CN118291415A - CRISPR (chemical reaction plasma enhanced surface plasmon resonance) Cr-P300M crotonylation acylation modification editing tool and application thereof - Google Patents

Abstract

The invention relates to a CRISPR (chemical reaction plasma enhanced chemical vapor deposition) Cr-P300M crotonylation acylation modification editing tool and application thereof. The crotonylation modification editing tool comprises: the amino acid sequence of the mutant P300 is shown as SEQ ID NO. 2, the mutant P300 targets transcription initiation sites of myogenic marker genes MYOG and MYMK, and the nucleotide sequence of the mutant sgRNA is as follows: the sg-MYOG-Bos is shown as SEQ ID NO:3 is shown in the figure; sg-MYMK-Bos is shown as SEQ ID NO:4 is shown in the figure; sg-MYOG-Mus is shown as SEQ ID NO:5 is shown in the figure; sg-MYMK-Mus is shown as SEQ ID NO: shown at 6. The tool promotes transcription and translation by increasing crotonylation modification of target genes, and can up-regulate gene expression and promote myotube differentiation by respectively carrying out site-directed crotonylation modification editing on transcription initiation sites of myogenic marker genes MYOG and MYMK of BMSC and C2C12 cells.

Description

CRISPR (chemical reaction plasma enhanced surface plasmon resonance) Cr-P300M crotonylation acylation modification editing tool and application thereof

Technical Field

The invention relates to the technical field of gene editing, in particular to preparation and application of a CRISPR (chemical and physical and chemical) Cr-P300M crotonylation acylation modification editing tool.

Background

Lysine crotonylation modification (Lysine crotonylation, kcr) refers specifically to a modification resulting from the transfer of crotonyl to lysine residues by the Histone Crotonyl Transferase (HCT) using crotonyl-CoA (Cr-CoA) as a substrate. Similar to most acylation modifications, kcr requires the involvement of writers, readers and Eraser (erases), where E1A binding protein P300 (P300) acts as the primary crotonyl transferase in mammalian cells for modification.

The P300 can catalyze acetylation and crotonylation modification simultaneously, and further research discovers that after isoleucine responsible for acetylation modification activity of the P300 protein is mutated into glycine, the P300 mutant loses the function of catalyzing acetylation and maintains the start of the crotonylation modification. In an in vitro transcription related experiment, after the P300 mutant replaces endogenous P300, the transcription level of the gene is obviously improved. However, the establishment of a gene site-directed crotonylation modification editing tool and related applications are lacking in the prior progress.

Disclosure of Invention

In order to achieve the aim of the invention, in a first aspect, the invention provides a P300 mutant, wherein the amino acid sequence of the mutant is shown as SEQ ID NO. 1.

Preferably, the mutant is obtained by mutating isoleucine at position 1395 of P300 into glycine, and the amino acid sequence of P300 is shown as SEQ ID NO. 1.

In a second aspect, the invention provides a CRISPR (chemical amplification of surface plasmon resonance) comprising a Cr-P300M gene site-directed crotonylation modification composition comprising: fusion proteins of dCas9 and P300 mutants and sgrnas.

Preferably, the amino acid sequence of the P300 mutant is shown as SEQ ID NO. 2.

Preferably, the dCas9 and P300 mutants are fused directly or via a linker peptide.

Preferably, the connecting peptide is P2A.

Preferably, the sgrnas target the transcription start sites of myogenic marker genes MYOG and MYMK, which myogenic marker genes MYOG and MYMK are of bovine or murine origin.

Preferably, the nucleotide sequence of the sgRNA is:

the sg-MYOG-Bos is shown as SEQ ID NO:3 is shown in the figure;

sg-MYMK-Bos is shown as SEQ ID NO:4 is shown in the figure;

sg-MYOG-Mus is shown as SEQ ID NO:5 is shown in the figure;

sg-MYMK-Mus is shown as SEQ ID NO: shown at 6.

In a preferred embodiment of the invention, a CRISPR (CRISPR) -Cr-P300M gene site-directed crotonylation modification editing vector is provided, comprising an sgRNA expression cassette and dCAS9 and P300 mutant fusion protein expression cassettes.

The amino acid sequence of the P300 mutant is shown as SEQ ID NO. 1, and the sgRNA targets transcription initiation sites of bovine myogenic marker genes MYOG and MYMK or targets transcription initiation sites of murine myogenic marker genes MYOG and MYMK.

In a third aspect, the invention provides a P300 mutant, CRISPR, cr-P300M gene site-directed crotonylation modification composition or CRISPR, cr-P300M gene site-directed crotonylation modification editing vector and application thereof in site-directed crotonylation acylation modification cells.

In a fourth aspect, the invention provides a method for preparing a gene site-directed crotonylation modification editing cell, comprising introducing a CRISPR (chemical amplification plasma enhanced surface plasmon resonance) Cr-P300M gene site-directed crotonylation acylation modification composition or a CRISPR (chemical amplification plasma enhanced surface plasmon resonance) Cr-P300M gene site-directed crotonylation acylation modification editing vector into a target cell to obtain the gene site-directed crotonylation modification editing cell.

Preferably, the cells of interest comprise bovine muscle satellite cells or mouse C2C12 myoblasts.

Preferably, the bovine muscle satellite cells are bovine fetal muscle satellite cells.

Preferably, the method of introduction comprises a liposome transfection method or an electroporation method.

Preferably, the method of introduction is a liposome transfection method.

The editing cell screening method is to separate and culture single cells after editing by a limiting dilution method.

The identification method is ChiP-qPCR of crotonylated pan antibody.

By means of the technical scheme, the invention has at least the following advantages and beneficial effects:

the invention provides a tool for mammal gene site-directed crotonylation modification editing, which can express sgRNA and dCAS9 and P300M fusion proteins, and promote transcription and translation by increasing the crotonylation modification of a target gene. Further taking BMSC and C2C12 cells as examples, the transcription initiation sites of myogenic marker genes MYOG and MYMK are respectively subjected to site-directed crotonylation modification and editing, so that gene expression can be up-regulated and myotube differentiation can be promoted. The invention of the gene site-directed crotonylation acylation modification system widens the way of gene apparent modification and editing, and provides a new idea for the cultivation of gene editing animals.

The conception, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.

Drawings

FIG. 1 is a schematic representation of the amino acid sequence of bovine and murine core P300 protein and bovine P300 mutants.

FIG. 2 is a CRISPR: cr-P300M crotonylation acylation modification editing plasmid map.

FIG. 3 shows the sgRNA sequences targeting the transcription initiation sites of the bovine myogenic marker genes MYOG and MYMK, and the sgRNA sequences targeting the transcription initiation sites of the murine myogenic marker genes MYOG and MYMK.

FIG. 4 shows the effect of CRISPR on Bovine Muscle Satellite Cell (BMSC) and mouse C2C12 myoblasts viability following transfection of cells with Cr-P300M plasmid.

FIG. 5 shows the intensity of the site-directed crotonylation modification editing of the myogenic marker genes MYOG and MYMK in Bovine Muscle Satellite Cells (BMSCs) and the transcription initiation sites of the myogenic marker genes MYOG and MYMK in mouse C2C12 myoblasts, respectively, by ChiP-qPCR detection of the crotonylation modification at the transcription initiation sites of bovine and mouse myogenic marker genes MYOG and MYMK.

FIG. 6 shows the intensity of site-directed crotonylation modification editing of myogenic marker genes MYOG and MYMK in Bovine Muscle Satellite Cells (BMSCs) and the transcription initiation sites of myogenic marker genes MYOG and MYMK in mouse C2C12 myoblasts, respectively, and the detection of acetylation modification at the transcription initiation sites of bovine and mouse myogenic marker genes MYOG and MYMK by ChiP-qPCR.

FIG. 7 shows the transcription levels of the myogenic marker genes MYOG and MYMK in Bovine Muscle Satellite Cells (BMSCs) and the transcription initiation sites of the myogenic marker genes MYOG and MYMK in mouse C2C12 myoblasts after site-directed crotonylation modification editing, respectively, by fluorescent quantitative PCR detection of the transcription levels of the bovine and mouse myogenic marker genes MYOG and MYMK after site-directed crotonylation modification editing.

FIG. 8 shows the translation level of bovine myogenic marker genes MYOG and MYMK after site-directed crotonylation modification editing of the transcription initiation sites of myogenic marker genes MYOG and MYMK in Bovine Muscle Satellite Cells (BMSCs) by immunoblotting.

FIG. 9 shows the translation level of mouse myogenic marker genes MYOG and MYMK after site-directed crotonylation modification editing of the transcription initiation sites of the myogenic marker genes MYOG and MYMK in mouse C2C12 myoblasts.

FIG. 10 shows the myotube formation after site-directed crotonylation modification editing of myogenic marker genes MYOG and MYMK in Bovine Muscle Satellite Cells (BMSCs) and detection of the site-directed crotonylation modification editing of bovine myogenic marker genes MYOG and MYMK.

FIG. 11 shows the myotube formation after site-directed crotonylation modification editing of the transcription initiation sites of the myogenic marker genes MYOG and MYMK in the myogenic cells of the mice C2C12, and the detection of the myogenic marker genes MYOG and MYMK.

Detailed Description

The following description of the preferred embodiments of the present invention refers to the accompanying drawings, which make the technical contents thereof more clear and easy to understand. This invention may be embodied in many different forms of embodiments and should not be construed as limited to the embodiments set forth herein; in the figure: muc is short for mice; bos is a bovine abbreviation; WT (wild-type), wild-type; m (mutant) is a mutant.

The invention provides a CRISPR (chemical reaction and physical amplification) Cr-P300M crotonylation modification editing tool, which comprises an sgRNA expression cassette and a dCAS9 and P300 mutant fusion protein expression cassette.

In the above system, sgRNA can target the region near the transcription initiation site of the gene, dCAS9 protein binds to the target sequence but does not cleave, and P300 mutant protein can increase the level of crotonylation modification of the gene without affecting the acetylation modification.

It should be noted that, the sgRNA can realize a transcription initiation site of a target gene, and the specific sequence is not limited as long as the accurate targeting function can be realized. dCAS9 protein can play a role of combining but not cutting DNA in various cells, and has the advantages of wide application range, high combining efficiency and the like. The P300 mutant protein will promote the level of crotonylation modification of the binding site, but will not affect its level of acetylation.

In a preferred embodiment, the nucleotide sequences targeting the transcription initiation site sgrnas of bovine myogenic marker genes MYOG and MYMK, and the nucleotide sequences targeting the transcription initiation site sgrnas of murine myogenic marker genes MYOG and MYMK are as set forth in seq id no: 3. 4, 5 and 6. MYOG (myogenin ) is a member of the myogenic regulatory factor family, MYMK (myomaker, myoblast fusion factor) is located in the plasma membrane, and participates in myoblast fusion, and myogenic marker genes MYOG and MYMK are highly expressed to promote myotube differentiation.

sg-MYOG-Bos: 5’-GGAGCTTGGGGGCTGGTGGCAGG -3’ (SEQ ID NO:3);

sg-MYMK-Bos: 5’-GCTCCACGCTCTGACTCAGCAGG-3’ (SEQ ID NO:4);

sg-MYOG-Mus: 5’-TTTAAATGGCACCCAGCAGTTGG-3’ (SEQ ID NO:5);

sg-MYMK-Mus: 5’-AGGAGGGCTTAAGTGCTCTGAGG-3’ (SEQ ID NO:6);

The tool provided by the invention can realize the site-directed crotonylation modification of genes, and can be used for constructing a cell line edited by the site-directed crotonylation modification of genes.

The invention also provides a preparation method of the gene site-directed crotonylation acylation modified editing cell. The target cells are bovine muscle satellite cells and mouse C2C12 myoblasts, and more preferably bovine fetal muscle satellite cells. The method of introduction is a liposome transfection method or an electroporation method, preferably a liposome transfection method, and has high transfection efficiency and little influence on the cell state.

In a preferred embodiment, a cell line is obtained by successful transfection of the CRISPR: cr-P300M editing vector by screening after introduction of the tool into the cells of interest. The monoclonal editing cell screening method is a limiting dilution method. The identification method preferably detects the crotonylation modification level near the transcription initiation site of the target gene by using crotonyl pan-resistance ChiP-qPCR.

The above-described cells modified and edited at a fixed point of a gene can be further used to detect the transcription and translation levels of the gene. Detecting transcript levels preferably real-time fluorescent quantitative PCR; immunoblots are preferred for detecting translation levels.

The main reagents used in the following examples:

Collagenase iv used to isolate bovine fetal muscle satellite cells was purchased from Sigma;

DMEM, FBS, PBS and Trypsase used for cell culture were both purchased from Gibco;

ChiP kit was purchased from Thermo;

the primer is synthesized by Shanghai biological engineering Co., ltd;

Fluorescent quantitative PCR kits were purchased from nuozhen biotechnology, inc;

Immunoblotting antibody: myogenic marker gene MYOG and MYMK antibodies were both purchased from Abcam, the α -Tubulin antibody was purchased from Proteintech.

The main instrument is as follows:

CO2 incubator (Thermo, USA);

fluorescent quantitative PCR instrument (Roche, switzerland);

exposure apparatus (Tanon, china).

Example 1CRISPR construction of Cr-P300M crotonylation modification editing tool and cytotoxicity detection

1.1 A bovine P300 gene sequence was synthesized, the P300 protein encoded by the gene sequence was a P300 mutant in which isoleucine (I) at position 1395 was mutated to glycine (G) (FIG. 1), the P300 gene sequence was ligated to dCAS9 sequence via linker P2A to form a sequence capable of expressing fusion protein, and dCAS9-P2A-P300 mutant was ligated between T7 promoter and hGHpoly signal (FIG. 2).

1.2 According to the DNA sequence of the transcription initiation site of each of the myogenic marker genes MYOG and MYMK of the cattle (figure 3), the DNA sequence of the transcription initiation site of each of the myogenic marker genes MYOG and MYMK of the mice (figure 3) is online) The sgrnas were designed and scored, and the sgrnas near the transcription initiation site and with high scores were selected as follows:

sg-MYOG-Bos: 5’-GGAGCTTGGGGGCTGGTGGCAGG -3’ (SEQ ID NO:3);

sg-MYMK-Bos: 5’-GCTCCACGCTCTGACTCAGCAGG-3’ (SEQ ID NO:4);

sg-MYOG-Mus: 5’-TTTAAATGGCACCCAGCAGTTGG-3’ (SEQ ID NO:5);

sg-MYMK-Mus: 5’-AGGAGGGCTTAAGTGCTCTGAGG-3’ (SEQ ID NO:6);

To facilitate ligation to the vector backbone, a linker sequence was added to the 4 sgRNA sequences and complementary paired oligonucleotide primers were synthesized.

1.3 Constructing vectors, namely P300M-MYOG-Bos, P300M-MYMK-Bos, P300M-MYOG-Mus and P300M-MYMK-Mus;

The oligonucleotide primers synthesized in step 1.2 were treated at 98℃for 10min, respectively, and then naturally cooled to room temperature for annealing.

A restriction enzyme MluI and KpnI pair containing CRISPR is used for carrying out enzyme digestion on a Cr-P300M carrier at 37 ℃ for 1.5 h, and the linearized fragment is recovered by cutting gel.

The annealed double-stranded fragments and the vector linear fragments are uniformly mixed for 16 ℃ overnight and are connected, the mixture is transformed into DH5 alpha competent cells, the DH5 alpha competent cells are coated on an LB plate containing ampicillin for growth, and then single colony expansion culture is selected and sequenced. The sequencing primers were as follows:

U6-promoter：5’-CCGTAACTTGAAAGTATTTCG-3’;

the positive clones are cultured and extracted to obtain P300M-MYOG-Bos, P300M-MYMK-Bos, P300M-MYOG-Mus and P300M-MYMK-Mus plasmids for subsequent cell transfection. Plasmid extraction uses a plasmid endotoxin removal extraction kit (TIANGEN, china).

1.4 Isolation and culture of bovine fetal muscle satellite cells (BMSCs)

The bovine muscle satellite cells used in this experiment were taken from the grassland livestock germplasm innovation and breeding base wild mongolian Niu Taier (around the age of march). Separating and culturing primary skeletal muscle satellite cells by tissue adherence method, washing fetal tissue with PBS containing double antibodies for 3 times, soaking in 75% alcohol for 5s, rapidly transferring into PBS, washing for 3 times, shearing to 1-3 mm3, spreading in culture dish, culturing in a culture dish at 38.5 deg.C in 5% CO2 incubator for 1-2 h, and adding complete culture solution after tissue blocks are adhered to the culture dish. Subculturing and freezing the cells when the cell growth reaches 80-90% confluence.

1.5 Culture of mouse C2C12 cells

The mouse C2C12 cells used in this test were purchased from Saiborin Biotechnology Co. C2C12 cells frozen in liquid nitrogen were rapidly thawed in a 39 ℃ water bath and the cell suspension 1500 rpm centrifuged for 5 minutes. The supernatant was discarded, the cell pellet was resuspended in fresh growth medium and inoculated into petri dishes at appropriate density and cultured in an incubator at 37℃with 5% CO 2. Subculturing is performed when the cell growth reaches 80-90% confluence.

1.6 Cell transfection

Primary bovine fetal muscle satellite cells or C2C12 cells were seeded into six well plates the day prior to transfection and transfection was performed when the cells reached 70-80% confluency. The transfection procedure was performed strictly according to the Lipofectamine 2000 (Invitrogen, USA) kit instructions. Specifically, 6. Mu.g of each of the recombinant plasmids P300M-MYOG-Bos, P300M-MYMK-Bos, P300M-MYOG-Mus and P300M-MYMK-Mus obtained in step 1.3 was transfected into BMSC and C2C12 cells, respectively, to obtain 8 transfected cells.

1.7 Limiting dilution method for screening edited cells

After liposome transfection of 48 h, cell suspensions were prepared and 96-well plates were seeded at a dilution ratio of 0.5 cells per well. After 4-5 days of culture, small cell clones were visible and used for subsequent experiments after cell growth.

1.8 Cell activity assay

According to the operation requirements of the CCK-8 (Beyotime, china) kit instruction, the cell viability after the fixed-point crotonylation modification editing of the myogenic marker genes MYOG and MYMK of the cattle or the mice is detected respectively, and an enzyme-labeled instrument is used for detecting the OD value at the position of a sample 450 and nm. Wild Type (WT) is unedited cells and P300 is gene site-directed editing of endogenous proteins. The results showed that the cell viability of the bovine and murine MYOG or MYMK site crotonylation modification edits were not significantly altered compared to the WT group, nor were the cell viability of endogenous P300 edits significantly altered (fig. 4).

Example 2 fixed-point crotonylation modification verification of myogenic marker Gene

2.1 ChiP-qPCR of crotonyl pantagraph

4X 10 ⁶ cells after editing were taken and crosslinked 10X min with 1% formaldehyde.

According to PIERCEMAGNETICCHIPKIT (Thermo, USA) instructions, the primary antibody selects Anti-Crotonyllysine Rabbit pAb (PTMBIO, china) and the secondary antibody selects rabit IgG (CST, USA).

The immunoprecipitated DNA fragments were purified by DNA purification kit (Beyotime, china) and used as templates for quantitative PCR (qPCR).

QPCR system: mu.l of cDNA, 1. Mu.M of upstream primer 1. Mu.l, 1. Mu.M of downstream primer 1. Mu.l, 12.5. Mu.l of SYBR GREEN MASTER mix (TaKaRa, china) and 25. Mu.l of RNase-freeH O8.5. Mu.l.

The reaction system: 95 ℃ 30 s,95 ℃ 5 s,60 ℃ 34s for 40 cycles.

The primer sequences were as follows:

MYOG-Bos-1F: 5’- GGCTATATTTATCTCTGGTTC-3’;

MYOG-Bos-1R: 5’- TGGAGGTGGACAGGCAGGTAG -3’;

MYMK-Bos-1F: 5’- TTCACACCAGCTTGGCAGGGC -3’;

MYMK-Bos-1R: 5’-GGGCGACTTGCAGTGTCTGG-3’;

MYOG-Mus-1F: 5’-CAGCTTAGAGGGGGGCTCAG-3’;

MYOG-Mus-1R: 5’- AGAAGTGGGGCTCCTGGTAG -3’;

MYMK-Mus-1F: 5’- AGAGAGAGAGAGAGAGAGAA-3’;

MYMK-Mus-1R: 5’-CCCTGGTCTCCAGGGCAGG-3’;

The ChiP-qPCR data were normalized using Fold enrichment (Fold enrichment) method: fold engineering=log2- ΔΔCt, ΔCt=Ct (IP) -Ct (Input) -log25, ΔΔct= delta Ct-delta Ct (IgG).

The results show that the crotonylation level is obviously improved compared with that of the WT group after the fixed-point crotonylation modification editing of the myogenic marker genes MYOG and MYMK and the fixed-point crotonylation modification editing of the murine myogenic marker genes MYOG and MYMK; the level of crotonylation was also increased in cells after endogenous P300 editing (fig. 5).

2.2 ChiP-qPCR of acetyl pantoea

Other ChiP-qPCR procedures were consistent with step 2.1 except for the selection of Anti-ACETYLLYSINE RABBIT MAB (PTMBIO, china) for the primary antibody. The results show that after the fixed-point crotonylation modification editing of the bovine myogenic marker genes MYOG and MYMK and the fixed-point crotonylation modification editing of the murine myogenic marker genes MYOG and MYMK, the acetylation level is not obviously changed compared with that of the WT group, and the cell acetylation level after the endogenous P300 editing is obviously increased (figure 6).

Example 3 Gene transcription and protein level detection after site-directed crotonylation editing modification

3.1 Real-time fluorescent quantitative PCR

Total RNA from the edited cells was extracted according to HiPuretotalRNAMiniKit (Magen, china) instructions and RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo, USA).

Total RNA was reverse transcribed into cDNA using GoScriptTM Reverse Transcription Mix Oligo (dT) (Promega, USA).

Primers were designed by PRIMER PREMIER 5.0.0 software and specific sequences were as follows:

MYOG-Bos-2F: 5’- GCGCAGACTCAAGAAGGTGA-3’;

MYOG-Bos-2R: 5’- TGCAGGCGCTCTATGTACTG-3’;

MYMK-Bos-2F: 5’-AGTCTCCCCATTCCTGACCC -3’;

MYMK-Bos-2R: 5’-TGATGAGGGTGAACAGGACA-3’；

MYOG-Mus-2F: 5’- CAGCCCAGCGAGGGAATTTA-3’;

MYOG-Mus-2R: 5’- AGAAGCTCCTGAGTTTGCCC-3’;

MYMK-Mus-2F: 5’- TATGGAGAGACAGGGGTCCA-3’;

MYMK-Mus-2R: 5’-GGCATGGGAGAAACCTTTGC-3’;

PCR system: 80 ng cDNA, 1. Mu.M upstream primer, 1. Mu.M downstream primer, SYBR GREEN MASTER mix (TaKaRa, china) and RNase-freeH O were added together in 20. Mu.l.

The reaction system: 95 ℃ 30 s,95 ℃ 5 s,60 ℃ 34s for 40 cycles.

GAPDH was used as an internal reference, and the primer sequences were as follows:

GAPDH-Bos-F: 5’-TGGTGAAGGTCGGAGTGAAC-3’;

GAPDH-Bos-R: 5’- ATGGCGACGATGTCCACTTT-3’;

GAPDH-Mus-F: 5’- CCCTTAAGAGGGATGCTGCC-3’;

GAPDH-Mus-R: 5’-TACGGCCAAATCCGTTCACA-3’;

The results showed that mRNA expression levels of myogenic marker genes MYOG and MYMK were significantly increased after editing the bovine and murine myogenic marker genes MYOG and MYMK site crotonylation modification (fig. 7).

3.2 Immunoblotting

The edited cells were lysed with RIPA Buffer (Beyotime, china) and protein concentration was determined with BCAProteinAssyKit (Beyotime, china).

Protein samples were separated by 10% SDS-PAGE and transferred to a membrane.

Primary antibody was incubated overnight at 4 ℃, secondary antibody was incubated at 37 ℃ for 1h, and ECL chemiluminescent detection kit (Biosharp, china) was exposed after extensive washing with 1 xtbst.

The results showed that protein expression of myogenic marker genes MYOG and MYMK site-directed crotonylation modification editing of myogenic genes MYOG (fig. 8) and MYMK (fig. 9) was significantly elevated.

EXAMPLE 4 myotube formation after fixed-point crotonylation acylation editing modification

Myogenic induction differentiation is carried out on the edited cells, myotube differentiation conditions of the third day (D3) of differentiation are observed, and the results show that after the myogenic marker genes MYOG and MYMK are subjected to site-directed crotonylation modification editing and after the myogenic marker genes MYOG and MYMK are subjected to site-directed crotonylation modification editing, the myotube forming capacity of BMSC (figure 10) and C2C12 (figure 11) is obviously enhanced.

The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims (10)

1. A P300 mutant is characterized in that the amino acid sequence of the mutant is shown as SEQ ID NO. 2.

2. A CRISPR (chemical reaction plasma enhanced surface plasmon resonance) Cr-P300M gene site-directed crotonylation acylation modified composition is characterized by comprising the following components: the fusion protein of dCAS9 and P300 mutant and sgRNA, the amino acid sequence of the P300 mutant is shown as SEQ ID NO. 2.

3. The composition of claim 2, wherein the dCas9 and P300 mutants are fused directly or via a linker peptide.

4. The composition of claim 3, wherein the linking peptide is P2A.

5. The composition of any one of claims 2-4, wherein the sgrnas target transcription initiation sites of myogenic marker genes MYOG and MYMK, which myogenic marker genes MYOG and MYMK are of bovine or murine origin.

6. The composition of claim 5, wherein the nucleotide sequence of the sgRNA is:

the sg-MYOG-Bos is shown as SEQ ID NO:3 is shown in the figure;

sg-MYMK-Bos is shown as SEQ ID NO:4 is shown in the figure;

sg-MYOG-Mus is shown as SEQ ID NO:5 is shown in the figure;

sg-MYMK-Mus is shown as SEQ ID NO: shown at 6.

7. A CRISPR (hybrid enhanced surface plasmon resonance) is characterized by comprising a Cr-P300M gene site-directed crotonylation acyl modification editing vector, and a sgRNA expression cassette and a dCAS9 and P300 mutant fusion protein expression cassette, wherein the amino acid sequence of the P300 mutant is shown as SEQ ID NO. 1, and the sgRNA targets transcription initiation sites of bovine myogenic marker genes MYOG and MYMK or targets transcription initiation sites of murine myogenic marker genes MYOG and MYMK.

8. The CRISPR of claim 7 wherein the Cr-P300M gene site-directed crotonylation modification editing vector is characterized in that the nucleotide sequence of the sgRNA is:

the sg-MYOG-Bos is shown as SEQ ID NO:3 is shown in the figure;

sg-MYMK-Bos is shown as SEQ ID NO:4 is shown in the figure;

sg-MYOG-Mus is shown as SEQ ID NO:5 is shown in the figure;

sg-MYMK-Mus is shown as SEQ ID NO: shown at 6.

9. The P300 mutant as claimed in claim 1, the CRISPR as claimed in claim 2 to 6, the composition of Cr-P300M gene site-directed crotonylation modification or the CRISPR as claimed in claim 7 to 8, the use of the Cr-P300M gene site-directed crotonylation modification editing vector in site-directed crotonylation modification cells.

10. The preparation method of the gene site-directed crotonylation acylation modified editing cell comprises the following steps:

introducing the CRISPR:: cr-P300M gene site-directed crotonylation modification composition of claim 2-6 or the CRISPR:: cr-P300M gene site-directed crotonylation modification editing vector of claim 7-8 into target cells to obtain the cells edited by the gene site-directed crotonylation acylation modification.