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1. WO2020141109 - METHOD FOR THE SELECTION OF CELLS BASED ON CRISPR/CAS-CONTROLLED INTEGRATION OF A DETECTABLE TAG TO A TARGET PROTEIN

Publication Number WO/2020/141109
Publication Date 09.07.2020
International Application No. PCT/EP2019/086666
International Filing Date 20.12.2019
IPC
C12N 15/11 2006.01
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
11DNA or RNA fragments; Modified forms thereof
C12N 15/90 2006.01
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12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
90Stable introduction of foreign DNA into chromosome
C12N 15/65 2006.01
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15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
65using markers
CPC
C12N 15/111
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15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
11DNA or RNA fragments; Modified forms thereof
111General methods applicable to biologically active non-coding nucleic acids
C12N 15/65
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15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
65using markers
C12N 15/907
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15Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
09Recombinant DNA-technology
87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
90Stable introduction of foreign DNA into chromosome
902using homologous recombination
907in mammalian cells
C12N 2310/20
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2310Structure or type of the nucleic acid
10Type of nucleic acid
20involving clustered regularly interspaced short palindromic repeats [CRISPRs]
Applicants
  • F. HOFFMANN-LA ROCHE AG [CH]/[CH] (AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BE, BF, BG, BH, BJ, BN, BR, BW, BY, BZ, CA, CF, CG, CH, CI, CL, CM, CN, CO, CR, CU, CY, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, FR, GA, GB, GD, GE, GH, GM, GN, GQ, GR, GT, GW, HN, HR, HU, ID, IE, IL, IN, IR, IS, IT, JO, JP, KE, KG, KH, KM, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LT, LU, LV, LY, MA, MC, MD, ME, MG, MK, ML, MN, MR, MT, MW, MX, MY, MZ, NA, NE, NG, NI, NL, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SI, SK, SL, SM, SN, ST, SV, SY, SZ, TD, TG, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, UZ, VC, VN, ZA, ZM, ZW)
  • HOFFMANN-LA ROCHE INC. [US]/[US] (US)
Inventors
  • HAAS, Alexander
Agents
  • SKOLAUT, Alexander
Priority Data
18215918.630.12.2018EP
Publication Language English (EN)
Filing Language English (EN)
Designated States
Title
(EN) METHOD FOR THE SELECTION OF CELLS BASED ON CRISPR/CAS-CONTROLLED INTEGRATION OF A DETECTABLE TAG TO A TARGET PROTEIN
(FR) PROCÉDÉ DE SÉLECTION DE CELLULES SUR LA BASE D'UNE INTÉGRATION CONTRÔLÉE PAR CRISPR/CAS D'UN MARQUEUR DÉTECTABLE À UNE PROTÉINE CIBLE
Abstract
(EN)
Herein is reported a method for providing cells that express a fusion protein from a marker protein and a cell-endogenous target protein, consisting of the steps a) transfecting the cells i) with a Cas9-encoding plasmid additionally containing a nucleic acid which is resistant to a first selection agent, ii) with a circular donor plasmid containing a first nucleic acid which confers resistance to a second selection reagent, and a second nucleic acid which encodes the marker protein and which is flanked 3' and 5' by nucleic acids homologous to the integration site in the cell, iii) a suitable synthetic crRNA, and iv) a suitable synthetic tracrRNA, b) cultivating the cells in the presence of the first and the second selection reagent, and c) selecting cells which under the conditions of step b) carry out cell division, and thereby providing/producing cells that express a fusion protein of a marker protein and a cell- endogenous target protein.
(FR)
La présente invention concerne un procédé de production de cellules exprimant une protéine de fusion à partir d'une protéine marqueur et d'une protéine cible endogène cellulaire, comprenant les étapes suivantes: a) transfection des cellules i) avec un plasmide codant pour Cas9 contenant en outre un acide nucléique qui est résistant à un premier agent de sélection, ii) avec un plasmide donneur circulaire contenant un premier acide nucléique qui confère une résistance à un second réactif de sélection, et un second acide nucléique codant pour la protéine marqueur et étant flanqué en 3' et 5'par des acides nucléiques homologues au site d'intégration dans la cellule, iii) avec un ARNcr synthétique approprié, et iv) avec un tracrARN synthétique approprié, b) culture des cellules en présence du premier et du second réactif de sélection, et c) sélection des cellules qui, dans les conditions de l'étape b), réalisent une division cellulaire, et ainsi fournir/produire des cellules exprimant une protéine de fusion à partir d'un protéine marqueur et d'une protéine cible endogène cellulaire.
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