Compositions and Methods for Targeted Gene Disruption in Prokaryotes
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Abstract
The present disclosure relates to engineered bacteriophage vector compositions comprising nucleic acids that express recombinant nucleases. Also provided are methods of using engineered bacteriophage vectors to effect genomic disruption or targeted gene disruption in prokaryotes. The disclosed compositions and methods are useful for reducing antibiotic resistance in bacteria cells.
46 Citations
27 Claims
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1-13. -13. (canceled)
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14. A bacteriophage comprising a polynucleotide that expresses:
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(a) an RNA-directed DNA-binding polypeptide comprising a nuclease module; and (b) a targeting module comprising a guide RNA, wherein the targeting module tethers the RNA-directed DNA-binding polypeptide to a target DNA sequence within a prokaryotic host cell, thereby producing a double-strand break within the target sequence. - View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22)
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23. A method of restricting growth of a host cell, comprising contacting the host cell to a bacteriophage, where in the bacteriophage specifically binds to the host cell and introduces a polynucleotide into the host cell, wherein the polynucleotide encodes:
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(i) an RNA-directed DNA-binding polypeptide comprising a nuclease module; and (ii) a targeting module comprising a guide RNA; wherein the introduction of the polynucleotide into the host cell induces the expression of the RNA-directed DNA-binding polypeptide and the targeting module within the host cell, and wherein the targeting module directs the RNA-directed DNA-binding polypeptide to a target DNA sequence within the host cell, thereby introducing a double-strand break at the target DNA sequence and restricting growth of the host cell. - View Dependent Claims (24, 25, 26)
wherein each guide RNA specifically directs an RNA-directed DNA-binding polypeptide to one of a plurality of DNA sequences within the host cell, thereby introducing a plurality of double-strand breaks within the plurality of DNA sequences within the host cell, thereby killing the host cell.
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27. A method of decreasing the relative representation of a specific strain of bacteria within a heterogenous population of bacteria, comprising contacting the heterogenous population of bacteria with a bacteriophage comprising a polynucleotide that expresses
(a) an RNA-directed DNA-binding polypeptide comprising a nuclease module; - and
(b) a targeting module comprising a guide RNA, wherein the targeting module tethers the RNA-directed DNA-binding polypeptide to a target DNA sequence within, thereby producing a double-strand break within the target sequence, wherein the target sequence is unique to the specific strain of bacteria, among the heterogenous population of bacteria.
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Specification