Targeted chromosomal genomic alterations in plants using modified single stranded oligonucleotides

Abstract

Presented are methods and compositions for targeted chromosomal genomic alterations with modified single-stranded oligonucleotides. The oligonucleotides of the invention have modified nuclease-resistant termini comprising LNA, phosphorothioate linkages or 2′-O-Me base analogues or combinations of such modifications.

What is claimed is:  
     
         1 . An oligonucleotide for targeted alteration of genetic sequence, comprising a single-stranded oligonucleotide having a DNA domain, said DNA domain having at least one mismatch with respect to the genetic sequence to be altered, and further comprising chemical modifications of the oligonucleotide, said chemical modifications selected from the group consisting of an o-methyl modification, an LNA modification including LNA derivatives and analogs, two or more phosphorothioate linkages on a terminus, and a combination of any two or more of these modifications.  
     
     
         2 . The oligonucleotide according to claim one that comprises two or more phosphorothioate linkages on at least the 3′ terminus.  
     
     
         3 . The oligonucleotide according to claim one that comprises a 2′-O-methyl analog.  
     
     
         4 . The oligonucleotide according to claim one that comprises an LNA nucleotide, including an LNA derivative or analog.  
     
     
         5 . The oligonucleotide according to claim one that comprises a combination of at least two modifications selected from the group of a phosphorothioate linkage, a 2′-O-methyl analog, a locked nucleotide analog and a ribonucleotide.  
     
     
         6 . The oligonucleotide according to any one of  claims 1  to  5  that comprises at least one unmodified ribonucleotide.  
     
     
         7 . The oligonucleotide according to any one of  claims 1  to  6 , wherein the sequence of said oligonucleotide is selected from the group consisting of SEQ ID NOS: 1-2672.  
     
     
         8 . A method of targeted alteration of genetic material, comprising combining the target genetic material with an oligonucleotide according to any one of  claims 1  to  7  in the presence of purified proteins.  
     
     
         9 . A method of targeted alteration of genetic material, comprising administering to a cell extract an oligonucleotide of any one of  claims 1  to  7 .  
     
     
         10 . A method of targeted alteration of genetic material, comprising administering to a cell an oligonucleotide of any one of  claims 1  to  7 .  
     
     
         11 . A method of targeted alteration of genetic sequence in callus, comprising administering to the callus an oligonucleotide of any one of  claims 1  to  7 .  
     
     
         12 . A method of targeted alteration of genetic sequence, comprising combining target genetic material with an oligonucleotide according to any one of  claims 1  to  7 , said target genetic material being a non-transcribed DNA strand of a duplex DNA.  
     
     
         13 . The genetic material obtained by any one of the methods of  claim 8 ,  9  or  claim 10 .  
     
     
         14 . A cell comprising the genetic material of  claim 13 .  
     
     
         15 . A plant organism comprising the cell according to  claim 14 .  
     
     
         16 . A plant or plant part produced by the method of  claim 11 .  
     
     
         17 . A method of determining whether an oligonucleotide is optimized for targeted alteration of a genetic sequence, which comprises: 
 (a) comparing the efficiency of alteration of a targeted genetic sequence by an oligonucleotide of any one of  claims 1  to  7  with the efficiency of alteration of the same targeted genetic sequence by a second oligonucleotide, said second oligonucleotide selected from the group of an oligonucleotide that lacks the mismatch, a fully modified phosphorothiolated oligonucleotide, a fully modified 2′-O-methylated oligonucleotide and a chimeric double-stranded double hairpin containing RNA and DNA nucleotides.    
     
     
         18 . The method of  claim 17  in which the alteration is produced in a plant cell extract.  
     
     
         19 . The method of  claim 17  in which the alteration is produced in a cell.  
     
     
         20 . A kit comprising the oligonucleotide according to any one of  claims 1  to  7  and a second oligonucleotide selected from the group of an oligonucleotide that lacks the mismatch, a fully modified phosphorothiolated oligonucleotide, a fully modified 2-O-methylated oligonucleotide and a chimeric double stranded double hairpin containing RNA and DNA nucleotides.