Recognition sequences for i-crei-derived meganucleases and uses thereof

Abstract

Methods of cleaving double-stranded DNA that can be recognized and cleaved by a rationally-designed, I-CreI-derived meganuclease are provided. Also provided are recombinant nucleic acids, cells, and organisms containing such recombinant nucleic acids, as well as cells and organisms produced using such meganucleases. Also provided are methods of conducting a custom-designed, I-CreI-derived meganuclease business.

1 . A method for cleaving a double-stranded DNA comprising:
 (a) identifying in said DNA at least one recognition site for a rationally-designed I-CreI-derived meganuclease with altered specificity relative to I-CreI, wherein said recognition site is not cleaved by a naturally-occurring I-CreI,   wherein said recognition site has a four base pair central sequence selected from the group consisting of TTGT, TTAT, TCTT, TCGT, TCAT, GTTT, GTCT, GGAT, GAGT, GAAT, ATGT, TTTC, TTCC, TGAC, TAAC, GTTC, ATAT, TCGA, TTAA, GGGC, ACGC, CCGC, CTGC, ACAA, ATAA, AAGA, ACGA, ATGA, AAAC, AGAC, ATCC, ACTC, ATTC, ACAT, GAAA, GGAA, GTCA, GTTA, GAAC, ATAT, TCGA, TTAA, GCCC, GCGT, GCGG and GCAG;   (b) providing said rationally-designed meganuclease; and   (c) contacting said DNA with said rationally-designed meganuclease;   whereby said rationally-designed meganuclease cleaves said DNA.   
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein said DNA cleavage is in vitro. 
     
     
         4 . The method of  claim 1 , wherein said DNA is selected from the group consisting of a PCR product; an artificial chromosome; genomic DNA isolated from bacteria, fungi, plants, or animal cells; and viral DNA. 
     
     
         5 . The method of  claim 1 , wherein said DNA cleavage is in vivo. 
     
     
         6 . The method of  claim 5 , wherein said DNA is present in a cell selected from the group consisting of a bacterial, fungal, plant and animal cell. 
     
     
         7 . The method of  claim 5 , wherein said DNA is present in a nucleic acid selected from the group consisting of a plasmid, a prophage and a chromosome. 
     
     
         8 . The method of  claim 1 , wherein said four base pair DNA sequence is selected from the group consisting of GTGT, GTAT, TTAG, GTAG, TTAC, TCTC, TCAC, GTCC, GTAC, TCGC, AAGC, GAGC, GCGC, GTGC, TAGC, TTGC, ATGC, ACAC, ATAC, CTAA, CTAC, GTAA, GAGA, GTGA, GGAC, GTAC, GCGA, GCTT, GCTC, GCGC, GCAC, GCTA, GCAA and GCAT. 
     
     
         9 . The method of  claim 1 , further comprising rationally-designing said I-CreI-derived meganuclease to recognize said recognition site. 
     
     
         10 . The method of  claim 1 , further comprising producing said rationally-designed I-CreI-derived meganuclease. 
     
     
         11 . A cell transformed with a nucleic acid comprising, in order:
 a) a first 9 base pair DNA sequence which can be bound by an I-CreI-derived meganuclease monomer or by a first domain from a single-chain I-CreI-derived meganuclease;   b) a four base pair DNA sequence selected from the group consisting of GTGT, GTAT, TTAG, GTAG, TTAC, TCTC, TCAC, GTCC, GTAC, TCGC, AAGC, GAGC, GCGC, GTGC, TAGC, TTGC, ATGC, ACAC, ATAC, CTAA, CTAC, GTAA, GAGA, GTGA, GGAC, GTAC, GCGA, GCTT, GCTC, GCGC, GCAC, GCTA, GCAA and GCAT; and   c) a second 9 base pair DNA sequence which can be bound by an I-CreI-derived meganuclease monomer or by a second domain from the single-chain I-CreI-derived meganuclease, wherein the second 9 base pair DNA sequence is in the reverse orientation relative to the first.   
     
     
         12 . A cell containing an exogenous nucleic acid sequence integrated into its genome, comprising, in order:
 a) a first exogenous 9 base pair DNA sequence which can be bound by an I-CreI-derived meganuclease monomer or by a first domain from a single-chain I-CreI-derived meganuclease;   b) an exogenous four base pair DNA sequence selected from the group consisting of GTGT, GTAT, TTAG, GTAG, TTAC, TCTC, TCAC, GTCC, GTAC, TCGC, AAGC, GAGC, GCGC, GTGC, TAGC, TTGC, ATGC, ACAC, ATAC, CTAA, CTAC, GTAA, GAGA, GTGA, GGAC, GTAC, GCGA, GCTT, GCTC, GCGC, GCAC, GCTA, GCAA and GCAT; and   a) a second exogenous 9 base pair DNA sequence which can be bound by an I-CreI-derived meganuclease monomer or by a second domain from the single-chain I-CreI-derived meganuclease, wherein the second 9 base pair DNA sequence is in the reverse orientation relative to the first.   
     
     
         13 . The cell of  claim 11 , wherein said nucleic acid is a plasmid. 
     
     
         14 . The cell of  claim 11 , wherein said nucleic acid is an artificial chromosome. 
     
     
         15 . The cell of  claim 11 , wherein said nucleic acid is integrated into the genomic DNA of said cell. 
     
     
         16 . The cell of  claim 11 , wherein said nucleic acid is a viral nucleic acid. 
     
     
         17 . The cell of  claim 11 , wherein said cell is selected from the group consisting of a human cell, a non-human animal cell, a plant cell, a bacterial cell, and a fungal cell. 
     
     
         18 . The cell of  claim 11 , wherein said four base pair DNA sequence is selected from the group consisting of TTGT, TTAT, TCTT, TCGT, TCAT, GTTT, GTCT, GGAT, GAGT, GAAT, ATGT, TTTC, TTCC, TGAC, TAAC, GTTC, ATAT, TCGA, TTAA, GGGC, ACGC, CCGC, CTGC, ACAA, ATAA, AAGA, ACGA, ATGA, AAAC, AGAC, ATCC, ACTC, ATTC, ACAT, GAAA, GGAA, GTCA, GTTA, GAAC, ATAT, TCGA, TTAA, GCCC, GCGT, GCGG and GCAG. 
     
     
         19 . The cell of  claim 18 , wherein said four base pair DNA sequence is selected from the group consisting of GTGT, GTAT, TTAG, GTAG, TTAC, TCTC, TCAC, GTCC, GTAC, TCGC, AAGC, GAGC, GCGC, GTGC, TAGC, TTGC, ATGC, ACAC, ATAC, CTAA, CTAC, GTAA, GAGA, GTGA, GGAC, GTAC, GCGA, GCTT, GCTC, GCGC, GCAC, GCTA, GCAA and GCAT. 
     
     
         20 . A method of conducting a custom-designed, I-CreI-derived meganuclease business comprising:
 (a) receiving a DNA sequence into which a double-strand break is to be introduced by a rationally-designed I-CreI-derived meganuclease;   (b) identifying in said DNA sequence at least one recognition site for a rationally-designed I-CreI-derived meganuclease with altered specificity relative to I-CreI, wherein said recognition site is not cleaved by a naturally-occurring I-CreI,   wherein said recognition site has a four base pair central sequence selected from the group consisting of TTGT, TTAT, TCTT, TCGT, TCAT, GTTT, GTCT, GGAT, GAGT, GAAT, ATGT, TTTC, TTCC, TGAC, TAAC, GTTC, ATAT, TCGA, TTAA, GGGC, ACGC, CCGC, CTGC, ACAA, ATAA, AAGA, ACGA, ATGA, AAAC, AGAC, ATCC, ACTC, ATTC, ACAT, GAAA, GGAA, GTCA, GTTA, GAAC, ATAT, TCGA, TTAA, GCCC, GCGT, GCGG and GCAG; and   (c) providing said rationally-designed meganuclease.   
     
     
         21 . The method of  claim 20 , further comprising rationally-designing said I-CreI-derived meganuclease to recognize said recognition site. 
     
     
         22 . The method of  claim 20 , further comprising producing said rationally-designed meganuclease. 
     
     
         23 . The method of  claim 20 , wherein the rationally-designed meganuclease is provided to the same party from which said DNA sequence has been received.