DK2764103T3 - Crispr-cas-systemer og fremgangsmåder til ændring af ekspressionen af genprodukter - Google Patents

Crispr-cas-systemer og fremgangsmåder til ændring af ekspressionen af genprodukter Download PDF

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DK2764103T3
DK2764103T3 DK13824232.6T DK13824232T DK2764103T3 DK 2764103 T3 DK2764103 T3 DK 2764103T3 DK 13824232 T DK13824232 T DK 13824232T DK 2764103 T3 DK2764103 T3 DK 2764103T3
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sequence
crispr
target
cell
expression
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Feng Zhang
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Broad Inst Inc
Massachusetts Inst Technology
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Claims (15)

1. Konstrueret, ikke-naturligt forekommende CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats)-Cas (CRISPR-associeret) (CRISPR-Cas)-vektorsystem, der omfatter en eller flere vektorer, der omfatter: a) et første regulatorisk element, der er operabelt koblet til en eller flere nukleotidsekvenser, der koder for et eller flere guide-RNA'er for CRISPR-Cas-systemet, der hybridiserer med målsekvenser i polynukleotidloci i en eukaryot celle, hvilket guide-RNA omfatter en guidesekvens, en tracr-sekvens og en tracr-matchende sekvens, b) et andet regulatorisk element, der er operabelt koblet til en nukleotidsekvens, der koder for et type II-Cas9-protein, hvilket protein omfatter et cellekernelokaliseringssignal (NLS); hvor bestanddel (a) og (b) er placeret på samme eller forskellige vektorer i systemet, hvor tracr-sekvensen er 30 eller flere nukleotider lang, og hvor det ene eller de flere guide-RNA'er er målrettet mod polynukleotidlociene i en eukaryot celle, og Cas9-proteinet spalter polynukleotidlociene, hvorved sekvens i polynukleotidlociene modificeres; og hvor Cas9-proteinet og det ene eller de flere guide-RNA'er ikke forekommer naturligt sammen.
2. Konstrueret, ikke-naturligt forekommende type II-CRISPR-Cas-vektorsystem ifølge krav 1, hvor Cas9-proteinet omfatter en eller flere mutationer i et katalytisk domæne, således at det muterede Cas9-protein mangler evnen til at spalte den ene streng af polynukleotidlociene og er en nickase.
3. System ifølge krav 1 eller 2, hvor vektorerne er virale vektorer.
4. System ifølge krav 3, hvor de virale vektorer er retrovirale, lentivirale, adenovirale, adenoassocierede eller herpes simplex-virale vektorer.
5. System ifølge et hvilket som helst af kravene 2 til 4, hvor Cas9-proteinet omfatter en eller flere mutationer i de katalytiske RuvC I-, RuvC II- eller RuvC III-domæner.
6. System ifølge et hvilket som helst af kravene 2 til 4, hvor Cas9-proteinet omfatter en mutation, der er udvalgt fra gruppen, der består af D10A, H840A, N854A og N863A med henvisning til positionsnummereringen i et Streptococcus pyogenes-Cas9 (SpCas9)-protein.
7. System ifølge et hvilket som helst af ovennævnte krav, hvor guide-RNA'et er et kimært RNA, der omfatter guidesekvensen, tracr-sekvensen, og en tracr-matchende sekvens.
8. System ifølge krav 7, hvor hybridisering mellem tracr- sekvensen og den tracr-matchende sekvens frembringer et transkript med en sekundær struktur.
9. System ifølge krav 8, hvor den sekundære struktur er en hårnålestruktur.
10. System ifølge et hvilket som helst af ovennævnte krav, hvor den eukaryote celle er en mammaliacelle eller en human celle.
11. System ifølge et hvilket som helst af ovennævnte krav, hvor Cas9-proteinet er kodonoptimeret til ekspression i en eukaryot celle.
12. System ifølge et hvilket som helst af ovennævnte krav, hvor der er indsat en reparationstemplate i de spaltede polynukleotidloci.
13. Anvendelse af et system ifølge et hvilket som helst af kravene 1 til 12: a) til stedspecifik gen-knockout; b) til stedspecifik genomredigering; c) til DNA-sekvensspecifik interferens; eller d) til multipleks genommodificering; forudsat at anvendelsen ikke omfatter en proces til modificering af kimcelle-genidentiteten hos mennesker; og hvor anvendelsen i) er in vitro eller ex vivo; eller ii) ikke er en fremgangsmåde til behandling af menneske- eller dyrekroppen ved hjælp af terapi.
14. Anvendelse ifølge krav 13, hvor anvendelsen yderligere omfatter reparation af det spaltede målpolynukleotid ved hjælp af homolog rekombination med et exogent templatepolynukleotid, hvor reparationen resulterer i en mutation, der omfatter en insertion, deletion eller substitution af et eller flere nukleotider i målpolynukleotidet.
15. Anvendelse af systemet ifølge et hvilket som helst af kravene 1 til 12 til frembringelse af et ikke-humant transgent dyr eller en transgen plante, forudsat at anvendelsen ikke er en fremgangsmåde til behandling af dyrekropppen ved hjælp af terapi.
DK13824232.6T 2012-12-12 2013-12-12 Crispr-cas-systemer og fremgangsmåder til ændring af ekspressionen af genprodukter DK2764103T3 (da)

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US201261736527P 2012-12-12 2012-12-12
US201361748427P 2013-01-02 2013-01-02
US201361791409P 2013-03-15 2013-03-15
US201361835931P 2013-06-17 2013-06-17
US201361842322P 2013-07-02 2013-07-02
US14/054,414 US8697359B1 (en) 2012-12-12 2013-10-15 CRISPR-Cas systems and methods for altering expression of gene products
PCT/US2013/074743 WO2014093661A2 (en) 2012-12-12 2013-12-12 Crispr-cas systems and methods for altering expression of gene products

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