Compositions and methods for genetic manipulation of methanotrophs
OwnerJeremy Minshull, Jana Stumpe +2inventors
InventorsJeremy Minshull, Jana Stumpe, Sonny Zhang, Megan Hsi
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Abstract
The present disclosure provides compositions and methods for the genetic manipulation of methanotrophs utilizing a site-specific polynucleotide modification system.
Claims33
A method of altering the genome of a methanotrophic bacterium, comprising culturing under conditions and for a time sufficient to allow expression in a methanotrophic bacterium of a site-specific polynucleotide modification system;
- wherein the methanotrophic bacterium contains a heterologous nucleic acid molecule encoding the site-specific polynucleotide modification system that is operably linked to a regulatory element in a vector, the nucleic acid molecule comprising:
- (a) a first heterologous nucleic acid molecule encoding a modification polypeptide, wherein the modification polypeptide comprises a targeting RNA binding domain and a site-specific nuclease domain, and
- (b) a second heterologous nucleic acid molecule encoding a targeting RNA, wherein the targeting RNA comprises a duplex-forming region and a DNA-targeting domain,
- wherein the complex of the expressed modification polypeptide with the expressed targeting RNA binds to and cleaves a genomic target sequence of the methanotrophic bacterium,
- thereby site-specifically altering the genome of the methanotrophic bacterium.
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A modified methanotrophic bacterium, comprising a heterologous nucleic acid molecule encoding a site-specific polynucleotide modification system that is operably linked to a regulatory element in a vector, the nucleic acid molecule comprising:
- (a) a first heterologous nucleic acid molecule encoding a modification polypeptide, wherein the modification polypeptide comprises a targeting RNA binding domain and a site-specific nuclease domain,
- (b) a second heterologous nucleic acid molecule encoding a targeting RNA, wherein the targeting RNA comprises a duplex-forming region and a DNA-targeting domain, and
- (c) a third heterologous nucleic acid molecule comprising an integration polynucleotide,
- wherein the expressed modification polypeptide can associate with the expressed targeting RNA to form a complex capable of binding to and cleaving a genomic target sequence of the methanotrophic bacterium.
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Dependencies are inferred from the claim text.