CN118291415A - CRISPR (chemical reaction plasma enhanced surface plasmon resonance) Cr-P300M crotonylation acylation modification editing tool and application thereof - Google Patents
CRISPR (chemical reaction plasma enhanced surface plasmon resonance) Cr-P300M crotonylation acylation modification editing tool and application thereof Download PDFInfo
- Publication number
- CN118291415A CN118291415A CN202410291699.5A CN202410291699A CN118291415A CN 118291415 A CN118291415 A CN 118291415A CN 202410291699 A CN202410291699 A CN 202410291699A CN 118291415 A CN118291415 A CN 118291415A
- Authority
- CN
- China
- Prior art keywords
- crotonylation
- mymk
- myog
- seq
- modification
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000004048 modification Effects 0.000 title claims abstract description 71
- 238000012986 modification Methods 0.000 title claims abstract description 71
- 238000010614 crotonylation reaction Methods 0.000 title claims abstract description 69
- 230000010933 acylation Effects 0.000 title claims abstract description 15
- 238000005917 acylation reaction Methods 0.000 title claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 7
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 title claims description 7
- 101001022911 Homo sapiens Protein myomaker Proteins 0.000 claims abstract description 50
- 102100035095 Protein myomaker Human genes 0.000 claims abstract description 50
- 230000001114 myogenic effect Effects 0.000 claims abstract description 50
- 101000589002 Homo sapiens Myogenin Proteins 0.000 claims abstract description 49
- 239000003550 marker Substances 0.000 claims abstract description 47
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 43
- 101150068427 EP300 gene Proteins 0.000 claims abstract description 37
- 102100038885 Histone acetyltransferase p300 Human genes 0.000 claims abstract description 36
- 108700009124 Transcription Initiation Site Proteins 0.000 claims abstract description 27
- 108091027544 Subgenomic mRNA Proteins 0.000 claims abstract description 16
- 230000014509 gene expression Effects 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 4
- 241000283690 Bos taurus Species 0.000 claims description 35
- 239000000203 mixture Substances 0.000 claims description 14
- 241001529936 Murinae Species 0.000 claims description 11
- 239000013598 vector Substances 0.000 claims description 11
- 102000037865 fusion proteins Human genes 0.000 claims description 7
- 108020001507 fusion proteins Proteins 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 3
- 125000002252 acyl group Chemical group 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 44
- 108091033409 CRISPR Proteins 0.000 abstract description 15
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 15
- 230000035897 transcription Effects 0.000 abstract description 8
- 238000013518 transcription Methods 0.000 abstract description 8
- 230000004069 differentiation Effects 0.000 abstract description 6
- 238000013519 translation Methods 0.000 abstract description 6
- TZSMWSKOPZEMAJ-UHFFFAOYSA-N bis[(2-methoxyphenyl)methyl] carbonate Chemical compound COC1=CC=CC=C1COC(=O)OCC1=CC=CC=C1OC TZSMWSKOPZEMAJ-UHFFFAOYSA-N 0.000 abstract 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 abstract 1
- 238000000623 plasma-assisted chemical vapour deposition Methods 0.000 abstract 1
- 238000000034 method Methods 0.000 description 18
- 210000003205 muscle Anatomy 0.000 description 15
- 210000001057 smooth muscle myoblast Anatomy 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 239000013615 primer Substances 0.000 description 10
- 238000011529 RT qPCR Methods 0.000 description 8
- 230000021736 acetylation Effects 0.000 description 8
- 238000006640 acetylation reaction Methods 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 210000003098 myoblast Anatomy 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 238000003753 real-time PCR Methods 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000001605 fetal effect Effects 0.000 description 5
- 238000001638 lipofection Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000003119 immunoblot Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 230000003833 cell viability Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000003113 dilution method Methods 0.000 description 3
- 229960000310 isoleucine Drugs 0.000 description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 102000008300 Mutant Proteins Human genes 0.000 description 2
- 108010021466 Mutant Proteins Proteins 0.000 description 2
- 102100032970 Myogenin Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 108090000992 Transferases Proteins 0.000 description 2
- 102000004357 Transferases Human genes 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- KFWWCMJSYSSPSK-PAXLJYGASA-N crotonoyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)/C=C/C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 KFWWCMJSYSSPSK-PAXLJYGASA-N 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 101150090724 3 gene Proteins 0.000 description 1
- 102000000132 Alpha tubulin Human genes 0.000 description 1
- 241000283725 Bos Species 0.000 description 1
- 108010075254 C-Peptide Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101000882390 Homo sapiens Histone acetyltransferase p300 Proteins 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010010416 Myogenic Regulatory Factors Proteins 0.000 description 1
- 102000015864 Myogenic Regulatory Factors Human genes 0.000 description 1
- 108010056785 Myogenin Proteins 0.000 description 1
- 102000004364 Myogenin Human genes 0.000 description 1
- 241000520272 Pantoea Species 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 125000000218 acetic acid group Chemical group C(C)(=O)* 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 108010030074 endodeoxyribonuclease MluI Proteins 0.000 description 1
- 238000011013 endotoxin removal Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000021268 myoblast fusion Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 210000000419 skeletal muscle satellite cell Anatomy 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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
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.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202410291699.5A CN118291415A (en) | 2024-03-14 | 2024-03-14 | CRISPR (chemical reaction plasma enhanced surface plasmon resonance) Cr-P300M crotonylation acylation modification editing tool and application thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202410291699.5A CN118291415A (en) | 2024-03-14 | 2024-03-14 | CRISPR (chemical reaction plasma enhanced surface plasmon resonance) Cr-P300M crotonylation acylation modification editing tool and application thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN118291415A true CN118291415A (en) | 2024-07-05 |
Family
ID=91683919
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202410291699.5A Pending CN118291415A (en) | 2024-03-14 | 2024-03-14 | CRISPR (chemical reaction plasma enhanced surface plasmon resonance) Cr-P300M crotonylation acylation modification editing tool and application thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN118291415A (en) |
-
2024
- 2024-03-14 CN CN202410291699.5A patent/CN118291415A/en active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN107502608B (en) | Construction method and application of sgRNA and ALDH2 gene-deleted cell strain for knocking out human ALDH2 gene | |
CN109750035B (en) | sgRNA for targeting and guiding Cas9 protein to efficiently cleave TCR and B2M gene locus | |
CN110628766B (en) | LncRNA coding gene related to sheep skeletal muscle development and application thereof | |
CN109295053B (en) | Method for regulating RNA splicing by inducing splice site base mutation or base substitution of polypyrimidine region | |
CN112111490B (en) | Method for visualizing endogenous low-abundance single-molecule RNA in living cells and application | |
CN113913532A (en) | miRNA marker related to Hu sheep papilla cell proliferation and detection primer and application thereof | |
CN111484993B (en) | Long-chain non-coding RNA IL21-AS1 and application thereof | |
CN115698301A (en) | Active DNA transposable systems and methods of use thereof | |
Wong et al. | A non-transmembrane form of Jagged-1 regulates the formation of matrix-dependent chord-like structures | |
CN114836418A (en) | CRISPR-Cas13d system for knocking down porcine epidemic diarrhea virus | |
Woo et al. | Overexpression of an alternatively spliced form of c-Myb results in increases in transactivation and transforms avian myelomonoblasts | |
EP2625267A2 (en) | Culture method for culturing pluripotent cells comprising an inhibitor of mirna-181a* | |
CN118291415A (en) | CRISPR (chemical reaction plasma enhanced surface plasmon resonance) Cr-P300M crotonylation acylation modification editing tool and application thereof | |
CN111424017A (en) | Exosome loading shRNA (short hairpin ribonucleic acid) and construction method and application thereof | |
CN116162609A (en) | Cas13 protein, CRISPR-Cas system and application thereof | |
WO2017036026A1 (en) | Genetically modified mesenchymal stem cell and bsab producing method by using same | |
CN109456995A (en) | Gene knockout plasmid, cell line and preparation method and application | |
CN114107253B (en) | System and method for gene editing by using engineering cells | |
CN116410999A (en) | Nucleic acid vector containing IRES and novel promoter and application thereof | |
CN109055429B (en) | Mouse osteogenic-like cell lentiviral vector targeting RunX2 gene and construction method thereof | |
CN114957439B (en) | Sheep PDGFD, PDGFD-encoding nucleic acid, recombinant lentivirus, host cell and application thereof | |
CN110946877A (en) | Stem cell biological product for treating liver cirrhosis and preparation method and application thereof | |
CN110564743A (en) | CircR-UQCC1 gene of Lianshan cattle, over-expression vector, construction method and application thereof | |
CN113584085B (en) | Lentiviral vector for suspension cells and application thereof | |
CN117844806B (en) | 5' -UTR element and application thereof in improving protein expression quantity |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination |