DK2412812T3 - Optimerede ikke-kanoniske zinkfingerproteiner - Google Patents
Optimerede ikke-kanoniske zinkfingerproteiner Download PDFInfo
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- DK2412812T3 DK2412812T3 DK11184941.0T DK11184941T DK2412812T3 DK 2412812 T3 DK2412812 T3 DK 2412812T3 DK 11184941 T DK11184941 T DK 11184941T DK 2412812 T3 DK2412812 T3 DK 2412812T3
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- sequence
- dna
- zinc finger
- gene
- plasmid
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- 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
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
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- 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
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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- 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
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C07K2319/81—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor containing a Zn-finger domain for DNA binding
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- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Claims (32)
1. Fusionsprotein, som omfatter: (a) et zinkfingerprotein, som omfatter en ikke-kanonisk zinkfinger, hvor den ik-ke-kanoniske zinkfinger er en ikke-C2H2-zinkfinger og har en spiralformet del, som er involveret i DNA-binding, og hvor den spiralformede dels zink-koordinerende region omfatter aminosyresekvensen HTRRCG; og hvor zinkfingerpro-teinet er modificeret til at binde sig til en målsekvens, og (b) et eller flere funktionelle domæner, hvor ét funktionelt domæne omfatter et spaltningshalvdomæne, og (c) en ZC-linker, som er placeret mellem spaltningshalvdomænet og zinkfin-gerproteinet, hvor aminosyresekvensen mellem zinkfingerproteinet og spaltningshalvdomænet betegnes ZC-linker.
2. Fusionsprotein ifølge krav 1, hvilket fusionsprotein omfatter en flerhed af zinkfingre, hvor mindst én af zinkfingrene omfatter en CCHC-zinkfinger.
3. Fusionsprotein ifølge krav 1 eller 2, hvor mindst én zinkfinger omfatter sekvensen Cys-(XA)2-4-Cys-(XB)i2-Flis-(Xc)3-5-Cys-(XD)i-io, hvor XA, Xb, Xc og XD kan være en hvilken som helst aminosyre.
4. Fusionsprotein ifølge et hvilket som helst af kravene 1-3, hvilket fusionsprotein omfatter en hvilken som helst af de sekvenser, som er vist i en hvilken som helst af tabellerne 1,2,3 eller 4.
5. Fusionsprotein ifølge et hvilket som helst af krav 3 eller krav 4, hvor (Xd)mo omfatter sekvensen QLV eller QKP.
6. Fusionsprotein ifølge krav 5, hvor sekvensen QLV eller QKP er de 3 C-ter-minale aminosyrerester i zinkfingeren.
7. Fusionsprotein ifølge et hvilket som helst af kravene 3-6, hvor (Xd)mo omfatter 1,2 eller 3 Gly (G)-rester.
8. Fusionsprotein ifølge et hvilket som helst af kravene 2-7, hvor fusionsproteinet omfatter 3, 4, 5 eller 6 zinkfingre.
9. Fusionsprotein ifølge et hvilket som helst af kravene 2-8, hvor finger 2 omfatter CCHC-zinkfingeren.
10. Fusionsprotein ifølge et hvilket som helst af kravene 2-9, hvor den C-termi-nale zinkfinger omfatter CCHC-fingeren.
11. Fusionsprotein ifølge et hvilket som helst af kravene 2-10, hvor mindst to zinkfingre omfatter CCFIC-zinkfingeren.
12. Fusionsprotein ifølge et hvilket som helst af kravene 2-11, hvor zinkfinger-proteinet omfatter en hvilken som helst af de sekvenser, som er vist i tabel 8, og er modificeret til at binde sig til en målsekvens i et IPP2-K-gen.
13. Fusionsprotein ifølge krav 1, hvor længden af ZC-linkeren er 5 aminosyrer.
14. Fusionsprotein ifølge krav 13, hvor aminosyresekvensen for ZC-linkeren er GLRGS (SEQ ID NO: 4).
15. Fusionsprotein ifølge krav 1, hvor længden af ZC-linkeren er 6 aminosyrer.
16. Fusionsprotein ifølge krav 15, hvor aminosyresekvensen for ZC-linkeren er GGLRGS (SEQ ID NO: 5).
17. Polynukleotid, som koder for et fusionsprotein ifølge et hvilket som helst af kravene 1-16.
18. Fremgangsmåde til målrettet spaltning af cellulært chromatin i en plantecelle, hvilken fremgangsmåde omfatter ekspression i cellen af et par af fusionsproteiner ifølge et hvilket som helst af kravene 1-16; hvor: (a) målsekvenserne for fusionsproteinerne er inden for ti nukleotider fra hinanden; og (b) fusionsproteinerne dimeriserer og spalter DNA, som er placeret mellem målsekvenserne.
19. Fremgangsmåde til målrettet genetisk rekombination i en værtsplantecelle, hvilken fremgangsmåde omfatter: (a) ekspression i værtscellen af et par af fusionsproteiner ifølge et hvilket som helst af kravene 1 -16, hvor målsekvenserne for fusionsproteinerne forefindes på et valgt værtsmållocus; og (b) identificering af en rekombinant værtscelle, som udviser en sekvensændring i værtsmållocuset.
20. Fremgangsmåde ifølge enten krav 18 eller krav 19, hvor sekvensændringen er en mutation valgt fra gruppen bestående af en deletion af genetisk materiale, en insertion af genetisk materiale, en substitution af genetisk materiale og en hvilken som helst kombination deraf.
21. Fremgangsmåde ifølge et hvilket som helst af kravene 18-20, hvilken fremgangsmåde endvidere omfatter indføring af et eksogent polynukleotid i værtscellen.
22. Fremgangsmåde ifølge krav 21, hvor det eksogene polynukleotid omfatter sekvenser, som er homologe med værtsmållocuset.
23. Fremgangsmåde ifølge et hvilket som helst af kravene 18-23, hvor planten er valgt fra gruppen bestående af en enkimbladet plante, en tokimbladet plante, nøgenfrøede planter og eukaryote alger.
24. Fremgangsmåde ifølge krav 23, hvor planten er valgt fra gruppen bestående af majs, ris, hvede, kartoffel, sojabønne, tomat, tobak, medlemmer af Brassi-ca-familien og Arabidopsis.
25. Fremgangsmåde ifølge et hvilket som helst af kravene 18-23, hvor planten er et træ.
26. Fremgangsmåde ifølge et hvilket som helst af kravene 18-25, hvor målsekvenserne forefindes i et IPP2-K-gen.
27. Fremgangsmåde til reduktion af niveauet af phytinsyre i frø, hvilken fremgangsmåde omfatter inaktivering eller ændring af et IPP2-K-gen ved fremgangsmåden ifølge krav 26.
28. Fremgangsmåde til at gøre phosphor mere metabolisk tilgængeligt i frø, hvilken fremgangsmåde omfatter inaktivering eller ændring af et IPP2-K-gen ved fremgangsmåden ifølge krav 26.
29. Plantecelle, som omfatter et fusionsprotein ifølge et hvilket som helst af kravene 1-16 eller et polynukleotid ifølge krav 17.
30. Plantecelle ifølge krav 29, hvor cellen er indeholdt i et frø.
31. Plantecelle ifølge krav 30, hvor frøet er et majsfrø.
32. Plantecelle ifølge et hvilket som helst af kravene 29-31, hvor IPP2-K er delvist eller helt inaktiveret.
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