Tuning microbial populations with programmable nucleases

Abstract

Various aspects and embodiments of the invention are directed to methods and compositions for reversing antibiotic resistance or virulence in and/or destroying pathogenic microbial cells such as, for example, pathogenic bacterial cells. The methods include exposing microbial cells to a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease targeted to one or multiple genes of interest.

What is claimed is: 
     
         1 . A method, comprising delivering to microbial cells in vivo a delivery vehicle with at least one nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease that targets a gene or nucleotide sequence of interest. 
     
     
         2 .- 24 . (canceled) 
     
     
         25 . A phagemid, a bacteriophage, or a donor bacterial cell comprising an engineered autonomously distributed circuit that contains at least one nucleic acid encoding a programmable nuclease that targets a gene or nucleotide sequence of interest. 
     
     
         26 . The phagemid, bacteriophage, or donor bacterial cell of  claim 25 , wherein the programmable nuclease is an RNA-guided nuclease. 
     
     
         27 . The phagemid, bacteriophage, or donor bacterial cell of  claim 26 , wherein the engineered autonomously distributed circuit contains a Cas9 nuclease, a guide RNA (gRNA), and/or a transactivating small RNA (tracrRNA). 
     
     
         28 . The phagemid, bacteriophage, or donor bacterial cell of  claim 27 , wherein the gRNA and the tracrRNA form a chimeric single-guide RNA molecule. 
     
     
         29 . The phagemid, bacteriophage, or donor bacterial cell of  claim 25 , wherein the programmable nuclease is a transcriptional activator like (TAL)-effector nuclease (TALEN). 
     
     
         30 . The phagemid, bacteriophage, or donor bacterial cell of  claim 25 , wherein the programmable nuclease is a zinc finger nuclease (ZFN). 
     
     
         31 . The phagemid, bacteriophage, or donor bacterial cell of  claim 25 , wherein the gene or nucleotide sequence of interest is located episomally. 
     
     
         32 . The phagemid, bacteriophage, or donor bacterial cell of  claim 25 , wherein the gene or nucleotide sequence of interest is located chromosomally. 
     
     
         33 . The phagemid, bacteriophage, or donor bacterial cell of  claim 25 , wherein the gene or nucleotide sequence of interest is a virulence factor gene or a toxin gene. 
     
     
         34 . The phagemid, bacteriophage, or donor bacterial cell of  claim 25 , wherein the gene or nucleotide sequence of interest is an antibiotic resistance gene. 
     
     
         35 . The phagemid, bacteriophage, or donor bacterial cell of  claim 25 , wherein the gene or nucleotide sequence of interest is a remodeling gene. 
     
     
         36 . The phagemid, bacteriophage, or donor bacterial cell of  claim 25 , wherein the gene or nucleotide sequence of interest is a modulatory gene. 
     
     
         37 . The phagemid, bacteriophage, or donor bacterial cell of  claim 25 , wherein the nucleotide sequence of interest comprises 16S ribosomal DNA (rDNA). 
     
     
         38 . The phagemid, bacteriophage, or donor bacterial cell of  claim 25 , wherein the programmable nuclease is operably linked to an inducible promoter. 
     
     
         39 .- 66 . (canceled) 
     
     
         67 . The donor bacterial cell of  claim 25 , wherein the donor bacterial cell is a probiotic and/or commensal bacterial cell. 
     
     
         68 . A nucleic acid encoding an engineered autonomously distributed circuit that contains a programmable nuclease that targets a virulence factor or an antibiotic resistance gene. 
     
     
         69 .- 76 . (canceled) 
     
     
         77 . A phagemid or bacteriophage comprising the nucleic acid of  claim 68 . 
     
     
         78 . (canceled) 
     
     
         79 . A library comprising a plurality of nucleic acids of  claim 68 , each encoding an engineered autonomously distributed circuit that contains a programmable nuclease that targets a virulence factor or an antibiotic resistance gene. 
     
     
         80 .- 88 . (canceled)