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1. (WO2018162702) NUCLEASE FUSIONS FOR ENHANCING GENOME EDITING BY HOMOLOGY-DIRECTED TRANSGENE INTEGRATION
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Pub. No.: WO/2018/162702 International Application No.: PCT/EP2018/055883
Publication Date: 13.09.2018 International Filing Date: 09.03.2018
IPC:
C12N 15/10 (2006.01) ,C12N 15/63 (2006.01) ,C12N 9/22 (2006.01) ,C07K 14/47 (2006.01)
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15
Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09
Recombinant DNA-technology
10
Processes for the isolation, preparation or purification of DNA or RNA
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15
Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09
Recombinant DNA-technology
63
Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
9
Enzymes, e.g. ligases (6.); Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating, or purifying enzymes
14
Hydrolases (3.)
16
acting on ester bonds (3.1)
22
Ribonucleases
C CHEMISTRY; METALLURGY
07
ORGANIC CHEMISTRY
K
PEPTIDES
14
Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
435
from animals; from humans
46
from vertebrates
47
from mammals
Applicants:
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE (INSERM) [FR/FR]; 101, rue de Tolbiac 75013 PARIS, FR
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE [FR/FR]; 3 rue Michel Ange 75794 PARIS CEDEX 16, FR
MUSEUM NATIONAL D'HISTOIRE NATURELLE [FR/FR]; 57, rue Cuvier 75005 PARIS, FR
UNIVERSITÉ DE NANTES [FR/FR]; 1, quai de Tourville 44000 NANTES, FR
Inventors:
ANEGON, Ignacio; FR
CHARPENTIER, Marine; FR
CONCORDET, Jean-Paul; FR
GIOVANNANGELI, Carine; FR
LOPEZ, Bernard; FR
Agent:
CABINET NONY; 11 rue Saint-Georges 75009 PARIS, FR
Priority Data:
17305260.610.03.2017EP
Title (EN) NUCLEASE FUSIONS FOR ENHANCING GENOME EDITING BY HOMOLOGY-DIRECTED TRANSGENE INTEGRATION
(FR) FUSIONS DE NUCLÉASE DESTINÉES À AMÉLIORER L’ÉDITION DE GÉNOME PAR INTÉGRATION DE TRANSGÈNE DIRIGÉE PAR HOMOLOGIE
Abstract:
(EN) The present invention relates to nuclease protein fusions for enhancing genome editing by homology-directed transgene integration (HDI). The inventors found that the rate of HDI mediated by the CRISPR/Cas9 system may be substantially improved by providing the Cas9 nuclease in the form of a fusion protein with at least the N-terminal domain of the CtIP protein. CtIP proteins are involved in the early steps of homologous recombination. In addition, the inventors identified the subdomains of the N-terminal domain of the CtIP protein that are important for improving the HDI rate. Thus, the invention relates to fusion proteins comprising a Cas9 protein, a tetramerization domain of a CtIP protein and a dimerization domain of a CtIP protein. Particularly, the inventors have tested these fusion proteins HEK293 cells, RG37DR cells and Sprague-Dawley rats.
(FR) La présente invention concerne des fusions de protéine de nucléase pour améliorer l’édition de génome par intégration de transgène dirigée par homologie (HDI). Les inventeurs ont découvert que la teneur en HDI à médiation dans le système (CRISPR/Cas9) peut être sensiblement améliorée en apportant la nucléase (Cas9) sous la forme d’une protéine de fusion avec au moins le domaine N-terminal de la protéine (CtIP). Les protéines (CtIP) sont impliquées dans les étapes précoces de la recombinaison homologue. De plus, les inventeurs ont identifié les sous-domaines du domaine N-terminal de la protéine (CtIP) qui sont importants pour améliorer la teneur en HDI. Ainsi, l’invention concerne des protéines de fusion comprenant une protéine (Cas9), un domaine de tétramérisation d’une protéine (CtIP) et un domaine de dimérisation d’une protéine (CtIP). En particulier, les inventeurs ont testé ces protéines de fusion cellules (HEK293), cellules (RG37DR) et rats Sprague-Dawley.
Designated States: AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW
African Regional Intellectual Property Organization (ARIPO) (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW)
Eurasian Patent Office (AM, AZ, BY, KG, KZ, RU, TJ, TM)
European Patent Office (EPO) (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR)
African Intellectual Property Organization (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG)
Publication Language: English (EN)
Filing Language: English (EN)