CN111235154A - sgRNA for targeted knockout of human MC1R gene and cell strain constructed by sgRNA - Google Patents
sgRNA for targeted knockout of human MC1R gene and cell strain constructed by sgRNA Download PDFInfo
- Publication number
- CN111235154A CN111235154A CN202010250731.7A CN202010250731A CN111235154A CN 111235154 A CN111235154 A CN 111235154A CN 202010250731 A CN202010250731 A CN 202010250731A CN 111235154 A CN111235154 A CN 111235154A
- Authority
- CN
- China
- Prior art keywords
- expression vector
- sgrna
- grna expression
- sgrna1
- sgrna2
- 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
- 108091027544 Subgenomic mRNA Proteins 0.000 title claims abstract description 38
- 101100346560 Homo sapiens MC1R gene Proteins 0.000 title claims abstract description 31
- 239000013604 expression vector Substances 0.000 claims abstract description 40
- 108020004999 messenger RNA Proteins 0.000 claims abstract description 28
- 108091033409 CRISPR Proteins 0.000 claims abstract description 23
- 238000013518 transcription Methods 0.000 claims abstract description 10
- 230000035897 transcription Effects 0.000 claims abstract description 10
- 238000012216 screening Methods 0.000 claims abstract description 8
- 229920001184 polypeptide Polymers 0.000 claims abstract description 6
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 6
- 210000004027 cell Anatomy 0.000 claims description 67
- 108020005004 Guide RNA Proteins 0.000 claims description 39
- 238000000338 in vitro Methods 0.000 claims description 29
- 108020004414 DNA Proteins 0.000 claims description 18
- 238000010276 construction Methods 0.000 claims description 15
- 239000013612 plasmid Substances 0.000 claims description 13
- 230000003321 amplification Effects 0.000 claims description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
- 238000003209 gene knockout Methods 0.000 claims description 10
- 238000000034 method Methods 0.000 claims description 9
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 8
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 claims description 7
- 239000012634 fragment Substances 0.000 claims description 7
- 102000053602 DNA Human genes 0.000 claims description 6
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 4
- 201000005202 lung cancer Diseases 0.000 claims description 4
- 208000020816 lung neoplasm Diseases 0.000 claims description 4
- 208000017520 skin disease Diseases 0.000 claims description 4
- 238000012546 transfer Methods 0.000 claims description 4
- 210000004881 tumor cell Anatomy 0.000 claims description 4
- 102000012410 DNA Ligases Human genes 0.000 claims description 3
- 108010061982 DNA Ligases Proteins 0.000 claims description 3
- 239000013600 plasmid vector Substances 0.000 claims description 3
- 230000008844 regulatory mechanism Effects 0.000 claims description 3
- 238000005138 cryopreservation Methods 0.000 claims description 2
- 101150015860 MC1R gene Proteins 0.000 abstract description 20
- 108090000623 proteins and genes Proteins 0.000 abstract description 9
- 230000003061 melanogenesis Effects 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 201000010099 disease Diseases 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 239000002773 nucleotide Substances 0.000 abstract 2
- 125000003729 nucleotide group Chemical group 0.000 abstract 2
- 230000010307 cell transformation Effects 0.000 abstract 1
- 230000002401 inhibitory effect Effects 0.000 abstract 1
- 101001134060 Homo sapiens Melanocyte-stimulating hormone receptor Proteins 0.000 description 16
- 102100034216 Melanocyte-stimulating hormone receptor Human genes 0.000 description 16
- 238000003197 gene knockdown Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 4
- 210000003491 skin Anatomy 0.000 description 4
- WHNFPRLDDSXQCL-UHFFFAOYSA-N α-melanotropin Chemical compound C=1N=CNC=1CC(C(=O)NC(CC=1C=CC=CC=1)C(=O)NC(CCCNC(N)=N)C(=O)NC(CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)NC(CCCCN)C(=O)N1C(CCC1)C(=O)NC(C(C)C)C(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(CCSC)NC(=O)C(CO)NC(=O)C(NC(=O)C(CO)NC(C)=O)CC1=CC=C(O)C=C1 WHNFPRLDDSXQCL-UHFFFAOYSA-N 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010362 genome editing Methods 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 238000010354 CRISPR gene editing Methods 0.000 description 2
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000012224 gene deletion Methods 0.000 description 2
- 238000012268 genome sequencing Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 210000002752 melanocyte Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108091006027 G proteins Proteins 0.000 description 1
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 1
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000003979 Mineralocorticoid Receptors Human genes 0.000 description 1
- 108090000375 Mineralocorticoid Receptors Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 108091028113 Trans-activating crRNA Proteins 0.000 description 1
- 210000004404 adrenal cortex Anatomy 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000002865 melanocortin Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000006452 multicomponent reaction Methods 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000002087 whitening effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Mycology (AREA)
- Endocrinology (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides sgRNA for targeted knockout of human MC1R gene and a cell strain constructed by the sgRNA. The sgRNA comprises sgRNA1 and sgRNA2, wherein the nucleotide sequence of sgRNA1 is shown in SEQ ID NO. 1, and the nucleotide sequence of sgRNA2 is shown in SEQ ID NO. 2. The sgRNA can accurately guide Cas9 protein to cut MC1R gene in a targeted manner, mRNA obtained by transcription of an expression vector constructed by the sgRNA is high in cell transformation rate, the success rate of knockout of the MC1R gene is greatly improved, and the obtained A549 cell strain can stably and lowly express melanogenesis-related protein, and has very important significance for researching melanogenesis and inhibiting screening of related polypeptides, melanogenesis and treatment of skin-related diseases and the like.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to sgRNA for targeted knockout of a human MC1R gene and a constructed cell strain thereof, in particular to a constructed human MC1R gene knockout A549 cell strain.
Background
The MC1R gene is encoded by an extension site in mammals and is one of members of the family of G protein-coupled receptors-melanocortin receptors (MCRs), has only one coding region, encodes a protein with 7 transmembrane domains, is mainly expressed in melanocytes of animals, and is the smallest one of the G protein-coupled receptors. The MC1R protein is one of the more important receptors and is a more thorough research on the whitening pathway. It is an important gene for controlling the synthesis of animal melanin, and shows different functions in melanocytes, adrenal cortex and other cells, and in nervous system and immune system.
Compared with the first two gene editing technologies, the CRISPR-Cas9 gene editing technology has the advantages of simple construction system, high accuracy, low cost, capability of editing a plurality of sites at fixed points and the like, and becomes a hotspot of current research. It mainly recognizes a specific DNA sequence through a short guide rna (guide rna), and then the Cas9 protein guided by it is positioned on the specific DNA sequence for cleavage, thereby playing a role in gene editing.
In the prior experimental technology, the most similar to the CRISPR-Cas9 system is siRNA targeted gene silencing technology, and the main defects are interference at the RNA level, low efficiency and unsuitability for long-term inhibition research. The MC1R gene deletion cell model is not available for melanogenesis research, so that the establishment of a MC1R gene deletion cell strain has important significance for researching melanogenesis, treating skin-related diseases and the like.
Disclosure of Invention
Based on the defects of the prior art, the first object of the present invention is to provide an sgRNA for targeted knockout of human MC1R gene; a second object of the present invention is to provide a gRNA expression vector for targeted knockout of the human MC1R gene; the third purpose of the invention is to provide a construction method of a gRNA expression vector; a fourth object of the present invention is to provide a primer for amplifying the sgRNA; the fifth object of the present invention is to provide a kit for knocking out the human MC1R gene; a sixth object of the present invention is to provide a CRISPR-Cas9 system; the seventh purpose of the invention is to provide a kit for constructing a cell line with a knockout human MC1R gene; the eighth purpose of the invention is to provide a method for constructing a cell line with a knockout human MC1R gene; the ninth purpose of the invention is to provide an A549 cell strain of human MC1R gene knockout; the tenth purpose of the invention is to provide the application of the human MC1R gene knockout A549 cell strain as a cell model in the research of skin color regulation mechanism, related polypeptide screening and related skin diseases.
The purpose of the invention is realized by the following technical means:
in one aspect, the invention provides a sgRNA for targeted knockout of a human MC1R gene, comprising sgRNA1 and sgRNA2, the nucleotide sequences of the sgRNA1 and the sgRNA2 are as follows:
a. sgRNA 1: CTGAGGGCGGCCACATGCCGGGG (shown as SEQ ID NO: 1)
b. sgRNA 2: AGACGGAGTGTCCCAGGAGTGGG (shown in SEQ ID NO: 2).
The sgRNA of the invention is obtained by inputting MC1R gene sequence (ID:4157) by using CRISPR on-line design tool (http:// criprpr. mit. edu /). Because MC1R only has one exon, the invention aims to knock out or knock down the MC1R gene as much as possible to reduce the expression quantity thereof, so that effector factors such as a-MSH which can be combined with the MC1R gene can not be combined to block a downstream melanogenesis pathway, therefore, in a plurality of designed and generated sgRNA sequences, in addition to considering high score, comprehensive evaluation is needed to judge whether the knock-out or knock-down site can damage MC1R protein coding, determine whether the possible combination site of a-MSH is contained in the knock-out sequence, simultaneously evaluate the risk of miss-target, and finally obtain the two sgRNA oligonucleotide sequences through screening.
The two sgrnas can accurately guide the Cas9 protein to target and cut the MC1R gene.
In another aspect, the present invention also provides a gRNA expression vector for targeted knockout of a human MC1R gene, the gRNA expression vector including a DNA sequence of the sgRNA1 described above and a gRNA expression vector including a DNA sequence of the sgRNA2 described above.
In another aspect, the present invention further provides a method for constructing the gRNA expression vector, which comprises: the sgRNA1 and sgRNA2 were amplified and annealed to obtain double-stranded DNA fragments, and the double-stranded DNA fragments were ligated to plasmid vectors by DNA ligase to obtain gRNA expression vectors.
In another aspect, the invention further provides a primer for amplifying the sgRNA, wherein the primer sequence of the primer comprises:
primer sequences for amplification of sgRNA 1:
CRISPR-F1：5’-CACCGCTGAGGGCGGCCACATGCCGGGG-3’(SEQ ID NO:3)
CRISPR-R1：5’-CCCCCGGCATGTGGCCGCCCTCAGCAAA-3’(SEQ ID NO:4)；
primer sequences for amplification of sgRNA 2:
CRISPR-F2：5’-CACCGAGACGGAGTGTCCCAGGAGTGGG-3’(SEQ ID NO:5)
CRISPR-R2：5’-CCCCACTCCTGGGACACTCCGTCTCAAA-3’(SEQ ID NO:6)。
in still another aspect, the present invention also provides a kit for knocking out human MC1R gene, which comprises the primer sequence.
In still another aspect, the invention further provides a CRISPR-Cas9 system, which comprises the gRNA expression vector and a Cas9 plasmid.
In still another aspect, the present invention further provides a kit for constructing a cell strain with a knockout human MC1R gene, the kit including mRNA transcribed in vitro by a gRNA expression vector constructed by the sgRNA1 and mRNA transcribed in vitro by a gRNA expression vector constructed by the sgRNA 2.
In still another aspect, the present invention provides a method for constructing a human MC1R gene knockout cell line, comprising the steps of:
mRNA transcribed in vitro by the gRNA expression vector constructed by the sgRNA1, mRNA transcribed in vitro by the gRNA expression vector constructed by the sgRNA2 and mRNA transcribed in vitro by the Cas9 plasmid are transfected into a host cell together, and then monoclonal culture and PCR identification are carried out;
and (4) performing amplification and cryopreservation on the positive clone which is correctly identified by the PCR to complete the construction of the knockout cell strain.
In the above construction method, preferably, the host cell comprises a tumor cell line.
In the above construction method, preferably, the tumor cell line includes a lung cancer cell line.
In the above construction method, preferably, the lung cancer cell strain includes an a549 cell strain.
In the above construction method, preferably, the sgRNA1 and the sgRNA2 respectively construct a gRNA expression vector for in vitro transcription using mMESSAGE mMACHINETMT7 ULTRA Transcription Kit.
In the above construction method, preferably, the in vitro transcription of Cas9 plasmid adopts megashort scriptTMT7Transcription Kit.
In the above construction method, preferably, when the electric transfer into the host cell is performed, the mass ratio of the total mass of the mRNA transcribed in vitro by the gRNA expression vector constructed by the sgRNA1 and the mRNA transcribed in vitro by the gRNA expression vector constructed by the sgRNA2 to the mRNA transcribed from the Cas9 plasmid is 1: (1-3).
In the above construction method, preferably, the mass ratio of mRNA transcribed in vitro by the gRNA expression vector constructed by the sgRNA1 to mRNA transcribed in vitro by the gRNA expression vector constructed by the sgRNA2 is 1: 1.
In still another aspect, the invention further provides a human MC1R gene knockout A549 cell strain.
On the other hand, the invention also provides application of the human MC1R gene knockout A549 cell strain as a cell model in researching a skin color regulation mechanism, relevant polypeptide screening and relevant skin diseases.
The invention has the beneficial effects that:
(1) the present invention providesThe sgRNA expressed by targeted knock-down MC1R gene can effectively target MC1R gene, accurately guide Cas9 protein to cut MC1R gene in a targeted manner, construct the gene into a CRISPR-Cas9 system, and transfect into cells to obtain low-expression cell strain; the mRNA transcribed in vitro in the invention is mMESSAGE mMACHINETMT7 ULTRA Transcription Kit and MEGAshortscriptTMCompared with the existing three-component Cas9 system, namely a system for directly synthesizing crRNA and tracrRNA in vitro and co-transforming cells with Cas9 plasmid, the T7Transcription Kit has the advantages of simple construction method, low cost and strong operability.
(2) The successfully constructed gRNA expression vector and the Cas9 plasmid are transcribed into mRNA in vitro, and then cells are electrically transferred, so that the transformation efficiency is high, and the success rate of gene knockout is greatly improved.
(3) Currently, RNA interference techniques are commonly used for reducing protein expression, which has the major disadvantage of low interference efficiency at the RNA level and is not suitable for long-term inhibition studies; cells with low expression stability of proteins can be constructed by using the CRISPR-Cas9 system; has great advantages in researching and researching molecular mechanisms involved in melanogenesis and related diseases.
(4) The A549 cell strain knocked out by the human MC1R gene constructed by the invention has important application prospect in researching skin color regulation and control mechanism, related polypeptide screening and related skin diseases as a cell model.
Drawings
FIG. 1 is a schematic diagram of the identification of amplified regions by monoclonal PCR in example 3 of the present invention; wherein the gRNA1, gRNA2 and E1(MC1R coding region) are respectively at the positions indicated by arrows, and three dashed boxes respectively indicate the three amplified regions.
FIG. 2 shows the result of identifying a monoclonal PCR-amplified region 1 in example 3 of the present invention; theoretical amplified band sizes were WT (wild type): 600 bp; heterozygote: 600 bp; a homozygote: 0 bp. 2G5, 3F10, 3H2 and 4B1 are 4 different monoclonals, M is Marker, WT is a wild type (i.e. an undamaged MC1R group), and water is a negative control group.
FIG. 3 shows the result of identifying the monoclonal PCR-amplified region 2 in example 3 of the present invention; the theoretical amplified band sizes were WT: 570 bp; heterozygote: 570 bp; a homozygote: 0 bp. 2G5, 3F10, 3H2 and 4B1 are 4 different monoclonals, M is Marker, WT is a wild type (i.e. an undamaged MC1R group), and water is a negative control group.
FIG. 4 shows the result of identifying the monoclonal PCR-amplified region 3 in example 3 of the present invention; the theoretical amplified band sizes were WT: 2169 bp; heterozygote: 2169/-441 bp; a homozygote: 441 bp. Wherein 2G5, 3C2, 3F10, 3H2, 4B1 and 4F2 are 6 different monoclonals, M is Marker, WT is wild type, i.e. MC1R is not knocked out, and water is a negative control group.
FIG. 5 shows the result of genome sequencing of clone 2G5 in example 3 of the present invention.
FIG. 6 shows the Western blot result expressed by the 2G5/A549/A375 MC1R protein in example 4 of the present invention.
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention. The experimental procedures not specified herein are generally carried out under the conventional conditions described in molecular cloning protocols or under the conditions recommended by the manufacturers of the reagents. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
Example 1 sgRNA screen for targeted knockout of the human MC1R gene
The MC1R gene sequence (ID:4157) was imported using the CRISPR on-line design tool (http:// criprpr. mit. edu /). Because MC1R only has one exon, the invention aims to knock out or knock down the MC1R gene as much as possible to reduce the expression quantity, so that effector factors such as a-MSH which can be combined with the MC1R gene can not be combined to block a downstream melanogenesis pathway, therefore, in a plurality of designed and generated sgRNA sequences, in addition to considering high score, comprehensive evaluation is needed to judge whether the knock-out or knock-down sites can damage MC1R protein codes, whether possible combination sites of a-MSH are contained in a knock-out sequence or not is determined, and the risk of miss-target is evaluated at the same time, and the following two sgRNA oligonucleotide sequences are finally obtained through screening with creative labor and are handed over to a Shanghai synthetic sequence.
sgRNA 1: CTGAGGGCGGCCACATGCCGGGG (shown as SEQ ID NO: 1)
sgRNA 2: AGACGGAGTGTCCCAGGAGTGGG (shown as SEQ ID NO: 2)
By adopting the two sgRNAs, the Cas9 protein can be accurately guided to cut the MC1R gene in a targeted manner.
Example 2 construction of expression vector for targeted knockout of human MC1R Gene
The sgrnas in example 1 are amplified and annealed respectively to obtain double-stranded DNA fragments, specifically as follows:
the sgRNA (sgRNA1 or sgRNA2) fragments were amplified with primers in which the CRISPR-F1 and CRISPR-R1 amplified sgRNA1, the CRISPR-F2 and CRISPR-R2 amplified sgRNA 2.
CRISPR-F1：5’-CACCGCTGAGGGCGGCCACATGCCGGGG-3’(SEQ ID NO:3)
CRISPR-R2：5’-CCCCCGGCATGTGGCCGCCCTCAGCAAA-3’(SEQ ID NO:4)。
CRISPR-F2：5’-CACCGAGACGGAGTGTCCCAGGAGTGGG-3’(SEQ ID NO:5)
CRISPR-R2：5’-CCCCACTCCTGGGACACTCCGTCTCAAA-3’(SEQ ID NO:6)。
Diluting the primers, and sterilizing ddH for CRISPR-F1, CRISPR-R1, CRISPR-F2 and CRISPR-R2 primers2O was diluted to a final concentration of 10. mu.M, and the PCR reaction system was as shown in Table 1 below:
table 1:
| composition of | x1 |
| genomicDNA | 1.5μl |
| Forward primer (10. mu.M) | 1.0μl |
| Reverse primer (10. mu.M) | 1.0μl |
| P112TaqDNA polymerase (Vazyme) | 12.5μl |
| ddH2O | 9.0μl |
| Total of | 25.0μl |
After the reagents in the table 1 are mixed uniformly, the mixture is put into a PCR instrument, and the PCR reaction conditions are shown in the table 2:
table 2:
the double-stranded DNA fragments of the two sgrnas were ligated to the respective plasmid vectors lenticrisprrv 2 by DNA ligase to obtain the respective gRNA expression vectors.
Example 3 construction of human MC1R Gene knock-out cell line
1. The respective mrnas were obtained:
the gRNA expression vector of example 2 and Cas9 plasmid (provided by seiko (guangzhou) biotechnology limited) were separately transcribed in vitro to obtain respective mrnas; the specific transcription method is as follows:
(1) reference to mMESSAGE mMACHINETMThe T7 ULTRATranscription Kit indicates that the in vitro transcription of the Cas9 plasmid is carried out to obtain the mRNA of the Cas9 plasmid.
(2) Refer to MEGAshortscriptTMThe T7Transcription Kit indicates that the gRNA expression vector is transcribed in vitro to obtain mRNA of sgRNA1 and mRNA of sgRNA 2.
(3) After LiCl precipitation and recovery, the nucleic acid concentration is measured by the Nanodrop 3000, and the in vitro transcription quality is detected by RNA gel electrophoresis.
2. Construction and identification of MC1R knockout A549 cell strain
DMEM high-sugar medium containing 10% fetal calf serum in 5% CO2Culturing A549 cells at 37 ℃; and taking cells in a logarithmic phase, and electrically transferring the mRNA of the Cas9, the sgRNA1 and the sgRNA2 into A549 cells by using an electrotransformation method under the following conditions:
BIO-RAD Gene Pulser Xcell electric transfer apparatus, 250v, 950uF, 200. omega., 2mm electric transfer cup, cell amount 1.0X 106In addition, 500ng each of mRNA of Cas9 and sgRNA1 and sgRNA2 was cultured for 48 hours after transfection, and the cells were collected by digestion.
Diluting the cells after the electric conversion into monoclonals, inoculating the monoclonals into a 96-well plate for culture, carrying out passage on the 96-well plate after the single cells are amplified to a certain number, continuing the culture of the monoclonals, and carrying out PCR identification on one 96-well plate. The schematic diagram of the amplification region is shown in FIG. 1, and the primers of the amplification region are as follows:
amplification region 1 (annealing temperature 62.0 ℃):
upstream primer TGACGAGGGGAGGGGTGAA (SEQ ID NO:7)
Downstream primer GCTTAGTTCATGGTGCTGCCA (SEQ ID NO:8)
Amplification region 2 (annealing temperature 62.0 ℃):
upstream primer TCAAGGAGGTGCTGACATGC (SEQ ID NO:9)
Downstream primer GAGGCAGGGATTTCACCTCC (SEQ ID NO:10)
Amplification region 3 (annealing temperature 62.0 ℃):
upstream primer TGACGAGGGGAGGGGTGAA (SEQ ID NO:11)
Downstream primer GAGGCAGGGATTTCACCTCC (SEQ ID NO:12)
The results showed that clone No. 2G5 was homozygous, completely knocking out the coding region of the MC1R gene, and the results are shown in FIG. 2, FIG. 3, and FIG. 4.
The results in FIG. 2 show that no band was amplified in region 1 of 4 single clones, the band of interest in the wild type, indicating that 4 single clones were detected as homozygotes in this region.
The results in FIG. 3 show that no band was amplified in region 2 of 4 single clones, the wild type band of interest, indicating that 4 single clones were detected as homozygotes in this region.
The results in FIG. 4 show that the wild type target band, 2G5 and 3H2 both have a single band of about 450bp, and the rest clones have a heteroband, which indicates that the clones No. 2G5 and 3H2 are homozygotes, the MC1R gene is knocked out, and the rest clones are identified as heterozygotes.
The clone numbered 2G5 was selected for genome sequencing, and the sequencing results are shown in fig. 5: this clone successfully knocked out the coding region of the MC1R gene.
Example 4MC1R knockout cell line sterility, morphological and functional characterization
A549 cells, MC1R gene-knocked-out 2G5 cells and melanoma A375 cells cultured in a T25 bottle are taken, culture solution is poured out, the cells are washed once by using a proper amount of PBS, then the cells are digested by 0.05% of pancreatin, and the cells are collected by centrifugation. Cells were washed 3 times with 4 ℃ pre-chilled PBS and residual liquid was aspirated. Adding a small amount of RIPA cell lysate containing phenylmethylsulfonyl fluoride and sodium fluoride, lysing the cells on ice for 10min, collecting the cells, and repeatedly blowing and beating the cells in a 1.5ml centrifuge tube. The tube was centrifuged at 15000r/min at 4 ℃ for 10min and the supernatant collected. The cellular protein concentration was measured by BCA method, and MC1R protein expression was detected by Western blot standard (MC1R antibody purchased from Sigma). The results are shown in FIG. 6.
From the results of FIG. 6, it can be seen that: the MC1R protein expression in the 2G5 cell strain is obviously reduced, which shows that the cell strain is successfully constructed and can be used as a screening model of polypeptide and protein which have interaction with MC1R subsequently.
The expression vector constructed by the sequences of sgRNA1 and sgRNA2 provided in the above description can effectively knock out the MC1R gene, and can be applied to cells, and particularly to a549 cells.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Shanxi Huikang Biotechnology Limited liability company
<120> sgRNA for targeted knockout of human MC1R gene and cell strain constructed by sgRNA
<130>GAI19CN4844
<160>12
<170>SIPOSequenceListing 1.0
<210>1
<211>23
<212>DNA
<213> Artificial sequence ()
<400>1
ctgagggcgg ccacatgccg ggg 23
<210>2
<211>23
<212>DNA
<213> Artificial sequence ()
<400>2
agacggagtg tcccaggagt ggg 23
<210>3
<211>28
<212>DNA
<213> Artificial sequence ()
<400>3
caccgctgag ggcggccaca tgccgggg 28
<210>4
<211>28
<212>DNA
<213> Artificial sequence ()
<400>4
cccccggcat gtggccgccc tcagcaaa 28
<210>5
<211>28
<212>DNA
<213> Artificial sequence ()
<400>5
caccgagacg gagtgtccca ggagtggg 28
<210>6
<211>28
<212>DNA
<213> Artificial sequence ()
<400>6
ccccactcct gggacactcc gtctcaaa 28
<210>7
<211>19
<212>DNA
<213> Artificial sequence ()
<400>7
tgacgagggg aggggtgaa 19
<210>8
<211>21
<212>DNA
<213> Artificial sequence ()
<400>8
gcttagttca tggtgctgcc a 21
<210>9
<211>20
<212>DNA
<213> Artificial sequence ()
<400>9
tcaaggaggt gctgacatgc 20
<210>10
<211>20
<212>DNA
<213> Artificial sequence ()
<400>10
gaggcaggga tttcacctcc 20
<210>11
<211>19
<212>DNA
<213> Artificial sequence ()
<400>11
tgacgagggg aggggtgaa 19
<210>12
<211>20
<212>DNA
<213> Artificial sequence ()
<400>12
gaggcaggga tttcacctcc 20
Claims (10)
1. A sgRNA for targeted knockout of a human MC1R gene, comprising a sgRNA1 and a sgRNA2, the nucleotide sequences of the sgRNA1 and the sgRNA2 are as follows:
a、sgRNA1：CTGAGGGCGGCCACATGCCGGGG(SEQ ID NO:1)
b、sgRNA2：AGACGGAGTGTCCCAGGAGTGGG(SEQ ID NO:2)。
2. a gRNA expression vector for targeted knockout of a human MC1R gene, the gRNA expression vector comprising a gRNA expression vector comprising the DNA sequence of sgRNA1 of claim 1 and a gRNA expression vector comprising the DNA sequence of sgRNA2 of claim 1.
3. A method of constructing a gRNA expression vector of claim 2, comprising: amplifying the sgRNA1 and the sgRNA2 of claim 1 respectively, annealing to obtain respective double-stranded DNA fragments, and ligating the respective double-stranded DNA fragments to a plasmid vector by using DNA ligase to obtain respective gRNA expression vectors.
4. A primer for amplifying the sgRNA of claim 1, the sequence of the primer comprising:
primer sequences for amplification of sgRNA 1:
CRISPR-F1：5’-CACCGCTGAGGGCGGCCACATGCCGGGG-3’(SEQ ID NO:3)
CRISPR-R1：5’-CCCCCGGCATGTGGCCGCCCTCAGCAAA-3’(SEQ ID NO:4)；
primer sequences for amplification of sgRNA 2:
CRISPR-F2：5’-CACCGAGACGGAGTGTCCCAGGAGTGGG-3’(SEQ ID NO:5)
CRISPR-R2：5’-CCCCACTCCTGGGACACTCCGTCTCAAA-3’(SEQ ID NO:6)。
5. a kit for knocking out human MC1R gene, the kit comprising the primer of claim 4.
6. A CRISPR-Cas9 system comprising the gRNA expression vector of claim 2 and a Cas9 plasmid.
7. A kit for constructing a cell strain with a knockout human MC1R gene, the kit comprising mRNA transcribed in vitro by a gRNA expression vector constructed by the sgRNA1 of claim 1, and mRNA transcribed in vitro by a gRNA expression vector constructed by the sgRNA2 of claim 1.
8. A method for constructing a cell strain with a knockout human MC1R gene comprises the following steps:
mRNA transcribed in vitro by a gRNA expression vector constructed by the sgRNA1 in claim 1, mRNA transcribed in vitro by a gRNA expression vector constructed by the sgRNA2 in claim 1 and mRNA transcribed in vitro by a Cas9 plasmid are jointly transfected into a host cell, and then monoclonal culture and PCR identification are carried out;
carrying out amplification cryopreservation on the positive clone which is correctly identified by the PCR to complete the construction of a knockout cell strain;
preferably, the host cell comprises a tumor cell line; preferably, the tumor cell line comprises a lung cancer cell line; preferably, the lung cancer cell strain comprises an a549 cell strain;
preferably, the sgRNA1 and the sgRNA2 respectively construct a gRNA expression vector for in vitro transcription by using mMESSAGENMAXIAPHINETMT7 ULTRATranscription Kit;
preferably, the in vitro transcription of the Cas9 plasmid adopts MEGAshortscriptTMT7Transcription Kit;
preferably, when the electric transfer into a host cell is carried out, the mass ratio of the total mass of mRNA transcribed in vitro by the gRNA expression vector constructed by the sgRNA1 and mRNA transcribed in vitro by the gRNA expression vector constructed by the sgRNA2 to the mass of mRNA transcribed by the Cas9 plasmid is 1: (1-3);
preferably, the mass ratio of mRNA transcribed in vitro by the gRNA expression vector constructed by the sgRNA1 to mRNA transcribed in vitro by the gRNA expression vector constructed by the sgRNA2 is 1: 1.
9. A549 cell strain of human MC1R gene knockout.
10. Use of the human MC1R gene knockout A549 cell strain of claim 9 as a cell model for studying skin color regulation mechanism, related polypeptide screening and related skin diseases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010250731.7A CN111235154A (en) | 2020-04-01 | 2020-04-01 | sgRNA for targeted knockout of human MC1R gene and cell strain constructed by sgRNA |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010250731.7A CN111235154A (en) | 2020-04-01 | 2020-04-01 | sgRNA for targeted knockout of human MC1R gene and cell strain constructed by sgRNA |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111235154A true CN111235154A (en) | 2020-06-05 |
Family
ID=70877258
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010250731.7A Pending CN111235154A (en) | 2020-04-01 | 2020-04-01 | sgRNA for targeted knockout of human MC1R gene and cell strain constructed by sgRNA |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111235154A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011011767A1 (en) * | 2009-07-24 | 2011-01-27 | Sigma-Aldrich Co. | Method for genome editing |
| CN106636201A (en) * | 2016-12-27 | 2017-05-10 | 安徽大学 | MC1R (melanocortin 1 receptor) gene carrier and construction method thereof |
| CN108660161A (en) * | 2017-03-31 | 2018-10-16 | 中国科学院上海生命科学研究院 | Method of the preparation based on CRISPR/Cas9 technologies without mosaic gene knock-out animal |
-
2020
- 2020-04-01 CN CN202010250731.7A patent/CN111235154A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011011767A1 (en) * | 2009-07-24 | 2011-01-27 | Sigma-Aldrich Co. | Method for genome editing |
| CN106636201A (en) * | 2016-12-27 | 2017-05-10 | 安徽大学 | MC1R (melanocortin 1 receptor) gene carrier and construction method thereof |
| CN108660161A (en) * | 2017-03-31 | 2018-10-16 | 中国科学院上海生命科学研究院 | Method of the preparation based on CRISPR/Cas9 technologies without mosaic gene knock-out animal |
Non-Patent Citations (3)
| Title |
|---|
| NING XIAO 等: ""A Novel Pale-Yellow Coat Color of Rabbits Generated via MC1R Mutation With CRISPR/Cas9 System"", 《FRONTIERS IN GENETICS》 * |
| ZORINA-LICHTENWALTER K 等: ""Homo sapiens melanocortin 1 receptor (MC1R), mRNA，ACCESSION: NM_002386.4"", 《GENBANK》 * |
| 赵爱春 等: "《家蚕转基因技术及应用》", 31 December 2017, 上海科学技术出版社 * |
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 | |
| CN108359712B (en) | Method for rapidly and efficiently screening SgRNA target DNA sequence | |
| CN105518135B (en) | Method for specifically knocking out pig CMAH gene by CRISPR-Cas9 and sgRNA for specifically targeting CMAH gene | |
| CN105518138B (en) | Method for specifically knocking out pig GFRA1 gene by CRISPR-Cas9 and sgRNA for specifically targeting GFRA1 gene | |
| CN105492608B (en) | Method for specifically knocking out pig PDX1 gene by CRISPR-Cas9 and sgRNA for specifically targeting PDX1 gene | |
| CN105518137B (en) | Method for specifically knocking out pig SALL1 gene by CRISPR-Cas9 and sgRNA for specifically targeting SALL1 gene | |
| CN105671080B (en) | Method for sheep MSTN gene knockout and site-specific integration exogenous gene mediated by CRISPR-Cas9 system | |
| WO2016187904A1 (en) | Method for pig cmah gene specific knockout by means of crispr-cas9 and sgrna for specially targeting cmah gene | |
| CN109880851B (en) | Screening report vector and screening method for enriching CRISPR/Cas 9-mediated homologous recombination repair cells | |
| CN111979243B (en) | Method for constructing TAP gene-deleted pig T2 cells by using CRISPR/Cas9 system | |
| CN110438128B (en) | Method for knocking out pig CCAR1 gene by using CRISPR/Cas9 system | |
| CN111534519B (en) | sgRNA for identifying eIF4G1 gene of pig, and coding DNA and application thereof | |
| CN110938629B (en) | Complete sgRNA for specifically recognizing pig Wip1 gene and application and product thereof | |
| CN110468133B (en) | Method for knocking out pig GOT1 gene by using CRISPR/Cas9 system | |
| CN111235153B (en) | sgRNA for targeted knockout of human MC1R gene and cell strain constructed by same | |
| CN110551762B (en) | CRISPR/ShaCas9 gene editing system and application thereof | |
| CN111235154A (en) | sgRNA for targeted knockout of human MC1R gene and cell strain constructed by sgRNA | |
| CN110747230A (en) | Method for promoting bovine skeletal muscle satellite cell myogenic differentiation | |
| CN114990093A (en) | Protein sequence MINI RFX-CAS13D with small amino acid sequence | |
| CN108085324B (en) | Amplification of full-length sequence of sheep B4GALNT2 gene and detection of expression level thereof | |
| CN114807126A (en) | Method for silencing expression of long non-coding RNA and application thereof | |
| CN110551763B (en) | CRISPR/SlutCas9 gene editing system and application thereof | |
| CN106701764B (en) | Promoter of 15kDa selenoprotein gene, core region and application thereof | |
| CN113308480B (en) | A-type Seneca virus SVA/HeB full-length infectious cDNA clone, and preparation method and application thereof | |
| CN113403342A (en) | Single base mutation method and system adopted by same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20200605 |
|
| RJ01 | Rejection of invention patent application after publication |