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US 2014/0004608 A1Published application
Meganuclease recombination system
AssigneeCellectis
InventorsJean-Pierre Cabaniols, Andre Choulika, Christophe Delenda
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Abstract
The invention relates to a set of genetic constructs which allow the efficient and reproducible introduction of a specific nucleotide sequence at a fixed position in the genome by generating a double strand break at a specific position in the genome using a meganuclease and so stimulating a homologous recombination event at this locus between the genomic site and a transfected donor sequence. The present invention also relates to methods using these constructs and to these materials in the form of a kit.
Claims20
A set of genetic constructs, comprising:
- a) construct (i) encoded by a nucleic acid molecule, which comprises components:
- A1-A2-A3-A4-A5 (i),
- wherein A1 is a first promoter, A2 is a first homologous portion, A3 is a meganuclease cleavage site, A4 is a first marker gene, A5 is a second homologous portion, and wherein construct (i) is configured to be stably integrated into the genome of at least one target cell;
- b) construct (ii) encoded by a nucleic acid molecule, which comprises components:
- A2′-B1-B2-B3-B4-A5′ (ii),
- wherein A2′ comprises a portion of said first homologous portion A2, B1 is a second marker gene different to said first marker gene, B2 is a second promoter, B3 is a multiple cloning site, B4 is a third promoter, A5′ comprises a portion of said second homologous portion A5;
- c) at least one construct selected from the group consisting of construct (iii) and construct (iv) encoded by nucleic acid molecules, which comprise components:
- C1-C2 (iii),
- C3 (iv), or
- construct (v) which is an isolated or recombinant protein which comprises component:
- C4 (v),
- wherein C1 is a fourth promoter, C2 is the open reading frame (ORF) of a meganuclease, C3 are messenger RNA (mRNA) versions of said meganuclease, C4 is an isolated or recombinant protein of said meganuclease,
- wherein said meganuclease from constructs (iii), (iv), or (v) recognizes and cleaves A3, and
- wherein constructs (iii), (iv), or (v) are configured to be co-transfected with construct (ii) into said at least one target cell.
11 more elements
A method for transforming by homologous recombination at least one cell, comprising:
- (a) stably transforming at least one cell by inserting construct (i), which is encoded by a nucleic acid molecule, which comprises:
- A1-A2-A3-A4-A5 (i),
- wherein A1 is a first promoter, A2 is a first homologous portion, A3 is a meganuclease cleavage site, A4 is a first marker gene, A5 is a second homologous portion, and wherein construct (i) is configured to be stably integrated into the genome of at least one target cell, into the genome of said at least one cell;
- (b) cloning a sequence coding for a gene of interest into position B3 of construct (ii), which is encoded by a nucleic acid molecule, which comprises at least the following components:
- A2′-B1-B2-B3-B4-A5′ (ii),
- wherein A2′ comprises a portion of said first homologous portion A2, B1 is a second marker gene different to said first marker gene, B2 is a second promoter, B3 is a multiple cloning site, B4 is a third promoter, A5′ comprises a portion of said second homologous portion A5;
- (c) co-transfecting said cell of (a), with said construct (ii) of (b) and constructs (iii), (iv) or (v), which are encoded by nucleic acid molecules, which comprise components:
- C1-C2 (iii),
- C3 (iv), or
- an isolated or recombinant protein which comprises component:
- C4 (v),
- wherein C1 is a fourth promoter, C2 is the open reading frame (ORF) of a meganuclease, C3 are messenger RNA (mRNA) versions of said meganuclease, C4 is an isolated or recombinant protein of said meganuclease,
- wherein a meganuclease from constructs (iii), (iv), or (v) recognizes and cleaves A3, and constructs (iii), (iv), or (v) are configured to be co-transfected with construct (ii) into said at least one target cell;
- (d) following homologous recombination between said construct (ii) and said stably inserted construct (i), selecting at least one cell from (c) based upon:
- the absence of a first marker gene encoded by component A4 of said construct (i) and the activity of a second marker gene encoded by component B1 and the activity of a third marker gene encoded by component A5.
12 more elements
Dependencies are inferred from the claim text.