Organelle genome modification using polynucleotide guided endonuclease
Inventors
Sakai, Hajime • Yoo, Byung-Chun • OROZCO, JR., Emil Meyer • Kishore, Ganesh • YADAV, Narendra Singh
Assignees
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Publication Number
US-12173295-B2
Publication Date
2024-12-24
Expiration Date
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.
Core Innovation
The invention relates to a method for altering a genome of a mitochondrion or a plastid in a cell. The method introduces into the mitochondrion or the plastid a polynucleotide encoding at least one guide ribonucleic acid and a polynucleotide guided polypeptide, wherein the guide RNA directs the polynucleotide guided polypeptide to cleave at least one target sequence present in the genome when the polynucleotide guided polypeptide is associated with the guide RNA.
The method further includes introducing a replacement deoxyribonucleic acid comprising at least two regions of homology to the mitochondrion or plastid genome, wherein each of the at least two regions of homology comprises at least 74 nucleotides. The replacement DNA is configured such that an altered mitochondrion genome or an altered plastid genome contains the replacement DNA after the method is performed.
The method includes selecting an altered cell in which the altered mitochondrion genome or the altered plastid genome comprises the replacement DNA. The disclosed approach supports organelle genome modification of mitochondrion or plastid genomes using guide RNA-directed cleavage combined with replacement DNA containing homologous regions.
Claims Coverage
The provided independent claim coverage centers on organelle genome modification in mitochondria or plastids using guide RNA-directed cleavage machinery and replacement DNA with homologous regions, followed by selection of altered cells. Two inventive features are consistently identified across the inputs.
Guide RNA-directed cleavage in mitochondrion or plastid genome
A polynucleotide encoding at least one guide ribonucleic acid and a polynucleotide guided polypeptide, wherein the guide RNA directs the polynucleotide guided polypeptide to cleave at least one target sequence present in the genome of the mitochondrion or the plastid when the polypeptide is associated with the guide RNA.
Replacement DNA with at least two regions of homology of at least 74 nucleotides
A replacement deoxyribonucleic acid comprising at least two regions of homology to the mitochondrion or plastid genome, wherein each of the at least two regions of homology comprises at least 74 nucleotides, followed by selecting an altered cell in which the altered mitochondrion genome or altered plastid genome comprises the replacement DNA.
The inventive scope centers on guide RNA-directed cleavage of a mitochondrion or plastid target sequence, replacement DNA having at least two homologous regions each comprising at least 74 nucleotides, and selection of an altered cell containing the replacement DNA.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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