CN104531633A - Cas9-scForkI fusion protein and application thereof - Google Patents
Cas9-scForkI fusion protein and application thereof Download PDFInfo
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- 108020001507 fusion proteins Proteins 0.000 title abstract 8
- 102000037865 fusion proteins Human genes 0.000 title abstract 8
- 108091033409 CRISPR Proteins 0.000 claims abstract description 15
- 238000012239 gene modification Methods 0.000 claims abstract description 6
- 238000007877 drug screening Methods 0.000 claims abstract description 4
- 230000004927 fusion Effects 0.000 claims description 22
- 230000002779 inactivation Effects 0.000 claims description 7
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 230000005017 genetic modification Effects 0.000 claims description 4
- 235000013617 genetically modified food Nutrition 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 2
- 238000013461 design Methods 0.000 abstract description 6
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- HKZAAJSTFUZYTO-LURJTMIESA-N (2s)-2-[[2-[[2-[[2-[(2-aminoacetyl)amino]acetyl]amino]acetyl]amino]acetyl]amino]-3-hydroxypropanoic acid Chemical compound NCC(=O)NCC(=O)NCC(=O)NCC(=O)N[C@@H](CO)C(O)=O HKZAAJSTFUZYTO-LURJTMIESA-N 0.000 abstract description 3
- 238000005520 cutting process Methods 0.000 abstract description 2
- 230000004913 activation Effects 0.000 abstract 1
- 238000010353 genetic engineering Methods 0.000 abstract 1
- 230000000694 effects Effects 0.000 description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 7
- 108020005004 Guide RNA Proteins 0.000 description 7
- 101100022252 Mus musculus Malt1 gene Proteins 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 6
- 239000003292 glue Substances 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 108091028113 Trans-activating crRNA Proteins 0.000 description 5
- 230000014509 gene expression Effects 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000009465 prokaryotic expression Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 2
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 238000001415 gene therapy Methods 0.000 description 2
- 150000002460 imidazoles Chemical class 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000003198 gene knock in Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 239000012145 high-salt buffer Substances 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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Abstract
The invention discloses a Cas9-scForkI fusion protein and application thereof, and belongs to the field of molecular biology. According to the Cas9-scForkI fusion protein and application, an inactivate Cas9 protein is transformed through a genetic engineering method; (G4S)10, namely (Gly-Gly-Gly-Gly-Ser)10, serves as a linker to be used for connecting two ForkIs; the ForkIs and the inactivate Cas9 are fused into Cas9-scForkI; and according to the Cas9-scForkI fusion protein, genomic editing can be carried out through sequence specificity. When the Cas9-scForkI fusion protein is used for drug screening or gene modification, more safety is obtained; and the fusion protein can further be used for gene treatment. Compared with the prior art, the design is more flexible; the cutting activation is higher; the input efficiency is higher when the fusion protein serves as a gene; and the fusion protein can be used for gene modification or gene treatment.
Description
Technical field
The invention belongs to technical field of molecular biology, be specifically related to a kind of Cas9-scForkI fusion 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.
ForkI is a kind of restriction endonuclease, just can cut DNA after ForkI dimerization.Have bibliographical information, Cas9 and the ForkI of inactivation merges, and namely Cas9-ForkI can be used for genomic editor.And Cas9-ForkI nickase can be more accurate for genomic editor.But these two technology need design two gRNA targets, design comparison is complicated.And the cutting efficiency of Cas9-ForkI nickase is also lower.The present invention's Cas9 albumen of an inactivation connects two ForkI, and these two ForkI are by (G4S)
10connect, the method design comparison is simple, only need design gRNA, and nicking activity is very high.The method is more suitable for mediated gene and knocks in.
Summary of the invention
The object of the invention is to solve the deficiencies in the prior art, a kind of Cas9-scForkI fusion rotein is provided.The present invention, by engineered way, transforms humanization Cas9 albumen, with (G4S)
10i.e. (Gly-Gly-Gly-Gly-Ser)
10be a linker, connect two ForkI by it and show, be then fused into Cas9-scForkI with the Cas9 of inactivation, this fusion rotein sequence-specificly can carry out genomic editor.
The technical solution used in the present invention is as follows:
Cas9-scForkI fusion rotein, the albumen of described fusion rotein for being made up of aminoacid sequence shown in SEQ ID NO.1.
To encode the nucleotide sequence of above-mentioned Cas9-scForkI fusion rotein.
Above nucleotide sequence, described nucleotide sequence is as shown in SEQ ID NO.2.
Described Cas9-scForkI fusion rotein, described Cas9 albumen is the Cas9 of inactivation.
Described Cas9-scForkI fusion rotein, described scForkI albumen is by two ForkI, middle by one (G4S)
10connect.
The described application of Cas9-scForkI fusion rotein in drug screening and genetic modification.
compared with prior art, its beneficial effect is in the present invention:the present invention, by engineered way, transforms the Cas9 albumen of inactivation, with (G4S)
10i.e. (Gly-Gly-Gly-Gly-Ser)
10be a linker, connect two ForkI by it, be then fused into Cas9-scForkI with the Cas9 of inactivation, this fusion rotein sequence-specificly can carry out genomic editor.Cas9-scForkI fusion rotein of the present invention does drug screening or genetic modification is safer, and this fusion rotein also can be used for gene therapy.
The present invention compared to the prior art comparatively, designs more flexible.And nicking activity is higher, the efficiency as gene knock-in is higher.Albumen not only may be used for genetic modification, also can be used for gene therapy.
Accompanying drawing explanation
Fig. 1 is the proof diagram of Cas9-scForkI protein-active;
Fig. 2 is that Cas9-scForkI albumen is to Malt1 gene nicking activity proof diagram.
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 the conventional products of commercial acquisition.
In the present invention, the gRNA sequence of sequence shown in SEQ ID NO.8, primer Primer_F1, primer Primer_ R1, Malt1 gene, primer Primer F, primer Primer R, primer Primer_F2 and primer Primer_ R2, be all purchased from Sangon Biotech (Shanghai) Co., Ltd.;
Glue reclaims and adopts glue to reclaim test kit, is purchased from TIANGEN Biotech (Beijing) Co., Ltd.;
HCas9 carrier is purchased from Addgene, Plasmid 41815;
Mouse ES cells, is purchased from Millipore;
With synthesize shown in SEQ ID NO.8 Cas9-ForkI-(Gly-Gly-Gly-Gly-Ser) 10-ForkI DNA sequence dna be student on commission's work biotechnology (Shanghai) limited-liability company synthesis.
the preparation of embodiment 1 Cas9-scForkI fusion rotein
The preparation method of described Cas9-scForkI fusion rotein, comprises step as follows:
First part, the structure of pCas9-scForkI carrier for expression of eukaryon:
To synthesize Cas9-ForkI-(Gly-Gly-Gly-Gly-Ser) 10-ForkI DNA sequence dna shown in SEQ ID NO.8, with Cas9-ForkI-(Gly-Gly-Gly-Gly-Ser) 10-ForkI DNA sequence dna for template, with primer Primer_F1:CAGCCTCCGGACTCTAGAGCCACCATGGACAAGAAGTACTC(SEQ ID NO.3) and primer Primer_ R1:AACTCATTACTAACCGGTTCATGAGCGGAAATTGATCTCGC(SEQ ID NO.4)
Increase, configuration reaction system is as shown in table 1:
Table 1 reaction system
| 5×primerSTAR Buffer | 10 ul |
| dNTP (2.5 mM) | 4μl |
| TaKaRa Taq HS (5 U/μl) | 0.5ul |
| Primer_F1 20pmol | 1ul |
| Primer_ R1 20pmol | 1ul |
| Cas9-ForkI-(Gly-Gly-Gly-Gly-Ser)10-ForkI (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, 5min, totally 30 circulations.
Glue reclaims Cas9-ForkI-(Gly-Gly-Gly-Gly-Ser) 10-Fork fragment, cuts hCas9 carrier with XbaI and AgeI enzyme, and glue reclaims skeleton carrier.Two fragments carry out In-Fusion reaction (Clothech), transform, clone identification, final acquisition pCas9-scForkI carrier for expression of eukaryon.This carrier for expression of eukaryon in order to verify the nicking activity of this albumen in eukaryotic cell.
Second section, the structure of pET-Cas9-scForkI prokaryotic expression carrier:
With Cas9-ForkI-(Gly-Gly-Gly-Gly-Ser) 10-ForkI DNA sequence dna for template, with primer Primer_F2:TTGTTAGCAGCCGGATCCATGGACAAGAAGTACTCCATTGGGC(SE Q ID NO.9) and primer Primer_ R2:ATCGAAGGTCGTCATATGTCATTATGAGCGGAAATTGATCTCG (SEQ ID NO.10)
Increase, configuration reaction system is as shown in table 2:
Table 2 reaction system
| 5×primerSTAR Buffer | 10 ul |
| dNTP (2.5 mM) | 4μl |
| TaKaRa Taq HS (5 U/μl) | 0.5ul |
| Primer_F2 20pmol | 1ul |
| Primer_ R2 20pmol | 1ul |
| Cas9-ForkI-(Gly-Gly-Gly-Gly-Ser)10-ForkI (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, 5min, totally 30 circulations.
Glue reclaims Cas9-ForkI-(Gly-Gly-Gly-Gly-Ser) 10-Fork fragment, cuts pET-16b carrier with BamHI and NdeI enzyme, and glue reclaims skeleton carrier.Two fragments carry out In-Fusion reaction (Clothech), transform, clone identification, final acquisition pET-Cas9-scForkI prokaryotic expression carrier.This prokaryotic expression carrier in order to express this albumen, for verifying the nicking activity of this albumen to DNA.
Part III, the expression and purification of Cas9-scForkI albumen:
Recombinant plasmid pET-Cas9-scForkI transformation of E. coli E. coli BL21.Transformed bacteria overnight incubation in the substratum of 5ml ammonia benzyl resistance, be transferred to next day in the identical substratum of 500ml and cultivate, to spend the night induction through IPTG 16 DEG C, collect the Cas9-scForkI albumen thalline obtained, 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-scForkI albumen of purifying.
the checking of embodiment 2 Cas9-scForkI Protein cleavage activity
By Cas9-scForkI albumen (300 nM) and linearizing plasmid pDNA3.1, tracrRNA, Mg
2+, crRNA different components is hatched.Find Cas9-scForkI albumen and linearizing plasmid pDNA3.1, tracrRNA, Mg
2+, gRNA hatches this group, and DNA has nicking activity.Illustrate that Cas9-scForkI albumen (300 nM) has activity.
Specific as follows: tracrRNA and crRNA of in-vitro transcription, 95 DEG C of sex change, then slowly cool to room temperature.By Cas9-scForkI albumen and linearizing and nonlinearized plasmid pDNA3.1 (200 ng), Mg
2+, tracrRNA (100 nM), crRNA (100 nM) different components 37 DEG C hatches one hour.The buffer of reaction is: 20 mM HEPES pH 7.5,150 mM KCl, 0.5 mM DTT, 0.1 mM EDTA, 10 mM MgCl
2.React after one hour, add 5X SDS loading buffer (30% glycerol, 1.2% SDS, 250 mM EDTA) electrophoresis.Result shows as shown in Figure 1, as seen from Figure 1, and Cas9-scForkI albumen and linearizing plasmid pDNA3.1, tracrRNA, Mg
2+, gRNA hatches this group, and DNA has nicking activity, and other groups do not have nicking activity.
embodiment 3 Cas9-scForkI albumen is to the checking of Malt1 gene nicking activity
Cas9-scForkI carrier for expression of eukaryon with for the gRNA:AACTGTGCTGCCGGGCAAC(SEQ ID NO.5 of Malt1 gene) electricity forwards mouse ES cells to.Electrotransfection is after 24 hours, and picked clones, is put into 96 orifice plates and cultivates.After cell covers with, every hole adds 47.5 ul ES cell pyrolysis liquids and 2.5 ul 10 mg/ml Proteinase Ks, plate is placed in 56 oC 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 ES cells genomic dna.Lysis liquid formula is: Tris-HCl pH 7.5(10 mM), EDTA pH 8.0(10 mM), NaCl(10 mM) and, 1% SDS.With primer Primer F:TGCTTTACAAAGTTTCCAGCTGAAG(SEQ ID NO.6) and primer Primer R:AACCAACCAACCAACCAGCTAA(SEQ ID NO.7) increase, configuration reaction system is as shown in table 3:
Table 3
| 10×Taq Buffer | 5 ul |
| dNTP (2.5 mM) | 4μl |
| TaKaRa Taq (5 U/μl) | 0.5ul |
| Primer F 20pmol | 1ul |
| Primer R20pmol | 1ul |
| Genomic dna 100ng/ul | 1ul |
| ddH 2O | 37.5 ul |
| Amount to | 50 ul |
Amplified reaction program: 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 Fig. 2, result shows, and Cas9-scForkI albumen has nicking activity to Malt1 gene.
The sequence of described fusion rotein and above-mentioned primer is as follows, and partial sequence is see table 5:
SEQ ID NO.1:
MDKKYSIGLA 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 NRLSDYDVAA 840
IVPQSFLKDD SIDNKVLTRS DKARGKSDNV 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 ADPKKKRKVG 1380
SQLVKSELEE KKSELRHKLK YVPHEYIELI EIARNSTQDR ILEMKVMEFF MKVYGYRGKH 1440
LGGSRKPDGA IYTVGSPIDY GVIVDTKAYS GGYNLPIGQA DEMQRYVEEN QTRNKHINPN 1500
EWWKVYPSSV TEFKFLFVSG HFKGNYKAQL TRLNHITNCN GAVLSVEELL IGGEMIKAGT 1560
LTLEEVRRKF NNGEINFRSG GGGSGGGGSG GGGSGGGGSG GGGSGGGGSG GGGSGGGGSG 1620
GGGSGGGGSG SQLVKSELEE KKSELRHKLK YVPHEYIELI EIARNSTQDR ILEMKVMEFF 1680
MKVYGYRGKH LGGSRKPDGA IYTVGSPIDY GVIVDTKAYS GGYNLPIGQA DEMQRYVEEN 1740
QTRNKHINPN EWWKVYPSSV TEFKFLFVSG HFKGNYKAQL TRLNHITNCN GAVLSVEELL 1800
IGGEMIKAGT LTLEEVRRKF NNGEINFRS 1829
SEQ ID NO.2:
atggacaaga agtactccat tgggctcgct 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 cgtggccgct 2520
atcgtgcccc agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc 2580
gataaagcta 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 gaaggtgggt 4140
tcccaactcg tgaagagtga acttgaggag aaaaagtcgg agctgcggca caaattgaaa 4200
tacgtaccgc atgaatacat cgaacttatc gaaattgcta ggaactcgac tcaagacaga 4260
atccttgaga tgaaggtaat ggagttcttt atgaaggttt atggataccg agggaagcat 4320
ctcggtggat cacgaaaacc cgacggagca atctatacgg tggggagccc gattgattac 4380
ggagtgatcg tcgacacgaa agcctacagc ggtgggtaca atcttcccat cgggcaggca 4440
gatgagatgc aacgttatgt cgaagaaaat cagaccagga acaaacacat caatccaaat 4500
gagtggtgga aagtgtatcc ttcatcagtg accgagttta agtttttgtt tgtctctggg 4560
catttcaaag gcaactataa ggcccagctc acacggttga atcacattac gaactgcaat 4620
ggtgcggttt tgtccgtaga ggaactgctc attggtggag aaatgatcaa agcgggaact 4680
ctgacactgg aagaagtcag acgcaagttt aacaatggcg agatcaattt ccgctcaggt 4740
ggcggaggtt caggcggagg tggatctgga ggaggcggtt ccggcggagg tggttcagga 4800
ggtggaggct ccggtggagg tggctcaggt ggcggaggtt caggcggagg tggatctgga 4860
ggaggcggtt ccggcggagg tggttcaggt tcccaactcg tgaagagtga acttgaggag 4920
aaaaagtcgg agctgcggca caaattgaaa tacgtaccgc atgaatacat cgaacttatc 4980
gaaattgcta ggaactcgac tcaagacaga atccttgaga tgaaggtaat ggagttcttt 5040
atgaaggttt atggataccg agggaagcat ctcggtggat cacgaaaacc cgacggagca 5100
atctatacgg tggggagccc gattgattac ggagtgatcg tcgacacgaa agcctacagc 5160
ggtgggtaca atcttcccat cgggcaggca gatgagatgc aacgttatgt cgaagaaaat 5220
cagaccagga acaaacacat caatccaaat gagtggtgga aagtgtatcc ttcatcagtg 5280
accgagttta agtttttgtt tgtctctggg catttcaaag gcaactataa ggcccagctc 5340
acacggttga atcacattac gaactgcaat ggtgcggttt tgtccgtaga ggaactgctc 5400
attggtggag aaatgatcaa agcgggaact ctgacactgg aagaagtcag acgcaagttt 5460
aacaatggcg agatcaattt ccgctcatga 5490
SEQ ID NO.8
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 gaaggtgggt 4140
tcccaactcg tgaagagtga acttgaggag aaaaagtcgg agctgcggca caaattgaaa 4200
tacgtaccgc atgaatacat cgaacttatc gaaattgcta ggaactcgac tcaagacaga 4260
atccttgaga tgaaggtaat ggagttcttt atgaaggttt atggataccg agggaagcat 4320
ctcggtggat cacgaaaacc cgacggagca atctatacgg tggggagccc gattgattac 4380
ggagtgatcg tcgacacgaa agcctacagc ggtgggtaca atcttcccat cgggcaggca 4440
gatgagatgc aacgttatgt cgaagaaaat cagaccagga acaaacacat caatccaaat 4500
gagtggtgga aagtgtatcc ttcatcagtg accgagttta agtttttgtt tgtctctggg 4560
catttcaaag gcaactataa ggcccagctc acacggttga atcacattac gaactgcaat 4620
ggtgcggttt tgtccgtaga ggaactgctc attggtggag aaatgatcaa agcgggaact 4680
ctgacactgg aagaagtcag acgcaagttt aacaatggcg agatcaattt ccgctcaggt 4740
ggcggaggtt caggcggagg tggatctgga ggaggcggtt ccggcggagg tggttcagga 4800
ggtggaggct ccggtggagg tggctcaggt ggcggaggtt caggcggagg tggatctgga 4860
ggaggcggtt ccggcggagg tggttcaggt tcccaactcg tgaagagtga acttgaggag 4920
aaaaagtcgg agctgcggca caaattgaaa tacgtaccgc atgaatacat cgaacttatc 4980
gaaattgcta ggaactcgac tcaagacaga atccttgaga tgaaggtaat ggagttcttt 5040
atgaaggttt atggataccg agggaagcat ctcggtggat cacgaaaacc cgacggagca 5100
atctatacgg tggggagccc gattgattac ggagtgatcg tcgacacgaa agcctacagc 5160
ggtgggtaca atcttcccat cgggcaggca gatgagatgc aacgttatgt cgaagaaaat 5220
cagaccagga acaaacacat caatccaaat gagtggtgga aagtgtatcc ttcatcagtg 5280
accgagttta agtttttgtt tgtctctggg catttcaaag gcaactataa ggcccagctc 5340
acacggttga atcacattac gaactgcaat ggtgcggttt tgtccgtaga ggaactgctc 5400
attggtggag aaatgatcaa agcgggaact ctgacactgg aagaagtcag acgcaagttt 5460
aacaatggcg agatcaattt ccgctcataa tga 5493
Table 4 primer sequence
| Sequence | SEQ ID NO. | |
| Primer Primer_F1 | CAGCCTCCGGACTCTAGAGCCACCATGGACAAGAAGTACTC | 3 |
| Primer Primer_ R1 | AACTCATTACTAACCGGTTCATGAGCGGAAATTGATCTCGC | 4 |
| The gRNA sequence of Malt1 gene | AACTGTGCTGCCGGGCAAC | 5 |
| Primer primer F | TGCTTTACAAAGTTTCCAGCTGAAG | 6 |
| Primer Primer R | AACCAACCAACCAACCAGCTAA | 7 |
| Primer Primer_F2 | ttgttagcag ccggatccat ggacaagaag tactccattg ggc | 9 |
| Primer Primer_ R2 | atcgaaggtc gtcatatgtc attatgagcg gaaattgatc tcg | 10 |
Claims (6)
1.Cas9-scForkI fusion 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 nucleotide sequence of the Cas9-scForkI fusion rotein described in coding requirement 1.
3. nucleotide sequence according to claim 2, is characterized in that, described nucleotide sequence is as shown in SEQ ID NO.2.
4. Cas9-scForkI fusion rotein according to claim 1, is characterized in that, described Cas9 albumen is the Cas9 of inactivation.
5. Cas9-scForkI fusion rotein according to claim 1, is characterized in that, described scForkI albumen is by two ForkI, middle by one (G4S)
10connect.
6. the application of Cas9-scForkI fusion rotein according to claim 1 in drug screening and genetic modification.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| WO2018024119A1 (en) * | 2016-08-03 | 2018-02-08 | 南京大学 | Method for editing target polynucleotide and application thereof |
| CN113604608A (en) * | 2021-08-05 | 2021-11-05 | 天益健康科学研究院（镇江）有限公司 | Buffer solution for cas12a editing DNA, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014144288A1 (en) * | 2013-03-15 | 2014-09-18 | The General Hospital Corporation | Using rna-guided foki nucleases (rfns) to increase specificity for rna-guided genome editing |
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2014
- 2014-11-18 CN CN201410656091.4A patent/CN104531633A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014144288A1 (en) * | 2013-03-15 | 2014-09-18 | The General Hospital Corporation | Using rna-guided foki nucleases (rfns) to increase specificity for rna-guided genome editing |
Non-Patent Citations (1)
| Title |
|---|
| JOHN P GUILINGER ET AL.: "Fusion of catalytically inactive cas9 to fokI nuclease improves the specificity of genome modification", 《NATURE BIOTECHNOLOGY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN106011104B (en) * | 2015-05-21 | 2019-09-27 | 清华大学 | Gene editing and expression regulation method are carried out using Cas system is split |
| WO2018024119A1 (en) * | 2016-08-03 | 2018-02-08 | 南京大学 | Method for editing target polynucleotide and application thereof |
| CN113604608A (en) * | 2021-08-05 | 2021-11-05 | 天益健康科学研究院（镇江）有限公司 | Buffer solution for cas12a editing DNA, preparation method and application thereof |
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