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 . 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 mutagenic nucleobase into the cell protoplasts using polyethylene glycol (PEG) mediated transformation. 
     
     
         2 . Method according to  claim 1 , wherein the mutagenic nucleobase is a ss mutagenic nucleobase. 
     
     
         3 . Method according to claim, wherein the mutagenic nucleobase comprises LNA substitutions that are at least one nucleotide removed from the targeted mismatch and, optionally, at least 3, 4 or 5 nucleotides removed form the 5′ and 3′ ends of the mutagenic nucleobase. 
     
     
         4 . Method according to  claim 1 , wherein the mutagenic nucleobase comprises propyne substitutions. 
     
     
         5 . Method according to  claim 1 , wherein the acceptor DNA is from genomic DNA, linear DNA, mammalian artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, plant artificial chromosomes, nuclear chromosomal DNA, organelle chromosomal DNA, episomal DNA. 
     
     
         6 . Method according to  claim 1 , for altering a cell, correcting a mutation by restoration to wild type, inducing a mutation, inactivating an enzyme by disruption of coding region, modifying bioactivity of an enzyme by altering coding region, modifying a protein by disrupting the coding region. 
     
     
         7 . Method for enhancing the efficiency of targeted mutagenesis in plant protoplasts comprising a step of PEG mediated transformation. 
     
     
         8 . Method according to  claim 7 , wherein the enhancement is at least 10-fold compared to transformation based on electroporation. 
     
     
         9 . Method according to  claim 7 , wherein the mutagenic nucleobase is a ss mutagenic nucleobase. 
     
     
         10 . Method according to  claim 7 , wherein the mutagenic nucleobase comprises LNA substitutions that are at least one nucleotide removed from the targeted mismatch and, optionally, at least 3, 4 or 5 nucleotides removed form the 5′ and 3′ ends of the oligonucleotide. 
     
     
         11 . Method according to  claim 7 , wherein the mutagenic nucleobase comprises propyne substitutions. 
     
     
         12 . Method according to  claim 7 , wherein the acceptor DNA is from genomic DNA, linear DNA, mammalian artificial chromosomes, bacterial artificial chromosomes, yeast artificial chromosomes, plant artificial chromosomes, nuclear chromosomal DNA, organelle chromosomal DNA, episomal DNA. 
     
     
         13 . Method according to  claim 7 , for altering a cell, correcting a mutation by restoration to wild type, inducing a mutation, inactivating an enzyme by disruption of coding region, modifying bioactivity of an enzyme by altering coding region, modifying a protein by disrupting the coding region.