Methods For Altering The Genome Of A Monocot Plant Cell

Abstract

Methods and compositions for altering the genome of a monocot plant cell, and a monocot plant are disclosed. The methods and compositions use a double-strand break inducing agent to alter a monocot plant or plant cell genomic sequence comprising a recognition sequence for the double-strand break inducing agent.

What is claimed is: 
     
         1 . A method to alter a monocot plant cell genome comprising:
 (a) contacting at least one monocot plant cell comprising a recognition sequence in its genome with (i) a phenotypic marker, (ii) a DNA fragment, and (ii) a double-strand break inducing agent capable of inducing a double-strand break at the recognition sequence;   (b) selecting cells comprising the phenotypic marker;   (c) identifying cells from step (b) having an alteration in their genome at the recognition sequence wherein the alteration is selected from the group consisting of (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii),   wherein identifying comprises any method that does not use a phenotypic marker; and,   (d) recovering a fertile monocot plant having the alteration in its genome.   
     
     
         2 . The method of  claim 1 , wherein the monocot plant cell is contacted with (i) the phenotypic marker, (ii) the DNA fragment, wherein the DNA fragment comprises a geminiviral origin of replication functional in the monocot plant cell, (iii) a replicase which binds to a geminiviral origin of replication and stimulates replication of the DNA fragment, and (iv) the double-strand break inducing agent capable of inducing a double-strand break at the recognition sequence. 
     
     
         3 . The method of  claim 1  wherein the alteration at the recognition sequence comprises insertion of a polynucleotide of interest. 
     
     
         4 . The method of  claim 1 , wherein the DNA fragment comprises at least a first region having homology to a genomic region flanking or comprising the recognition sequence. 
     
     
         5 . The method of  claim 1 , wherein the DNA fragment comprises in the following order the first region of homology, the polynucleotide of interest, and a second region of homology to the genomic region flanking or comprising the recognition sequence. 
     
     
         6 . The method of  claim 1 , wherein the alteration at the recognition sequence comprises replacement, wherein the alteration is produced by homologous recombination. 
     
     
         7 . The method of  claim 1 , wherein recovering a fertile monocot plant having the alteration in its genome occurs at a higher frequency as compared to a control method without an inducing agent. 
     
     
         8 . The method of  claim 1 , wherein identifying in step (c) comprises at least one method selected from the group consisting of PCR, Southern blot, restriction digest, and DNA sequencing. 
     
     
         9 . The method of  claim 1 , wherein the DNA fragment comprises a T-DNA. 
     
     
         10 . The method of  claim 2 , wherein the geminiviral origin of replication is from a wheat dwarf virus and the replicase is RepA. 
     
     
         11 . The method of  claim 1 , wherein the inducing agent is an endonuclease, a zinc finger nuclease, a transposase, or a site-specific recombinase. 
     
     
         12 . The method of  11 , wherein the endonuclease is I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, or PI-TliII. 
     
     
         13 . The method of  claim 12 , wherein the endonuclease is modified to specifically bind an endogenous plant target polynucleotide sequence, wherein the modified endonuclease no longer binds to its wild type endonuclease recognition sequence. 
     
     
         14 . The method of  claim 13 , wherein the modified endonuclease is derived from I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, or PI-TliII. 
     
     
         15 . The method of  claim 11 , wherein the inducing agent is a site-specific recombinase selected from the group consisting of FLP, Cre, SSV1, R, Gin, lambda Int, phiC31 Int, and HK022 Int. 
     
     
         16 . The method of  claim 1  wherein the inducing agent is encoded by a polynucleotide modified to increase the number of maize preferred codons. 
     
     
         17 . The method of  claim 16 , wherein the inducing agent is encoded by the polynucleotide of SEQ ID NO: 1. 
     
     
         18 . The method of  claim 1 , wherein the recognition sequence is an endogenous monocot plant genome sequence. 
     
     
         19 . The method of  claim 1 , wherein the monocot cell is from maize, rice, sorghum, barley, wheat, millet, oats, sugarcane, turfgrass, or switch grass. 
     
     
         20 . The method of  claim 19 , wherein the monocot cell is from maize. 
     
     
         21 . A method of modifying a specific endogenous genomic target sequence comprising:
 (a) contacting at least one monocot plant cell comprising the target sequence in its genome with (i) a phenotypic marker, (ii) a DNA fragment, and (ii) an engineered double-strand break inducing agent capable of inducing a double-strand break at the endogenous target sequence;   (b) selecting cells comprising the phenotypic marker; and,   (c) identifying cells from step (b) having an alteration in their genome at the target sequence wherein the alteration is selected from the group consisting of (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii),   wherein identifying comprises any method that does not use a phenotypic marker.   
     
     
         22 . The method of  claim 21 , wherein the engineered double-strand break inducing agent is derived from I-CreI. 
     
     
         23 . The method of  claim 22 , wherein the engineered double-strand break inducing agent is expressed as a fusion protein. 
     
     
         24 . The method of  claim 23 , wherein the engineered double-strand break inducing agent comprises SEQ ID NO: 21. 
     
     
         25 . The method of  claim 20  further comprising recovering a fertile monocot plant having the alteration in its genome. 
     
     
         26 . A maize plant comprising an exogenous phenotypic marker and further comprising an alteration of the LIG3-4 sequence of SEQ ID NO: 15 in its genome. 
     
     
         27 . A method of modifying a monocot plant cell genome comprising:
 (a) crossing (i) a first monocot plant comprising in its genome a recognition sequence and a recombinase and (ii) a second monocot plant comprising in its genome a replicon flanked by excision sites recognized by the recombinase, wherein the replicon comprises a double-strand break inducing agent capable of inducing a double-strand break at the recognition sequence;   (b) recovering progeny produced by step (a);   (c) identifying progeny from step (b) having an alteration in their genome at the recognition sequence wherein the alteration is selected from the group consisting of (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, and (iv) any combination of (i)-(iii),   wherein identifying comprises any method that does not use a marker; and,   (d) recovering a fertile monocot plant having the alteration in its genome.   
     
     
         28 . The method of  claim 27 , wherein the replicon further comprises a DNA fragment comprising at least one region having homology to a genomic region flanking or comprising the recognition sequence. 
     
     
         29 . The method of  claim 27  wherein identifying in step (c) comprises at least one method selected from the group consisting of PCR, Southern blot, restriction digest, and DNA sequencing. 
     
     
         30 . The method of  claim 27  further comprising outcrossing the plant from step (d) to produce segregating progeny, whereby a segregated progeny comprising only the alteration in its genome can be recovered. 
     
     
         31 . The method of  claim 27  wherein the recombinase is FLP and the excision sites are FRT sites. 
     
     
         32 . The method of  claim 27  wherein the replicon is a geminiviral replicon. 
     
     
         33 . The method of  claim 27  wherein the double-strand break inducing agent is an endonuclease, a zinc finger nuclease, a transposase, or a site-specific recombinase. 
     
     
         34 . The method of  claim 33 , wherein the endonuclease is (a) I-SceI, I-SceII, I-SceIII, I-SceIV, I-SceV, I-SceVI, I-SceVII, I-CeuI, I-CeuAIIP, I-CreI, I-CrepsbIP, I-CrepsbIIP, I-CrepsbIIIP, I-CrepsbIVP, I-TliI, I-PpoI, PI-PspI, F-SceI, F-SceII, F-SuvI, F-TevI, F-TevII, I-AmaI, I-AniI, I-ChuI, I-CmoeI, I-CpaI, I-CpaII, I-CsmI, I-CvuI, I-CvuAIP, I-DdiI, I-DdiII, I-DirI, I-DmoI, I-HmuI, I-HmuII, I-HsNIP, I-LlaI, I-MsoI, I-NaaI, I-NanI, I-NcIIP, I-NgrIP, I-NitI, I-NjaI, I-Nsp236IP, I-PakI, I-PboIP, I-PcuIP, I-PcuAI, I-PcuVI, I-PgrIP, I-PobIP, I-PorI, I-PorIIP, I-PbpIP, I-SpBetaIP, I-ScaI, I-SexIP, I-SneIP, I-SpomI, I-SpomCP, I-SpomIP, I-SpomIIP, I-SquIP, I-Ssp6803I, I-SthPhiJP, I-SthPhiST3P, I-SthPhiSTe3bP, I-TdeIP, I-TevI, I-TevII, I-TevIII, I-UarAP, I-UarHGPAIP, I-UarHGPA13P, I-VinIP, I-ZbiIP, PI-MtuI, PI-MtuHIP PI-MtuHIIP, PI-PfuI, PI-PfuII, PI-PkoI, PI-PkoII, PI-Rma43812IP, PI-SpBetaIP, PI-SceI, PI-TfuI, PI-TfuII, PI-ThyI, PI-TliI, or PI-TliII; or (b) a derivative thereof. 
     
     
         35 . The method of  claim 27  wherein the recognition is an endogenous monocot plant genome sequence. 
     
     
         36 . The method of  claim 27  wherein the monocot plant is maize, rice, sorghum, barley, wheat, millet, oats, sugarcane, turfgrass, or switch grass. 
     
     
         37 . The method of  claim 36  wherein the monocot plant is maize. 
     
     
         38 . The method of  claim 27  wherein the first monocot plant is the female parent in the cross.