DK3155099T3 - Nukleasemedieret dna-samling - Google Patents
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- DK3155099T3 DK3155099T3 DK15735807.8T DK15735807T DK3155099T3 DK 3155099 T3 DK3155099 T3 DK 3155099T3 DK 15735807 T DK15735807 T DK 15735807T DK 3155099 T3 DK3155099 T3 DK 3155099T3
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- nucleic acid
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Claims (21)
1. Fremgangsmåde til samling af mindst to nukleinsyrer, hvilken fremgangsmåde omfatter: (a) etablering af kontakt mellem en første nukleinsyre og et første nukleasemiddel, hvor det første nukleasemiddel omfatter et Cas-protein og et førings-RNA (gRNA) (gRNA-Cas kompleks), en zinkfingemuklease, eller en transskriptionsaktivatorlignende effektomuklease (Transcription Activator-Like Effector Nuclease - TALEN), hvor det første nukleasemiddel spalter den første nukleinsyre på et første målsted for at frembringe en første fordøjet nukleinsyre med en overlappende endesekvens delt af en anden nukleinsyre; (b) etablering af kontakt mellem den første fordøjede nukleinsyre og den anden nukleinsyre med en exonuklease for at eksponere komplementære sekvenser mellem den første fordøjede nukleinsyre og den anden nukleinsyre; og (c) samling af de to nukleinsyrefragmenter, der er genereret fra trin (b).
2. Fremgangsmåde ifølge krav 1, hvor trin (c) omfatter: (i) annealing af de eksponerede komplementære sekvenser; (ii) forlængelse af 3 ’-enderne af de annealede komplementære sekvenser og (iii) ligering af den første og den anden nukleinsyre.
3. Fremgangsmåde ifølge krav 1 eller 2, hvor de mindst to nukleinsyrer er dobbeltstrengede; og/eller den overlappende endesekvens ligger i intervallet fra 20 bp til 200 bp i længden.
4. Fremgangsmåde ifølge et hvilket som helst af kravene 1-3, hvor trin (a) endvidere omfatter etablering af kontakt mellem den anden nukleinsyre og et andet nukleasemiddel, hvor det andet nukleasemiddel spalter den anden nukleinsyre på et andet målsted for at frembringe en anden fordøjet nukleinsyre med den overlappende endesekvens, og hvor den anden nukleinsyre fra trin (b) er den anden fordøjede nukleinsyre.
5. Fremgangsmåde ifølge et hvilket som helst af kravene 1-4, hvor det første nukleasemiddel omfatter Cas-proteinet og gRNA’et, hvor Cas-proteinet er et Cas9-protein, gRNA’et omfatter en nukleinsyresekvens, der koder for et CRISPR- (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA) og et transaktiverende CRISPR-RNA (tracrRNA), og det første målsted er umiddelbart flankeret af en PAM- (Protospacer Adjacent Motif) sekvens, eventuelt hvor Cas9-proteinet omfatter et RuvC-domæne og et HNH-domæne, hvoraf mindst ét mangler endonukleaseaktivitet.
6. Fremgangsmåde ifølge et hvilket som helst af kravene 1-5, hvor den første nukleinsyre, den anden nukleinsyre eller begge nukleinsyrer er afledt af et kunstigt bakteriekromosom.
7. Fremgangsmåde ifølge krav 6, hvor det kunstige bakteriekromosom omfatter et humant DNA, et gnaver-DNA, et syntetisk DNA eller en kombination deraf.
8. Fremgangsmåde til samling af to eller flere nukleinsyrer, hvilken fremgangsmåde omfatter: (a) etablering af kontakt mellem en første nukleinsyre og mindst ét nukleasemiddel, hvor det mindst ene nukleasemiddel omfatter et Cas-protein og et førings-RNA (gRNA) (gRNA-Cas-kompleks), en zinkfmgemuklease, eller en transskriptionsaktivatorlignende effektomuklease (Transcription Activator-Like Effector Nuclease - TALEN), hvor det mindst ene nukleasemiddel spalter den første nukleinsyre på et første målsted for at generere en første fordøjet nukleinsyre; (b) etablering af kontakt mellem den første fordøjede nukleinsyre og en anden nukleinsyre, en forbindelses-oligo og en exonuklease, hvor forbindelses-oligoen omfatter: (i) en første komplementær sekvens, der er komplementær til den første fordøjede nukleinsyre; (ii) en spacer og (iii) en anden komplementær sekvens, der er komplementær til den anden nukleinsyre; hvor exonukleasen eksponerer den første og den anden komplementære sekvens; og (c) samling af forbindelses-oligoen med den første fordøjede nukleinsyre og den anden nukleinsyre.
9. Fremgangsmåde ifølge krav 8, hvor samling i trin (c) omfatter: (i) annealing af den første komplementære sekvens af forbindelses-oligoen til den første fordøjede nukleinsyre og den anden komplementære sekvens af forbindelses-oligoen til den anden nukleinsyre; og (ii) ligering af forbindelses-oligoen til den første fordøjede nukleinsyre og den anden nukleinsyre.
10. Fremgangsmåde ifølge krav 9, hvor trin (i) endvidere omfatter forlængelse af 3’-enden af den første fordøjede nukleinsyre og/eller den anden nukleinsyre.
11. Fremgangsmåde ifølge et hvilket som helst af kravene 8-10, hvor den første komplementære sekvens af forbindelses-oligoen er mellem 15 og 120 komplementære baser, og den anden komplementære sekvens af forbindelses-oligoen er mellem 15 og 120 komplementære baser; og/eller forbindelses-oligoens spacer omfatter ikke-komplementære nukleinsyrer.
12. Fremgangsmåde ifølge et hvilket som helst af kravene 8-11, hvor de to eller flere nukleinsyrer er dobbeltstrengede.
13. Fremgangsmåde ifølge et hvilket som helst af kravene 8-12, hvor den første fordøjede nukleinsyre er sømløst samlet med den anden nukleinsyre.
14. Fremgangsmåde ifølge krav 13, hvor det mindst ene nukleasemiddel er designet til at spalte et mindst 20 bp fragment fra enden af den første nukleinsyre, hvor den sømløse samling vil forekomme, hvor forbindelses-oligoens spacer omfatter en sekvens, der er identisk med det mindst 20 bp fragment, hvor ingen nukleinsyrebaser er til stede mellem den første komplementære sekvens og det mindst 20 bp fragment, og ingen nukleinsyrebaser er til stede mellem den anden komplementære sekvens og det mindst 20 bp fragment, således at samling af den første fordøjede nukleinsyre med forbindelses-oligoen og den anden nukleinsyre rekonstruerer det mindst 20 bp fragment og sømløst samler den første nukleinsyre og den anden nukleinsyre, eventuelt hvor det mindst 20 bp fragment er dobbeltstrenget.
15. Fremgangsmåde ifølge krav 13 eller 14, hvor forbindelses-oligoen omfatter et lineært dobbeltstrenget DNA-ffagment, eventuelt hvor det lineære dobbeltstrengede DNA-ffagment ikke omfatter en udvælgelseskassette.
16. Fremgangsmåde ifølge et hvilket som helst af kravene 13-15, hvor forbindelses-oligoen er fra ca. 50 bp til ca. 400 bp, eventuelt hvor forbindelses-oligoen er fra ca. 100 bp til ca. 300 bp.
17. Fremgangsmåde ifølge et hvilket som helst af kravene 8-16, hvor spaceren er mellem ca. 20 bp til ca. 120 bp; og/eller forbindelses-oligoen samles med den første nukleinsyre og den anden nukleinsyre i den samme reaktion eller forbindelses-oligoen samles med den første nukleinsyre og den anden nukleinsyre sekventielt.
18. Fremgangsmåde ifølge et hvilket som helst af kravene 8-17, hvor trin (a) endvidere omfatter: (i) etablering af kontakt mellem den anden nukleinsyre og et andet nukleasemiddel, hvor det andet nukleasemiddel spalter den anden nukleinsyre for at frembringe en anden fordøjet nukleinsyre, der omfatter en nukleotidsekvens, der er komplementær til den anden komplementære sekvens af forbindelses-o li goen, hvor den første fordøjede nukleinsyre samles med den anden fordøjede nukleinsyre; eller (ii) etablering af kontakt mellem den anden nukleinsyre og et restriktionsenzym eller en meganuklease, hvor restriktionsenzymet eller meganukleasen spalter den anden nukleinsyre for at frembringe en anden fordøjet nukleinsyre, der omfatter en nukleotidsekvens, der er komplementær til den anden komplementære sekvens i forbindelses-oligoen, hvor den første fordøjede nukleinsyre samles med den anden fordøjede nukleinsyre.
19. Fremgangsmåde ifølge et hvilket som helst af kravene 8-18, hvor det mindst ene nukleasemiddel omfatter Cas-proteinet og gRNA’et, hvor Cas-proteinet er et Cas9-protein, hvor gRNA’et omfatter en nukleinsyresekvens, der koder for et CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA) og et transaktiverende CRISPR-RNA (tracrRNA), og hvor det første målsted er umiddelbart flankeret af en PAM- (Protospacer Adjacent Motif) sekvens, eventuelt hvor Cas9-proteinet omfatter et RuvC-domæne og et HNH-domæne, hvoraf mindst ét mangler endonukleaseaktivitet.
20. Fremgangsmåde ifølge et hvilket som helst af kravene 8-19, hvor den første nukleinsyre, den anden nukleinsyre eller begge nukleinsyrer er afledt af et kunstigt bakteriekromosom; og/eller den første nukleinsyre, den anden nukleinsyre eller begge nukleinsyrer omfatter et humant DNA, et gnaver-DNA, et syntetisk DNA eller en kombination deraf.
21. Fremgangsmåde ifølge et hvilket som helst af kravene 1 -20, hvor den første nukleinsyre, den anden nukleinsyre eller begge nukleinsyrer er mindst 10 kb.
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| PCT/US2015/037199 WO2015200334A1 (en) | 2014-06-23 | 2015-06-23 | Nuclease-mediated dna assembly |
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