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AssigneeCellectis
InventorsSylvain Arnould, Patrick Chames, Andre Choulika, Jean-Charles Epinat, Emmanuel Lacroix
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This patent application relates to hybrid and/or single-chain rare-cutting endonucleases, called meganucleases, which recognize and cleave a specific nucleotide sequence, to polynucleotide sequences encoding for said rare-cutting endonucleases, to a vector comprising one of said polynucleotide sequences, to a cell or animal comprising one of said polynucleotide sequences or said rare-cutting endonucleases, to a process for producing one of said rare-cuuting endonucleases and any use of the disclosed products and methods. More particularly, this invention contemplates any use of such rare-cutting endonuclease for genetic engineering and gene therapy.
1- A hybrid meganuclease comprising a first domain and a second domain in the orientation N-terminal toward C-terminal, said first and second domains being derived from two different initial dodecapeptide meganucleases and wherein said hybrid meganuclease is capable of causing DNA cleavage.
2- Hybrid meganuclease according to claim 1, wherein said first domain is derived from a first di-dodecapeptide meganuclease and said second domain is derived from a second di-dodecapeptide meganuclease, said first and second domains being bound by a convenient linker.
3- Hybrid meganuclease according to claim 1, wherein said first domain is derived from a mono-dodecapeptide meganuclease and said second domain is derived from a di-dodecapeptide meganuclease, said first and second domains being bound by a convenient linker.
4- Hybrid meganuclease according to claim 1, wherein said first domain is derived from a di-dodecapeptide meganuclease and said second domain is derived from a mono-dodecapeptide meganuclease, said first and second domains being bound by a convenient linker.
5- Hybrid meganuclease according to claim 1, wherein said first domain is derived from a mono-dodecapeptide meganuclease and said second domain is derived from another mono-dodecapeptide meganuclease, said first and second domains being bound by a convenient linker.
6- Hybrid meganuclease according to claim 2, wherein said first domain is derived from the N-terminal domain of said first di-dodecapeptide meganuclease and said second domain is derived from the C-terminal domain of said second di-dodecapeptide meganuclease.
7- Hybrid meganuclease according to claim 2, wherein said first domain is derived from the N-terminal domain of said first di-dodecapeptide meganuclease and said second domain is derived from the N-terminal domain of said second di-dodecapeptide meganuclease.
8- Hybrid meganuclease according to claim 2, wherein said first domain is derived from the C-terminal domain of said first di-dodecapeptide meganuclease and said second domain is derived from the C-terminal domain of said second di-dodecapeptide meganuclease.
9- Hybrid meganuclease according to claim 2, wherein said first domain is derived from the C-terminal domain of said first di-dodecapeptide meganuclease and said second domain is derived from the N-terminal domain of said second di-dodecapeptide meganuclease.
10- Hybrid meganuclease according to claim 3, wherein said second domain is derived from the N-terminal domain of said di-dodecapeptide meganuclease.
11- Hybrid meganuclease according to claim 3, wherein said second domain is derived from the C-terminal domain of said di-dodecapeptide meganuclease.
12- Hybrid meganuclease according to claim 4, wherein said first domain is derived from the N-terminal domain of said di-dodecapeptide meganuclease.
13- Hybrid meganuclease according to claim 4, wherein said first domain is derived from the C-terminal domain of said di-dodecapeptide meganuclease.
14- Hybrid meganuclease according to any of claims 6, 7, and 12, wherein said convenient linker comprises a loop derived from said first di-dodecapeptide meganuclease or a part thereof.
15- Hybrid meganuclease according to any of claims 6, 8, and 11, wherein said convenient linker comprises a loop derived from said second di-dodecapeptide meganuclease or a part thereof.
16- Hybrid meganuclease according to claim 1, wherein said dodecapeptide meganuclease is selected from the group consisting of:
17- Hybrid meganuclease according to claim 1, wherein said dodecapeptide meganuclease is selected from the group consisting of:
18- Hybrid meganuclease according to claim 4, wherein said di-dodecapeptide meganuclease is I-Dmo I and said mono-dodecapeptide meganuclease is I-Cre I.
19- Hybrid meganuclease according to claim 18, wherein said hybrid meganuclease comprises the sequence of SEQ ID No 4.
20- A hybrid meganuclease comprising a first and a second domains in the orientation N-terminal toward C-terminal, wherein each domain are derived from the same di-dodecapeptide meganuclease and said first domain is derived from the C-terminal domain and said second domain is derived from the N-terminal domain and wherein said hybrid meganuclease is capable of causing DNA cleavage.
21- A single-chain meganuclease comprising a first and a second domain in the orientation N-terminal toward C-terminal, wherein said first and second domains are derived from the same mono-dodecapeptide meganuclease and wherein said single-chain meganuclease is capable of causing DNA cleavage.
22- Single-chain meganuclease according to claim 21, wherein said mono-dodecapeptide meganuclease is I-Cre.
23- Single-chain meganuclease according to claim 24, wherein said single-chain meganuclease comprises the sequence of SEQ ID No 6.
24- A purified or recombinant polynucleotide encoding a meganuclease according to any of claims 1, 20 and 21.
25- A vector comprising a polynucleotide according to claim 24.
26- A host cell comprising a polynucleotide according to claim 24.