 'Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing', Innovation (Camb), 3: 100264.

[0033] Based on the above advantages, the present application provides the application of the crRNA or the expression vector or the kit in knocking out the bovine MSTN gene and / or constructing a bovine MSTN gene knockout cell line.

[0034] As an embodiment, the bovine includes Huaxi cattle; and the original cell of the bovine MSTN gene knockout cell line includes a fibroblast cell.

[0035] Based on the above advantages, the present application provides a method for constructing a bovine MSTN gene knockout cell line, comprising the following steps:

[0036] The expression vector and the Cas12i nuclease expression vector are introduced into bovine fibroblast cells to obtain positive knockout cells; and the positive knockout cells are cultured and subcultured to obtain the bovine MSTN gene knockout cell line.

[0037] As an embodiment, the mass ratio of the expression vector and the Cas12i nuclease expression vector is 2:3. As an embodiment, the bovine includes Huaxi cattle.

[0038] The construction method provided by the present application can obtain a Huaxi cattle MSTN gene knockout cell line, and the obtained positive monoclonal cell line can be used for nuclear transfer experiments in the subsequent to produce cloned embryos carrying target gene editing. The ultimate goal is to obtain Huaxi cattle with a double muscle phenotype through the development of nuclear transfer embryos, thereby providing new genetic resources and improvement approaches for the field of agricultural biotechnology.

[0039] In order to further illustrate the present application, the crRNA mediated by CRISPR / Cas12i and the application thereof in knocking out the bovine MSTN gene provided by the present application are described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0040] Example 1

[0041] (I) Screening of high-efficiency crRNA for MSTN gene knockout in CRISPR / Cas12i-mediated West-Cattle

[0042] 1. Design of crRNA for 7 natural mutation sites of MSTN gene

[0043] For the C→A mutation at nt282bp in the first exon of MSTN gene, which leads to the mutation of phenylalanine (F) at position 94 of the amino acid sequence to leucine (L), named F94L, two crRNAs, 94-1 and 94-2, were designed.

[0044] For the C→T mutation at nt238bp in the second exon of MSTN gene, which leads to the mutation of glutamine (Q) at position 204 of the amino acid sequence to a stop codon, named Q204X, two crRNAs, 204-1 and 204-2, were designed; for the insertion of 10bp at the 419th nucleotide of the second exon, which replaced the original 7bp (del7-ins10), leading to the mutation of phenylalanine (F) at position 140 of the amino acid sequence to a stop codon, named nt419, four crRNAs, 419-1, 419-2, 419-3, and 419-4, were designed; for the G→T mutation at position 676 of the second exon, which leads to the mutation of glutamic acid (E) at position 226 of the amino acid sequence to a stop codon, named E226X, four crRNAs, 226-1, 226-2, 226-3, and 226-4, were designed.

[0045] For the deletion of 11 bases (del11) at nt821-831 of the third exon of MSTN gene, which leads to the loss of 3 amino acids at positions 275, 276, and 277, the reading frame shifts from position 274, and the stop codon is advanced to position 287, named nt821, three crRNAs, 821-1, 821-2, and 821-3, were designed; for the mutation from G to A at position 938 of the third exon, which leads to the mutation of cysteine (C) at position 313 of the amino acid sequence to tyrosine (Y), named C313Y, four crRNAs, 313-1, 313-2, 313-3, and 313-4, were designed; for the g.874G>T mutation in the third exon, which leads to the mutation of glutamic acid (E) at position 291 of the amino acid sequence to a stop codon, named E291X, three crRNAs, 291-1, 291-2, and 291-3, were designed.

[0046] The crRNA consists of two parts, one is a 20-base targeting sequence, which is complementary to the target DNA; the other is a domain, which binds to the tracrRNA. See Table 1 for details.

[0047] Table 1 Sequence of crRNA at each site

[0048]

[0049]

[0050] Note: The bold sequence is the domain, and the non-bold sequence is the targeting sequence.

[0051] 2. Construction of Puc19-crRNA expression vector

[0052] (1) Synthesize the positive and negative strands of each crRNA according to Table 1, wherein the nucleotide sequence of the positive strand is: add a BsaI enzyme cutting site (5'-GCAG-3') at the 5' end of the coding sequence of the crRNA shown in Table 1, and the negative strand is the reverse complement of the positive strand. Take 94-1 as an example, the nucleotide sequence of the corresponding positive strand is 5'-GCAGGTTCGATGTCCAGAGAGATGCCAG-3' (SEQ ID NO. 37), and the nucleotide sequence of the negative strand is 5'-CTGGCATCTCTCTGGACATCGAACCTGC-3' (SEQ ID NO. 38). Dissolve the positive and negative strands with appropriate amount of ddH2O to make their final concentration 100 μM. Add 10x PCR Buffer 5 μL, positive strand 22.5 μL, and negative strand 22.5 μL into a PCR tube, mix well, and anneal at 99°C for 10 min to form double-stranded DNA.

[0053] (2) Use Solution I ligase (Takara, 6022Q) to connect the BsaI enzyme cut Puc19 vector skeleton and the double-stranded DNA obtained in step (1) to obtain Puc19-crRNA expression vectors containing different crRNAs.

[0054] (3) After transferring the Puc19-crRNA expression vectors obtained in step (2) into DH5α competent cells, respectively, pick single colony bacterial plaques, perform streak culture, and send to the company for sequencing. Extract plasmids from bacterial plaques with correct sequencing results to obtain amplified Puc19-crRNA expression vectors.

[0055] 3. Electroporation-mediated DNA transfection of bovine fibroblasts

[0056] (1) Observe the morphology and confluence of bovine fibroblasts in a 6-well plate, and the cell confluence reaches 80% (about 2.5 x 10 6The medium is removed.

[0057] (2) Wash once with 1 mL of PBS, and remove the PBS.

[0058] (3) Add 1 mL of 0.25% trypsin, digest at 37°C for 2 min, add 2 mL of DMEM containing 10% FBS to terminate digestion, and gently blow the cells with a pipette to disperse them, and then transfer them into a 15 mL centrifuge tube.

[0059] (4) Centrifuge at 1200 rpm for 3 min, and remove the medium.

[0060] (5) Add 100 μL of electrotransfection liquid, resuspend the cells, and then add the electrotransformation cup, 2 μg of Puc19-crRNA expression vector, and 3 μg of Cas12i nuclease expression vector, and gently knock the bottom of the electrotransformation cup to make it uniform.

[0061] (6) Perform electrotransformation using a single-well cell nucleus transfection instrument (Lonza), and the electrotransformation program is CZ-167.

[0062] (7) Use a matched pipette to completely suck out the liquid in the electrotransformation cup, and add it to one well of a 6-well plate containing 2 mL of mesenchymal stem cell medium (MSC), and shake well; the formula of MSC is 83% MEM Alpha (Gibco, 12571063) + 15% fetal bovine serum (FBS) (Gibco, 12664025) + 1% glutamine (Gibco, 25030081) + 1% sodium pyruvate (Gibco, 11360070) + 1% non-essential amino acids (MEM NEAA) (Gibco, 11140050) + 1% penicillin-streptomycin (PS) (Gibco, 15140122).

[0063] (8) After 24 h of electrotransformation of fibroblasts, observe the electrotransformation efficiency under a fluorescence microscope (part of the results are shown in Figure 1 ).

[0064] (9) 48 h after electrotransformation, remove the culture solution, wash once with 1 mL of PBS, and remove the PBS; add 1 mL of 0.25% trypsin, and place it at 37°C for 2 min, add 2 mL of fibroblast culture solution to terminate digestion, centrifuge at 1200 rpm for 3 min, remove the supernatant, and resuspend the cell pellet using 300 μL of DMEM containing 1% FBS, for use in the following flow sorting experiment.

[0065] 4. Flow sorting of positive cells and detection of editing efficiency

[0066] (1) Resuspend the cells from step 3 with a high-speed multi-color flow cytometer (Beckman Coulter, MoFlo XDP) to sort the cells, collect the green fluorescent cells for subsequent experiments.

[0067] (2) Detection of editing efficiency

[0068] 1) Extract genomic DNA from the green fluorescent cells, and use the genomic DNA as a template for the first round of nested PCR. The primer sequences are shown in Table 2. The reaction system is: DNA template 1 μL, primer F1 11 μL, primer R1 11 μL, KOD enzyme 12.5 μL, and ddH2O 9.5 μL. The reaction program includes: 53°C annealing for 5s, 68°C extension for 1s.

[0069] Table 2: Primer for the first round of nested PCR

[0070]

[0071] Use the product of the first round of nested PCR as a template for the second round of nested PCR. The primer sequences are shown in Table 3. The reaction system is: one round of PCR product template 2 μL, primer F2 2 μL, primer R2 2 μL, KOD enzyme 25 μL, and ddH2O 19 μL. The reaction program includes: 53°C annealing for 5s, 68°C extension for 1s.

[0072] Table 3: Primer for the second round of nested PCR

[0073]

[0074] 2) Anneal the product of the second round of nested PCR to obtain the nested PCR annealing product. The annealing conditions are: 95°C for 10 min, then reduce the temperature by 10°C per min until the temperature reaches 4°C.

[0075] 3) Perform T7E1 enzyme treatment on the nested PCR annealing product to obtain the T7E1 enzyme cleavage product. The reaction system is: nested PCR annealing product 15 μL, T7E1 enzyme 0.5 μL, NEBuffer 22.5 μL, and ddH2O 2 μL. The reaction conditions are: 37°C for 1h, 75°C for 3 min.

[0076] 4) Perform agarose gel electrophoresis detection on the nested PCR two-round product as the control group (WT) and the T7E1 enzyme cleavage product as the experimental group. The agarose gel concentration is 2.5%, and the electrophoresis is performed at 120v for 30 min. Observe the electrophoresis map and take photos (and Figure 2 Figure 3 ). If multiple bands appear in the T7E1 enzyme cleavage experimental group, it represents better editing effect, such as 94-1 in the box. ​

[0077] 5) Add 3'-A overhang to the 3' end of the nested PCR second round product obtained in step 2) to obtain an A-added product, and the system is as follows: 14.5 μL of the column-recovered product of the nested PCR second round, 2 μL of 10x Taq buffer, 3 μL of dNTP Mix, and 0.5 μL of r-Taq Mix, and the reaction condition is 72 °C for 15 min. Then, the A-added product is connected with pMD 19-T plasmid (Takara) to obtain a connection product, and the connection system is as follows: 5 μL of Solution I (Takara, 6022Q), 1 μL of pMD 19-T plasmid, and 4 μL of the A-added product.

[0078] 6) After the connection product of step 5) is transferred into DH5α competent cells, solid culture medium is cultured for 12-14 h, and 15 single colonies are picked from the culture plate and subjected to bacterial liquid PCR, respectively. The reaction system is as follows: 1 single colony template, 1 μL of M13-F, 1 μL of M13-R, 12.5 μL of Taq Mix, and 10.5 μL of ddH2O, and the primer sequences are as follows:

[0079] M13-F: 5'-TGTAAAACGACGGCCAGT-3' (SEQ ID NO. 35);

[0080] M13-R: 5'-CAGGAAACAGCTATGACC-3' (SEQ ID NO. 36).

[0081] 7) The bacterial liquid PCR products obtained in step 6) are respectively detected by agarose gel electrophoresis, and the samples with the purpose fragment size are sequenced, and one sample is subjected to sequencing of 15 single colony PCR products.

[0082] 8) The sequencing data obtained in step 7) are analyzed by using snapgene, the number of bacteria with editing effect (see Figure 5 ) in 15 single colony PCR products of one sample is counted, the proportion of the number of bacteria with editing effect in 15 single colonies is calculated, the editing efficiency of different sites is obtained, and the crRNA with the highest editing efficiency is screened. Each sample is subjected to three repeated experiments, and the average value is obtained. The results are shown in Table 4 and Figure 4 (* indicates significant difference, P < 0.05; ns indicates no significant difference).

[0083] Table 4 Average editing efficiency of crRNA at each site in three experiments

[0084] crRNA Average editing efficiency crRNA Average editing efficiency crRNA Average editing efficiency 94-1 89.22％ 226-3 32.83％ 291-2 27.60％ 94-2 60.01％ 226-4 25.88％ 291-3 17.59％ 419-1 7.68％ 204-1 27.11％ 313-1 17.28％ 419-2 12.67％ 204-2 51.66％ 313-2 14.19％ 419-3 35.73％ 821-1 33.48％ 313-3 42.17％ 419-4 31.47％ 821-2 26.13％ 313-4 33.00％ 226-1 61.14％ 821-3 45.13％ / / 226-2 44.76％ 291-1 19.83％ / /

[0085] Table 4 Average editing efficiency of crRNA at each site in three experiments Figure 4It can be seen that the crRNA editing efficiency of 94-1, 419-3, 226-1, 204-2, 821-3, 291-2 and 313-3 in different sites is the highest, and the order is 89.22%, 35.73%, 61.14%, 51.66%, 45.13%, 27.60% and 42.17% respectively.

[0086] Example 2

[0087] Construction of MSTN gene knockout cell line mediated by CRISPR / Cas12i

[0088] The above seven crRNAs with the highest editing efficiency and Cas12i nuclease expression vectors are co-transfected into fibroblasts again to construct the MSTN gene knockout cell line. According to Example 1, the crRNA is constructed into a puc19 plasmid, and 2ug and 3ug of Puc19-crRNA and Cas12i nuclease expression vectors are respectively transferred. Figure 1 After 24h, observe the fluorescence and take pictures Figure 5 ), 48h later, perform flow sorting, plate single cells into 96-well plates, and observe the cell condition after 7 days of culture; when the cells grow to 70%-80%, subculture them into 48-well plates; observe the cell condition after 2 days of culture in the 48-well plates, and when the cells grow to 70%-80%, subculture them into 24-well plates; observe the cell condition after 1 day of culture in the 24-well plates, and when the cells grow to 70%-80%, take 1 / 4 of the cells for identification, and the remaining 3 / 4 of the cells are cryopreserved for later use. The cell genotype identification method is the same as step 4 in Example 1, and the identified editing types, base sequences (BaseSeq) and amino acid sequence (AA Seq) changes are shown in Figure 5 . The boxed font represents the PAM sequence, the underlined font represents the Spacer sequence, the bold font and underlined font represent the edited amino acid sequence, * represents the stop codon generated after editing, WT is the wild type sequence, and MUT is the edited sequence.

[0089] According to the sequencing results, 94-1, 419-3, 226-1, 204-2, 821-3, 291-2 and 313-3 all knockout different sites of the MSTN gene sequence.

[0090] (1) The 94-1 editing site is located in the first exon of the MSTN gene, and a 5bp deletion is generated from the 534th bp, resulting in a change from serine (S) at position 178 to arginine (R), and a stop codon is generated in advance.

[0091] (2) The 419-3 editing site is located in the second exon of the MSTN gene, and a 28bp deletion is generated from the 2514th bp, resulting in a change from tyrosine (Y) at position 24 to leucine (L), and a stop codon is generated in advance.

[0092] (3) The 226-1 editing site is located in the second exon of the MSTN gene, and a 5-bp deletion is generated from the 2752nd bp, resulting in a premature stop codon.

[0093] (4) The 204-2 editing site is located in the second exon of the MSTN gene, and a 18-bp deletion is generated from the 2662nd bp, resulting in a change of threonine (T) at the 88th position to isoleucine (I).

[0094] (5) The 821-3 editing site is located in the third exon of the MSTN gene, and a 15-bp deletion is generated from the 4902nd bp, resulting in a change of asparagine (N) to leucine (L).

[0095] (6) The 291-2 editing site is located in the third exon of the MSTN gene, and a 13-bp deletion is generated from the 4979th bp, resulting in a change of asparagine (N) to proline (P).

[0096] (7) The 313-3 editing site is located in the third exon of the MSTN gene, and a 27-bp deletion is generated from the 5023rd bp, resulting in a change of glutamine (Q) to alanine (A).

[0097] Among them, the 94-1, 419-3 and 226-1 editing sites generate a premature stop codon, resulting in that the MSTN gene protein cannot be translated and the protein function is affected, so the three types of mutations are more likely to obtain the double-muscle phenotype of the Chuanxi cattle.

[0098] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments, and people can also obtain other embodiments according to the present embodiment without creativity, and these embodiments all belong to the protection scope of the present application.

Claims

1. A crRNA targeting the MSTN gene, characterized in that, The crRNA is 94-1; the nucleotide sequence of 94-1 is shown in SEQ ID NO.

1.

2. The crRNA according to claim 1, characterized in that, The crRNA further includes: 419-3, 226-1, 204-2, 821-3, 291-2, and 313-3; the nucleotide sequence of 419-3 is shown in SEQ ID NO.5; the nucleotide sequence of 226-1 is shown in SEQ ID NO.7; the nucleotide sequence of 204-2 is shown in SEQ ID NO.12; the nucleotide sequence of 821-3 is shown in SEQ ID NO.15; the nucleotide sequence of 291-2 is shown in SEQ ID NO.17; and the nucleotide sequence of 313-3 is shown in SEQ ID NO.

21.

3. An expression carrier, characterized in that, A double-stranded nucleic acid molecule comprising a base vector and an insertion base vector; the double-stranded nucleic acid molecule consists of a positive strand and a negative strand, the positive strand and the negative strand being in opposite complementary pairs; the positive strand comprises a nucleic acid molecule encoding the crRNA of claim 1 or 2.

4. The expression vector according to claim 3, characterized in that, The underlying vector includes the Puc19 vector.

5. The expression vector according to claim 4, characterized in that, The double-stranded nucleic acid molecule is located between the BsaI restriction sites of the Puc19 vector.

6. A kit for knocking out the MSTN gene, characterized in that, Includes the crRNA as described in claim 1 or 2 and / or the expression vector as described in any one of claims 3 to 5.

7. The reagent kit according to claim 6, characterized in that, The kit also includes Cas12i nuclease and / or Cas12i nuclease expression vector.

8. The use of the crRNA according to claim 1 or 2, the expression vector according to any one of claims 3 to 5, or the kit according to claim 6 or 7 in knocking out the bovine MSTN gene and / or constructing a bovine MSTN gene knockout cell line.

9. The application according to claim 8, characterized in that, The original cells of the bovine MSTN gene knockout cell line include fibroblasts.

10. A method for constructing a bovine MSTN gene knockout cell line, characterized in that, Includes the following steps: The expression vector and Cas12i nuclease expression vector according to any one of claims 3 to 5 are transformed into bovine fibroblasts to obtain positive knockout cells; the positive knockout cells are cultured and passaged to obtain the bovine MSTN gene knockout cell line.

11. The construction method according to claim 10, characterized in that, The mass ratio of the expression vector to the Cas12i nuclease expression vector is 2:3.

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