CN105647885A - Cas9 fusion protein and coding sequence thereof - Google Patents
Cas9 fusion protein and coding sequence thereof Download PDFInfo
- Publication number
- CN105647885A CN105647885A CN201610038907.6A CN201610038907A CN105647885A CN 105647885 A CN105647885 A CN 105647885A CN 201610038907 A CN201610038907 A CN 201610038907A CN 105647885 A CN105647885 A CN 105647885A
- Authority
- CN
- China
- Prior art keywords
- cas9
- ubiquitin
- gene
- fusion protein
- cell
- 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.)
- Granted
Links
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
-
- 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
- C12N2810/00—Vectors comprising a targeting moiety
- C12N2810/10—Vectors comprising a non-peptidic targeting moiety
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of biology, and relates to fusion protein and the DNA sequence for coding the fusion protein, in particular to the Cas9 fusion protein with a Cas9 segment and a Ubiquitin segment, and the coding sequence of the Cas9 fusion protein. On one hand, the Cas9 protein can be rapidly degraded after playing a targeting role in an embryonic cell, and therefore the chimerism mutation effect on embryonic development is reduced. More importantly, by means of the Ubiquitin-i-cas9 protein, the homozygosis efficiency of a gene targeting embryo can be improved, wherein i is the first-place amino acid of a connector for connecting Ubiquitin and Cas9 and can be arginine (R) or proline (P) or leucine (L). Especially, the targeting efficiency of the particularly-preferential Ubiquitin-R-Cas9 protein for a wild type Cas9 protein gene is improved by 3.51 times and is beyond the expectation of technicians.
Description
Technical field
The invention belongs to biological technical field, relate to the DNA sequence of a kind of fusion protein and encoding fusion protein.
Technical background
Animal disease model serves pivotal role in research human diseases pathogenesis and drug screening. Non-human primate is highly similar to the mankind on biology, hereditism, if macaque and human genome homology are up to 93% (Gibbsetal., 2007), thus becomes the important animal model of mankind's major disease research and drug screening. But, owing to lacking the embryonic stem cell line of big animal, traditional gene targeting is difficult to for setting up primate disease model. And gene is carried out having shown huge potentiality in accurately modification in orientation by the gene editing new technique CRISPR/Cas9 of development in recent years, have been widely used for different genera and carry out genome manipulation and genetic modification. Meanwhile, this new technique be also attempted for treat some diseases caused because of genetic flaw (Schwanketal., 2013; Wuetal., 2013). But, CRISPR/Cas9 technology still awaits improving on the precision of genetic modification.
CRISPR/Cas9 is a kind of new technique being instructed Cas albumen gene to be edited and modifies by RNA deriving from antibacterial acquired immunity. CRISPR/Cas9 system is carried out artificial reconstructed allowing the system to more simply and easily for gene editing (Jineketal., 2013) by Jinek etc. The DNA of specific site is cut by Cas9 restriction endonuclease under the guiding of guide RNA molecule, forms double-stranded DNA breach, and then the DNA of fracture can be repaired by cell by homologous recombination machinery or non-homologous end joining mechanism. If cell is repaired by homologous recombination machinery, the DNA breach of fracture can be filled up by other section of DNA fragment, thus one section of " new " hereditary information can be introduced. At present, the Cas9 system of RNA mediation can success mediating bacterial, plant cell, zebrafish embryo, mice, human archeocyte, the discarded embryo of non-human primate machin embryo, the even mankind etc. genome editor (Choetal., 2013;Congetal., 2013; Jineketal., 2013; Malietal., 2013; Niuetal., 2014; Plattetal., 2014; Shanetal., 2013; Chenetal., 2015). And this technology successfully correct also by gene knock-in diseased n animal dcc gene (Schwanketal., 2013; Wuetal., 2013) or be reorganized as viral vector and set up animal model for cancer (Plattetal., 2014) etc., previous achievement in research is all that the treatment that this technology will be applied to human inheritance's deficiency disorders future provides hope.
Developing rapidly so that the foundation of animal disease model is no longer limited to the model organism of minority of CRISPR/Cas9 system so that realize genome editor in all species and be possibly realized. This technology is easy to use, simple and target practice efficiency is higher, it is possible to realizes that pair of alleles is carried out shearing and obtains homozygous mutation, also can design multiple gRNA in multiple sites of genes of interest to reach to knock out completely purpose. But currently also there is the weak points such as the latent gene chimeric polymorphism of sudden change, effect of missing the target, gene knock-in homologous recombination efficiency be relatively low in this technology. Chimeric effect problem for gene targeting, owing to small animal model growth cycle is shorter, can be screened by embryonic stem cell or constantly hybridization is gone down to posterity and obtained Mutants homozygous, but for the primate being closer to the mankind, its sexual maturity time is accomplished by 4 to 5 years, pregnant time was up to 165 days, and still lacked at present monkey embryonic stem cell line, and therefore chimeric effect has huge challenge on homozygous gene sudden change monkey model setting up.
Chimeric mutational effect is likely to be due to Cas9mRNA and the sgRNA of injection still continuous expression after germ cell embryo divides, and injection Cas9 albumen also occur this chimerism (Plattetal., 2014; Sungetal., 2014) (SungYH, 2014; KimS, 2014), show that Cas9 albumen is longer thus when causing that fertilized egg cell splits into four cells, eight cells even blastaea periods in the intracellular half-life, each cell forms several genes modified types in various degree because of random assortment Cas9 albumen in various degree, and the embryonal tissue formed and the animal of new life can be carried different genetic modification types in different cell or tissues or have the genetic modification of (strand or double-stranded DNA) in various degree. Additionally, the DNA damage repair mechanism of cell self or the non-homogeneous recombination repair of DNA etc. also can affect gene targeting efficiency and chimeric effect.
Every kind of protein in protokaryon and eukaryote body has its life characteristics, is also called the half-life. the half-life of protein is relevant with the aminoacid of its polypeptide chain N-terminal specific, i.e. N-hold-carrying then (Bachmairetal., 1986). therefore available N-hold-carrying is then with the amino acid whose ubiquitin fusion protein (ubiquitinfusiondegradation of variety classes, UFD) degraded signal changes the protein half life depending on ubiquitination degradation pathway, such as N end is connected with Arginine (Ub-R-tragetedprotein), Leucine (Ub-L--tragetedprotein), Proline (Ub-P--tragetedprotein) or Lys (Ub-K--tragetedprotein) can substantially reduce the stability of destination protein thus accelerated degradation, and it is connected with Met, Ser, Ala then makes the more stable (Johnsonetal. of destination protein, 1995). and meeting its degraded of N-end gauage albumen then, to depend on aminoacid of the 15th or 17 be lysine, therefore the lysine of destination protein can be made to occur in the 17th going to insert one section of random amino acid sequence between stable degraded signal and destination protein.If the 3rd lysine connected on the linker going between stable degraded signal and destination protein then can further enhance ubiquitination efficiency (Bachmairetal., 1986; Dantumaetal., 2000).
Summary of the invention
Present invention aim at providing a kind of Cas9 fusion protein, its coded sequence, plasmid containing coded sequence and expression vector.
Cas9 fusion protein provided by the invention includes Cas9 fragment and Ubiquitin fragment. Connexon the 1st amino acids of Ubiquitin and Cas9 is different obtains different experiment effects. Described Ubiquitin is Ubiquitin-L, Ubiquitin-P or Ubiquitin-R abbreviation of connexon the 1st amino acids of Ubiquitin and Cas9 (L, P, the R are).
The DNA encoding sequence of Ubiquitin is connected in the DNA encoding sequence of Cas9 by the mode transformed by plasmid, thus reaching to accelerate the effect of Cas9 protein degradation. Connexon the 1st amino acids of Ubiquitin and Cas9 is different obtains different practical functions, we have found that Ubiquitin-R (R represents that connexon the 1st amino acids of Ubiquitin and Cas9 is arginine) best results by testing, Ubiquitin-L, Ubiquitin-P take second place.
In a preferred embodiment in accordance with this invention, Ubiquitin is Ubiquitin-R.
As preferred embodiment, described fusion protein is containing, for example aminoacid sequence shown in SEQIDNO.1. Invention provides a kind of Cas9 fusion protein coded sequence simultaneously, it is characterised in that containing Cas9DNA fragment and UbiquitinDNA fragment.
We have found that Ubiquitin-R (R represents that connexon the 1st amino acids of Ubiquitin and Cas9 is arginine) best results by testing, Ubiquitin-L, Ubiquitin-P take second place.
In a preferred embodiment in accordance with this invention, described Ubiquitin is Ubiquitin-R.
As preferred embodiment, described coded sequence is containing, for example DNA sequence shown in SEQIDNO.2.
Invention provides simultaneously and comprises the plasmid of above-mentioned coded sequence, expression vector or host cell; Preferably, described carrier is Ubiquitin-R-Cas9.
A kind of gene targeting method, utilizes described fusion protein or described coded sequence to carry out gene targeting. Preferably, described gene is mammalian genes; It is highly preferred that described gene is non-human mammal gene; It is further preferred that described gene is non-human primate's gene.
A kind of method reducing the chimeric effect of zooblast gene targeting, it utilizes above-mentioned fusion protein or coded sequence to carry out gene targeting; Preferably, described cell is mammalian cell; It is highly preferred that described cell is non-human mammalian cell; It is further preferred that described cell is non-human primate's cell
As one embodiment of the present invention, the invention provides a kind of CRISPR/Cas9 of significantly reducing new method in the chimeric effect of non-human primate machin gene targeting: namely it only plays gene targeting effect at development of fertilized ova early stage (two cell stage or four cell stage) with fixing quantity by adding the ubiquitination degraded signal Ubiquitin half-life shortening Cas9 albumen.
In a particularly preferred embodiment of the present invention, disclose the transformation carrier of a kind of Cas9 protein sequence containing the special degraded signal of Ubiquitin and the application in primate machin thereof. Specifically include: prepare above-mentioned improved Cas9 protein sequence, build improved plasmid vector;Design the genome edit tool sgRNA of specific recognition, cutting machin genome specific bit point sequence, the Cas9mRNA that the plasmid vector of (Ub-i-Cas9) after (WT-Cas9) before described genome edit tool sgRNA, described transformation and transformation generates through in vitro transcription is imported the unicellular germ cell embryo of monkey by the method using common microinjection, utilizes the singe-cell PCR technical appraisement gene targeting efficiency of single blastomere isolation technics and optimization, homozygous mutation efficiency.
Hereinafter, with Ubiquitin-i represent Ubquitin-L, Ubquitin-P or Ubquitin-R any one.
Based on CRISPR/Cas9 mediated gene shooting method, fusion protein provided by the invention is utilized to carry out gene targeting, it is possible to the problem solving the chimeric effect of gene targeting that CRISPR/Cas9 technology exists. The method is particularly applicable in the vector construction process knocking out machin PINK1, Parkin and ASPM gene by we. Our experiment includes 1) Cas9 protein expression vector MLM3613-Cas9 before transformation, 2) Ubiquitin-i-Cas9 protein expression vector and carry the sgRNA expression vector of PINK1, Parkin and ASPM gene targeting sequence after transformation; 3) and use and have the method for microinjection altogether described genome edit tool { being included: sgRNA, the Cas9mRNA} importing unicellular germ cell embryo of monkey generated through in vitro transcription by the plasmid vector of (Ub-i-Cas9) after (WT-Cas9) before described transformation and transformation; 4) the singe-cell PCR technical appraisement gene targeting efficiency of single blastomere isolation technics and optimization, homozygous mutation efficiency are utilized.
Due to chimeric effect occur with injection after expressed Cas9 albumen longer half-life there is important relation. It is capable of while not affecting Cas9 cutting efficiency by the method for the present invention being shortened by Cas9 protein half-life to control it with quantitative timing and only play gene targeting effect in germ cell fetal development early stage, thus reducing chimeric mutational effect, the disease model and genetic flaw repairing and treating efficiently setting up non-human primate homozygous mutation is significant.
One aspect of the present invention can so that Cas9 albumen is degraded rapidly after playing target practice effect in embryonic cell, thus reducing the chimeric mutational effect of fetal development. Especially particularly preferred Ubiquitin-R-Cas9 albumen, target practice mutation rate of isozygotying improves 3.51 times than wild type Cas9.
The more important thing is, Ubiquitin-i-Cas9 albumen provided by the present invention can improve embryonic gene target practice homozygosity. Especially particularly preferred Ubiquitin-R-Cas9 albumen, wild type Cas9 albumen embryo's gene targeting homozygosity efficiency is improve 350% by it. This is outside technical staff expects.
Compared with prior art, importance is the fact that of the present invention:
First, the invention discloses the transformation carrier of a kind of Cas9 protein sequence containing the special degraded signal of Ubiquitin and the application in primate machin thereof, experiment proves that this improved Cas9 can to PINK1, Parkin and ASPM gene carries out effective gene target practice, and target practice rate is up to 73.83%; Further, comparing with WT-Cas9 (8.25%), improved Ub-R-Cas9 gene targeting homozygous mutation rate is up to 28.97%, and its target practice mutation rate of isozygotying improves 3.51 times than WT-Cas9.
Second, utilize whole embryo genomic DNA can not Accurate Analysis Cas9 practice shooting after the chimeric mutational type of each cell and mutation rate in embryo, injection is had four cells of target practice RNA or eight cell stages to isolate single blastomere by the present invention, and utilize different cell mutation situations and the mutation rate of the singe-cell PCR technology same embryonic origin of more precisely Analysis and Identification, reliably confirm Ubiquitin-Cas9 fusion protein remarkable result in improving target practice homozygous mutation rate.
3rd, for verifying that Ub-i-Cas9 albumen used in the present invention makes development of fertilized ova to four cells or just no longer play target practice effect before this period is earlier because of fast degradation ability, the single blastomere that the present invention utilizes embryo's single blastomere isolation technics to be separated by four cell stages and comes again is cultivated and is carried out singe-cell PCR when growth is four cell, and qualification result proves that the fast degradation of Ub-i-Cas9 can ensure the homogeneity of the genetic modification type in four cell stage later stages further.
The use of Ub-i-Cas9 provided by the present invention can substantially reduce the chimeric effect of the CRISPR/Cas9 gene targeting mediated, improve its genetic modification precision, provide new method for genetic flaw repairing and treating and for efficiently setting up the big animal model with homozygous mutation, there is potential Important Economic value and significance.
Accompanying drawing explanation
Fig. 1 is for including the Ub-i-Cas9 structural representation of different aminoacids degraded signal (black matrix).
Fig. 2 is Ub-i-Cas9 plasmid construction flow chart.
Fig. 3 is Ub-i-Cas9 expression in 293 cells and half-life detection; Wherein, A.Ub-i-Cas9 expression in 293 cells; B.WT-Cas9 and the UbR-Cas9 half-life; C. the relative quantification statistics of B is schemed.
Fig. 4 is WT-Cas9 and Ub-R-Cas9 expression in machin embryo; Wherein, a.WT-Cas9 and Ub-R-Cas9 expression in machin embryo; B.Ub-R-Cas9 gene targeting PCR in 293 cells identifies and sequencing result.
Fig. 5 is that WT-Cas9 and Ub-R-Cas9 Parkin gene targeting efficiency in machin embryo compares; Wherein, a.WT-Cas9 and Ub-R-Cas9 target practice efficiency statistical table in machin embryo; The PCR representative result that b.WT-Cas9 and Ub-R-Cas9 practices shooting in machin embryo; C.Ub-R-Cas9 is target practice sequencing result in machin embryo.
Fig. 6 is that singe-cell PCR enzyme action identifies target practice result; Injection has the germ cell embryonic cell of WT-Cas9 and Ub-R-Cas9 to be developed to, and that four cell stage separate after single blastomere separates and is further cultured for developing into new four cells is unicellular.
Detailed description of the invention
The invention will be further described below, but embodiments of the present invention are not limited to below example introduction, and all equivalent changes made according to principles of the invention or theory or accommodation are regarded as the category of present invention protection.
The ubiquitin protein degraded signal Ubiquitin-R-myc sequence such as SEQIDNO.3 that invention relates to; Ubiquitin-R sequence such as SEQIDNO.4; Myc sequence such as SEQIDNO.5; Cas9 protein sequence such as SEQIDNO.6.
Coded sequence such as SEQIDNO.2. Ubquitin-L-Cas9, Ubquitin-P-Cas9 of the Ubquitin-R-Cas9 fusion protein of code book invention is then that the cgc on SEQIDNO.2. the 229th��231 replaces with cuc, ccc respectively in the particular embodiment.
Embodiment 1
Building purpose plasmid Ub-i-Cas9 (i represents R, L, the P) expression vector comprising different protein degradation signals, schematic diagram is Fig. 1 such as, described structure flow and method following (Fig. 2).
For ease of building, first with MLM3613Cas9 (Addgene, Plasmid#42251, it being called WT-Cas9) two restriction enzyme sites of NotI and EcoRI on skeleton plasmid are connected on pGEX4T-1 plasmid will clone the segment cut of restriction enzyme site, translation initiation site and part Cas9 protein sequence containing T7 promoter, NcoI after (Fig. 2 a, b), pGEX4T1-NotI-T7-NcoI-Cas9-EcoRI-.Subsequently, the design forward primer with NotI restriction enzyme site and T7 promoter sequence I and the downstream primer (sequence is respectively as shown in SEQIDNO.22, SEQIDNO.23-25) with Myc-sequence label, L/P/L site and NocI restriction enzyme site, it is simultaneously introduced the amino acid whose DNA sequence of L/P/R at downstream primer, from HEK293 cell cDNA, amplifies Ubiquitin sequence; , T7-Ubiquitin-L/P/R-myc (being expanded by people's HEK293 cell cDNA) sequence is cloned restriction enzyme site by NotI and NcoI and is connected to pGEX4T1-NotI-T7-NcoI-Cas9-EcoRI-carrier, such as Fig. 2 c, 2d. Again NotI-T7-ubiquitin-L/P/R-Myctag-Cas9-EcoRI-fragment is connected to the linearizing MLM3613-Cas9 skeleton plasmid of NotI and EcoRI two restriction enzyme site, successfully constructs Ubquitin-R/L/P-Myctag-Cas9 expression vector (Fig. 2 e).
SEQIDNO.22:Ub5 '-1Not1:5 '-ATCCGCGGCCGCTAATACGACTCACTATAGGGCCATGCAGATCTTCGTGAAGAC-3 ' T7promoter
3��Primers:
UbR3��-204Nco1(SEQIDNO.23):
5��-ATCCATGGTCTTAGCATGTACCAGATCTTCTTCAGAAATAAG
TTTTTGTTCTTTACCTCGCCCACCTCTGAGACGGAG-3��
UbL3��-204Nco1(SEQIDNO.24):
5��-ATCCATGGTCTTAGCATGTACCAGATCTTCTTCAGAAATAAG
TTTTTGTTCTTTACCTAGCCCACCTCTGAGACGGAG-3��
UbP3��-204Nco1(SEQIDNO.25):
5��-ATCCATGGTCTTAGCATGTACCAGATCTTCTTCAGAAATAAG
TTTTTGTTCTTTACCTGGCCCACCTCTGAGACGGAG-3��
The expression plasmid carrier containing Ub-i-Cas9 built uses primer extension method that its sequence is carried out sequence verification, and sequencing result demonstrates and successfully constructs.
Embodiment 2
Build the sgRNA carrier of the CRISPR/Cas9 containing PINK1, Parkin and ASPM gene target sequence.
Table 1. target sequence site
Note: dashed part sequence is the PAM sequence of sgRNA, i.e. its binding site with target dna sequence.
By before above-mentioned constructed sgRNA target sequence carrier and transformation, after improved Cas9 plasmid vector PmeI enzyme linearisation, glue reclaims, in vitro transcription synthesis RNA.
Described sgRNA expression vector, the expression of sgRNA is controlled containing a T7 promoter, can be inserted into the specific target sequence of 20bpsgRNA after with BbsI restriction digest, the recognition site of this specific target sequence is positioned at PINK1 gene Second Exon, Parkin gene Second Exon, ASPM gene the 3rd exon and the tenth exon. In order to screen the sgRNA sequence that can carry out gene editing and less PINK1 and the ASPM gene of effect of missing the target, it is low and meet the CRISPR/Cas9 target sequence site in conjunction with genome signature (5 '-G (19N)-NGG3 ') or (5 '-CCN (19N)-C-3 ') that applicant utilizes the Blast software of NCBI to screen effect of missing the target on the PINK1 gene of macaque and the ASPM gene order of machin respectively, such as table 1, construction method is with reference to existing document (Changetal., 2013).
Embodiment 3
Ub-i-Cas9 (i represents LP, R) the plasma half-life detection built.
First, by WT-Cas9 and Ub-i-Cas9 plasmid transfection to 293 cells, after transfecting 48 hours, Westernblot detects Cas9 expressing quantity result and shows, with WT-Cas9 for 100%, Ub-L-Cas9, Ub-P-Cas9, Ub-R-Cas9 degrade respectively to 84.76%, 80.92% and 63.89%.
Especially Ub-R-Cas9 therein, although intersect the difference of only one of which base in its sequence with Ub-L-Cas9, Ub-P-Cas9, but having surprisingly found that is that its degradation rate is significantly faster than other fusion protein, has reached very rare degree.
Subsequently, we compare the half-life of WT-Cas9 and Ub-R-Cas9, namely after plasmid transfection 24 hours, add 50 micromole's cycloheximides to culture medium, respectively at 0h, 2h, 4h, 8h, 12h, 24h collects cell, Westernblot testing result is such as shown in Fig. 3 B, C, compared with WT-Cas9, Ub-R-Cas9 protein half-life significantly shortened, and just degraded about percent 50 at 4 hours.
Secondly, WT-Cas9 and Ubquitin-R-Cas9 in vitro transcription synthesizing mRNA microinjection in machin embryo, as shown in table 2, Showed by immune group result Ubquitin-R-Cas9 substantially accelerates than WT-Cas9 degradation speed in machin embryo. Ub-R-Cas9 in the embryo after injection 48 hours content than WT-Cas9 few 69.18%.
Table 2.Ubquitin-R-Cas9 and WT-Cas9 Cas9 residual quantity in embryo compares
Residual quantity after injection 24h | Residual quantity after injection 48h | |
Ub-R-Cas9 | 63.38 | 18.96 |
WT-Cas9 | 100% | 61.53 |
Embodiment 4
The Ub-R-Cas9 gene targeting function of described success structure and Efficiency testing.
First; WT-Cas9/Pink2-2sgRNA and Ub-R-Cas9/Pink2-2sgRNA plasmid is transfected respectively to 293 cells; select individual cells by stepwise dilution method after 48 hours or multiple cell carries out PCR qualification; confirm that Ub-R-Cas9 has normal gene target practice function through T7E1 detection and order-checking, such as Fig. 4 b.
Secondly, WT-Cas9 or Ub-R-Cas9mRNA and sgRNA microinjection altogether is entered monkey one cell embryos. Treat that fetal development to four cells or eight cell stage separate single blastomere and carry out singe-cell PCR qualification (pcr amplification primer sequence is in Table 3). Additionally, the single blastomere that injection is had four cell stages of WT-Cas9 and Ub-R-Cas9 to separate and comes by available embryo's single blastomere separate mode is again cultivated and carried out singe-cell PCR when growth is four cell, qualification result is Fig. 6 such as.
Table 3. amplimer sequence
Result shows: PINK1, Parkin and ASPM gene can be carried out gene targeting by this improved Ub-R-Cas9 carrier, and target practice rate is up to 73.83%. And Ub-R-Cas9 and WT-Cas9 (8.25%) compares the probability that can significantly improve homozygous knockout up to 28.97%, improve about 3.5 times, such as Fig. 5. Additionally, Fig. 6 result also further demonstrate that Ub-R-Cas9 because having fast degradation ability thus ensureing the homogeneity of the genetic modification type in four cell stage later stages, hence it is evident that reduce the chimeric effect caused by the WT-Cas9 lasting target practice caused because the half-life is longer.
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention are also not restricted to the described embodiments; the change made under other any spirit without departing from the present invention and principle, modification, replacement, combination, simplification; all without departing from present disclosure, it is included in protection scope of the present invention.
Claims (10)
1. a Cas9 fusion protein, it is characterised in that include Cas9 fragment and Ubquitin fragment.
2. fusion protein as claimed in claim 1, it is characterised in that described Ubiquitin is Ubiquitin-L, Ubiquitin-P or Ubiquitin-R; It is highly preferred that described Ubiquitin is Ubiquitin-R.
3. fusion protein as claimed in claim 1, it is characterised in that containing, for example aminoacid sequence shown in SEQIDNO.1.
4. a Cas9 fusion protein coded sequence, it is characterised in that containing Cas9DNA fragment and UbquitinDNA fragment.
5. coded sequence as claimed in claim 4, it is characterised in that described Ubiquitin is Ubiquitin-L, Ubiquitin-P or Ubiquitin-R; It is highly preferred that described Ubiquitin is Ubiquitin-R.
6. coded sequence as claimed in claim 4, it is characterised in that containing, for example DNA sequence shown in SEQIDNO.2.
7. the plasmid containing sequence as described in as arbitrary in claim 4-6.
8. the expression vector containing sequence as described in as arbitrary in claim 4-6.
9. a gene targeting method, it is characterised in that utilize as arbitrary in claim 1-3 as described in fusion protein or claim 4-6 arbitrary as described in coded sequence carry out gene targeting; Preferably, described gene is mammalian genes; It is highly preferred that described gene is non-human mammal gene; It is further preferred that described gene is non-human primate's gene.
10. the method reducing the chimeric effect of zooblast gene targeting, it is characterised in that utilize as arbitrary in claim 1-3 as described in fusion protein or claim 4-6 arbitrary as described in coded sequence carry out gene targeting; Preferably, described cell is mammalian cell; It is highly preferred that described cell is non-human mammalian cell; It is further preferred that described cell is non-human primate's cell.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610038907.6A CN105647885B (en) | 2016-01-20 | 2016-01-20 | Cas9 fusion protein and coding sequence thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201610038907.6A CN105647885B (en) | 2016-01-20 | 2016-01-20 | Cas9 fusion protein and coding sequence thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN105647885A true CN105647885A (en) | 2016-06-08 |
CN105647885B CN105647885B (en) | 2017-08-18 |
Family
ID=56487808
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201610038907.6A Active CN105647885B (en) | 2016-01-20 | 2016-01-20 | Cas9 fusion protein and coding sequence thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN105647885B (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108998452A (en) * | 2018-07-11 | 2018-12-14 | 暨南大学 | A kind of method that substantia nigra gene knockout quickly establishes animal model for parkinsonism |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA3111479A1 (en) | 2017-09-26 | 2019-04-04 | The Board Of Trustees Of The University Of Illinois | Crispr/cas system and method for genome editing and modulating transcription |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103725712A (en) * | 2014-01-17 | 2014-04-16 | 南京大学 | Intermediate vector for conditional gene knockout without species limits as well as preparation method and application of intermediate vector |
CN103981215A (en) * | 2014-05-23 | 2014-08-13 | 安徽省农业科学院水稻研究所 | Backbone plasmid carrier for genetic engineering and application thereof |
WO2015048690A1 (en) * | 2013-09-27 | 2015-04-02 | The Regents Of The University Of California | Optimized small guide rnas and methods of use |
CN104531632A (en) * | 2014-11-18 | 2015-04-22 | 李云英 | Rapidly-degraded Cas9-ODC422-461 fusion protein and application thereof |
-
2016
- 2016-01-20 CN CN201610038907.6A patent/CN105647885B/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015048690A1 (en) * | 2013-09-27 | 2015-04-02 | The Regents Of The University Of California | Optimized small guide rnas and methods of use |
CN103725712A (en) * | 2014-01-17 | 2014-04-16 | 南京大学 | Intermediate vector for conditional gene knockout without species limits as well as preparation method and application of intermediate vector |
CN103981215A (en) * | 2014-05-23 | 2014-08-13 | 安徽省农业科学院水稻研究所 | Backbone plasmid carrier for genetic engineering and application thereof |
CN104531632A (en) * | 2014-11-18 | 2015-04-22 | 李云英 | Rapidly-degraded Cas9-ODC422-461 fusion protein and application thereof |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108998452A (en) * | 2018-07-11 | 2018-12-14 | 暨南大学 | A kind of method that substantia nigra gene knockout quickly establishes animal model for parkinsonism |
Also Published As
Publication number | Publication date |
---|---|
CN105647885B (en) | 2017-08-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN105647969B (en) | Method for breeding zebra fish with stat1a gene deletion by gene knockout | |
CN110551759B (en) | Composition and method for improving recombination efficiency of transgenic cells | |
CN104531705A (en) | Method for knocking off animal myostatin gene by using CRISPR-Cas9 system | |
CN107058320A (en) | The preparation and its application of IL7R gene delection zebra fish mutant | |
CN104531704A (en) | Method for knocking off animal FGF5 gene by using CRISPR-Cas9 system | |
CN106467909A (en) | A kind of method that acquisition glyphosate-resistant rice is replaced by nucleotide fixed point | |
CN111926017B (en) | Preparation and application of csf1ra gene deletion zebra fish mutant | |
CN104611368B (en) | The carrier of frameshift mutation is not generated after restructuring, the gene site-directed method knocked in and application are carried out in pawl frog genome | |
CN106282231B (en) | Construction method and application of mucopolysaccharide storage disease type II animal model | |
CN103820452B (en) | A kind of sgRNA fragment and application thereof | |
CN112410341B (en) | Mouse model construction method capable of inducing specific elimination of neutrophils | |
CN112608939B (en) | Construction method and application of mouse model with conditional mTERT overexpression | |
CN111718933B (en) | Preparation method and application of rrbp1 gene knockout hot claw frog model | |
CN105505879B (en) | Method and culture medium for culturing transgenic animal embryonic cells or transgenic animals | |
Ohde et al. | CRISPR/Cas9-based heritable targeted mutagenesis in Thermobia domestica: a genetic tool in an apterygote development model of wing evolution | |
CN110199020A (en) | For quickly generating the DNA plasmid of the homologous recombination vector for cell line exploitation | |
CN105647885A (en) | Cas9 fusion protein and coding sequence thereof | |
CN110066805A (en) | The method of gene knockout breeding adgrf3b Gene Deletion zebra fish | |
US11202419B2 (en) | Soybeans with reduced antinutritional factor content | |
CN113907042B (en) | Method for constructing neutrophil-loss mouse model | |
CN113373150B (en) | sgRNA of targeting dat gene and application thereof | |
CN114591957A (en) | Construction method and application of severe hemophilia A animal model | |
CN105440111A (en) | Pair of transcription activator-like effector nucleases (CTFs), coding sequences and application thereof | |
CN114592011B (en) | Construction method of PTDSS2 conditional gene knockout mouse model | |
CN114606270B (en) | Construction method of Mia3 conditional gene knockout mouse model |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |