Nucleotide sequence encoding the enzyme I-SceI and the uses thereof

Abstract

An isolated DNA encoding the enzyme I-SceI is provided. The DNA sequence can be incorporated in cloning and expression vectors, transformed cell lines and transgenic animals. The vectors are useful in gene mapping and site-directed insertion of genes.

1 - 22 . (canceled) 
     
     
         23 . A method for inducing at least one site directed double-stranded break in the DNA of an organism comprising:
 (a) providing an isolated cell of said organism comprising at least one endonuclease recognition site comprising 18 base pairs at a location in the DNA of the cell,   (b) providing said endonuclease to said cell by genetically modifying the cell with a nucleic acid comprising said endonuclease or by introducing said endonuclease protein into the cell such that the endonuclease cleaves said endonuclease site at the location in the DNA of the cell.   
     
     
         24 . The method of  claim 23 , wherein said method further comprises providing to said cell
 a plasmid comprising a DNA sequence homologous to the sequence of the chromosome, which allows homologous recombination, and   a modified sequence,   wherein said endonuclease cleaves the endonuclease recognition site,   whereby said cleavage promotes the insertion of said modified sequence into said DNA of said cell at a specific site by homologous recombination.   
     
     
         25 . The method of  claim 23 , wherein said endonuclease cleaves the DNA at a unique location in the genome. 
     
     
         26 . The method of  claim 23 , wherein said endonuclease recognition site is located between two DNA direct repeats,
 wherein said endonuclease cleaves the endonuclease recognition site,   whereby said cleavage promotes the recombination between the two direct repeats leading to the deletion of one repeat and sequences between the repeats.   
     
     
         27 . The method of  claim 26 , wherein said sequences between the repeats comprises a selectable marker. 
     
     
         28 . A method for deleting a viral nucleic acid sequence from a target DNA in a cell comprising:
 providing a cell containing a viral nucleic acid sequence in said target DNA with at least one Group I intron encoded endonuclease sites within said viral nucleic acid sequence and   expressing the Group I intron encoded endonuclease in the cell by genetically modifying the cell with a nucleic acid comprising said endonuclease or introducing said endonuclease protein into the cell such that the endonuclease cleaves the Group I intron encoded endonuclease site in the viral sequence,   wherein a nucleic acid sequence is formed in the cell resulting a deletion of a viral nucleic acid sequence.   
     
     
         29 . The method of  claim 28 , wherein the nucleic acid sequence having a deletion of a viral nucleic acid sequence is formed by end-joining. 
     
     
         30 . The method of  claim 28 , wherein the nucleic acid sequence having a deletion of a viral nucleic acid sequence is formed by intrachromosomal homologous recombination. 
     
     
         31 . The method of any of  claims 28 - 30 , wherein said target nucleic acid sequence comprises a retroviral nucleic acid sequence. 
     
     
         32 . The method of any of  claims 28 - 30 , wherein said target nucleic acid sequence comprises a retroviral LTR. 
     
     
         33 . The method of  claim 32 , wherein said target nucleic acid sequence comprises two retroviral LTRs 
     
     
         34 . The method of  claim 32 , wherein said target nucleic acid sequence comprises a selectable marker. 
     
     
         35 . A method for deleting a viral nucleic acid sequence in a target nucleic acid sequence in an isolated cell, wherein said method comprises:
 providing an isolated cell containing a viral nucleic acid sequence in said target DNA with a Group I intron encoded endonuclease site within said viral nucleic acid sequence;   expressing the Group I intron encoded endonuclease in the cell by genetically modifying the cell with a nucleic acid comprising said Group I intron encoded endonuclease or introducing said Group I intron encoded endonuclease protein into the cell;   cleaving the Group I intron encoded endonuclease site with the endonuclease; and   forming in the cell a nucleic acid sequence having a deletion of a viral nucleic acid sequence proximal to the Group I intron encoded endonuclease site.   
     
     
         36 . The method of  claim 35 , wherein the nucleic acid sequence having a deletion of a viral nucleic acid sequence proximal to the Group I intron encoded endonuclease site is formed by interchromosomal homologous recombination. 
     
     
         37 . The method of  claim 35 , wherein the nucleic acid sequence having a deletion of a viral nucleic acid sequence proximal to the Group I intron encoded endonuclease site is formed by intrachromosomal homologous recombination. 
     
     
         38 . The method of any of  claims 35 - 37 , wherein said target nucleic acid sequence comprises a retroviral nucleic acid sequence. 
     
     
         39 . The method of any of  claims 35 - 37 , wherein said target nucleic acid sequence comprises a retroviral LTR. 
     
     
         40 . The method of  claim 39 , wherein said target nucleic acid sequence comprises two retroviral LTRs. 
     
     
         41 . The method of  claim 39 , wherein said target nucleic acid sequence comprises a selectable marker. 
     
     
         42 . A method for deleting a nucleic acid sequence from a target DNA in a cell comprising:
 providing an isolated cell containing a target DNA with two Group I intron encoded endonuclease sites within said target nucleic acid sequence; and   expressing the Group I intron encoded endonuclease in the cell by genetically modifying the cell with a nucleic acid comprising said endonuclease or introducing said endonuclease protein into the cell such that the endonuclease cleaves the two Group I intron encoded endonuclease sites in the target nucleic acid sequence;   wherein a nucleic acid sequence is formed in the cell having a deletion of a nucleic acid sequence between the two Group I intron encoded endonuclease sites.   
     
     
         43 . The method of  claim 37 , wherein the nucleic acid sequence having a deletion of a nucleic acid sequence between the two Group I intron encoded endonuclease sites is formed by end-joining. 
     
     
         44 . The method of  claim 37 , wherein the nucleic acid sequence having a deletion of a nucleic acid sequence between the two Group I intron encoded endonuclease sites is formed by intrachromosomal homologous recombination. 
     
     
         45 . The method of any of  claims 42 - 44 , wherein said target nucleic acid sequence comprises a transgene. 
     
     
         46 . The method of any of  claims 42 - 44 , wherein said target nucleic acid sequence comprises a selectable marker. 
     
     
         47 . The method of  claim 46 , wherein said target nucleic acid sequence comprises a positive selectable marker. 
     
     
         48 . The method of  claim 46 , wherein said target nucleic acid sequence comprises a negative selectable marker. 
     
     
         49 . A method for deleting a nucleic acid sequence in a target nucleic acid sequence in an isolated cell, wherein said method comprises:
 providing an isolated cell containing a Group I intron encoded endonuclease site within said target nucleic acid sequence;   expressing the Group I intron encoded endonuclease in the cell by genetically modifying the cell with a nucleic acid comprising said Group I intron encoded endonuclease or introducing said Group I intron encoded endonuclease protein into the cell;   cleaving the Group I intron encoded endonuclease site with the endonuclease; and   forming in the cell a nucleic acid sequence having a deletion of a nucleic acid sequence proximal to the Group I intron encoded endonuclease site.   
     
     
         50 . The method of  claim 49 , wherein the nucleic acid sequence having a deletion of a nucleic acid sequence proximal to the Group I intron encoded endonuclease site is formed by interchromosomal homologous recombination. 
     
     
         51 . The method of  claim 49 , wherein the nucleic acid sequence having a deletion of a nucleic acid sequence proximal to the Group I intron encoded endonuclease site is formed by intrachromosomal homologous recombination. 
     
     
         52 . The method of any of  claims 49 - 51 , wherein said target nucleic acid sequence comprises a transgene. 
     
     
         53 . The method of any of  claims 49 - 51 , wherein said target nucleic acid sequence comprises a selectable marker. 
     
     
         54 . The method of  claim 53 , wherein said target nucleic acid sequence comprises a positive selectable marker. 
     
     
         55 . The method of  claim 53 , wherein said target nucleic acid sequence comprises a negative selectable marker.