Use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof

Abstract

The present invention provides a method for detecting nucleic acid cleavage induced by a modified rare-cutting endonuclease derived from an initial rare-cutting endonuclease, said modified rare-cutting endonuclease being able to cleave a DNA target sequence, which may be different from the recognition and cleavage site of the initial rare-cutting endonuclease. In one embodiment of the invention expression of a marker gene is used as an indicator of target DNA cleavage.

1 . A method for detecting a nucleic acid cleavage induced by a modified rare-cutting endonuclease derived from an initial rare-cutting endonuclease, said modified rare-cutting endonuclease being able to cleave a DNA target sequence, said method comprising:
 (i) providing a host cell containing a genetic construct comprising an inactivated marker gene, said marker gene being inactivated by an internal duplication thereof, the internal duplication sequences being separated by an intervening sequence comprising a target DNA sequence to be tested for cleavage, wherein said target DNA sequence is identical to or different from that recognized and cleaved by the initial rare-cutting endonuclease; and   (ii) introducing into said cell one or more nucleic acid molecules encoding the modified rare-cutting endonuclease, and   (iii) detecting expression of said marker gene, wherein expression of said gene indicates its activation by genetic recombination upon cleavage of said target DNA sequence by said modified rare-cutting endonuclease.   
     
     
         2 . The method of  claim 1 , wherein the marker gene is integrated into the genome of the cell. 
     
     
         3 . The method of  claim 1 , wherein the internal duplication is at least 50 base pairs in length. 
     
     
         4 . The method of  claim 1 , wherein the internal duplication is at least 200 base pairs in length. 
     
     
         5 . The method of  claim 1 , wherein the marker gene encodes:
 (i) a positive marker; or   (ii) a negative marker.   
     
     
         6 . The method of  claim 1 , wherein said marker gene encodes beta-galactosidase or a fluorescent protein. 
     
     
         7 . The method of  claim 1 , wherein the intervening sequence comprises a second marker gene. 
     
     
         8 . The method of  claim 1 , wherein the modified rare-cutting endonuclease is a double-stranded endonuclease having a target DNA sequence of at least 12 base pairs. 
     
     
         9 . The method of  claim 1 , wherein the modified rare-cutting endonuclease is:
 (i) a group I intron encoded endonuclease;   (ii) a zinc-finger protein; or   (iii) a nuclease fused to a DNA binding domain.   
     
     
         10 . The method of  claim 9 , wherein the modified rare-cutting endonuclease is a homing endonuclease. 
     
     
         11 . The method of  claim 1 , wherein said intervening sequence comprises a target DNA sequence that is different from that cleaved by said initial rare-cutting endonuclease. 
     
     
         12 . The method of  claim 1 , wherein said intervening sequence comprises a target DNA sequence that is identical to that cleaved by said initial rare-cutting endonuclease. 
     
     
         13 . The method of  claim 1 , further comprising mutating the nucleic acid molecules encoding said modified rare-cutting endonuclease to modify the cleavage specificity thereof. 
     
     
         14 . The method of  claim 1 , wherein the modified rare-cutting endonuclease recognizes and cleaves a target DNA sequence that is different from that of the initial rare-cutting endonuclease. 
     
     
         15 . The method of  claim 1 , wherein said DNA target sequence is derived from a genome where cleavage is desired. 
     
     
         16 . The method of  claim 1 , wherein said cell is a yeast cell. 
     
     
         17 . The method according to  claim 16 , wherein the cell of step (i) is a yeast cell having a construct comprising said target DNA sequence is cloned into a yeast replicative plasmid. 
     
     
         18 . The method of  claim 17 , wherein step (ii) consists of mating said yeast cell of step i) with another yeast cell comprising said nucleic acid molecule encoding the modified rare-cutting endonuclease. 
     
     
         19 . The method of  claim 18 , wherein said nucleic acid molecule encoding the modified rare-cutting endonuclease is included in expression vector. 
     
     
         20 . Use of the method according to  claim 1 , for the screening of a modified rare-cutting endonuclease library, in order to detect cleavage of a target DNA by one or more modified rare-cutting endonucleases included within said library. 
     
     
         21 . Use of the method according to  claim 1 , for the screening of a target DNA library, in order to detect cleavage of a target DNA by a modified rare-cutting endonucleases. 
     
     
         22 . The method according to  claim 20 , further comprising the step of isolating the cells, in which expression of the marker gene is detected. 
     
     
         23 . The method according to  claim 22 , further comprising the step of isolating the nucleic acid encoding said modified rare-cutting endonuclease from said isolated cell. 
     
     
         24 . The method of  claim 21 , further comprising the step of isolating the target DNA cleaved by the modified rare-cutting endonuclease.