Organelle genome modification using polynucleotide guided endonuclease

Abstract

Provided herein are methods and systems for altering the genome of an organelle. In some embodiments, the method comprises introducing into an organelle a recombinant DNA construct comprising a first polynucleotide encoding at least one guide RNA and a second polynucleotide encoding a polynucleotide guided polypeptide; and growing a cell comprising the organelle under conditions in which the first polynucleotide and the second polynucleotide are each expressed.

1 . A method for altering the genome of an organelle, the method comprising:
 a. introducing into an organelle a recombinant DNA construct comprising the following:
 i. a first polynucleotide encoding at least one guide RNA, wherein the at least one guide RNA directs a polynucleotide guided polypeptide to cleave at least one target sequence present in an organelle genome; 
 ii. a second polynucleotide encoding a polynucleotide guided polypeptide, wherein the polynucleotide guided polypeptide, when associated with the guide RNA, cleaves the at least one target sequence; 
 iii. optionally, a third polynucleotide encoding at least one homologous organelle DNA sequence, wherein the at least one homologous organelle DNA is of sufficient size for homologous recombination, wherein integration of the at least one homologous organelle DNA sequence into the organelle genome results in removal of the at least one target sequence; 
 iv. optionally, a fourth polynucleotide encoding at least one selectable marker or at least one screenable marker, or both; wherein the fourth polynucleotide is operably linked to a promoter that is functional in the organelle; and 
 v. optionally, a fifth polynucleotide encoding an origin of replication that is functional in the organelle; 
   b. growing a cell comprising the organelle of (a) under conditions in which the first polynucleotide of (i) and the second polynucleotide of (ii) are each expressed; and   c. Selecting a cell that is homoplasmic for the altered genome of the organelle.   
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the organelle comprises a plastid. 
     
     
         5 . The method of  claim 1 , wherein the organelle comprises a mitochondrion. 
     
     
         6 . The method of  claim 1 , comprising the third polynucleotide of (iii), wherein the third polynucleotide of (iii) comprises a sixth and a seventh polynucleotide, wherein the sixth and the seventh polynucleotides correspond to two adjacent regions of homology in the organelle genome, wherein the sixth and seventh polynucleotides are separated by a sequence that is heterologous to the organelle DNA. 
     
     
         7 . The method of  claim 6 , wherein the sequence that is heterologous to the organelle DNA comprises at least one selected from the group consisting of: the first polynucleotide, the second polynucleotide, the fourth polynucleotide, an eighth polynucleotide, and any combination thereof, wherein the eighth polynucleotide encodes an RNA that is heterologous to the organelle. 
     
     
         8 . The method of  claim 1 , wherein the at least one guide RNA is present on a polycistronic transcription unit. 
     
     
         9 . The method of  claim 8 , wherein the at least one guide RNA is processed from a polycistronic RNA after transcription of the polycistronic transcription unit by use of at least one selected from the group consisting of: an RNA cleavage site, a Csy4 cleavage site, a ribozyme cleavage site, a polynucleotide guided polypeptide cleavage site, the presence of a tRNA sequence, and any combination thereof. 
     
     
         10 . The method of  claim 9 , wherein the polycistronic RNA comprises a first tRNA sequence 5′ to the at least one guide RNA and a second tRNA sequence 3′ to the at least one guide RNA. 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . The method of  claim 6 , wherein at least one selected from the group consisting of: the first polynucleotide, the second polynucleotide, the fourth polynucleotide, the fifth polynucleotide, and any combination thereof, is located outside the region bounded by the sixth and the seventh polynucleotide. 
     
     
         14 . The method of  claim 6 , comprising the fourth and the fifth polynucleotides, wherein both the fourth and the fifth polynucleotides are located outside the region bounded by the sixth and the seventh polynucleotides. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . (canceled) 
     
     
         18 . The method of  claim 6 , wherein said polynucleotide-guided polypeptide is selected from the group consisting of: a Cas9 protein, a MAD2 protein, a MAD7 protein, a CRISPR nuclease, a nuclease domain of a Cas protein, a Cpf1 protein, an Argonaute, modified versions thereof, and any combination thereof. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 6 , wherein the method further comprises introducing into the organelle a polynucleotide encoding at least one selectable marker selected from the group consisting of: a positive selectable marker, a negative selectable marker, and any combination thereof. 
     
     
         22 . The method of  claim 21 , wherein the method further involves growing the cell in the presence of a positive selection agent and selecting a cell that is homoplasmic for the altered genome of the organelle. 
     
     
         23 . The method of  claim 22 , wherein the method further involves growing the cell in the absence of the positive selection agent, followed by selecting a cell that lacks a non-integrated recombinant DNA construct. 
     
     
         24 . The method of  claim 22 , wherein the method further involves growing the cell in the absence of the positive selection agent, followed by growing the cell in the presence of a negative selection agent, followed by selecting a cell that lacks a non-integrated recombinant DNA construct. 
     
     
         25 . The method of  claim 6 , wherein the cell is a plant cell, wherein the organelle is a plastid or a mitochondrion, and wherein the method further comprises regenerating a plant from the plant cell comprising an altered organelle genome. 
     
     
         26 . The method of  claim 6 , wherein the cell is a yeast cell or an algal cell. 
     
     
         27 . The method of  claim 25 , wherein the method further comprises introducing a cytoplasmic male sterility gene into the organelle of the plant cell. 
     
     
         28 . (canceled) 
     
     
         29 . (canceled) 
     
     
         30 . A cell produced by the method of  claim 6 , wherein the cell is selected from the group consisting of: a yeast cell, an algal cell, a plant cell, an insect cell, a non-human animal cell, an isolated and purified human cell, and a mammalian tissue culture cell. 
     
     
         31 . A plant, seed, root, stem, leaf, flower, fruit, or bean produced by the method of  claim 25 , wherein the plant, seed, root, stem, leaf, flower, fruit, or bean comprises an organelle with an altered genome. 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . A method for altering the genome of an organelle, the method comprising:
 a. introducing into a cell:
 i. a polynucleotide encoding an RNA sequence comprising an organelle targeting RNA operably linked to a guide polynucleic acid, wherein the guide polynucleic acid directs a polynucleotide guided polypeptide to cleave a target sequence present in an organelle genome, wherein the polynucleotide is operably linked to at least one regulatory element; and either 
 ii. a second polynucleotide encoding a modified polynucleotide guided polypeptide, wherein the second polynucleotide is operably linked to at least one regulatory element, and wherein the modified polynucleotide guided polypeptide comprises a polynucleotide guided polypeptide operably linked to an organelle targeting peptide; wherein the organelle targeting RNA of (i) and the organelle targeting peptide of (ii) each target the same organelle; or 
 iii. a third polynucleotide, wherein the third polynucleotide is operably linked to at least one regulatory element, wherein the third polynucleotide encodes an RNA molecule comprising an organelle targeting RNA operably linked to an RNA sequence encoding a polynucleotide guided polypeptide; wherein the organelle targeting RNA of (i) and the organelle targeting RNA of (iii) each target the same organelle; and 
   b. growing the cell under conditions in which the polynucleotide of (i) and the second polynucleotide of (ii) or the third polynucleotide of (iii) are both expressed.   
     
     
         35 . The method of  claim 34 , further comprising introducing a polynucleotide comprising at least one donor polynucleotide into the organelle, wherein the at least one donor polynucleotide comprises at least one homologous sequence with respect to the organelle genome, wherein integration of all or part of the at least one donor polynucleotide into the organelle genome results in removal of the target site of the guide polynucleic acid. 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . A cell produced by the method of  claim 35 , wherein the cell is selected from the group consisting of: a yeast cell, an algal cell, a plant cell, an insect cell, a non-human animal cell, an isolated and purified human cell, and a mammalian tissue culture cell. 
     
     
         41 . A plant, seed, root, stem, leaf, flower, fruit, or bean produced by the method of  claim 35 , wherein the plant, seed, root, stem, leaf, flower, fruit, or bean comprises an organelle with an altered genome. 
     
     
         42 . A method for altering a genome of an organelle, the method comprising:
 (a) introducing into an organelle of a cell:
 (i) at least one guide RNA, wherein the at least one guide RNA directs a polynucleotide guided polypeptide to cleave at least one target sequence present in the genome of the organelle; 
 (ii) a polynucleotide guided polypeptide, wherein the polynucleotide guided polypeptide, when associated with the at least one guide RNA, cleaves the at least one target sequence; and 
 (iii) a replacement DNA; and 
   (b) selecting a cell comprising an organelle comprising the replacement DNA.   
     
     
         43 . The method of  claim 42 , wherein the replacement DNA of step (a) part (iii) comprises fragments of organellar DNA or a complete organellar DNA from a cultivar, line, sub-species and other species and is distinct from the genome of the organelle of step (a). 
     
     
         44 . The method of  claim 42 , wherein the at least one target sequence is not present in the replacement DNA. 
     
     
         45 . The method of  claim 42 , wherein after step (a) part (ii) and prior to step (a) part (iii), a cell is selected in which the genome of the organelle has been eliminated. 
     
     
         46 . A cell produced by the method of  claim 42 , wherein the cell is selected from the group consisting of: a yeast cell, an algal cell, a plant cell, an insect cell, a non-human animal cell, an isolated and purified human cell, and a mammalian tissue culture cell. 
     
     
         47 . A plant, seed, root, stem, leaf, flower, fruit, or bean produced by the method of  claim 42 , wherein the plant, seed, root, stem, leaf, flower, fruit, or bean comprises an organelle with an altered genome. 
     
     
         48 . The method of  claim 6 , wherein the recombinant DNA construct is linear and single-stranded, wherein the recombinant DNA construct is operably linked to a modified VirD2 protein, wherein the modified VirD2 protein comprises a VirD2 protein operably linked to an organelle targeting peptide, wherein the modified VirD2 protein has also been modified such that at least one native nuclear localization sequence of the VirD2 protein is no longer functional. 
     
     
         49 . The method of  claim 48 , wherein the recombinant DNA construct is operably linked to at least one modified VirE2 protein, wherein the at least one modified VirE2 protein comprises a VirE2 protein operably linked to an organelle targeting peptide, wherein the at least one modified VirE2 protein has also been modified such that at least one native nuclear localization sequence of the VirE2 protein is no longer functional. 
     
     
         50 . The method of  claim 6 , wherein the recombinant DNA construct is operably linked to at least one modified RecA protein, wherein the at least one modified RecA protein comprises a RecA protein operably linked to an organelle targeting peptide. 
     
     
         51 . The method of  claim 6 , wherein the recombinant DNA construct is operably linked to at least one chimeric polypeptide, wherein the at least one chimeric polypeptide comprises an organelle targeting peptide and a cell penetrating peptide. 
     
     
         52 . The method of  claim 35 , wherein the donor polynucleotide is introduced into the organelle by:
 a. introducing into the cell a polynucleotide encoding a modified RNA donor sequence, wherein the modified RNA donor sequence comprises an organelle targeting RNA operably linked to a donor RNA, wherein the modified RNA donor sequence further comprises a reverse transcriptase primer site, wherein the polynucleotide is operably linked to at least one regulatory element;   b. introducing into the cell a polynucleotide encoding a modified reverse transcriptase, wherein the modified reverse transcriptase comprises a reverse transcriptase operably linked to an organelle targeting peptide, wherein the polynucleotide is operably linked to at least one regulatory element, wherein the organelle targeting RNA of (a) and the organelle targeting peptide of (b) each target the same organelle; and   c. growing the cell under conditions wherein the polynucleotides of (a) and (b) are both expressed.