Mutagenesis method using polyethylene glycol mediated introduction of mutagenic nucleobases into plant protoplasts

Abstract

Method for targeted alteration of a duplex acceptor DNA sequence in a plant cell protoplast, comprising combining the duplex acceptor DNA sequence with a donor mutagenic nucleobase, wherein the duplex acceptor DNA sequence contains a first DNA sequence and a second DNA sequence which is the complement of the first DNA sequence and wherein the donor mutagenic nucleobase comprises at least one mismatch with respect to the duplex acceptor DNA sequence to be altered, preferably with respect to the first DNA sequence, wherein the method further comprises a step of introducing the donor mutagenic nucleobase into the cell protoplasts using polyethylene glycol (PEG) mediated transformation and the use of PEG protoplast transformation for enhancing the rate of targeted mutagenesis.

1 . A composition comprising polyethylene glycol and a synthetic single-stranded nucleobase having a length of between 10-60 nucleotides,
 wherein the nucleobase comprises a sequence complementary to a target sequence in the plant genome.   
     
     
         2 . The composition of  claim 1 , further comprising one or more plant protoplasts. 
     
     
         3 . The composition of  claim 1 , wherein the nucleobase is a single-stranded DNA nucleobase. 
     
     
         4 . The composition of  claim 1 , wherein the single-stranded nucleobase comprises one or more modified nucleotides. 
     
     
         5 . The composition of  claim 1 , wherein the nucleobase comprises a mismatch with respect to the target sequence in the plant genome. 
     
     
         6 . The composition of  claim 1 , wherein the single-stranded nucleobase is conjugated to a protein. 
     
     
         7 . The composition of  claim 1 , wherein the single-stranded nucleobase is chemically modified for protection against nuclease degradation. 
     
     
         8 . The composition of  claim 4 , wherein the single-stranded nucleobase comprises one or more Locked Nucleic Acid (LNA) substitutions. 
     
     
         9 . The composition of  claim 5 , wherein the single-stranded nucleobase comprises one or more LNA substitutions that are at least one nucleotide removed from the mismatch. 
     
     
         10 . The composition of  claim 9 , wherein the single-stranded nucleobase comprises two LNAs located at least one nucleotide removed from either side of the mismatch. 
     
     
         11 . The composition of  claim 8 , wherein the LNA substitutions are at least 3 nucleotides removed from the 5′ and 3′ ends of the single-stranded nucleobase. 
     
     
         12 . The composition of  claim 8 , wherein the LNA substitutions are at least 4 nucleotides removed from the 5′ and 3′ ends of the single-stranded nucleobase. 
     
     
         13 . The composition of  claim 8 , wherein the LNA substitutions are at least 5 nucleotides removed from the 5′ and 3′ ends of the single-stranded nucleobase. 
     
     
         14 . The composition of  claim 4 , wherein the single-stranded nucleobase comprises one or more propyne substitutions. 
     
     
         15 . The composition of  claim 4 , wherein the single-stranded nucleobase comprises a phosphorothioate linkage. 
     
     
         16 . The composition of  claim 4 , wherein the single-stranded nucleobase comprises a 2′-O-methyl analog.