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.
Claims12
A set of genetic constructs, comprising:
- a) construct (i) comprising SEQ ID NO:
- 6,
- wherein construct (i) is configured to be stably integrated into the genome of at least one target cell;
- b) construct (ii) comprising SEQ ID NO:
- 22;
- and
- c) at least one construct selected from the group consisting of construct (iii), construct (iv), and construct (v), which respectively comprise components:
- C1-C2 (iii),
- C3 (iv), and
- C4 (v),
- wherein constructs (iii) and (iv) are nucleic acid molecules and construct (v) is an isolated or recombinant protein,
- wherein C1 is a promoter, C2 is an open reading frame (ORF) of a meganuclease, C3 is messenger RNA (mRNA) encoding of said meganuclease, and C4 is an isolated or recombinant protein of said meganuclease,
- wherein said meganuclease from constructs (iii), (iv), or (v) recognizes and cleaves a meganuclease cleavage site in construct (i), and
- wherein constructs (iii), (iv), or (v) are configured to be co-transfected with construct (ii) into said at least one target cell.
A method for transforming by homologous recombination at least one isolated cell, comprising:
- (a) stably transforming at least one isolated cell by inserting construct (i) comprising SEQ ID NO:
- 6, into the genome of said at least one isolated cell;
- (b) cloning a sequence coding for a gene of interest into a multiple cloning site of construct (ii) comprising SEQ ID NO:
- 22;
- (c) co-transfecting said cell of step (a) with said construct (ii) of step (b) and construct (iii), (iv), or (v), which respectively comprise components:
- C1-C2 (iii),
- C3 iv), and
- C4 (v),
- wherein constructs (iii) and (iv) are nucleic acid molecules and construct (v) is an isolated or recombinant protein,
- wherein C1 is a, C2 is an open reading frame (ORF) of a meganuclease, C3 is a messenger RNA (mRNA) encoding said meganuclease, and C4 is an isolated or recombinant protein of said meganuclease,
- wherein a meganuclease from constructs (iii), (iv), or (v) recognizes and cleaves a meganuclease cleavage site in construct (i);
- and
- (d) following homologous recombination between said construct (ii) and said stably inserted construct (i), selecting at least one cell from step (c) based upon:
- absence of hygromycin resistance, presence of puromycin resistance, and presence of neomycin resistance.
Dependencies are inferred from the claim text.