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
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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
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caccgagacg gagtgtccca ggagtggg 28
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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.
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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 * |
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