CN104531632A - Rapidly-degraded Cas9-ODC422-461 fusion protein and application thereof - Google Patents
Rapidly-degraded Cas9-ODC422-461 fusion protein and application thereof Download PDFInfo
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- CN104531632A CN104531632A CN201410656081.0A CN201410656081A CN104531632A CN 104531632 A CN104531632 A CN 104531632A CN 201410656081 A CN201410656081 A CN 201410656081A CN 104531632 A CN104531632 A CN 104531632A
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Abstract
The invention discloses a rapidly-degraded Cas9-ODC422-461 fusion protein and application thereof, and belongs to the technical field of molecular biology. According to the rapidly-degraded Cas9-ODC422-461 fusion protein and the application, a humanization Cas9 protein is transformed with a genetic engineering method, a PEST protein degradation sequence is added, and therefore the rapidly-degraded Cas9-ODC422-461l fusion protein is obtained. The Cas9-ODC422-461l fusion protein can conduct induced control over an expression of Cas9; the staying time of the Cas9-ODC422-461l fusion protein in cells is shortened; and nonspecific cutting toxicity of Cas9-ODC422-461l overexpression to the cells is accordingly reduced. The rapidly-degraded Cas9-ODC422-461l fusion protein is safer when being used for drug screening or gene modification; and the fusion protein can be further used for gene therapy.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to a kind of Cas9-ODC of fast degradation
422-461fusion rotein and application thereof.
Background technology
CRISPR-Cas is derived from the immunity system of bacterium and archeobacteria, target spot specific RNA can be utilized Cas9 nuclease to be taken to concrete target spot on genome, thus modify specific gene site.RNA guiding Cas9 can play function, in specific site cracking complete genome group in various kinds of cell and organism.The potential of this genome editor of Cas9 makes this technology be widely used in the clone of people, mouse, rat, the gene knockout of several species and knocking in.But the Cas9 technology of this RNA guiding has a very large defect to be exactly serious effect of missing the target.This effect of missing the target, it is long that one side is wherein exactly transformation period of Cas9.
Summary of the invention
The amino acid of mouse ornithine decarboxylase (MODC) C-end comprises the degraded territory of a PEST sequence, and this degraded territory promotes the degraded of this albumen, makes the transformation period of albumen very short.This feature has very large application in gene functional research.
The present invention utilizes amino acid PEST sequence and the Cas9 protein fusion of mouse ornithine decarboxylase (MODC) C-end, invents a kind of Cas9-ODC of fast degradation
422-461fusion rotein.This albumen reduces Cas9-ODC
422-461the time that fusion rotein exists in cell, the transformation period shortens greatly.At eukaryotic expression system, it can inducibility transient expression, reaches the genomic effect of clean cut, the effect of missing the target of generation is reduced greatly.
The technical solution used in the present invention is as follows:
The Cas9-ODC of fast degradation
422-461fusion rotein, the albumen of described fusion rotein for being made up of aminoacid sequence shown in SEQ ID NO.1.
Encode the Cas9-ODC of above-mentioned fast degradation
422-461the nucleotide sequence of fusion rotein.
Above nucleotide sequence, described nucleotide sequence is as shown in SEQ ID NO.2.
The Cas9-ODC of fast degradation of the present invention
422-461fusion rotein, the Cas9-ODC of described fast degradation
422-461fusion rotein is that the amino acid fusion of Cas9 albumen and mouse ornithine decarboxylase (MODC) C-end 422 – 461 forms, amino acid due to (MODC) C-end 422 – 461 comprises the degraded that a degraded territory promotes this albumen, makes the transformation period of albumen very short.
The Cas9-ODC of described fast degradation
422-461the application of fusion rotein in drug screening and genetic modification.
Described Cas9-ODC
422-461the gene clone of fusion rotein to carrier for expression of eukaryon for the genetic modification of cell and animal.
compared with prior art, its beneficial effect is in the present invention:the present invention, by engineered way, transforms humanization Cas9 albumen, adds PEST proteolytic degradation sequence, obtains the Cas9-ODC of fast degradation
422 – 461lfusion rotein; Cas9-ODC
422 – 461lfusion rotein can the expression of Induction Control Cas9, reduces Cas9-ODC
422 – 461lthe time that albumen exists in cell, thus reduce Cas9-ODC
422 – 461lprocess LAN to the non-specific cutting toxicity of cell.The Cas9-ODC of fast degradation of the present invention
422 – 461lfusion rotein does drug screening or genetic modification is safer, and this fusion rotein also can be used for gene therapy.
Compared to the prior art, Cas9-ODC
422 – 461lfusion rotein may be used for abduction delivering inside cell because transformation period very short, can the promotor of transcriptional control so add one, add inductor, just can control Cas9-ODC in time
422 – 461lthe expression of fusion rotein, thus can modulability carry out genome editor.Another one advantage is, this albumen is used for gene therapy, because the transformation period of this fusion rotein is very short, the time that it exists at cell is many, as medicine, and its side effect smaller.
Accompanying drawing explanation
Fig. 1 is Cas9-ODC
422 – 461land Cas9-ubiquitin
g75A/G76Vthe detection of expression figure of albumen inside eukaryotic cell; Wherein, 1 is transfected wild-type Cas9, and 2 is transfection Cas9-ODC
422 – 461l, 3 is transfection Cas9-ubiquitin
g75A/G76V;
Fig. 2 is pTRE-Cas9, pTRE-Cas9-ODC
422 – 461and pTRE-Cas9-ubiquitin
g75A/G76V2at the expression of HEK 293 Tet-On clone; Wherein, 1 is the pTRE-Cas9 of transfected wild-type, and 2 is pTRE-Cas9-ODC
422 – 461l, 3 is pTRE-Cas9-ubiquitin
g75A/G76V;
Fig. 3 is that electricity turns wild-type Cas9-gRNA1, picked clones, pcr amplification Malt1 gene, sequencing result figure;
Fig. 4 is that electricity turns pCas9-ODC
422 – 461-gRNA, picked clones, pcr amplification Malt1 gene, sequencing result figure;
Fig. 5 is that electricity turns pCas9-ubiquitin
g75A/G76V-gRNA1, picked clones, pcr amplification Malt1 gene, sequencing result figure.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by buying the conventional products obtained.
Primer Primer_Cas9_F, primer Primer_ODC_R, primer Ubiquitin R, Cas9-ODC
422 – 461l_ F:NdeI, primer Cas9-ODC
422 – 461l_ R:BamHI, primer Cas9-ubiquitin
g75A/G76V_ F:NdeI, primer Cas9-ubiquitin
g75A/G76V_ R:BamHI, Primer F and Primer R are all purchased from Sangon Biotech (Shanghai) Co., Ltd.;
GRNA1 carrier is purchased from Addgene, Plasmid 41824
HCas9 carrier is purchased from Addgene, Plasmid 41815;
Glue reclaims and adopts glue to reclaim test kit, is purchased from TIANGEN Biotech (Beijing) Co., Ltd.;
Prokaryotic expression carrier pET-16b, is purchased from Novagen;
Intestinal bacteria E. coli BL21, is purchased from Takara;
IPTG, is purchased from Takara;
Cycloheximide cycloheximide, is purchased from Sigma;
HEK 293 Tet-On clone, is purchased from life technologies company;
Mouse ES cells, is purchased from Millipore;
293T cell, is purchased from Clontech company;
G418, is purchased from Sigma;
Primary antibodie, Cas9 antibody, mouse-anti, is purchased from Abcam company;
Two of HRP mark resists, and rabbit against murine, is purchased from Abcam company;
Cas9-ubiquitin
g75A/G76Vthe synthesis of DNA sequence dna and Cas9-ODC
422 – 461synthesis equal student on commission's work biotechnology (Shanghai) limited-liability company synthesis of DNA sequence dna.
embodiment 1 Cas9-ODC
422 – 461l
and Cas9-ubiquitin
g75A/G76V
the preparation of fusion rotein
Method is as follows:
First part: Cas9-ODC
422-461the structure of carrier for expression of eukaryon:
The PEST sequence of proline rich, L-glutamic acid, Serine, Threonine is relevant with the fast degradation of protein, we analyze Cas9 albumen with PESTFind (http://emboss.bioinformatics.nl/cgi-bin/emboss/pestfind), do not find potential PEST sequence.Cas9 has the very long transformation period.In order to reduce its transformation period.We merge PEST sequence ODC at the C end of Cas9 albumen
422 – 461and Ub-G75A/G76V, be built into following carrier, pCas9-ODC
422 – 461, pCas9-ubiquitin
g75A/G76V, build pCas9-ubiquitin
g75A/G76Vas a contrast.
Concrete construction process is as follows:
(1) with Cas9-ODC
422 – 461dNA sequence dna is template (SEQ ID NO.2), with primer Primer_Cas9_F:CAGCCTCCGGACTCTAGAGGATCGA(SEQ ID NO.3) and primer Primer_ODC_R:AACTCATTACTAACCGGTTCACACATTGATCCTAGCAGAAGCA (SEQ ID NO.4) increase Cas9-ODC
422 – 461, be cloned into the XbaI & AgeI site of hCas9 carrier, be built into pCas9-ODC
422 – 461carrier.
Detailed amplification step is as follows:
Pcr amplification Cas9-ODC
422 – 461, shown in configuration reaction system table 1.
Table 1
Amplified reaction program: 98 DEG C of 10s, 61 DEG C of 30s, 72 DEG C, 4.2min, totally 30 circulations.
Glue reclaims Cas9-ODC
422 – 461fragment, cuts hCas9 carrier with XbaI & AgeI enzyme, and glue reclaims skeleton carrier.Two fragments carry out In-Fusion reaction (Clothech), and transform, clone identification, finds pCas9-ODC
422 – 461lpositive colony.
(2) with Cas9-ubiquitin
g75A/G76VdNA sequence dna is template (SEQ ID NO.5), with primer Primer_Cas9_F:CAGCCTCCGGACTCTAGAGGATCGA(SEQ ID NO.3) and primer Ubiquitin R:AACTCATTACTAACCGGTACCTCCGCGCAAACGTAAGACTAA(SEQ ID NO.6) increase Cas9-ubiquitin
g75A/G76V, be cloned into the XbaI & AgeI site of hCas9 carrier, be built into pCas9-ubiquitin
g75A/G76Vcarrier.
Detailed amplification step is as follows:
Pcr amplification Cas9-ubiquitin
g75A/G76V, configuration reaction system is as shown in table 2.
Table 2
| 5×primerSTAR Buffer | 10 ul |
| dNTP (2.5 mM) | 4μl |
| TaKaRa Taq HS (5 U/μl) | 0.5ul |
| Primer_F1 20pmol (final conc. 0.4 μM) | 1ul |
| Primer_ R1 20pmol (final conc. 0.4 μM) | 1ul |
| Cas9-ubiquitin G75A/G76V (20ng/ul) | 1ul |
| ddH2O | 32.5 ul |
| Amount to | 50 ul |
Amplified reaction program: 98 DEG C of 10s, 61 DEG C of 30s, 72 DEG C, 4.2min, totally 30 circulations.
Glue reclaims Cas9-ubiquitin
g75A/G76Vfragment, cuts hCas9 carrier with XbaI & AgeI enzyme, and glue reclaims skeleton carrier.Two fragments carry out In-Fusion reaction (Clothech), and transform, clone identification, finds Cas9-ubiquitin
g75A/G76Vpositive colony.
Second section: pET-16b-Cas9-ODC
422 – 461land pET-16b-Cas9-ubiquitin
g75A/G76Vthe structure of prokaryotic expression carrier:
(1) primer Cas9-ODC is used
422 – 461l_ F:NdeI:
TCGAAGGTCGTCATATG ATGGACAAGAAGTACTCCATTGGGCT(SEQ ID NO.7) and primer Cas9-ODC
422 – 461l_ R:BamHI:
TTGTTAGCAGCCGGATCCTCACACATTGATCCTAGCAGAAGC(SEQ ID NO.8) remove the Cas9-ODC that increases
422 – 461l, configuration reaction system is as shown in table 3.
Table 3
| 5×primerSTAR Buffer | 10 ul |
| dNTP (2.5 mM) | 4μl |
| TaKaRa Taq HS (5 U/μl) | 0.5ul |
| Cas9-ODC 422–461l_F: NdeI 20pmol | 1ul |
| Cas9-ODC 422–461l _R：BamHI20pmol | 1ul |
| Cas9-ODC 422–461l (20ng/ul) | 1ul |
| ddH2O | 32.5 ul |
| Amount to | 50 ul |
Amplified reaction program: 98 DEG C of 10s, 61 DEG C of 30s, 72 DEG C, 4.5min, totally 30 circulations.
Glue reclaims Cas9-ODC
422 – 461lfragment, cuts prokaryotic expression carrier pET-16b with NdeI and BamHII enzyme, and glue reclaims skeleton carrier.Two fragments carry out In-Fusion reaction (Clothech), and transform, clone identification, finally obtains pET16b-Cas9-ODC
422 – 461lprokaryotic expression carrier.
(2) primer Cas9-ubiquitin is used
g75A/G76V_ F:NdeI:
TCGAAGGTCGTCATATGATGGACAAGAAGTACTCCATTGGGCT(SEQ ID NO.9) and primer Cas9-ubiquitin
g75A/G76V_ R:BamHI:
TTGTTAGCAGCCGGATCCCTAACCGGTACCTCCGCGCAAA(SEQ ID NO.10) remove the Cas9-ubiquitin that increases
g75A/G76V, configuration reaction system is as shown in table 4.
Table 4
| 5×primerSTAR Buffer | 10 ul |
| dNTP (2.5 mM) | 4μl |
| TaKaRa Taq HS (5 U/μl) | 0.5ul |
| Cas9-ubiquitin G75A/G76V_F: NdeI 20pmol | 1ul |
| Cas9-ubiquitin G75A/G76V_R：BamHI20pmol | 1ul |
| Cas9-ubiquitin G75A/G76V (20ng/ul) | 1ul |
| ddH2O | 32.5 ul |
| Amount to | 50 ul |
Amplified reaction program: 98 DEG C of 10s, 61 DEG C of 30s, 72 DEG C, 4.5min, totally 30 circulations.
Glue reclaims Cas9-ubiquitin
g75A/G76Vfragment, cuts prokaryotic expression carrier pET-16b with NdeI and BamHII enzyme, and glue reclaims skeleton carrier.Two fragments carry out In-Fusion reaction (Clothech), and transform, clone identification, finally obtains pET16b-Cas9-ubiquitin
g75A/G76Vprokaryotic expression carrier.
Part III: Cas9-ODC
422 – 461land Cas9-ubiquitin
g75A/G76Vthe expression and purification of albumen
Recombinant plasmid pET-16b-Cas9-ODC
422 – 461land pET-16b-Cas9-ubiquitin
g75A/G76Vtransformation of E. coli E. coli BL21.Transformed bacteria overnight incubation in the substratum of 5ml ammonia benzyl resistance, is transferred in the identical substratum of 500ml and cultivates next day, to spend the night induction through IPTG 16 DEG C, collects the Cas9-ODC obtained respectively
422 – 461land Cas9-ubiquitin
g75A/G76Valbumen thalline, the supernatant of centrifugal acquisition solubility after high pressure fragmentation, carries out Ni post affinity chromatography.After treating that target protein is combined with Ni post, first use rinsing liquid (20mM Tris-HCl, 500mM NaCl, 5%(v/v) glycerine, 60mM imidazoles, pH8.0) wash, use elution buffer (20mM Tris-HCl, 500mM NaCl, 5%(v/v) glycerine afterwards, 500mM imidazoles, pH8.0) wash away albumen.Utilize PD-10 desalting column to be replaced as the high-salt buffer in elution fraction containing volume percent to be the PBS solution of 20% glycerine, carry out the detection of SDS-PAGE afterwards, gained is the Cas9-ODC of purifying
422 – 461land Cas9-ubiquitin
g75A/G76Valbumen.
embodiment 2 Cas9-ODC
422 – 461l
and Cas9-ubiquitin
g75A/G76V
the expression of albumen inside eukaryotic cell and degraded
By wild-type pCas9, pCas9-ODC
422 – 461and pCas9-ubiquitin
g75A/G76Vbe transfected in 293T cell, after transfection 24 hours, process 6 hours respectively with cycloheximide cycloheximide (50 mg/mL), stop the synthesis of new albumen.Collecting cell, cracking, then do western blot test, get fermented liquid supernatant stoste 10ul loading, after SDS-PAGE protein electrophoresis completes, albumen is transferred on pvdf membrane, in conjunction with two anti-(rabbit against murine of primary antibodie (Cas9 antibody, mouse-anti) and HRP mark,), then develop on film.
As shown in Figure 1, compare with wild-type Cas9, other fusion protein expressions are lower, illustrate, Cas9-ODC in result display
422 – 461land Cas9-ubiquitin
g75A/G76Vfusion rotein has degraded, and the transformation period is shorter.
embodiment 3
PCas9, pCas9-ODC
422 – 461and pCas9-ubiquitin
g75A/G76Vthe promotor of plasmid changes TRE into, is built into carrier pTRE-Cas9, pTRE-Cas9-ODC
422 – 461with p TRE-Cas9-ubiquitin
g75A/G76V2, three carriers are transfected into HEK 293 Tet-On clone, then go screening with G418.Find and express Cas9, Cas9-ODC
422 – 461and Cas9-ubiquitin
g75A/G76V2the cell of expressing.Tsiklomitsin process is thin 1 hour, after then removing tsiklomitsin, spends six hours, goes the expression of detection three albumen, as shown in Figure 2 with western blot.This result illustrates, Cas9-ODC
422 – 461and Cas9-ubiquitin
g75A/G76V2the transformation period of fusion rotein is shorter.
embodiment 4
Electricity turns wild-type Cas9-gRNA1 to mouse ES cells, and electricity turns pCas9-ODC
422 – 461-gRNA1 is to mouse ES cells, and electricity turns pCas9-ubiquitin
g75A/G76V-gRNA1 is to mouse ES cells.The target practice site of gRNA1 carrier is: AACTGTGCTGCCGGGCAAC(SEQ ID NO.11), screen with G418, find positive colony, picking mono-clonal.Be put into 96 orifice plates to cultivate.After cell covers with, every hole adds 47.5 ul cell pyrolysis liquids and 2.5 ul 10 mg/ml Proteinase Ks, plate is placed in 56 DEG C of incubators digestion and spends the night.Next day, the mixed solution of 10 ml precooling dehydrated alcohol+150 ul 5M NaCl prepared by every plate, and room temperature leaves standstill 2 hrs, allows DNA precipitate, and centrifugal, wash-out obtains mouse cell genomic dna.Extract cell genomic dna, with primer Primer F:TGCTTTACAAAGTTTCCAGCTGAAG(SEQ ID NO.12) and primer Primer R:AACCAACCAACCAACCAGCTAA(SEQ ID NO.13) increase, configuration reaction system is as shown in table 5.
Table 5
| 10×Taq Buffer | 5 ul |
| dNTP (2.5 mM) | 4μl |
| TaKaRa Taq (5 U/μl) | 0.5ul |
| primer F 20pmol | 1ul |
| Primer R 20pmol | 1ul |
| Genomic dna 100ng/ul | 1ul |
| ddH2O | 37.5 ul |
| Amount to | 50 ul |
Amplification condition: 95 DEG C of 3min, 95 DEG C of 10s, 60 DEG C of 30s, 72 DEG C of 30s, totally 30 circulations.
Reclaim PCR primer and send order-checking, as shown in Figure 3-Figure 5, result shows sequencer map: compared with turning wild-type Cas9-gRNA1 with electricity, electricity turns pCas9-ODC
422 – 461-gRNA1 and pCas9-ubiquitin
g75A/G76Vthe cell of-gRNA1, Malt1 gene has nicking activity.
The sequence of fusion rotein of the present invention and above-mentioned primer is as follows, and partial sequence is see table 6.
SEQ ID NO.1
MDKKYSIGLD IGTNSVGWAV ITDEYKVPSK KFKVLGNTDR HSIKKNLIGA LLFDSGETAE 60
ATRLKRTARR RYTRRKNRIC YLQEIFSNEM AKVDDSFFHR LEESFLVEED KKHERHPIFG 120
NIVDEVAYHE KYPTIYHLRK KLVDSTDKAD LRLIYLALAH MIKFRGHFLI EGDLNPDNSD 180
VDKLFIQLVQ TYNQLFEENP INASGVDAKA ILSARLSKSR RLENLIAQLP GEKKNGLFGN 240
LIALSLGLTP NFKSNFDLAE DAKLQLSKDT YDDDLDNLLA QIGDQYADLF LAAKNLSDAI 300
LLSDILRVNT EITKAPLSAS MIKRYDEHHQ DLTLLKALVR QQLPEKYKEI FFDQSKNGYA 360
GYIDGGASQE EFYKFIKPIL EKMDGTEELL VKLNREDLLR KQRTFDNGSI PHQIHLGELH 420
AILRRQEDFY PFLKDNREKI EKILTFRIPY YVGPLARGNS RFAWMTRKSE ETITPWNFEE 480
VVDKGASAQS FIERMTNFDK NLPNEKVLPK HSLLYEYFTV YNELTKVKYV TEGMRKPAFL 540
SGEQKKAIVD LLFKTNRKVT VKQLKEDYFK KIECFDSVEI SGVEDRFNAS LGTYHDLLKI 600
IKDKDFLDNE ENEDILEDIV LTLTLFEDRE MIEERLKTYA HLFDDKVMKQ LKRRRYTGWG 660
RLSRKLINGI RDKQSGKTIL DFLKSDGFAN RNFMQLIHDD SLTFKEDIQK AQVSGQGDSL 720
HEHIANLAGS PAIKKGILQT VKVVDELVKV MGRHKPENIV IEMARENQTT QKGQKNSRER 780
MKRIEEGIKE LGSQILKEHP VENTQLQNEK LYLYYLQNGR DMYVDQELDI NRLSDYDVDH 840
IVPQSFLKDD SIDNKVLTRS DKNRGKSDNV PSEEVVKKMK NYWRQLLNAK LITQRKFDNL 900
TKAERGGLSE LDKAGFIKRQ LVETRQITKH VAQILDSRMN TKYDENDKLI REVKVITLKS 960
KLVSDFRKDF QFYKVREINN YHHAHDAYLN AVVGTALIKK YPKLESEFVY GDYKVYDVRK 1020
MIAKSEQEIG KATAKYFFYS NIMNFFKTEI TLANGEIRKR PLIETNGETG EIVWDKGRDF 1080
ATVRKVLSMP QVNIVKKTEV QTGGFSKESI LPKRNSDKLI ARKKDWDPKK YGGFDSPTVA 1140
YSVLVVAKVE KGKSKKLKSV KELLGITIME RSSFEKNPID FLEAKGYKEV KKDLIIKLPK 1200
YSLFELENGR KRMLASAGEL QKGNELALPS KYVNFLYLAS HYEKLKGSPE DNEQKQLFVE 1260
QHKHYLDEII EQISEFSKRV ILADANLDKV LSAYNKHRDK PIREQAENII HLFTLTNLGA 1320
PAAFKYFDTT IDRKRYTSTK EVLDATLIHQ SITGLYETRI DLSQLGGDSR ADPKKKRKVH 1380
GFPPEVEEQD DGTLPMSCAQ ESGMDRHPAA CASARINV 1418
SEQ ID NO.2
atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540
gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600
atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660
cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720
cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780
gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840
cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700
actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080
gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgcat 4140
ggcttcccgc cggaggtgga ggagcaggat gatggcacgc tgcccatgtc ttgtgcccag 4200
gagagcggga tggaccgtca ccctgcagcc tgtgcttctg ctaggatcaa tgtgtga 4257
SEQ ID NO.5
atggacaaga agtactccat tgggctcgat atcggcacaa acagcgtcgg ctgggccgtc 60
attacggacg agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc 120
cacagcataa agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa 180
gccacgcggc tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc 240
tacctgcagg agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg 300
ctggaggagt cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc 360
aatatcgtgg acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag 420
aagcttgtag acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat 480
atgatcaaat ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat 540
gtcgacaaac tctttatcca actggttcag acttacaatc agcttttcga agagaacccg 600
atcaacgcat ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg 660
cggctcgaaa acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat 720
cttatcgccc tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa 780
gatgccaagc ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc 840
cagatcggcg accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt 900
ctgctgagtg atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt 960
atgatcaagc gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga 1020
cagcaactgc ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc 1080
ggatacattg acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg 1140
gaaaaaatgg acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc 1200
aaacagcgca ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac 1260
gctatcctca ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt 1320
gagaaaatcc tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc 1380
agattcgcgt ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa 1440
gtcgtggata agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa 1500
aatctgccta acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt 1560
tataacgagc tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg 1620
tctggagagc agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc 1680
gtgaaacagc tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc 1740
agcggagtgg aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc 1800
attaaagaca aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc 1860
ctcaccctta cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct 1920
catctcttcg acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg 1980
cggctgtcaa gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg 2040
gattttctta agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac 2100
tctctcacct ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt 2160
cacgagcaca tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc 2220
gttaaggtcg tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt 2280
atcgagatgg cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg 2340
atgaagagga ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca 2400
gttgaaaaca cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg 2460
gacatgtacg tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat 2520
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
gataaaaata gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa 2640
aattattggc ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg 2700
actaaggctg aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag 2760
cttgttgaga cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac 2820
accaagtacg atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct 2880
aagctggtct cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat 2940
taccaccatg cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa 3000
tatcccaagc ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa 3060
atgatcgcaa agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc 3120
aatattatga attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga 3180
ccacttatcg aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc 3240
gcgacagtcc ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta 3300
cagaccggag gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc 3360
gcacgcaaaa aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct 3420
tacagtgtac tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc 3480
aaggaactgc tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac 3540
tttctcgagg cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag 3600
tactctctct ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg 3660
cagaaaggta acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc 3720
cactatgaaa agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa 3780
caacacaaac actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg 3840
atcctcgccg acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag 3900
cccatcaggg agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg 3960
cctgcagcct tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag 4020
gaggtcctgg acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc 4080
gacctctctc agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgatg 4140
cagatattcg tgaagaccct gacaggaaaa accatcaccc tggaggtgga gcccagtgac 4200
acaatcgaga acgtgaaggc aaaaatccag gacaaggaag gcatccacca aaacgaggaa 4260
cgcctcatct tcgccggaaa gcaactcgag gacggccgaa cactcagcga ttataacatc 4320
cagaaggaga gcacattgca cttagtctta cgtttgcgcg gaggtaccgg ttag 4374
Table 6 sequence
| Sequence | SEQ ID NO. | |
| Primer Primer_Cas9_F | CAGCCTCCGGACTCTAGAGGATCGA | 3 |
| Primer Primer_ODC_R | AACTCATTACTAACCGGTTCACACATTGATCCTAGCAGAAGCA | 4 |
| Primer Ubiquitin R | AACTCATTACTAACCGGTACCTCCGCGCAAACGTAAGACTAA | 6 |
| Primer Cas9-ODC 422–461l_F: NdeI | tcgaaggtcgtcatatg ATGGACAAGAAGTACTCCATTGGGCT | 7 |
| Cas9-ODC 422–461l _R：BamHI | ttgttagcagccggatccTCACACATTGATCCTAGCAGAAGC | 8 |
| Cas9-ubiquitin G75A/G76V_F: NdeI | tcgaaggtcgtcatatg ATGGACAAGAAGTACTCCATTGGGCT | 9 |
| Cas9-ubiquitin G75A/G76V_R：BamHI | ttgttagcagccggatccCTAACCGGTACCTCCGCGCAAA | 10 |
| The target practice site of gRNA1 carrier | AACTGTGCTGCCGGGCAAC | 11 |
| Primer F | TGCTTTACAAAGTTTCCAGCTGAAG | 12 |
| Primer R | AACCAACCAACCAACCAGCTAA | 13 |
Claims (6)
1. the Cas9-ODC of fast degradation
422-461fusion rotein, is characterized in that, the albumen of described fusion rotein for being made up of aminoacid sequence shown in SEQ ID NO.1.
2. the Cas9-ODC of coding fast degradation according to claim 1
422-461the nucleotide sequence of fusion rotein.
3. nucleotide sequence according to claim 2, is characterized in that, described nucleotide sequence is as shown in SEQ ID NO.2.
4. the Cas9-ODC of fast degradation according to claim 1
422-461fusion rotein, is characterized in that the Cas9-ODC of described fast degradation
422-461fusion rotein is that the amino acid fusion of Cas9 albumen and mouse ornithine decarboxylase C-end 422 – 461 forms.
5. the Cas9-ODC of fast degradation according to claim 1
422-461the application of fusion rotein in drug screening and genetic modification.
6. the Cas9-ODC of fast degradation according to claim 5
422-461the application of fusion rotein in drug screening and genetic modification, is characterized in that described Cas9-ODC
422-461the gene clone of fusion rotein to carrier for expression of eukaryon for the genetic modification of cell and animal.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105647885A (en) * | 2016-01-20 | 2016-06-08 | 广州元曦生物科技有限公司 | Cas9 fusion protein and coding sequence thereof |
| CN106011104A (en) * | 2015-05-21 | 2016-10-12 | 清华大学 | Method for carrying out gene editing and expression regulation by utilizing Cas splitting system |
| WO2018081978A1 (en) * | 2016-11-03 | 2018-05-11 | 深圳华大基因研究院 | Method and system for improving gene editing efficiency |
| WO2019103020A1 (en) * | 2017-11-22 | 2019-05-31 | 国立大学法人神戸大学 | Complex for genome editing having stability and few side-effects, and nucleic acid coding same |
| EP3481956A4 (en) * | 2016-07-05 | 2020-07-08 | The Johns Hopkins University | Compositions and methods comprising improvements of crispr guide rnas using the h1 promoter |
| US11788088B2 (en) | 2017-09-26 | 2023-10-17 | The Board Of Trustees Of The University Of Illinois | CRISPR/Cas system and method for genome editing and modulating transcription |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1414108A (en) * | 2002-07-24 | 2003-04-30 | 杭州华大基因研发中心 | cell test system, construction method and application |
| WO2013176772A1 (en) * | 2012-05-25 | 2013-11-28 | The Regents Of The University Of California | Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription |
-
2014
- 2014-11-18 CN CN201410656081.0A patent/CN104531632A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1414108A (en) * | 2002-07-24 | 2003-04-30 | 杭州华大基因研发中心 | cell test system, construction method and application |
| WO2013176772A1 (en) * | 2012-05-25 | 2013-11-28 | The Regents Of The University Of California | Methods and compositions for rna-directed target dna modification and for rna-directed modulation of transcription |
Non-Patent Citations (3)
| Title |
|---|
| LE CONG, ET AL.: "Multiplex Genome Engineering Using CRISPR/Cas Systems", 《SCIENCE》 * |
| PATRICK D HSU,ET AL.: "DNA targeting specificity of RNA-guided Cas9 nucleases", 《NAT BIOTECHNOL.》 * |
| XIANQIANG LI,ET AL.: "Generation of Destabilized Green Fluorescent Protein as a Transcription Reporter", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
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| CN106011104A (en) * | 2015-05-21 | 2016-10-12 | 清华大学 | Method for carrying out gene editing and expression regulation by utilizing Cas splitting system |
| CN105647885B (en) * | 2016-01-20 | 2017-08-18 | 广州元曦生物科技有限公司 | Cas9 fusion proteins and its coded sequence |
| CN105647885A (en) * | 2016-01-20 | 2016-06-08 | 广州元曦生物科技有限公司 | Cas9 fusion protein and coding sequence thereof |
| US11766488B2 (en) | 2016-07-05 | 2023-09-26 | The Johns Hopkins University | Compositions and methods comprising improvements of CRISPR guide RNAS using the H1 promoter |
| EP3481956A4 (en) * | 2016-07-05 | 2020-07-08 | The Johns Hopkins University | Compositions and methods comprising improvements of crispr guide rnas using the h1 promoter |
| WO2018081978A1 (en) * | 2016-11-03 | 2018-05-11 | 深圳华大基因研究院 | Method and system for improving gene editing efficiency |
| CN109689693B (en) * | 2016-11-03 | 2022-06-28 | 深圳华大生命科学研究院 | Method and system for improving gene editing efficiency |
| CN109689693A (en) * | 2016-11-03 | 2019-04-26 | 深圳华大生命科学研究院 | Improve the method and system of gene editing efficiency |
| US11788088B2 (en) | 2017-09-26 | 2023-10-17 | The Board Of Trustees Of The University Of Illinois | CRISPR/Cas system and method for genome editing and modulating transcription |
| WO2019103020A1 (en) * | 2017-11-22 | 2019-05-31 | 国立大学法人神戸大学 | Complex for genome editing having stability and few side-effects, and nucleic acid coding same |
| JPWO2019103020A1 (en) * | 2017-11-22 | 2021-01-21 | 国立大学法人神戸大学 | Genome editing complex with stable and few side effects and nucleic acid encoding it |
| JP7328695B2 (en) | 2017-11-22 | 2023-08-17 | 国立大学法人神戸大学 | Stable genome editing complex with few side effects and nucleic acid encoding the same |
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