WO2019213592A1 - Methods and compositions for killing a target bacterium - Google Patents

Methods and compositions for killing a target bacterium Download PDF

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Publication number
WO2019213592A1
WO2019213592A1 PCT/US2019/030695 US2019030695W WO2019213592A1 WO 2019213592 A1 WO2019213592 A1 WO 2019213592A1 US 2019030695 W US2019030695 W US 2019030695W WO 2019213592 A1 WO2019213592 A1 WO 2019213592A1
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WIPO (PCT)
Prior art keywords
bacteriophage
crispr
cas system
target
bacterium
Prior art date
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PCT/US2019/030695
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English (en)
French (fr)
Inventor
Paul M. GAROFOLO
David G. Ousterout
Kurt SELLE
Sandi WONG
Hannah Hewitt TUSON
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Locus Biosciences Inc
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Locus Biosciences Inc
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Publication date
Priority to CA3099316A priority Critical patent/CA3099316A1/en
Application filed by Locus Biosciences Inc filed Critical Locus Biosciences Inc
Priority to JP2021510294A priority patent/JP2021522857A/ja
Priority to CN201980044965.1A priority patent/CN112424363A/zh
Priority to KR1020207035088A priority patent/KR20210018273A/ko
Priority to AU2019262200A priority patent/AU2019262200A1/en
Priority to EP19796615.3A priority patent/EP3788152A4/en
Publication of WO2019213592A1 publication Critical patent/WO2019213592A1/en
Priority to US16/899,436 priority patent/US20200354690A1/en
Anticipated expiration legal-status Critical
Priority to US17/469,648 priority patent/US20220002681A1/en
Priority to US17/858,899 priority patent/US20220387531A1/en
Priority to US17/858,885 priority patent/US20220380736A1/en
Priority to US17/932,812 priority patent/US20230038106A1/en
Ceased legal-status Critical Current

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Definitions

  • the method for killing a target bacterium comprises introducing into a target bacterium a bacteriophage comprising: a first nucleic acid encoding a spacer sequence or a crRNA transcribed therefrom, wherein the spacer sequence is complementary to a target nucleotide sequence from a target gene in the target bacterium; and a gene that is capable of inducing lysis of the target bacterium.
  • the target bacterium is killed by lytic activity of the bacteriophage or activity of a CRISPR-Cas system using the spacer sequence or the crRNA transcribed therefrom.
  • the first nucleic acid sequence is a CRISPR array further comprising at least one repeat sequence.
  • the bacteriophage further comprises a second nucleic acid encoding a transcriptional activator for the CRISPR-Cas system.
  • the gene is endogenous or exogenous. In some embodiments, the
  • transcriptional activator is regulated by quorum sensing (QS) signals.
  • the transcriptional activator is a protein involved in sensing stress of a bacterium membrane.
  • the protein involved in sensing stress is response regulator BaeSR.
  • the transcriptional activator is a protein that stabilizes Cas.
  • the protein that stabilizes Cas is heat shock protein G (HtpG).
  • HtpG heat shock protein G
  • transcriptional activator is a metabolic sensing protein.
  • the metabolic sensing protein is cAMP receptor protein (CRP).
  • the CRP is sensitive to cyclic AMP (cAMP).
  • the metabolic sensing protein is a sigma factor.
  • the sigma factor is RpoN (s 54 ).
  • the transcriptional activator disrupts the activity of an inhibitory element.
  • the inhibitory element comprises heat-stable nucleoid-structuring protein (H-NS), leucine responsive regulatory protein (LRP), or CodY.
  • the inhibitory element is a transcriptional repressor.
  • the transcriptional repressor is a global transcriptional repressor.
  • the transcriptional activator comprises LeuO or a polypeptide having at least 75% sequence homology with SEQ ID NO: 1.
  • the transcriptional activator comprises CD2983 or a polypeptide having at least 75% sequence homology with SEQ ID NO: 2.
  • the CRISPR-Cas system is endogenous to the target bacterium. In some embodiments, the CRISPR-Cas system is exogenous to the target bacterium.
  • the CRISPR-Cas system is a type I CRISPR-Cas system, a type II CRISPR-Cas system, or a type III CRISPR-Cas system. In some embodiments, the CRISPR-Cas system comprises the type I CRISPR-Cas system.
  • the target nucleotide sequence comprises all or a part of a promoter sequence for the target gene. In some embodiments, the target nucleotide sequence comprises all or a part of a nucleotide sequence located on a coding strand of a transcribed region of the target gene.
  • the target nucleotide sequence is at least a portion of an essential gene that is needed for survival of the target bacterium.
  • the essential gene is Jsf a cpP, gapA, infA , sec K, csrA, Irml) ftsA , fits A , glyQ, eno, or nusG.
  • the at least one repeat sequence is operably linked to the at least one spacer sequence at either its 5’ end or its 3’ end.
  • the target bacterium is killed solely by the lytic activity of the bacteriophage. In some embodiments, the target bacterium is killed solely by the activity of the CRISPR-Cas system.
  • the target bacterium is killed by both the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system in combination. In some embodiments, the target bacterium is killed by the activity of the CRISPR- Cas system independently of the lytic activity of the bacteriophage. In some embodiments, the activity of the CRISPR-Cas system supplements or enhances the lytic activity of the bacteriophage. In some embodiments, the spacer nucleotide sequence overlaps with a second spacer sequence. In some embodiments, the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic.
  • the lytic activity of the bacteriophage, the activity of the CRISPR-Cas system, or both is modulated by a concentration of the bacteriophage.
  • the bacteriophage infects multiple bacterial strains.
  • the bacteriophage is an obligate lytic bacteriophage.
  • the bacteriophage is a temperate bacteriophage that is rendered lytic.
  • the bacteriophage does not confer any new properties onto the target bacterium beyond cellular death caused by the lytic activity of the bacteriophage and/or the activity of the CRISPR-Cas array.
  • the target bacterium is C. difficile.
  • the bacteriophage is fO ⁇ 146 or fO ⁇ 24- 2.
  • the target bacterium is E. coli.
  • the bacteriophage is T4, T7, or T7m.
  • the first nucleic acid encoding a spacer sequence or a crRNA is inserted into a non-essential bacteriophage gene.
  • the non-essential gene is gp49 , gp75, or hoc.
  • the non-essential gene is gpO.7, gp4.3, gp4.5, or gp4.7.
  • the non-essential gene is gp0.6, gp0.65, gpO.7, gp4.3, or gp4.5.
  • methods for killing a plurality of target bacteria such as in a mixed population of bacteria comprising the target bacteria and non-target bacteria (e.g., in therapeutic and/or environmental treatment processes, such as described herein).
  • the target bacteria are treated according to any process described herein (e.g., for killing target bacterium), and a first population of the target bacteria is killed by lytic activity of the bacteriophage and a second population of the target bacteria is killed by activity of a CRISPR-Cas system using the spacer sequence or the crRNA transcribed therefrom (e.g., wherein the non-target bacteria is not killed (e.g., killed at a lesser rate than the target bacteria, such as at 50%, the rate, less than 25% the rate, less than 10% the rate, or less than 20% killed, less than 10% killed, less than 5% killed, or the like).
  • the method comprises:
  • the transcriptional activator is regulated by quorum sensing (QS) signals.
  • QS quorum sensing
  • the transcriptional activator is a protein involved in sensing stress to a bacterium membrane.
  • the protein involved in sensing stress is response regulator BaeSR.
  • the transcriptional activator is a protein that stabilizes Cas.
  • the protein that stabilizes Cas is heat shock protein G (HtpG).
  • the transcriptional activator is a metabolic sensing protein.
  • the metabolic sensing protein is cAMP receptor protein (CRP). In some embodiments, the CRP is sensitive to cyclic AMP (cAMP). In some embodiments, the metabolic sensing protein is a sigma factor. In some embodiments, the sigma factor is RpoN (s54).
  • the transcriptional activator disrupts the activity of an inhibitory element.
  • the inhibitory element is heat-stable nucleoid-structuring protein (H-NS), leucine responsive regulatory protein (LRP), or CodY. In some embodiments, the inhibitory element is a transcriptional repressor. In some embodiments, the transcriptional repressor is a global transcriptional repressor.
  • the transcriptional activator comprises LeuO or a polypeptide having at least 75% sequence homology with SEQ ID NO: 1. In some embodiments, the transcriptional activator comprises CD2983 or a polypeptide having at least 75% sequence homology with SEQ ID NO: 2. In some embodiments, the CRISPR-Cas system is endogenous. In some embodiments, the CRISPR-Cas system is exogenous. In some embodiments, the CRISPR-Cas system is a type I CRISPR-Cas system, a type II CRISPR-Cas system, or a type III CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type I CRISPR-Cas system.
  • the bacteriophage infects multiple bacterial strains. In some embodiments, the bacteriophage is an obligate lytic bacteriophage. In some embodiments, the bacteriophage is a temperate bacteriophage that is rendered lytic. In some embodiments, the target bacterium is C. difficile. In some embodiments, the bacteriophage is c
  • the non-essential gene is gp49. In some embodiments, the non-essential gene is gp75. In some embodiments, the non-essential gene is hoc. In some embodiments, the non-essential gene is gpO.7, gp4.3, gp4.5, or gp4.7. In some
  • the non-essential gene is gp0.6, gp0.65, gpO.7, gp4.3, or gp4.5.
  • the method comprises introducing into a target bacterium a bacteriophage comprising: lytic activity, and a first nucleic acid sequence encoding an anti-CRISPR polypeptide.
  • the anti-CRISPR polypeptide enhances the lytic activity of the bacteriophage (e.g., as determined by how fast the target bacterium is killed).
  • the anti-CRISPR polypeptide inactivates a CRISPR-Cas system.
  • the anti-CRISPR polypeptide inactivates the CRISPR-Cas system using a process comprising gene regulation interference.
  • the anti-CRISPR polypeptide inactivates the CRISPR-Cas system using a process comprising nuclease recruitment interference.
  • the CRISPR-Cas system is a type I CRISPR-Cas system, a type II CRISPR-Cas system, or a type III CRISPR-Cas system.
  • the CRISPR-Cas system is a type I CRISPR-Cas system.
  • the anti-CRISPR polypeptide binds directly or indirectly to a Cascade or Cascade like complex.
  • the anti-CRISPR polypeptide is a truncated protein, a fusion protein, a dimer protein, or mutated protein.
  • the bacteriophage further comprises a second nucleic acid encoding a CRISPR array.
  • the CRISPR array comprises at least one repeat sequence and at least one spacer sequence that is complementary to a target nucleotide sequence from a target gene in the target bacterium.
  • methods of killing target bacteria e.g., in a mixed population of bacteria comprising target bacteria and non-target bacteria).
  • the method comprises introducing into target bacteria a bacteriophage having lytic activity and comprising a first nucleic acid sequence encoding an anti-CRISPR polypeptide.
  • the anti-CRISPR polypeptide enhances the lytic activity of the bacteriophage (e.g., as measured by number of target bacteria killed in a given amount of time).
  • bacteriophages comprising: a first nucleic acid encoding a spacer sequence or a crRNA transcribed therefrom, wherein the spacer sequence is complementary to a (e.g., target) nucleotide sequence from a (e.g., target) gene in a (e.g., target) bacterium; and a gene that is capable of inducing lysis of the (e.g., target) bacterium.
  • the target bacterium is killed by the lytic activity of the bacteriophage or activity of a CRISPR-Cas system using the spacer sequence or the crRNA transcribed therefrom.
  • the bacteriophage further comprises a second nucleic acid encoding a transcriptional activator for the CRISPR-Cas system.
  • the transcriptional activator is regulated by quorum sensing (QS) signals.
  • the transcriptional activator is a protein involved in sensing stress of a bacterium membrane.
  • the protein is response regulator BaeSR.
  • the transcriptional activator is a protein that stabilizes Cas.
  • the protein that stabilizes Cas is heat shock protein G (HtpG).
  • the transcriptional activator is a metabolic sensing protein.
  • the metabolic sensing protein is cAMP receptor protein (CRP).
  • the CRP is sensitive to cyclic AMP (cAMP).
  • the metabolic sensing protein is a sigma factor.
  • the sigma factor is RpoN (s 54 ).
  • the transcriptional activator disrupts the activity of an inhibitory element of the target bacterium.
  • the inhibitory element is heat-stable nucleoid-structuring protein (H-NS), leucine responsive regulatory protein (LRP), or CodY.
  • the inhibitory element is a transcriptional repressor.
  • the transcriptional repressor is a global transcriptional repressor.
  • the transcriptional activator comprises LeuO or a polypeptide having at least 75% sequence homology with SEQ ID NO: 1. In some embodiments, the transcriptional activator comprises CD2983 or a polypeptide having at least 75% sequence homology with SEQ ID NO: 2. In some embodiments, the CRISPR-Cas system is endogenous. In some embodiments, the CRISPR-Cas system is exogenous. In some embodiments, the CRISPR-Cas system is a type I CRISPR-Cas system, a type II CRISPR-Cas system, or a type III CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type I CRISPR-Cas system.
  • the target nucleotide sequence comprises all or a part of a promoter sequence for the target gene. In some embodiments, the target nucleotide sequence comprises all or a part of a nucleotide sequence located on a coding strand of a transcribed region of the target gene. In some embodiments, the target nucleotide sequence is essential. In some embodiments, the essential gene is Jsf a cpP, gapA, infA , sec K, csrA, Irml) ftsA , fits A , glyQ, eno, or nusG. In some embodiments, the target nucleotide sequence is a non-essential gene.
  • the first nucleic acid sequence is a CRISPR array comprising at least one repeat sequence.
  • the at least one repeat sequence is operably linked to the spacer sequence at either its 5’ end or its 3’ end.
  • the bacteriophage infects multiple bacterial strains.
  • the bacteriophage is an obligate lytic bacteriophage.
  • the bacteriophage is a temperate bacteriophage that is rendered lytic.
  • the temperate bacteriophage is rendered lytic by the removal, replacement, or inactivation of one or more lysogeny genes.
  • the target bacterium is C. difficile.
  • the bacteriophage is c
  • the target bacterium is E. coli.
  • the bacteriophage is T4, T7, or T7m.
  • the first nucleic acid encoding a spacer sequence or a crRNA is inserted into a non- essential gene.
  • the non-essential gene is gp49.
  • the non-essential gene is gp75.
  • the non-essential gene is hoc.
  • the non-essential gene is gpO.7, gp4.3, gp4.5 , or gp4.7.
  • non- essential gene is gp0.6, gp0.65, gpO.7, gp4.3, or gp4.5. Also disclosed herein, in some
  • compositions comprising the bacteriophage disclosed herein, and a pharmaceutically acceptable excipient.
  • the pharmaceutical compositions is in a form of a tablet, a liquid, a syrup, an oral formulation, an intravenous formulation, an intranasal formulation, an ocular formulation, an otic formulation, a subcutaneous formulation, an inhalable respiratory formulation, a suppository, or any combination thereof.
  • methods of treating a disease in a subject comprising administering the bacteriophage disclosed herein to the subject.
  • the subject is a mammal.
  • the disease is a bacterial infection.
  • a bacteria causing the bacterial infection is an Acinetobacter species, an Actinomyces species, Burkholderia cepacia complex, a Campylobacter species, a Candida species, Clostridium difficile , Corynebacterium minutissium, Corynebacterium pseudodiphtheriae, Corynebacterium stratium, Corynebacterium group Gl, Corynebacterium group G2, Enter obacteriaceae, an Enterococcus species, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, a Moraxella species, Mycobacterium tuberculosis complex, Neisseria gonorrhoeae , Neisseria meningitidis , a non-tuberculous mycobacteria species, a Porphyromonas species, Prevotella melaninogenicus, a Pseudomonas species, Salmonella typhimurium
  • the bacterium is a drug resistant bacterium that is resistant to at least one antibiotic. In some embodiments, the bacterium is a multi drug resistant bacterium that is resistant to at least one antibiotic. In some embodiments, the bacterium is Pseudomonas. In some embodiments, the bacterium is staphylococcus.
  • the bacterium is Escherichia coli. In some embodiments, the bacterium is Clostridium difficile. In some embodiments, the bacterium is methicillin resistant. In some embodiments, the bacterium is methicillin resistant staphylococcus aureus. In some embodiments, the bacterium is multidrug resistant Pseudomonas Aeruginosa.
  • the antibiotic comprises a cephalosporin, a fluoroquinolone, a carbapenem, a colistin, an aminoglycoside, vancomycin, streptomycin, or methicillin. In some embodiments, the administering is intra-arterial, intravenous, intramuscular, oral, subcutaneous, topical, inhalation, intravesical or any combination thereof.
  • bacteriophages comprising a nucleic acid encoding a transcriptional activator for a CRISPR-Cas system in a (e.g., target) bacterium.
  • the transcriptional activator is regulated by quorum sensing (QS) signals.
  • QS quorum sensing
  • the transcriptional activator is a protein involved in sensing stress to a bacterium membrane.
  • the protein involved in sensing stress is response regulator BaeSR.
  • the transcriptional activator is a protein that stabilizes Cas.
  • the protein that stabilizes Cas is heat shock protein G (HtpG).
  • the transcriptional activator is a metabolic sensing protein.
  • the metabolic sensing protein is cAMP receptor protein (CRP).
  • the CRP is sensitive to cyclic AMP (cAMP).
  • the metabolic sensing protein is a sigma factor.
  • the sigma factor is RpoN (s54).
  • the transcriptional activator disrupts the activity of an inhibitory element.
  • the inhibitory element is heat-stable nucleoid-structuring protein (H-NS), leucine responsive regulatory protein (LRP), or CodY.
  • the inhibitory element is a transcriptional repressor.
  • the transcriptional repressor is a global transcriptional repressor.
  • the transcriptional activator comprises LeuO or a polypeptide having at least 75% sequence homology with SEQ ID NO: 1.
  • the transcriptional activator comprises CD2983 or a polypeptide having at least 75% sequence homology with SEQ ID NO: 2.
  • the CRISPR-Cas system is endogenous. In some embodiments, the
  • the CRISPR-Cas system is exogenous.
  • the CRISPR-Cas system is a type I CRISPR-Cas system, a type II CRISPR-Cas system, or a type III CRISPR-Cas system.
  • the CRISPR-Cas system is a type I CRISPR-Cas system.
  • the bacteriophage infects multiple bacterial strains.
  • the bacteriophage is an obligate lytic bacteriophage.
  • the bacteriophage is a temperate bacteriophage that is rendered lytic.
  • the target bacterium is C. difficile. In some
  • the bacteriophage is fO ⁇ 146 or fO ⁇ 24-2.
  • the target bacterium is E. coli.
  • the bacteriophage is T4, T7, or T7m.
  • the nucleic acid encoding a transcriptional activator is inserted into a non-essential bacteriophage gene.
  • the non-essential gene is gp49.
  • the non-essential gene is gp75.
  • the non-essential gene is hoc.
  • the non-essential gene is gpO.7, gp4.3, gp4.5 , or gp4.7.
  • the non-essential gene is gp0.6, gp0.65, gpO.7, gp4.3 , or gp4.5.
  • pharmaceutical compositions comprising the bacteriophage disclosed herein, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is in a form of a tablet, a liquid, a syrup, an oral formulation, an intravenous formulation, an intranasal formulation, an ocular formulation, an otic formulation, a subcutaneous formulation, an inhalable respiratory formulation, a suppository, and any combination thereof.
  • methods of treating a disease in a subject comprising administering the bacteriophage to the subject.
  • the subject is a mammal.
  • the disease is a bacterial infection.
  • a bacteria causing the bacterial infection is an infectious organism.
  • Acinetobacter species an Actinomyces species, Burkholderia cepacia complex, a Campylobacter species, a Candida species, Clostridium difficile , Corynebacterium minutissium, Corynebacterium pseudodiphtheriae, Corynebacterium stratium, Corynebacterium group Gl, Corynebacterium group G2, Enter obacteriaceae, an Enterococcus species, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, a Moraxella species, Mycobacterium tuberculosis complex, Neisseria gonorrhoeae , Neisseria meningitidis , a non-tuberculous mycobacteria species, a Porphyromonas species, Prevotella melaninogenicus, a Pseudomonas species, Salmonella typhimurium, Serratia marcescens Staphylococcus aure
  • the bacterium is a drug resistant bacterium that is resistant to at least one antibiotic.
  • the bacterium is a multi-drug resistant bacterium that is resistant to at least one antibiotic.
  • the bacterium is Pseudomonas.
  • the bacterium is staphylococcus.
  • the bacterium is Escherichia coli.
  • the bacterium is Clostridium difficile.
  • the bacterium is methicillin resistant.
  • the bacterium is methicillin resistant staphylococcus aureus.
  • the bacterium is multidrug resistant Pseudomonas Aeruginosa.
  • the antibiotic comprises a cephalosporin, a fluoroquinolone, a carbapenem, a colistin, an aminoglycoside, vancomycin, streptomycin, or methicillin.
  • the administering is intra-arterial, intravenous, intramuscular, oral, subcutaneous, topical, inhalation, or any combination thereof.
  • bacteriophages comprising: lytic activity, and a first nucleic acid sequence encoding an anti-CRISPR polypeptide.
  • the anti-CRISPR polypeptide enhances the lytic activity of the bacteriophage.
  • the anti-CRISPR polypeptide inactivates a CRISPR-Cas system.
  • the anti- CRISPR polypeptide inactivates the CRISPR-Cas system using a process comprising gene regulation interference.
  • the anti-CRISPR polypeptide inactivates the CRISPR-Cas system using a process comprising nuclease recruitment interference.
  • the CRISPR-Cas system is a type I CRISPR-Cas system, a type II CRISPR-Cas system, or a type III CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type I CRISPR-Cas system.
  • the anti-CRISPR polypeptide binds directly or indirectly to a Cascade or Cascade-like complex. In some embodiments, the anti-CRISPR polypeptide is a truncated protein, a fusion protein, a dimer protein, or mutated protein.
  • the bacteriophage further comprises a second nucleic acid encoding a CRISPR array.
  • CRISPR array comprises at least one repeat sequence and at least one spacer sequence that is complementary to a target nucleotide sequence from a target gene in the target bacterium.
  • pharmaceutical compositions comprising the bacteriophage disclosed herein, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is in a form of a tablet, a liquid, a syrup, an oral formulation, an intravenous formulation, an intranasal formulation, an ocular formulation, an otic formulation, a subcutaneous formulation, an inhalable respiratory formulation, a suppository, and any combination thereof.
  • a disease in a subject comprising administering the bacteriophage disclosed herein to the subject.
  • the subject is a mammal.
  • the disease is a bacterial infection.
  • a bacterium causing the bacterial infection is an
  • Acinetobacter species an Actinomyces species, Burkholderia cepacia complex, a Campylobacter species, a Candida species, Clostridium difficile , Corynebacterium minutissium, Corynebacterium pseudodiphtheriae, Corynebacterium stratium, Corynebacterium group Gl, Corynebacterium group G2, Enter obacteriaceae, an Enterococcus species, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, a Moraxella species, Mycobacterium tuberculosis complex, Neisseria gonorrhoeae , Neisseria meningitidis , a non-tuberculous mycobacteria species, a Porphyromonas species, Prevotella melaninogenicus, a Pseudomonas species, Salmonella typhimurium, Serratia marcescens Staphylococcus aure
  • the bacterium is a drug resistant bacterium that is resistant to at least one antibiotic. In some embodiments, the bacterium is a multi-drug resistant bacterium that is resistant to at least one antibiotic. In some embodiments, the bacterium is Pseudomonas. In some embodiments, the bacterium is staphylococcus. In some embodiments, the bacterium is Escherichia coli. In some embodiments, the bacterium is Clostridium difficile. In some embodiments, the bacterium is methicillin resistant. In some embodiments, the bacterium is methicillin resistant staphylococcus aureus.
  • the bacterium is multi drug resistant Pseudomonas Aeruginosa.
  • the antibiotic comprises a cephalosporin, a fluoroquinolone, a carbapenem, a colistin, an aminoglycoside, vancomycin, streptomycin, or methicillin.
  • the administering is intra-arterial, intravenous, intramuscular, oral, subcutaneous, topical, inhalation, or any combination thereof.
  • a temperate bacteriophage comprising a removal, replacement, or inactivation of at least one lysogeny gene, wherein the temperate bacteriophage is rendered lytic thereby killing the target bacterium.
  • the lysogeny gene is a repressor gene.
  • the lysogeny gene is cl phage repressor gene.
  • the bacteriophage infects multiple bacterial strains.
  • the target bacterium is C. difficile.
  • the bacteriophage is c
  • the bacteriophage further comprises a first nucleic acid encoding a spacer sequence or a crRNA transcribed therefrom.
  • the spacer sequence is complementary to a target nucleotide sequence from a target gene in the target bacterium.
  • the first nucleic acid sequence is a CRISPR array further comprising at least one repeat sequence.
  • the at least one repeat sequence is operably linked to the spacer sequence at either its 5’ end or its 3’ end.
  • the bacteriophage further comprises a second nucleic acid encoding a transcriptional activator for a CRISPR-Cas system.
  • the CRISPR-Cas system is endogenous to the target bacterium.
  • the CRISPR-Cas system is endogenous to the target bacterium.
  • the CRISPR-Cas system is exogenous to the target bacterium.
  • the CRISPR-Cas system is a type I CRISPR-Cas system, a type II CRISPR-Cas system, or a type III CRISPR-Cas system.
  • the CRISPR-Cas system comprises a type I CRISPR-Cas system.
  • the target nucleotide sequence comprises all or a part of a promoter sequence for the target gene.
  • the target nucleotide sequence comprises all or a part of a nucleotide sequence located on a coding strand of a transcribed region of the target gene.
  • the target nucleotide sequence comprises at least a portion of an essential gene that is needed for survival of the target bacterium.
  • the target bacterium is killed by both lytic activity of the bacteriophage and activity of the CRISPR-Cas system in combination.
  • activity of the CRISPR-Cas system supplements or enhances lytic activity of the bacteriophage.
  • the target bacterium is killed by activity of a CRISPR- Cas system independently of lytic activity of the bacteriophage.
  • lytic activity of the bacteriophage and activity of a CRISPR-Cas system are synergistic.
  • lytic activity of the bacteriophage, activity of a CRISPR-Cas system, or both is modulated by a concentration of the bacteriophage.
  • compositions comprising: (a) a temperate bacteriophage comprising a removal, replacement, or inactivation of at least one lysogeny gene; and (B) a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is in a form of a tablet, a liquid, a syrup, an oral formulation, an intravenous formulation, an intranasal formulation, an ocular formulation, an otic formulation, a subcutaneous formulation, an inhalable respiratory formulation, a suppository, and any combination thereof.
  • a pharmaceutical composition disclosed herein treats microbiome imbalance.
  • Figure 1 illustrates the workflow process for engineering a CRISPR-enhanced
  • Figure 2A exemplifies a schematic diagram of the linear alignment of CRISPR-Cas systems from within five strains of C. difficile with identification of the various CRISPR-Cas constituent components.
  • Figure 2B further exemplifies a schematic diagram of the CRISPR-Cas system operon structures for C. difficile strains 630 and R20291.
  • Figure 3A exemplifies the reduction in cell population for E. coli strain BW25113 when treated with a native wild-type bacteriophage T7m or a corresponding engineered crPhage T7m. Native wild-type bacteriophage shows bacteria killing by lytic activity.
  • Corresponding crT7m comprising LeuO and a CRISPR array for ftsA shows an additional 5-log improvement in bacterial killing activity over the wild-type bacteriophage T7m.
  • Figure 3B exemplifies the lethality of the CRISPR array for ftsA (a 7-log reduction) when administered directly to the bacteria independent of phage delivery.
  • Figure 4A exemplifies the reduction in cell population for C. difficile strain R2029lwhen treated with a native wild-type bacteriophage c
  • Native wild-type bacteriophage shows bacteria killing by lytic activity.
  • )CDl46 shows an additional l-log improvement in bacterial killing activity over the wild-type bacteriophage.
  • Figure 4B exemplifies the lethality of the CRISPR array for R20291-3 (a 3.5-log reduction) when administered directly to the bacteria independent of phage delivery.
  • Figure 5 exemplifies a bacterial lawn of C. difficile strain 069 which are insensitive to lysis by wild-type phage c
  • the lack of phage plaques on the left of the image indicates lack of killing by the wild-type phage c
  • )CDl46 on the right of the image is indicative of bacterial death due to the activity of the CRISPR array targeting R20291-3.
  • Figure 6A- Figure 6V exemplify the enhanced lethality of crPhage c
  • Figure 6A exemplifies crPhage c
  • Population growth of the various strains of C. difficile was monitored by optical density at 600nm for up to 6 hours following treatment with either the wild-type or CRISPR-array containing crPhage c
  • Figure 6B exemplifies crPhage c
  • Figure 6C exemplifies crPhage c
  • Figure 6D exemplifies crPhage c
  • Figure 6E exemplifies crPhage c
  • Figure 6F exemplifies crPhage c
  • Figure 6G
  • Figure 6S exemplifies crPhage c
  • Figure 6T exemplifies crPhage c
  • Figure 6U exemplifies crPhage c
  • Figure 6V exemplifies crPhage c
  • Figure 7A- Figure 7C exemplify the enhanced lethality of crPhage c
  • Population growth of the various strains of C. difficile was monitored by optical density at 600nm for up to 6 hours following treatment with either the wild-type or CRISPR-array containing crPhage c
  • Figure 7A exemplifies crPhage c
  • Figure 7B exemplifies crPhage c
  • Figure 7C exemplifies crPhage c
  • Figure 8A- Figure 8E exemplify the enhanced lethality of crPhage c
  • Population growth of the various strains of C. difficile was monitored using CFU reduction assay for up to 6 hours following treatment with either the wild-type or CRISPR-array containing crPhage c
  • CFU reduction assays provide enhanced quantitative sensitivity over optical density measurements.
  • Figure 8A exemplifies crPhage c
  • Figure 8B exemplifies crPhage c
  • Figure 8C exemplifies crPhage c
  • Figure 8D exemplifies crPhage c
  • Figure 8E exemplifies crPhage c
  • Figure 9 exemplifies the enhanced lethality of crPhage c
  • Population growth of C. difficile strain CD 19 was monitored using CFU reduction assay for up to 6 hours following treatment with either the wild- type or CRISPR-array containing crPhage c
  • the use of CFU reduction assay provides enhanced quantitative sensitivity over optical density measurements.
  • Figure 10 exemplifies a combinatorial comparison of crPhage c
  • )CD24-2 anti-bacterial activity was conducted for each crPhage individually as well as the when administered together. Co-administration showed improved killing efficacy as compared to treatment with a combination of both wild-type phages together.
  • Figure 11 exemplifies that the killing activity of the crPhage c
  • Figure 12A- Figure 12B exemplify an in-silico model predicting the number of resistant clones that emerge over time due to target site mutation as a function of the number of independent genes targeted by crRNAs. These models assume highly conservative assumptions that (1) mutational rate is independent of gene target and (2) that all 32 bases of crRNA match for activity. Two types of infection were modeled: an acute infection rising to a total burden of 10 10 CFU by doubling every 6 hours as seen in Figure 12A or an aggressive infection to a total burden of 10 14 CFU by doubling every 20 minutes as seen in Figure 12B. Both models show that 3 independent gene targets are sufficient to prevent mutational escape up to 28 days of infection length.
  • FIG. 13 exemplifies the strain coverage for a series of individual Type I-E crRNAs targeted to conserved regions of the E. coli genome.
  • crRNA array targets include the following genes in order of highest to lowest percentage of strain coverage: /. ' s/fl 00%), a cpP (99%), gapA (99%), inf A (99%), secY (99%), secY’2 (99%), csrA (99%), trmD (99 %),ftsA (99%), nusG (99%), fusA’2 (99 %),fusA (98%), glyQ (98%), eno (95%), gap A’2 (91%), eno’2 (89%), and nusG’2 (73%).
  • Figure 14 exemplifies the functional lethality assessment for a series of individual type I-E crRNAs with spacers targeted to conserved regions of the E. coli genome.
  • crRNA targets include the following genes: acpP, csrA, eno,fusA, gapA, glyQ, inf A, nusG, secY, , trmD, and Tsf
  • Figure 15 illustrates a schematic overview of three engineered CRISPR-enhanced bacteriophages against E. coli developed as a cocktail of three distinct obligate lytic bacteriophages that contain an identical DNA sequence encoding the transcriptional activator LeuO and a CRISPR- array.
  • the three engineered LeuO enhanced bacteriophages include crT4, crT7, and crT7m.
  • Figure 16A exemplifies the relative prevalence and distribution of canonical Type I-E or Type I-F CRISPR-Cas systems in E. coli.
  • Six hundred and twenty-five publicly available E. coli genomes were analyzed, spanning a diversity of strains including: uropathogenic . coli (UPEC), Shiga toxin producing E. coli , (STEC), Ol57:IT7 serotype . coli , diarrheagenic . coli (DEC), non- 157 O antigen type . coli , and enteropathogenic . coli (EPEC).
  • UPEC uropathogenic . coli
  • STEC Shiga toxin producing E. coli
  • Ol57:IT7 serotype . coli , diarrheagenic . coli (DEC), non- 157 O antigen type . coli , and enteropathogenic . coli (EPEC).
  • Figure 16B shows that approximately 78% (487/625) of all tested strains in Figure 16A have a complete CRISPR-Cas3 system, either type I-E or type I-F.
  • Figure 17 exemplifies non-lytic Ml3-derived phagemid delivery of LeuO enhanced CRISPR array constructs using a validated ftsA spacer sequence designed to test the dependence on LeuO expression for CRISPR-mediated lethality. Lethality of LeuO enhanced phagemid vectors was tested via transduction of M13 bacteriophages into a range of strains including a parent EMG2 containing a wild-type H-NS repressed E.
  • Type I-E CRISPR-Cas3 operon a BW25113- derivative lacking the H-NS repression motifs in the CRISPR-Cas3 operon (Ahns), a BW25113- derivative containing an overexpressed CRISPR-Cas3 operon (BW+Cas) and a BW25H3- derivative lacking Cas3 genes (BWACas).
  • Figure 18A- Figure 18C exemplify the improved lethality kinetics for the three LeuO enhanced crPhages (Figure 18A - crT7m, Figure 18B - crT4, and Figure 18C - crT7) comprising the transcriptional activator LeuO along with a CRISPR array as compared to their wild-type variants.
  • Target E. coli were incubated for 2 or 5 hours for crT7m, crT4 and crT7, respectively, in growth media at the indicated multiplicity-of-infection (ratio of phage to bacteria) for each phage. Significant differences were observed in CFU reduction across all three crPhages.
  • Figure 19A- Figure 19E exemplify dose-response in vitro kill curves for LeuO enhanced crPhages crT4, crT7, and crT7m against . coli strain MG1655 for each crPhage or crPhage cocktail and resultant changes in population were measured by optical density. E.
  • coli was grown to mid-log phase and treated with multiplicity-of-infection (MOI; ratio of phage to bacteria) as follows:
  • Figure 19A crT7 was incubated at MOIs of 0.0001, 0.01, and 1.0;
  • Figure 19B crT7m was incubated at MOIs of 0.0009, 0.09, and 9.0;
  • Figure 19C crT4 was incubated at MOIs of 0.0006, 0.06, and 6.0.
  • Each phage was mixed in equal amounts to create a crPhage cocktail (‘Cocktail’) and was incubated at MOIs (for each crPhage) of 0.0006, 0.06, and 6.0, as seen in Figure 19D.
  • Figure 19E is a zoomed in graph from Figure 19D.
  • FIG. 20 exemplifies the dose-dependent relationship observed between concentrations of LeuO enhanced crPhages and the resultant time-to-lysis in E. coli MG1655.
  • E. coli was grown to mid-log phase and treated with multiplicity-of-infection (MOI; ratio of phage to bacteria) as indicated for each crPhage.
  • MOI multiplicity-of-infection
  • MOI in excess of 1.0 resulted in the fastest time-to-lysis, presumably being limited by the lytic period of each phage.
  • the observed time-to-lysis for each phage was approximately 15-20 minutes for crT7m and crT7 and
  • Figure 21A- Figure 21G illustrate a schematic timeline representation of the dosing parameters for an in vivo tolerability of the three LeuO enhanced crPhages crT4, crT7, and crT7m. No overt toxicity was observed during veterinary observation and no measurable changes in body temperature or body weight were noted after dosing with each crPhage preparation as shown in Figure 21B- Figure 21G.
  • Figure 21B and Figure 21E illustrates crT7 body temperature and body weight after dosing, respectively.
  • Figure 21C and Figure 21F illustrates crT7M body temperature and body weight after dosing, respectively.
  • Figure 21D and Figure 21G illustrates crT4 body temperature and body weight after dosing, respectively.
  • Figure 22A- Figure 22D illustrate a schematic timeline representation for the treatment parameters for a murine in-vivo peritonitis model with E. coli with the LeuO enhanced crPhages and the results.
  • Female CD-l mice were injected intraperitoneally with a lethal dose of E. coli ( ⁇ 5xl0 7 CFU/ mouse of ATCC 8739) followed within 30 minutes by intraperitoneal injections of saline or crPhages.
  • Figure 23A- Figure 23E illustrate a schematic timeline representation for the treatment parameters for a murine in-vivo thigh infection model with E. coli treated with the LeuO enhanced crPhages for monitoring the effect upon bacterial bioburden reduction and the results. Mice were inoculated with 10 5 CFU of E.
  • Figure 24 illustrates in-vivo persistence and distribution of LeuO enhanced crPhages.
  • Female CD-l mice were treated with approximately l .OxlO 9 PFU/dose/phage of a crT7/crT7m cocktail by intraurethral instillation directly into the bladder.
  • 3 mice per time point were sacrificed and collected bladder, kidney, blood, liver and spleen whole tissue homogenates were diluted and subjected to phage titration analysis to quantify the total combined amount of crT7 and crT7m. Presence of active crPhage was detected up to 72 hours after dosing.
  • Figure 26A- Figure 26B exemplify reduction in lysogeny formation rate and reduction in viable CD 19 cells treated with a cl-knockout bacteriophage.
  • Figure 26A exemplifies a reduction in viable CD 19 cells when treated with Ac I CD24-2 as compared to CD 19 cells treated with WT CD24-2.
  • Figure 26B exemplifies a reduction in the percent lysogens in the surviving CD 19 cells that were treated with Acl CD24-2 as compared to in CD 19 cells treated with WT CD24-2.
  • Figure 27A- Figure 27B exemplify comparative CFU reduction in bladder via i.v. delivery ( Figure 27A) or local delivery (Figure 27B) of wild type phage (wtPhage), crPhage and ciprofloxacin. Results exemplify improved CFU reduction with crPhage compared to wtPhage.
  • Figure 28A- Figure 28B exemplify dose response of crPhage treatment in bladder (Figure 28A) and in kidney (Figure 28B). crPhage were delivered intraurethrally.
  • Figure 29 illustrates a schematic of an exemplary UTI efficacy study with research-grade material compared WT versus engineered cocktail via intravenous (IV), intra-urethral (IU), or concurrent IV and IU delivery.
  • IV intravenous
  • IU intra-urethral
  • IV and IU delivery concurrent IV and IU delivery.
  • Figure 30A - Figure 30D exemplify a UTI efficacy study demonstrating reduction in E.coli in bladder (Figure 30A and Figure 30B) and kidney ( Figure 30C and Figure 30D) following intraurethral (IU) or intravenous (IV) administration. Measurements were taken 54 hour post infection in Figure 30A and Figure 30C. Measurements were taken 102 hour post infection in Figure 30B and Figure 30D.
  • the results exemplify that crPhage cocktail has 1.5 to 3.5-log improved kill over wtPhage cocktail at l20h in the bladder. The results also exemplify that regardless of delivery route, at l20h crPhage cocktail performs comparable with ciprofloxacin in the bladder.
  • Figure 31A - Figure 31D illustrate route-dependent penetration of phage into different tissues, such as in urine 78 hour post infection (Figure 31 A), into kidney 102 hour post infection (Figure 31B), into bladderl02 hour post infection (Figure 31C), and into spleen 102 hour post infection (Figure 31D).
  • Figure 32 illustrates a schematic of an exemplary UTI efficacy study with research-grade material compared WT versus engineered cocktail via intravenous (IV), intra-urethral (IU), or concurrent IV and IU delivery.
  • Figure 33A - Figure 33D illustrate IV dosing of crPhage cocktail requires high doses for efficacy, while IU delivery is effective even at low doses and high dose crPhage outperforms ciprofloxacin.
  • CFUs in Figure 33A (bladder) and Figure 33C (kidney) are analyzed 54 hour post infection.
  • CFUs in Figure 33B (bladder) and Figure 33D (kidney) are analyzed 102 hour post infection.
  • Figure 34A is a schematic of an exemplary human study conducted in adults with reoccurring and asymptomatic E.coli colonization of the urinary tract.
  • Figure 34B is an exemplary study participant inclusion and exclusion criteria for the UTI Phase lb study.
  • Figure 35A - Figure 35F illustrate engineered phage (p33s and p33s-6) show increased killing against both Type IE and Type IF E.coli strains.
  • Figure 36A - Figure 36F illustrate engineered phage (CRISPR phage crp0046) show increased killing against both Type IE and Type IF E.coli strains.
  • Figure 37A - Figure 37C illustrate switching phage cocktails overcomes target bacterial resistance in E.coli.
  • Figure 38A - Figure 38B illustrate a comparison of wildtype phage PB1 and CRISPR- enhanced PB1 (cr-PBl) against P. aeruginosa strains.
  • Figure 39A - Figure 39B illustrate plasmid based killing in E.coli and P. aeruginosa by Type I CRISPR-Cas systems.
  • phrases such as“between X and Y” and“between about X and Y” should be interpreted to include X and Y.
  • phrases such as“between about X and Y” mean“between about X and about Y” and phrases such as“from about X to Y” mean“from about X to about Y.”
  • transitional phrase“consisting essentially of’ means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed disclosure.
  • the term “consisting essentially of’ when used in a claim of this disclosure is not intended to be interpreted to be equivalent to“comprising.”
  • chimeric refers to a nucleic acid molecule or a polypeptide in which at least two components are derived from different sources (e.g., different organisms, different coding regions).
  • “Complement” as used herein mean 100% complementarity or identity with the comparator nucleotide sequence or it mean less than 100% complementarity (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like, complementarity).
  • Complement or complementable may also be used in terms of a “complement” to or“complementing” a mutation.
  • the terms“complementary” or“complementarity”, as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.
  • sequence“A-G-T” binds to the complementary sequence“T-C-A.”
  • Complementarity between two single-stranded molecules is“partial,” in which only some of the nucleotides bind, or it is complete when total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.
  • the term“gene” refers to a nucleic acid molecule capable of being used to produce mRNA, tRNA, rRNA, miRNA, anti-microRNA, regulatory RNA, and the like. Genes may or may not be capable of being used to produce a functional protein or gene product. Genes include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and/or 5' and 3' untranslated regions).
  • a gene is "isolated” by which is meant a nucleic acid that is substantially or essentially free from components normally found in association with the nucleic acid in its natural state. Such components include other cellular material, culture medium from recombinant production, and/or various chemicals used in chemically synthesizing the nucleic acid.
  • a“target nucleotide sequence” refers to the portion of a target gene that is complementary to the spacer sequence of the recombinant CRISPR array.
  • a“target DNA,”“target nucleotide sequence,”“target region,” or a“target region in the genome” refers to a region of an organism's genome that is fully complementary or substantially complementary (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a spacer sequence in a CRISPR array.
  • 70% complementary e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9
  • a target region is about 10 to about 40 consecutive nucleotides in length located immediately adjacent to a PAM sequence (PAM sequence located immediately 3' of the target region) in the genome of the organism.
  • a target nucleotide sequence is located adjacent to or flanked by a PAM (protospacer adjacent motif). While PAMs are often specific to the particular CRISPR- Cas system, a PAM sequence is determined by a suitable method.
  • experimental approaches include targeting a sequence flanked by all possible nucleotides sequences and identifying sequence members that do not undergo targeting, such as through in vitro cleavage of target DNA or the transformation of target plasmid DNA.
  • a computational approach includes performing BLAST searches of natural spacers to identify the original target DNA sequences in bacteriophages or plasmids and aligning these sequences to determine conserved sequences adjacent to the target sequence.
  • the term“protospacer adjacent motif’ or“PAM” refers to a DNA sequence present on the target DNA molecule adjacent to the sequence matching the guide RNA spacer. This motif is found in the target gene next to the region to which a spacer sequence binds as a result of being complementary to that region and identifies the point at which base pairing with the spacer nucleotide sequence begins.
  • the PAM is located immediately 5' to the sequence that matches the spacer, and thus is 3' to the sequence that base pairs with the spacer nucleotide sequence.
  • Non-limiting examples of PAMs include CCA, CCT, CCG, CCT, CCA, TTC, AAG, AGG, ATG, GAG, and/or CC.
  • PAM is directly recognized by Cascade. The exact PAM sequence that is required varies between each different CRISPR-Cas system and is identified through established bioinformatics and experimental procedures. Once a protospacer is recognized, Cascade generally recruits the endonuclease Cas3, which cleaves and degrades the target DNA.
  • the PAM is required for a Cas9/sgRNA to form an R-loop to interrogate a specific DNA sequence through Watson-Crick pairing of its guide RNA with the genome.
  • the PAM specificity is a function of the DNA-binding specificity of the Cas9 protein (e.g., a— protospacer adjacent motif recognition domain at the C-terminus of Cas9).
  • CRISPR CRISPR-associated complex for antiviral defense
  • Cascade refers to a complex of polypeptides involved in processing of pre-crRNAs and subsequent binding to the target DNA in type I
  • CRISPR-Cas systems CRISPR-Cas systems.
  • These polypeptides include, but are not limited to, the Cascade polypeptides of type I subtypes l-A, l-B, l-C, l-D, l-E and l-F.
  • Non-limiting examples of type I -A polypeptides include Cas7 (Csa2), Cas8al (Csxl3), Cas8a2 (Csx9), Cas5, Csa5, Cas6a, Cas3' and/or a Cas3".
  • Non-limiting examples of type l-B polypeptides include Cas6b, Cas8b (Cshl), Cas7 (Csh2) and/or Cas5.
  • Non-limiting examples of type-IC polypeptides include Cas5d, Cas8c (Csdl), and/or Cas7 (Csd2).
  • Non-limiting examples of type-ID polypeptides include CaslOd (Csc3), Csc2, Cscl, and/or Cas6d.
  • Non-limiting examples of type l-E polypeptides include Csel (CasA), Cse2 (CasB), Cas7 (CasC), Cas5 (CasD) and/or Cas6e (CasE).
  • Non- limiting examples of type I-F polypeptides include Cysl, Cys2, Cas7 (Cys3) and/or Cas6f (Csy4).
  • a recombinant nucleic acid described herein comprises, consists essentially of, or consists of, a nucleotide sequence encoding a subset of type-l Cascade polypeptides that function to process a CRISPR array and subsequently bind to a target DNA using the spacer of the processed CRISPR RNA as a guide.
  • A“CRISPR array” as used herein means a nucleic acid molecule that comprises at least two repeat sequences, or a portion of each of said repeat sequences, and at least one spacer sequence.
  • One of the two repeat sequences, or a portion thereof, is linked to the 5' end of the spacer sequence and the other of the two repeat sequences, or portion thereof, is linked to the 3' end of the spacer sequence.
  • the combination of repeat sequences and spacer sequences is synthetic, made by man and not found in nature.
  • a "CRISPR array” refers to a nucleic acid construct that comprises from 5' to 3' at least one repeat-spacer sequences (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more repeat-spacer sequences, and any range or value therein), wherein the 3' end of the 3' most repeat-spacer sequence of the array are linked to a repeat sequence, thereby all spacers in said array are flanked on both the 5' end and the 3' end by a repeat sequence.
  • repeat-spacer sequences e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more repeat-spacer sequences, and any range or value therein
  • “spacer sequence” or“spacer refers to a nucleotide sequence that is complementary to a target DNA (i.e., target region in the genome or the“protospacer sequence,” which is adjacent to a protospacer adjacent motif (PAM) sequence).
  • a target DNA i.e., target region in the genome or the“protospacer sequence,” which is adjacent to a protospacer adjacent motif (PAM) sequence.
  • PAM protospacer adjacent motif
  • the spacer sequence is fully complementary or substantially complementary (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a target DNA.
  • 70% complementary e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more
  • A“repeat sequence” as used herein refers to, for example, any repeat sequence of a wild- type CRISPR locus or a repeat sequence of a synthetic CRISPR array that are separated by "spacer sequences" (e.g., a repeat-spacer-repeat sequence).
  • a repeat sequence useful with this disclosure is any known or later identified repeat sequence of a CRISPR locus or it is a synthetic repeat designed to function in a CRISPR system, for example CRISPR Type I system.
  • the term“CRISPR phage”,“CRISPR enhanced phage”, and“crPhage” refers to a bacteriophage particle comprising bacteriophage DNA comprising at least one heterologous polynucleotide.
  • the polynucleotide encodes at least one component of a CRISPR-Cas system (e.g., CRISPR array, crRNA; e.g., PI bacteriophage comprising an insertion of crRNA targeting).
  • the polynucleotide encodes at least one transcriptional activator of a CRISPR-Cas system.
  • the term“CRISPR phage”,“CRISPR enhanced phage”, and“crPhage” refers to a bacteriophage particle comprising bacteriophage DNA comprising at least one heterologous polynucleotide.
  • the polynucleotide encodes at least one component of a CRISPR-Cas system (e.g.,
  • polynucleotide encodes at least one component of an anti-CRISPR polypeptide of a CRISPR-Cas system.
  • the phrase“substantially identical,” or“substantial identity” in the context of two nucleic acid molecules, nucleotide sequences or protein sequences refers to two or more sequences or subsequences that have at least about 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and/or 100% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for aligning a comparison window are conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA).
  • An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence.
  • Percent sequence identity is represented as the identity fraction multiplied by 100.
  • the comparison of one or more polynucleotide sequences is to a full- length polynucleotide sequence or to a portion thereof, or to a longer polynucleotide sequence.
  • “Percent identity” is determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.
  • the recombinant nucleic acids molecules, nucleotide sequences and polypeptides disclosed herein are“isolated.”
  • An“isolated” nucleic acid molecule, an“isolated” nucleotide sequence or an "isolated” polypeptide is a nucleic acid molecule, nucleotide sequence or polypeptide that exists apart from its native environment.
  • an isolated nucleic acid molecule, nucleotide sequence or polypeptide exists in a purified form that is at least partially separated from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polynucleotide.
  • the isolated nuclei c acid molecule, the isolated nucleotide sequence and/or the isolated polypeptide is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more pure.
  • anti-CRISPR or“Acr” refers to any protein or gene product with functional anti-CRISPR activity. Due to a lack of consistency in the literature, one of skill in the art will understand the interchangeability of terms designating the various anti-CRISPR proteins. For example, as used herein the designation of Acr 1 -Bo is interchangeable with
  • An anti-CRISPR protein is any bacteriophage protein with activity that prevents the function of a bacterial CRISPR-Cas system. Activity of an anti-CRISPR protein prevents a host bacterium from mounting a CRISPR-Cas system based defense against the invading bacteriophage.
  • treat By the terms “treat,” “treating,” or “treatment,” it is intended that the severity of the subject's condition is reduced or at least partially improved or modified and that some alleviation, mitigation or decrease in at least one clinical symptom is achieved, and/or there is a delay in the progression of the disease or condition, and/or delay of the onset of a disease or illness.
  • a disease or a condition the term refers to a decrease in the symptoms or other manifestations of the infection, disease or condition.
  • treatment provides a reduction in symptoms or other manifestations of the infection, disease or condition by at least about 5%, e.g., about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more.
  • an“infection”,“a disease”, or“a condition”, used herein refer to any adverse, negative, or harmful physiological condition in a subject.
  • the source of an“infection”,“a disease”, or“a condition” is the presence of a target bacterial population in and/or a subject.
  • the bacterial population comprises one or more target bacterial species.
  • the one or more bacteria in the bacterial population comprise one or more strains of one or more bacteria.
  • the target bacterial population causing an“infection”,“a disease”, or“a condition” is acute or chronic.
  • the target bacterial population causing an“infection”,“a disease”, or“a condition” is localized or systemic.
  • the target bacterial population causing an“infection”,“a disease”, or“a condition” is idiopathic.
  • the target bacterial population causing an“infection”,“a disease”, or“a condition” is acquired through means, including but not limited to, respiratory inhalation, ingestion, skin and wound infections, blood stream infections, middle-ear infections, gastrointestinal tract infections, peritoneal membrane infections, urinary tract infections, urogenital tract infections, oral soft tissue infections, intra abdominal infections, epidermal or mucosal absorption, eye infections (including contact lens contamination), endocarditis, infections in cystic fibrosis, infections of indwelling medical devices such as joint prostheses, dental implants, catheters and cardiac implants, sexual contact, and/or hospital-acquired and ventilator-associated bacterial pneumonias.
  • biofilm means an accumulation of microorganisms embedded in a matrix of polysaccharide. Biofilms form on solid biological or non-biological surfaces and are medically important, accounting for over 80 percent of microbial infections in the body.
  • the terms“prevent,”“preventing,” and“prevention” (and grammatical variations thereof) refer to prevention and/or delay of the onset of an infection, disease, condition and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset of the infection, disease, condition and/or clinical symptom(s) relative to what would occur in the absence of carrying out the methods disclosed herein prior to the onset of the disease, disorder and/or clinical symptom(s).
  • to prevent infection food, surfaces, medical tools and devices are treated with compositions and by methods disclosed herein.
  • A“subject” disclosed herein includes any animal that has or is susceptible to an infection, disease or condition involving bacteria.
  • subjects are mammals, avians, reptiles, amphibians, or fish.
  • Mammalian subjects include but are not limited to humans, non human primates (e.g., gorilla, monkey, baboon, and chimpanzee, etc.), dogs, cats, goats, horses, pigs, cattle, sheep, and the like, and laboratory animals (e.g., rats, guinea pigs, mice, gerbils, hamsters, and the like).
  • Avian subjects include but are not limited to chickens, ducks, turkeys, geese, quail, pheasants, and birds kept as pets (e.g., parakeets, parrots, macaws, cockatoos, canaries, and the like).
  • suitable subjects include both males and females and subjects of any age, including embryonic (e.g., in-utero or in-ovo ), infant, juvenile, adolescent, adult and geriatric subjects.
  • a subject is a human.
  • pharmaceutically acceptable it is meant a material that is not biologically or otherwise undesirable, i.e., the material are administered to a subject without causing any undesirable biological effects such as toxicity.
  • bacteriophages characterized and/or comprising any nucleic acid described herein.
  • a temperate bacteriophage e.g., a bacteriophage having lytic activity, such as described herein.
  • the temperate bacteriophage comprises a removal, replacement, or inactivation of at least one lysogeny gene.
  • bacteriophages having lytic activity and comprising (a) a nucleic acid encoding a spacer sequence and/or (b) a crRNA transcribed therefrom.
  • provided in certain embodiments herein are
  • bacteriophages comprising (i) a first nucleic acid encoding a spacer sequence and/or a crRNA transcribed therefrom, and (ii) a gene that is capable of inducing lysis of a bacterium (e.g., a target bacterium).
  • the spacer sequence is complementary to a nucleic acid sequence of a target gene of or in a bacterium (e.g., target bacterium).
  • bacteriophages comprising a nucleic acid encoding a transcriptional activator for the CRISPR-Cas system in a bacterium (e.g., a target bacterium).
  • a bacterium e.g., a target bacterium.
  • bacteriophages having lytic activity and a first nucleic acid sequence encoding an anti-CRISPR polypeptide (and/or comprising an anti-CRISPR polypeptide).
  • a bacteriophage provided herein has any one or more of the above references characteristics and/or activities.
  • such bacteriophages comprise any one or more characteristic or activity described in the summary or detailed description herein.
  • such bacteriophages are utilized and various compositions (e.g., pharmaceutical compositions) and methods, such as described herein.
  • such bacteriophages are useful in any number of applications and methods (e.g., of tuning the microbiome of an individual or subject, such as one in need thereof), such as those described herein.
  • such bacteriophages are utilized in methods of or that involve killing (e.g., selectively killing) a bacterium (e.g., a target bacterium).
  • the target bacterium is in a mixed population of bacteria, such as in an individual, environment, or other suitable location, such as described herein.
  • a bacteriophage selectively kills a target bacteria or bacterium, e.g., such that the bacteria that is not the target bacterium or bacteria is killed at a lesser rate than the target bacteria, such as at less than 50% the rate, less than 25% the rate, less than 10% the rate, or about 0% the rate (i.e., not at all) relative to the target bacterium or bacteria.
  • a target bacteria or bacterium e.g., such that the bacteria that is not the target bacterium or bacteria is killed at a lesser rate than the target bacteria, such as at less than 50% the rate, less than 25% the rate, less than 10% the rate, or about 0% the rate (i.e., not at all) relative to the target bacterium or bacteria.
  • less than 50% of the non-target bacterium is killed, less than 25%, less than 20%, less than 10%, less than 5% killed, or the like is killed.
  • a nucleic acid encoding a CRISPR array comprises at least one repeat sequence and at least one spacer sequences
  • a CRISPR array is of any length and comprises any number of spacer nucleotide sequences alternating with repeat nucleotide sequences necessary to achieve the desired level of killing of the target bacterium by use of one or more target genes.
  • the CRISPR array comprise, consist essentially of, or consist of 1 to about 100 spacer nucleotide sequences, each linked on its 5' end and its 3' end to a repeat nucleotide sequence.
  • a recombinant CRISPR array of disclosed herein consist essentially of, or consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, or more, spacer nucleotide sequences
  • the spacer sequence described herein comprises one, two, three, four, or five mismatches as compared to the target DNA. In some embodiments, mismatches are contiguous. In some embodiments, mismatches are noncontiguous. In some embodiments, the spacer sequence has 70% complementarity to a target DNA. In some embodiments, the spacer nucleotide sequence has 80% complementarity to a target DNA. In some embodiments, the spacer nucleotide sequence is 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementarity to a target nucleotide sequence of a target gene. In some embodiments, the spacer sequence has 100% complementarity to the target DNA.
  • a spacer sequence has complete complementarity or substantial complementarity over a region of a target nucleotide sequence that are at least about 8 nucleotides to about 150 nucleotides in length. In some embodiments, a spacer sequence have complete complementarity or substantial complementarity over a region of a target nucleotide sequence that is at least about 20 nucleotides to about 100 nucleotides in length. In some embodiments, the 5 ' region of a spacer sequence is 100% complementary to a target DNA while the 3' region of the spacer is substantially complementary to the target DNA and therefore the overall complementarity of the spacer sequence to the target DNA is less than 100%.
  • the first 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides in the 3' region of a 20 nucleotide spacer sequence is 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA.
  • the first 7 to 12 nucleotides of the 3' end of the spacer sequence is 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 50% complementary (e.g., 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
  • the first 7 to 10 nucleotides in the 3' end of the spacer sequence is 75%-99% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are at least about 50% to about 99% complementary to the target DNA. In some embodiments, the first 7 to 10 nucleotides in the 3' end of the spacer sequence is 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA.
  • the first 10 nucleotides (within the seed region) of the spacer sequence is 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA.
  • the 5' region of a spacer sequence e.g., the first 8 nucleotides at the 5' end, the first 10 nucleotides at the 5' end, the first 15 nucleotides at the 5' end, the first 20 nucleotides at the 5' end
  • the first 8 nucleotides at the 5' end of a spacer sequence have 100%
  • a spacer sequence described herein is about 15 nucleotides to about 150 nucleotides in length.
  • a spacer nucleotide sequence is about 15 nucleotides to about 100 nucleotides in length (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,
  • a spacer nucleotide sequence is a length of about 8 to about 150 nucleotides, about 8 to about 100 nucleotides, about 8 to about 50 nucleotides, about 8 to about 40 nucleotides, about 8 to about 30 nucleotides, about 8 to about 25 nucleotides, about 8 to about 20 nucleotides, about 10 to about 150 nucleotides, about 10 to about 100 nucleotides, about 10 to about 80 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40, about 10 to about 30, about 10 to about 25, about 10 to about 20, about 15 to about 150, about 15 to about 100, about 15 to about 50, about 15 to about 40, about 15 to about 30, about 20 to about 150 nucleotides, about 20 to about 100 nucleotides, about 20 to about 80 nucleotides, about 20 to about 50 nucleotides, about 20 to about 40, about 20 to about 30, about 20 to about 25, at least about 8,
  • the identity of two or more spacer nucleotide sequences of a CRISPR array disclosed herein is different. In some embodiments, the identity of two or more spacer nucleotide sequences of a CRISPR array is different but are complementary to one or more target nucleotide sequences. In some embodiments, the identity of two or more spacer nucleotide sequences of a CRISPR array is different and are complementary to one or more target nucleotide sequences that are overlapping sequences. In some embodiments, the identity of two or more spacer nucleotide sequences of a CRISPR array is different and are complementary to one or more target nucleotide sequences that are not overlapping sequences.
  • a polynucleotide, nucleotide sequence and/or recombinant nucleic acid molecule described herein e.g., polynucleotides comprising a CRISPR array, Cascade polypeptides, Cas9 polypeptides, Cas3 polypeptides, Cas3’ polypeptides, Cas3" polypeptides, recombinant Type I or Type II, Type III, Type IV, Type V, Type VI CRISPR-Cas systems of the disclosure, polynucleotides encoding transcriptional activators, and the like) is codon optimized for expression in any species of interest.
  • Codon optimization involves modification of a nucleotide sequence for codon usage bias using species-specific codon usage tables.
  • the codon usage tables are generated based on a sequence analysis of the most highly expressed genes for the species of interest.
  • the codon usage tables are generated based on a sequence analysis of highly expressed nuclear genes for the species of interest.
  • the modifications of the nucleotide sequences are determined by comparing the species specific codon usage table with the codons present in the native polynucleotide sequences.
  • Codon optimization of a nucleotide sequence results in a nucleotide sequence having less than 100% identity (e.g., 50%, 60%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and the like) to the native nucleotide sequence but which still encodes a polypeptide having the same function as that encoded by the original nucleotide sequence.
  • the nucleotide sequence and/or recombinant nucleic acid molecule of this disclosure are codon optimized for expression in the organism/species of interest.
  • a repeat nucleotide sequence of a CRISPR array comprises a nucleotide sequence of any known repeat nucleotide sequence of a CRISPR-Cas system.
  • the CRISPR-Cas system is a type-I CRISPR-Cas system.
  • a repeat nucleotide sequence is of a synthetic sequence comprising the secondary structure of a native repeat from a type-I CRISPR-Cas system (e.g., an internal hairpin).
  • a spacer nucleotide sequence of a CRISPR array described herein is linked at its 5' end to the 3’ end of a repeat sequence.
  • the spacer nucleotide sequence is linked at its 5’ end to about 1 to about 8, about 1 to about 10, or about 1 to about 15 nucleotides of the 3’ end of a repeat nucleotide sequence.
  • the about 1 to about 8, about 1 to about 10, about 1 to about 15 nucleotides of the repeat nucleotide sequence are a portion of the 3’ end of a repeat nucleotide sequence.
  • spacer nucleotide sequence is linked at its 3' end to the 5’ end of a repeat nucleotide sequence. In some embodiments, the spacer is linked at its 3’ end to about 1 to about 8, about 1 to about 10, or about 1 to about 15 nucleotides of the 5’ end of a repeat nucleotide sequence. In some embodiments, the about 1 to about 8, about 1 to about 10, about 1 to about 15 nucleotides of the repeat nucleotide sequence are a portion of the 5’ end of a repeat nucleotide sequence.
  • a spacer nucleotide sequence described herein is linked at its 5' end to a first repeat nucleotide sequence and linked at its 3' end to a second repeat nucleotide sequence to form a repeat-spacer-repeat sequence.
  • a spacer described herein is linked at its 5’ end to about 1 to about 8, about 1 to about 10, or about 1 to about 15 nucleotides of the 3’ end of a first repeat sequence and is linked at its 3’ end to about 1 to about 8, about 1 to about 10, or about 1 to about 15 nucleotides of the 5’ end of a second repeat sequence.
  • the about 1 to about 8, about 1 to about 10, about 1 to about 15 nucleotides of the first repeat sequence are a portion of the 3’ end of the first repeat nucleotide sequence. In some embodiments, the about 1 to about 8, about 1 to about 10, about 1 to about 15 nucleotides of the first second sequence are a portion of the 3’ end of the second repeat nucleotide sequence. In some
  • a spacer nucleotide sequence disclosed herein is linked at its 5' end to the 3’ end of a first repeat nucleotide sequence and is linked at its 3' end to the 5’ of a second repeat nucleotide sequence where the spacer nucleotide sequence and the second repeat nucleotide sequence are repeated to form a repeat-(spacer-repeat)n sequence such that n is any integer from 1 to 100.
  • a repeat-(spacer-repeat)n sequence disclosed herein comprise, consist essentially of, or consist of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 ,
  • a repeat sequence is identical to or substantially identical to a repeat sequence from a wild-type CRISPR Type I, II, or III loci.
  • a repeat sequence comprises a portion of a wild type repeat sequence (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15 or more contiguous nucleotides of a wild type repeat sequence).
  • a repeat sequence comprises, consists essentially of, or consists of at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
  • nucleotide e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
  • nucleotides 100, or more nucleotides, or any range therein).
  • recombinant CRISPR arrays, nucleotide sequences, and/or nucleic acid molecules disclosed herein are operatively associated with a variety of promoters, terminators and other regulatory elements for expression in various organisms or cells.
  • at least one promoter and/or terminator is operably linked to a recombinant nucleic acid molecule and/or a recombinant CRISPR array disclosed herein.
  • Any promoter useful with this disclosure is used and includes, for example, promoters functional with the organism of interest as well as constitutive, inducible, developmental regulated, tissue-specific/preferred- promoters, and the like, as described herein.
  • a regulatory element as used herein is endogenous or heterologous.
  • an endogenous regulatory element derived from the subject organism is inserted into a genetic context in which it does not naturally occur (e.g. a different position in the genome than as found in nature), thereby producing a recombinant or non-native nucleic acid.
  • expression of a construct disclosed herein is constitutive, inducible, temporally regulated, developmentally regulated, or chemically regulated.
  • a construct is made constitutive, inducible, temporally regulated, developmentally regulated, or chemically regulated by operatively linking the construct to a promoter functional in an organism of interest.
  • repression is made reversible by operatively linking a recombinant nucleic acid construct disclosed herein to an inducible promoter that is functional in an organism of interest.
  • the choice of promoter described herein will vary depending on the quantitative, temporal and spatial requirements for expression, and also depending on the host cell to be transformed.
  • Exemplary promoters for use with the methods, bacteriophage and composition disclosed herein include promoters that are functional in bacteria.
  • L-arabinose inducible (araBAD, READ) promoter any lac promoter, L-rhamnose inducible (rhaPBAD) promoter, T7 RNA polymerase promoter, trc promoter, tac promoter, lambda phage promoter (p L p L -9G-50), anhydrotetracycline-inducible (let A) promoter, trp , Ipp, phoA , recA, pro l /, cst- 1, cad A , nar , Ipp- lac, cspA, 11-lac operator, T3 -lac operator, T4 gene 32, T5 -lac operator, nprM- lac operator, Vhb, Protein A, cory n eb acteri al -E.
  • arabin inducible (araBAD, READ) promoter
  • coli like promoters thr, horn, diphtheria toxin promoter, sig A, sig B, nusG, SoxS, katb, a-amylase (Pamy), Ptms, P43 (comprised of two overlapping RNA polymerase s factor recognition sites, sA, sB), Ptms, P43, rplK-rplA, ferredoxin promoter, and/or xylose promoter.
  • Ptms P43 (comprised of two overlapping RNA polymerase s factor recognition sites, sA, sB), Ptms, P43, rplK-rplA, ferredoxin promoter, and/or xylose promoter.
  • inducible promoters are used.
  • chemical- regulated promoters are used to modulate the expression of a gene in an organism through the application of an exogenous chemical regulator.
  • the use of chemically regulated promoters enables RNAs and/or the polypeptides disclosed herein to be synthesized only when, for example, an organism is treated with the inducing chemicals.
  • the application of a chemical induces gene expression.
  • the application of the chemical represses gene expression is used.
  • the promoter is a light-inducible promoter, where application of specific wavelengths of light induces gene expression. In some embodiments, a promoter is a light- repressible promoter, where application of specific wavelengths of light represses gene expression.
  • nucleotide sequences, constructs, and expression cassettes disclosed herein are expressed transiently and/or stably incorporated into the genome of a host organism.
  • a polynucleotide disclosed herein is introduced into a cell by any method known to those of skill in the art. Exemplary methods of transformation include
  • transformation of a cell comprises nuclear transformation. In some embodiments, transformation of a cell comprises plasmid transformation and conjugation.
  • nucleotide sequences when more than one nucleotide sequence is introduced, are assembled as part of a single nucleic acid construct, or as separate nucleic acid constructs, and are located on the same or different nucleic acid constructs. In some embodiments, nucleotide sequences are introduced into the cell of interest in a single nucleic acid construct, or as separate nucleic acid constructs, and are located on the same or different nucleic acid constructs. In some embodiments, nucleotide sequences are introduced into the cell of interest in a single
  • transformation event or in separate transformation events.
  • a nucleic acid construct is an "expression cassette” or in an expression cassette.
  • expression cassette means a recombinant nucleic acid molecule comprising a nucleotide sequence of interest (e.g., the recombinant nucleic acid molecules and CRISPR arrays disclosed herein), wherein the nucleotide sequence is operably associated with at least a control sequence (e.g., a promoter).
  • the expression cassettes are designed to express the recombinant nucleic acid molecules and/or the recombinant CRISPR arrays disclosed herein.
  • an expression cassette comprising a nucleotide sequence of interest is chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • an expression cassette is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • an expression cassette includes a transcriptional and/or translational termination region (i.e. termination region) that is functional in the selected host cell.
  • termination regions are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and for correct mRNA polyadenylation.
  • the termination region is native to the transcriptional initiation region, is native to the operably linked nucleotide sequence of interest, is native to the host cell, or is derived from another source (i.e., foreign or heterologous to the promoter, to the nucleotide sequence of interest, to the host, or any combination thereof).
  • terminators are operably linked to the recombinant nucleic acid molecule and CRISPR array disclosed herein.
  • an expression cassette includes a nucleotide sequence for a selectable marker.
  • selectable marker means a nucleotide sequence that when expressed imparts a distinct phenotype to the host cell expressing the marker and thus allows such transformed cells to be distinguished from those that do not have the marker.
  • a nucleotide sequence encode either a selectable or screenable marker, depending on whether the marker confers a trait that is selected for by chemical means, such as by using a selective agent (e.g. an antibiotic), or on whether the marker is simply a trait that one identifies through observation or testing, such as by screening (e.g., fluorescence).
  • a selective agent e.g. an antibiotic
  • screening e.g., fluorescence
  • nucleic acid molecules and nucleotide sequences described herein are used in connection with vectors.
  • vector refers to a composition for transferring, delivering or introducing a nucleic acid (or nucleic acids) into a cell.
  • a vector comprises a nucleic acid molecule comprising the nucleotide sequence(s) to be transferred, delivered or introduced.
  • Non-limiting examples of general classes of vectors include but are not limited to a viral vector, a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a fosmid vector, a bacteriophage, an artificial chromosome, or an agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self-transmissible or mobilizable.
  • a vector as defined herein transforms prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms.
  • a shuttle vector replicates in actinomycetes and bacteria and/or eukaryotes.
  • the nucleic acid in the vector are under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell.
  • the vector is a bi-functional expression vector which functions in multiple hosts.
  • CRISPR-Cas systems are naturally adaptive immune systems found in bacteria and archaea.
  • the CRISPR system is a nuclease system involved in defense against invading phages and plasmids that provides a form of acquired immunity.
  • a Type I, Type II, Type II, Type IV, Type V, or Type VI CRISPR- Cas system is used herein.
  • processing of a CRISPR-array disclosed herein includes, but is not limited to, the following processes: 1) transcription of the nucleic acid encoding a CRISPR array into a pre-crRNA and optional tracrRNA; 2) pre-crRNA processing by either Cas6 or Cas9/Rnase III into mature crRNAs; 3) mature crRNA complexation Cas9 or Cascade; 4) target recognition by the complexed mature crRNA/Cas9 or crRNA/Cascade complexes; and 5) nuclease activity at the target leading to double or single stranded DNA breakage.
  • Type I systems are divided into seven subtypes including: type I-A, type I-B, type I-C, type I-D, type I-E, type I-F, and type I-U.
  • Type I CRISPR-Cas systems include a multi-subunit complex called Cascade (for complex associated with antiviral defense), Cas3 (a protein with nuclease, helicase, and exonuclease activity that is responsible for degradation of the target DNA), and crRNA (stabilizes Cascade complex and directs Cascade and Cas3 to DNA target).
  • Cascade for complex associated with antiviral defense
  • Cas3 a protein with nuclease, helicase, and exonuclease activity that is responsible for degradation of the target DNA
  • crRNA stabilizes Cascade complex and directs Cascade and Cas3 to DNA target.
  • Cascade forms a complex with the crRNA, and the protein-RNA pair recognizes its genomic target by complementary base pairing between the 5’ end of the crRNA sequence and a predefined protospacer.
  • This complex is directed to homologous loci of pathogen DNA via regions encoded within the crRNA and protospacer-adjacent motifs (PAMs) within the pathogen genome.
  • Base pairing occurs between the crRNA and the target DNA sequence leading to a conformational change.
  • the PAM is recognized by the CasA protein within Cascade, which then unwinds the flanking DNA to evaluate the extent of base pairing between the target and the spacer portion of the crRNA.
  • Sufficient recognition leads Cascade to recruit and activate Cas3. Cas3 then nicks the non -target strand and begins degrading the strand in a 3’-to-5’ direction.
  • a CRISPR array disclosed herein comprises a nucleic acid that encodes a processed, mature crRNA.
  • a mature crRNA is introduced into a phage or a target bacterium described herein.
  • a phage comprises a nucleic acid that encodes a processed, mature crRNA.
  • an endogenous or exogenous Cas6 processes a CRISPR array into mature crRNA.
  • an exogenous Cas6 is introduced into a phage.
  • a phage comprises an exogenous Cas6.
  • an exogenous Cas6 is introduced into a target bacterium.
  • the CRISPR-Cas system is endogenous to the target bacterium. In some embodiments, the CRISPR-Cas system is exogenous to the target bacterium. In some embodiments, the CRISPR-Cas system is a type II CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type III CRISPR-Cas system.
  • a Type I CRISPR-Cas system is used herein.
  • Type-I Cascade polypeptides process CRISPR arrays to produce a processed RNA that is then used to bind the complex to a DNA that is complementary to a spacer in the processed RNA.
  • a first nucleic acid that is introduced into the bacteriophage encodes the Cascade polypeptides that are involved in processing of the first nucleic acid disclosed herein.
  • a type-I Cascade polypeptide disclosed herein have an amino acid sequence having substantial identity to a wild-type type-I Cascade polypeptide.
  • a Cascade polypeptide described herein is a functional fragment of any full length type-l Cascade polypeptides.
  • the type I Cascade complex is a type I-A Cascade polypeptides, a type I-B Cascade polypeptides, a type I-C Cascade polypeptides, a type I- D Cascade polypeptides, a type I-E Cascade polypeptides, a type I-F Cascade polypeptides, or a type I-U Cascade polypeptides.
  • the type I Cascade complex comprises: (a) a nucleotide sequence encoding a Cas6b polypeptide, a nucleotide sequence encoding a Cas8b (Cshl) polypeptide, a nucleotide sequence encoding a Cas7 (Csh2) polypeptide, and a nucleotide sequence encoding a Cas5 polypeptide (Type l-B); (b) a nucleotide sequence encoding a Cas5d polypeptide, a nucleotide sequence encoding a Cas8c (Csdl ) polypeptide, and a nucleotide sequence encoding a Cas7 (Csd2) polypeptide (Type I- C); (c) a nucleotide sequence encoding a Csel (CasA) polypeptide, a nucleotide sequence encoding a Cse2 (Cas
  • Bacteriophages or“phages” represent a group of bacterial viruses and are engineered or sourced from environmental sources. Individual bacteriophage host ranges are usually narrow, meaning, phages are highly specific to one strain or few strains of a bacterial species and this specificity makes them unique in their antibacterial action. Bacteriophages are bacterial viruses that rely on the host's cellular machinery to replicate. Generally, phages generally fall into three categories: lytic, lysogenic, and temperate. Lytic bacteriophages infect a host cell, undergo numerous rounds of replication, and trigger cell lysis to release newly made bacteriophage particles. In some embodiments, the lytic bacteriophages disclosed herein retain their replicative ability.
  • the lytic bacteriophages disclosed herein retain their ability to trigger cell lysis. In some embodiments, the lytic bacteriophages disclosed herein retain both they replicative ability and the ability to trigger cell lysis. In some embodiments, the bacteriophages disclosed herein comprise a CRISPR array. In some embodiments, the CRISPR array does not affect the bacteriophages ability to replicate and/or trigger cell lysis. Lysogenic bacteriophages permanently reside within the host cell, either within the bacterial genome or as an
  • Temperate bacteriophages are capable of being lytic or lysogenic, and choose one versus the other depending on growth conditions and the physiological state of the cell. Anytime a lysogenic bacterium is exposed to adverse conditions, the lysogenic state is terminated. This process is called induction. Adverse conditions which favor the termination of the lysogenic state include desiccation, exposure to UV or ionizing radiation, and exposure to mutagenic chemicals. This leads to the expression of the phage genes, reversal of the integration process, and lytic multiplication.
  • Bacteriophages package and deliver synthetic DNA using three general approaches. Under the first approach, the synthetic DNA is randomly recombined into the bacteriophage genome, which usually involves a selectable marker. Under the second approach, restriction sites within the phage are used to introduce synthetic DNA in-vitro. Under the third approach, a plasmid generally encoding the phage packaging sites and lytic origin of replication is packaged as part of the assembly of the bacteriophage particle. The resulting plasmids have been coined“phagemids.”
  • Phages are limited to a given bacterial strain for evolutionary reasons. In some cases, injecting their genetic material into an incompatible strain is counterproductive. Phages have therefore evolved to specifically infect a limited cross-section of strains. However, some phages have been discovered that inject their genetic material into a wide range of bacteria. The classic example is the PI phage, which has been shown to inject DNA in a range of gram-negative bacteria.
  • the bacteriophage or phagemid DNA is from a lysogenic or temperate bacteriophage. In some embodiments, the bacteriophage or phagemid DNA is from an obligate lytic bacteriophage.
  • the bacteriophages or phagemids include but are not limited to PI phage, a Ml 3 phage, a l phage, a T4 phage, a c
  • the bacteriophage is fO ⁇ 146 C.
  • the bacteriophage is fO ⁇ 24-2 C. difficile bacteriophage. In some embodiments, the bacteriophage is T4 E. coli bacteriophage. In some embodiments, the bacteriophage is T7 E. coli bacteriophage. In some embodiments, the bacteriophage is T7m E. coli bacteriophage.
  • a plurality of bacteriophages are used together.
  • the plurality of bacteriophages used together targets the same or different bacteria within a sample or subject.
  • the bacteriophages used together comprises T4 phage, T7 phage, T7m phage, or any combination of bacteriophages described herein.
  • bacteriophages of interest are obtained from environmental sources or commercial research vendors. In some embodiments, obtained bacteriophages are screened for lytic activity against a library of bacteria and their associated strains. In some embodiments, the bacteriophages are screened against a library of bacteria and their associated strains for their ability to generate primary resistance in the screened bacteria.
  • a target bacterium comprising introducing into a target bacterium a bacteriophage comprising: a nucleic acid encoding a spacer sequence or a crRNA transcribed therefrom, wherein the spacer sequence is complementary to a target nucleotide sequence from a target gene in the target bacterium; and a gene that is capable of inducing lysis of the target bacterium, wherein the target bacterium is killed by lytic activity of the bacteriophage or activity of a CRISPR-Cas system using the spacer sequence or the crRNA transcribed therefrom.
  • bacteriophages comprising: a nucleic acid encoding a spacer sequence or a crRNA transcribed therefrom, wherein the spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium; and a gene that is capable of inducing lysis of the target bacterium, wherein the target bacterium is killed by the lytic activity of the bacteriophage or activity of a CRISPR-Cas system using the spacer sequence or the crRNA transcribed therefrom.
  • the introduction of a nucleic acid encoding a CRISPR array into a bacteriophage does not disrupt the lytic activity of the bacteriophage. In some embodiments, the introduction of a nucleic acid encoding a CRISPR array into a bacteriophage preserves the lytic activity of the bacteriophage. In some embodiments, the nucleic acid is inserted into the
  • the nucleic acid is inserted into the bacteriophage genome at a transcription terminator site at the end of an operon of interest. In some embodiments, the nucleic acid is inserted into the bacteriophage genome as a replacement for one or more removed non-essential genes. In some embodiments, the nucleic acid is inserted into the
  • the replacement of non-essential and/or lysogenic genes with the nucleic acid does not affect the lytic activity of the bacteriophage. In some embodiments, the replacement of non- essential and/or lysogenic genes with the nucleic acid preserves the lytic activity of the
  • the replacement of non-essential and/or lysogenic genes with the nucleic acid enhances the lytic activity of the bacteriophage. In some embodiments, the replacement of non-essential and/or lysogenic genes with the nucleic acid renders a lysogenic bacteriophage lytic.
  • the nucleic acid is introduced into the bacteriophage genome at a first location while one or more non-essential and/or lysogenic genes are separately removed and/or inactivated from the bacteriophage genome at a separate location.
  • the removal and/or inactivation of one or more non-essential and/or lysogenic genes does not affect the lytic activity of the bacteriophage.
  • the removal and/or inactivation of one or more non-essential and/or lysogenic genes preserves the lytic activity of the bacteriophage.
  • the removal of one or more non-essential and/or lysogenic genes renders a lysogenic bacteriophage into a lytic bacteriophage.
  • one or more lytic genes are introduced into the bacteriophage so as to render a non-lytic, lysogenic
  • the bacteriophage is a temperate bacteriophage which has been rendered lytic by any of the aforementioned means. In some embodiments, a temperate
  • bacteriophage is rendered lytic by the removal, replacement, or inactivation of one or more lysogenic genes.
  • the lytic activity of the bacteriophage is due to the removal, replacement, or inactivation of at least one lysogeny gene.
  • a temperate bacteriophage is rendered lytic by the removal, replacement, or inactivation of one or more lysogenic gene and comprises a CRISPR array comprising at least one spacer that is
  • a temperate bacteriophage is rendered lytic by the removal, replacement, or inactivation of one or more lysogenic gene via a CRISPR array comprising a spacer directed to the one or more lysogenic gene and comprises a CRISPR array comprising at least one spacer that is complementary to a target nucleotide sequence in a target gene in a target bacterium.
  • the lysogenic gene plays a role in the maintenance of lysogenic cycle in the bacteriophage.
  • the lysogenic gene plays a role in establishing the lysogenic cycle in the bacteriophage.
  • the lysogenic gene plays a role in both establishing the lysogenic cycle and in the maintenance of the lysogenic cycle in the bacteriophage.
  • the lysogenic gene is a repressor gene.
  • the lysogenic gene is cl repressor gene.
  • the lysogenic gene is an activator gene.
  • the lysogenic gene is ell gene.
  • the lysogenic gene is lexA gene.
  • the lysogenic gene is int (integrase) gene.
  • two or more lysogeny genes are removed, replaced, or inactivated to cause arrest of a bacteriophage lysogeny cycle and/or induction of a lytic cycle.
  • a temperate bacteriophage is rendered lytic by the insertion of one or more lytic genes.
  • a temperate bacteriophage is rendered lytic by the insertion of one or more genes that contribute to the induction of a lytic cycle.
  • a temperate bacteriophage is rendered lytic by altering the expression of one or more genes that contribute to the induction of a lytic cycle.
  • a temperate bacteriophage phenotypically changes from a lysogenic bacteriophage to a lytic bacteriophage.
  • a temperate bacteriophage is rendered lytic by environmental alterations.
  • environmental alterations include, but are not limited to, alterations in temperature, pH, or nutrients, exposure to antibiotics, hydrogen peroxide, foreign DNA, or DNA damaging agents, presence of organic carbon, and presence of heavy metal (e.g. in the form of chromium (VI).
  • a temperate bacteriophage that is rendered lytic is prevented from reverting to lysogenic state.
  • a temperate bacteriophage that is rendered lytic is prevented from reverting back to lysogenic state by way of introducing an additions CRIPSR array.
  • the bacteriophage does not confer any new properties onto the target bacterium beyond cellular death cause by lytic activity of the bacteriophage and/or the activity of the CRISPR array.
  • the replacement, removal, inactivation, or any combination thereof, of one or more non-essential and/or lysogenic genes is achieved by chemical, biochemical, and/or any suitable method.
  • the insertion of one or more lytic genes is achieved by any suitable chemical, biochemical, and/or physical method by homologous recombination.
  • the bacteriophage is an obligate lytic bacteriophage. In some embodiments, the bacteriophage is c
  • the non-essential gene to be removed and/or replaced from the bacteriophage is a gene that is non-essential for the survival of the bacteriophage. In some embodiments, the non-essential gene to be removed and/or replaced from the bacteriophage is a gene that is non-essential for the induction and/or maintenance of lytic cycle. In some
  • the non-essential gene to be removed and/or replaced from the bacteriophage is gp49 from c
  • the non-essential gene to be removed and/or replaced from the
  • bacteriophage is the hoc gene from a T4 E. coli bacteriophage.
  • the non- essential gene to be removed and/or replaced include gpO.7, gp4.3 , gp4.5 , gp4.7, or any
  • the non-essential gene to be removed and/or replaced is gp0.6 , gp0.65 , gpO.7, gp4.3 , gp4.5 , or any combination thereof from a T7m . coli bacteriophage.
  • a bacteriophage disclosed herein further comprises a transcriptional activator.
  • the transcriptional activator encoded regulates the expression of genes of interest within the target bacterium.
  • the transcriptional activator activates the expression of genes of interest within the target bacterium whether exogenous or endogenous.
  • the transcriptional activator activates the expression genes of interest within the target bacterium by disrupting the activity of one or more inhibitory elements within the target bacterium.
  • the inhibitory element comprises a
  • the inhibitory element comprises a global transcriptional repressor.
  • the inhibitory element is a histone-like nucleoid- structuring (H-NS) protein or homologue or functional fragment thereof.
  • the inhibitory element is a leucine responsive regulatory protein (LRP).
  • the inhibitory element is a CodY protein.
  • the CRISPR-Cas system is poorly expressed and considered silent under most environmental conditions.
  • the regulation of the CRISPR-Cas system is the result of the activity of transcriptional regulators, for example histone-like nucleoid-structuring (H- NS) protein which is widely involved in transcriptional regulation of the host genome.
  • H-NS exerts control over host transcriptional regulation by multimerization along AT-rich sites resulting in DNA bending.
  • the regulation of the type-I CRISPR-Cas3 operon is regulated by H-NS.
  • LRP leucine responsive regulatory protein
  • LRP activity of LRP in regulating expression of the CRISPR-Cas system varies from bacteria to bacteria. Unlike, H-NS which has broad inter-species repression activity, LRP has been shown to differentially regulate the expression of the host CRISPR-Cas system. As such, in some instances, LRP reflects a host-specific means of regulating CRISPR-Cas system expression in different bacteria.
  • CodY is a GTP-sensing transcriptional repressor that acts through DNA binding.
  • the intracellular concentration of GTP acts as an indicator for the environmental nutritional status. Under normal culture conditions, GTP is abundant and binds with CodY to repress transcriptional activity. However, as GTP concentrations decreases, CodY becomes less active in binding DNA, thereby allowing transcription of the formerly repressed genes to occur. As such, CodY acts as a stringent global transcriptional repressor.
  • the transcriptional activator is a LeuO polypeptide, any homolog or functional fragment thereof, a nucleic acid sequence encoding the same, or an agent that upregulates LeuO.
  • the transcriptional activator comprises any ortholog or functional equivalent of LeuO.
  • LeuO acts in opposition to H-NS by acting as a global transcriptional regulator that responds to environmental nutritional status of a bacterium. Under normal conditions, LeuO is poorly expressed. However, under amino acid starvation and/or reaching of the stationary phase in the bacterial life cycle, LeuO is upregulated. Increased expression of LeuO leads to it antagonizing H-NS at overlapping promoter regions to effect gene expression.
  • LeuO upregulates the expression of the CRISPR-Cas system.
  • LeuO drives increased expression of the casABCDE operon which has predicted LeuO and H-NS binding sequences upstream of CasA.
  • the expression of LeuO leads to disruption of an inhibitory element.
  • the disruption of an inhibitory element due to expression of LeuO removes the transcriptional repression of a CRISPR-Cas system.
  • the expression of LeuO removes transcriptional repression of a CRISPR-Cas system due to activity of H-NS.
  • the expression of LeuO removes transcriptional repression of a CRISPR-Cas system due to activity of LRP.
  • the disruption of an inhibitory element due to the expression of LeuO causes an increase in the expression of a CRISPR-Cas system.
  • the increase in the expression of a CRISPR-Cas system due to the disruption of an inhibitory element caused by the expression of LeuO causes an increase in the CRISPR-Cas processing of a nucleic acid encoding a CRISPR array. In some embodiments, the increase in the expression of a CRISPR-Cas system due to the disruption of an inhibitory element by the expression of LeuO causes an increase in the CRISPR-Cas processing of a nucleic acid encoding a CRISPR array so as to increase the level of lethality of the CRISPR array against a bacterium. In some embodiments, transcriptional activator described herein, causes increase activity of a bacteriophage and/or CRISPR-Cas system described herein.
  • sequence for LeuO or any homolog or functional fragments thereof from E. coli strain K12 includes but is not limited to GenBank accession number:
  • the transcriptional activator is a CD2983 polypeptide or any homolog or functional fragment thereof, or a nucleic acid encoding the same.
  • the transcriptional activator is any ortholog or functional equivalent of CD29883.
  • CD2983 act as a specific transcriptional regulator that responds to environmental nutritional status of a bacterium.
  • the CRISPR-Cas system is regulated by the environmental nutritional status of glucose in a ccpA dependent manner.
  • CodY Under nutritional stress, CodY becomes less active to allow expression of CD2983.
  • Upregulation of CD2983 is associated with CRISPR-Cas system upregulation.
  • the expression of CD2983 leads to disruption of an inhibitory element.
  • the disruption of an inhibitory element due to expression of CD2983 removes the transcriptional repression of a CRISPR-Cas system.
  • the expression of CD2983 removes transcriptional repression of a CRISPR-Cas system due to activity of CodY.
  • the disruption of an inhibitory element due to the expression of CD2983 causes an increase in the expression of a CRISPR-Cas system.
  • the increase in the expression of a CRISPR-Cas system due to the disruption of an inhibitory element caused by the expression of CD2983 causes an increase in the CRISPR-Cas processing of the first nucleic acid encoding a CRISPR array. In some embodiments, the increase in the expression of a CRISPR-Cas system due to the disruption of an inhibitory element by the expression of CD2983 causes an increase in the CRISPR-Cas processing of the first nucleic acid encoding a CRISPR array so as to increase the level of lethality of the CRISPR array against a bacterium.
  • sequence for CD2983 or any homolog or functional fragments thereof from C. difficile strain 630 includes but is not limited to GenBank accession number:
  • killing of the target bacterium is achieved by the lytic activity of the bacteriophage. In some embodiments, killing of the target bacterium is achieved by the activity of the first nucleic acid encoding a CRISPR array comprising at least one spacer that is
  • killing of the bacterium is achieved by the processing of the CRISPR array by a type I, type II, or a type III CRISPR-Cas system to produce a processed crRNA capable of directing CRISPR-Cas based endonuclease activity and/or cleavage at the target nucleotide sequence in the target gene of the bacterium.
  • the killing of the bacterium is achieved by the enhanced processing of the CRISPR array due to the expression of a second nucleic acid encoding a CRISPR-Cas system transcriptional activator.
  • killing of the bacterium is achieved by the lytic activity of the bacteriophage and by the activity of a nucleic acid encoding a CRISPR array comprising at least one spacer that is complementary to a target nucleotide sequence in a target gene in the target bacterium in combination. In some embodiments, killing of the bacterium is achieved by the lytic activity of the bacteriophage and by the enhanced activity of the nucleic acid encoding a CRISPR array due to the activity of the expressed transcriptional activator.
  • the killing of the bacterium by a combination of the lytic activity of the bacteriophage and by the activity of the nucleic acid encoding a CRISPR array is synergistic. In some embodiments, the killing of the bacterium by a combination of the lytic activity of the bacteriophage and by the activity of the nucleic acid encoding a CRISPR array is synergistic due to the expression of the CRISPR-Cas transcriptional activator encoded by the second nucleic acid.
  • bacteriophages that comprises a nucleic acid encoding a transcriptional activator for a CRISPR-Cas system. Also, disclosed herein, are bacteriophages that comprise a nucleic acid encoding a transcriptional activator for a CRISPR-Cas system.
  • the introduction of a nucleic acid encoding a transcriptional activator for a CRISPR-Cas into a bacteriophage is used to modulate the activity of a CRISPR-Cas system in the target bacterium.
  • the transcriptional activator introduced by the bacteriophage increases the expression of a CRISPR-Cas system in the target bacterium.
  • the increased expression of a CRISPR-Cas system in the target bacterium due to the introduction of a transcriptional activator by a first bacteriophage enhances the lethality of a second different bacteriophage comprising a CRISPR array as described by previous embodiments.
  • the increased expression of a CRISPR-Cas system in the target bacterium due to the introduction of a transcriptional activator by a first bacteriophage enhances the lethality of a second different bacteriophage comprising a pre-processed immature or a processed mature crRNA as described by previous embodiments.
  • Quorum sensing is the chemical communication between bacteria within a bacterial population which permits the coordination of gene expression with respect to the population density. QS relies upon chemical signals that are produced and accumulate during bacterial growth. Upon hitting a threshold level, QS signals bind to transcriptional regulators to influence bacterial gene expression. In some bacteria, QS signaling enhances the CRISPR-Cas system for bacterial defense by de-repressing its expression. In addition to QS signaling, the regulation of CRISPR-Cas system expression is believed to be sensitive to perturbations in the host bacterium’s membrane integrity.
  • BaeSR is a two component response regulator system that links host membrane envelope stress to the activation of Cas genes.
  • heat shock protein G HtpG
  • metabolic sensing proteins such as the cAMP metabolite sensing cAMP receptor protein (CRP) are able to activate CRISPR-Cas expression.
  • Other metabolic sensing proteins which regulate Cas expression includes sigma factor RpoN (s 54 ) which responds to nitrogen starvation.
  • the transcriptional activator comprises a QS signal.
  • the transcriptional activator comprises a protein involved in sensing stress to the membrane of the host bacterium. In some embodiments, this protein comprises BaeSR. In some embodiments, the transcriptional activator comprises a protein which stabilizes Cas. In some embodiments, this protein comprises HtpG. In some embodiments, the transcriptional activator is a metabolic sensing protein. In some embodiments, the metabolic sensing protein comprises CRP or RpoN (s 54 ). In some embodiments, a nucleic acid encoding a transcriptional activator or a functional fragment thereof is introduced into the target bacteria.
  • a nucleic acid encoding a transcriptional activator or a functional fragment thereof is introduced into the target bacteria via a CRISPR array described herein.
  • the methods disclosed herein comprises: introducing a bacteriophage comprising a nucleic acid encoding a transcriptional activator for the CRISPR-Cas system in the target bacterium.
  • bacteriophages comprising a nucleic acid encoding a transcriptional activator for a CRISPR-Cas system in a target bacterium.
  • a bacteriophage disclosed herein further comprises an Anti- CRISPR.
  • a method disclosed herein comprises introducing into a target bacterium a bacteriophage comprising: lytic activity, and a first nucleic acid sequence encoding an anti-CRISPR polypeptide, wherein the anti-CRISPR polypeptide enhances the lytic activity of the bacteriophage.
  • bacteriophages comprising: lytic activity, and a first nucleic acid sequence encoding an anti-CRISPR polypeptide, wherein the anti- CRISPR polypeptide enhances the lytic activity of the bacteriophage.
  • the nucleic acid encoding an anti-CRISPR polypeptide directly enhances the lytic activity of the bacteriophage or another bacteriophage.
  • enhancement of the lytic activity of the bacteriophage is due to the anti-CRISPR polypeptide inhibiting, inactivating, and/or repressing the activity of a CRISPR-Cas system in the host target bacterium.
  • An anti-CRISPR polypeptide is any bacteriophage protein with activity that prevents the function of a bacterial CRISPR-Cas system.
  • an anti-CRISPR protein prevents a host bacterium from mounting a CRISPR-Cas system based defense against the invading bacteriophage.
  • the anti-CRISPR polypeptide inactivates the host bacterium’s CRISPR-Cas system using a process comprising gene regulation interference.
  • the anti- CRISPR polypeptide inactivates the host bacterium’s CRISPR-Cas system using a process comprising nuclease recruitment interference.
  • the anti-CRISPR polypeptide inhibits, inactivates, and/or represses the activity of a type I CRISPR-Cas system, type II CRISPR- Cas system, or a type III CRISPR-Cas system, Type IV CRISPR-Cas system, Type V CRISPR-Cas system, or Type VI CRISPR-Cas system.
  • the protein product of a nucleic acid encoding an anti-CRISPR polypeptide or the introduced anti-CRISPR polypeptide binds directly or indirectly to a Cascade or a Cascade-like complex.
  • the anti-CRISPR polypeptide is a truncated, mutated, or fused to another protein of interest. In some embodiments, the anti-CRISPR polypeptide is a dimer protein. In some embodiments, the anti-CRISPR polypeptide is a homodimer or heterodimer protein. In one embodiment, the anti-CRISPR polypeptide comprises AcrIIClBoe, AcrIIClNme, AcrIIC2Nme, AcrIIC3Nme, AcrIIC4Hpa, AcrIIC5Smu, or any functional fragments thereof. In one embodiment, the anti-CRISPR polypeptide binds with specific affinity to a specific binding site upon the CRISPR-Cas system.
  • the anti-CRISPR polypeptide inhibits, inactivates, or represses the activity of a CRISPR-Cas system in the target bacterium, wherein said CRISPR-Cas system targets the bacteriophage comprising the nucleic acid encoding the anti-CRISPR polypeptide. In some embodiments, the anti-CRISPR polypeptide inhibits, inactivates, or represses the activity of a CRISPR-Cas system in the target bacterium, wherein said CRISPR-Cas system targets a second orthogonal bacteriophage different than a first bacteriophage.
  • the second orthogonal bacteriophage is different than the first bacteriophage.
  • the inhibition, inactivation, or repression of the CRISPR-Cas system activity in the target bacterium by the anti-CRISPR polypeptide from a first bacteriophage enhances the activity of the first bacteriophage or a second orthogonal bacteriophage.
  • the second orthogonal bacteriophage has lytic activity.
  • the second orthogonal bacteriophage comprises a bacteriophage of any of the embodiments disclosed herein.
  • killing bacteria is achieved by the lytic activity of the bacteriophage.
  • the lytic activity of the bacteriophage is due to the removal, replacement, or inactivation of at least one lysogeny gene.
  • the lysogenic gene plays a role in the maintenance of lysogenic cycle in the bacteriophage.
  • the lysogenic gene plays a role in establishing the lysogenic cycle in the bacteriophage.
  • the lysogenic gene plays a role in both establishing the lysogenic cycle and in the maintenance of the lysogenic cycle in the bacteriophage.
  • the lysogenic gene is a repressor gene.
  • the lysogenic gene is cl repressor gene.
  • the lysogenic gene is an activator gene. In some embodiments, the lysogenic gene is ell gene. In some embodiments, the lysogenic gene is lexA gene. In some embodiments, the lysogenic gene is ini (integrase) gene. In some embodiments, a temperate bacteriophage is rendered lytic by the insertion of one or more genes that contribute to the induction of a lytic cycle. In some
  • a temperate bacteriophage is rendered lytic by altering the expression of one or more genes that contribute to the induction of a lytic cycle.
  • a temperate bacteriophage phenotypically changes from a lysogenic bacteriophage to a lytic bacteriophage.
  • a temperate bacteriophage is rendered lytic by environmental alterations.
  • environmental alterations include, but are not limited to, alterations in temperature, pH, or nutrients, exposure to antibiotics, hydrogen peroxide, foreign DNA, or DNA damaging agents, presence of organic carbon, and presence of heavy metal (e.g. in the form of chromium (VI).
  • a temperate bacteriophage that is rendered lytic is prevented from reverting to lysogenic state. In some embodiments, a temperate bacteriophage that is rendered lytic is prevented from reverting back to lysogenic state by way of introducing an additions CRIPSR array. In some embodiments, killing of a target bacterium is achieved by the activity of a CRISPR array comprising at least one spacer that is complementary to a target nucleotide sequence in a target gene in the target bacterium. In some embodiments, killing of the target bacterium is achieved by the activity of a mature crRNA.
  • killing of the bacterium is achieved by the processing of the CRISPR array by a Type I, Type II, Type III, Type IV, Type V, or a Type VI CRISPR-Cas system to produce a processed crRNA capable of directing CRISPR-Cas based endonuclease activity and/or cleavage at the target nucleotide sequence in the target gene of the bacterium.
  • killing of a target bacterium is achieved by the activity of the CRISPR array independent to the lytic and/or non-lytic activity of the bacteriophage.
  • the killing of a target bacterium is by any method or combination of methods disclosed herein.
  • killing of the bacterium are achieved solely by the lytic activity of the bacteriophage. In some embodiments, killing of the bacterium is achieved solely by the activity of the nucleic acid encoding a CRISPR array comprising at least one spacer. In some embodiments, killing of the bacterium is achieved solely by the activity of the nucleic acid encoding a mature crRNA. In some embodiments, killing of the bacterium is achieved by a combination of the lytic activity of the bacteriophage and the activity of the CRISPR array or mature crRNA.
  • killing of the bacterium by a combination of the lytic activity of the bacteriophage and by the activity of the first nucleic acid encoding a CRISPR array is synergistic. In some embodiments, the killing activity of the CRISPR array or mature crRNA supplements or enhances the lytic activity of the bacteriophage. In some embodiments, killing of a target bacterium is a synergistic effect of two or more systems.
  • the synergistic killing of the bacterium is modulated by the concentration of the bacteriophage and/or the design of the CRISPR array. In some embodiments, the synergistic killing of the bacterium is modulated to favor killing by the lytic activity of the bacteriophage over the activity of the CRISPR array by increasing the concentration of
  • the synergistic killing of the bacterium is modulated to disfavor killing by the lytic activity of the bacteriophage over the activity of the CRISPR array by decreasing the concentration of bacteriophage administered to the bacterium.
  • lytic replication allows for amplification and killing of the target bacteria.
  • amplification of a phage is not required.
  • the synergistic killing of the bacterium is modulated to favor killing by the activity of the CRISPR array over the lytic activity of the bacteriophage by altering the number, the length, the composition, the identity, or any combination thereof, of the spacers so as to increase the lethality of the CRISPR array. In some embodiments, the synergistic killing of the bacterium is modulated to disfavor killing by the activity of the CRISPR array over the lytic activity of the bacteriophage by altering the number, the length, the composition, the identity, or any combination thereof, of the spacers so as to decrease the lethality of the CRISPR array.
  • the target nucleotide sequence in the bacterium to be killed is any essential target nucleotide sequence of interest.
  • the target nucleotide sequence is a non-essential sequence.
  • a target nucleotide sequence comprises, consists essentially of or consist of all or a part of a nucleotide sequence encoding a promoter, or a complement thereof, of a target gene.
  • the spacer nucleotide sequence is complementary to a promoter, or a part thereof, of a target gene.
  • the target nucleotide sequence comprises all or a part of a nucleotide sequence located on a coding or a non-coding strand of DNA. In some embodiments, the target nucleotide sequence comprises all or a part of a nucleotide sequence located on a coding of a transcribed region of a target gene.
  • An essential gene is any gene of an organism that is critical for its survival. However, being essential is highly dependent on the circumstances in which an organism lives. For instance, a gene required to digest starch is only essential if starch is the only source of energy. In some
  • the essential gene includes but is not limited to: acpP, csrA, eno,fusA, gapA, gfyQ, inf A, nusG, secY, trmD, Tsf and ftsA.
  • a non-essential gene is any gene of an organism that is not critical for survival. However, being non-essential is highly dependent on the circumstances in which an organism lives.
  • non-limiting examples of a target gene of interest includes a gene encoding a transcriptional regulator, a translational regulator, a polymerase gene, a metabolic enzyme, a transporter, an RNase, a protease, a DNA replication enzyme, a DNA modifying or degrading enzyme, a regulatory RNA, a transfer RNA, or a ribosomal RNA.
  • a target gene is a gene involved in cell-division, cell structure, metabolism, motility, pathogenicity or virulence.
  • a target gene includes a hypothetical gene whose function is not yet characterized. Thus, for example, the target genes are any gene from any bacterium.
  • a bacteriophage disclosed herein is further genetically modified to express an antibacterial peptide, a functional fragment of an antibacterial peptide or a lytic gene.
  • a bacteriophage disclosed herein express at least one antimicrobial agent or peptide disclosed herein.
  • a bacteriophage disclosed herein comprises a nucleic acid sequence that encodes an enzybiotic where the protein product of the nucleic acid sequence targets phage resistant bacteria.
  • the bacteriophage comprises nuclei c acids which encode enzymes which assist in breaking down or degrading biofilm matrix.
  • a bacteriophage disclosed herein comprises nucleic acids encoding Dispersin D aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta- galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase or
  • the enzyme is selected from the group consisting of cellulases, such as glycosyl hydroxylase family of cellulases, such as glycosyl hydroxylase 5 family of enzymes also called cellulase A; polyglucosamine (PGA) depolymerases; and colonic acid depolymerases, such as l,4-L-fucodise hydrolase Characterisation of a 1, 4-beta- fucoside hydrolase degrading colanic acid, depolymerazing alginase, DNase I, or combinations thereof.
  • a bacteriophage disclosed herein secretes an enzyme disclosed herein.
  • an antimicrobial agent or peptide is expressed and/or secreted by a bacteriophage disclosed herein.
  • a bacteriophage disclosed herein secretes and expresses an antibiotic such as ampicillin, penicillin, penicillin derivatives, cephalosporins, monobactams, carbapenems, ofloxacin, ciproflaxacin, levofloxacin, gatifloxacin, norfloxacin, lomefloxacin, trovafloxacin, moxifloxacin, sparfloxacin, gemifloxacin, pazufloxacin or any antibiotic disclosed herein.
  • an antibiotic such as ampicillin, penicillin, penicillin derivatives, cephalosporins, monobactams, carbapenems, ofloxacin, ciproflaxacin, levofloxacin, gatifloxacin, norfloxacin, lomefloxacin, trovafloxacin, moxifloxacin
  • a bacteriophage disclosed herein comprises a nucleic acid sequence encoding an antibacterial peptide, expresses an antibacterial peptide, or secretes a peptide that aids or enhances killing of a target bacterium. In some embodiments, a bacteriophage disclosed herein comprises a nucleic acid sequence encoding a peptide, a nucleic acid sequence encoding an antibacterial peptide, expresses an antibacterial peptide, or secretes a peptide that aids or enhances the activity of a CRISPR-Cas system.
  • the bacteriophages disclosed herein treat or prevent diseases or conditions mediated or caused by bacteria as disclosed herein in a human or animal subjects. Such bacteria are typically in contact with tissue of the subject including: gut, oral cavity, lung, armpit, ocular, vaginal, anal, ear, nose or throat tissue.
  • a bacterial infection is treated by modulating the activity of the bacteria and/or by directly killing of the bacteria.
  • one or more target bacteria present in a bacterial population are pathogenic.
  • the pathogenic bacteria are uropathogenic.
  • the pathogenic bacterium is uropathogenic E. Coli (UPEC). In some embodiments, the pathogenic bacteria are diarreagenic. In some embodiments, the pathogenic bacteria are diarreagenic E.coli (DEC). In some embodiments, the pathogenic bacteria are Shiga-toxin producing. In some embodiments, the pathogenic bacterium is Shiga-toxin producing E.coli (STEC). In some embodiments, the pathogenic bacteria are Shiga-toxin producing. In some embodiments, the pathogenic bacterium is Shiga-toxin producing E.coli (STEC). In some embodiments, the pathogenic bacterium is Shiga-toxin producing E.coli (STEC). In some embodiments, the pathogenic bacterium is Shiga-toxin producing E.coli (STEC).
  • the pathogenic bacteria are various 0-antigen:H-antigen serotype . coli. In some embodiments, the pathogenic bacteria are entereopathogenic. In some embodiments, the pathogenic bacterium is entereopathogenic E.coli (EPEC).
  • the pathogenic bacteria are various strains of C. difficile including: CD043, CD05, CD073, CD093, CD180, CD106, CD128, CD199, CD111, CD108, CD25, CD148, CD 154, FOBT195, CD03, CD038, CD112, CD196, CD105, UK1, UK6, BI-9, CD041, CD042, CD046, CD 19, or R2029l.
  • the bacteriophages disclosed herein are used to treat an infection, a disease, or a condition, in the gastrointestinal tract of a subject. In some embodiments, the bacteriophages are used to modulate and/or kill target bacteria within the microbiome or gut flora of a subject. In some embodiments, the bacteriophages are used to selectively modulate and/or kill one or more target bacteria from a plurality of bacteria within the microbiome or gut flora of a subject. In some embodiments, the bacteriophages are used to selectively modulate and/or kill one or more target entereopathogenic bacteria from a plurality of bacteria within the microbiome or gut flora of a subject. In some embodiments, the target entereopathogenic bacterium is
  • the bacteriophages are used to selectively modulate and/or kill one or more target diarreagenic bacteria from a plurality of bacteria within the microbiome or gut flora of a subject.
  • the target diarreagenic bacterium is diarreagenic E.coli (DEC).
  • the bacteriophages are used to selectively modulate and/or kill one or more target Shiga-toxin producing bacteria from a plurality of bacteria within the microbiome or gut flora of a subject.
  • the target Shiga- toxin producing bacterium is Shiga-toxin producing E.coli (STEC).
  • the bacteriophages are used to selectively modulate and/or kill one or more target enteropathogenic C. difficile bacteria strains within the microbiome or gut flora of a subject including: CD043, CD05, CD073, CD093, CD180, CD106, CD128, CD199, CD111, CD108, CD25, CD148, CD154, FOBT195, CD03, CD038, CD112, CD196, CD105, UK1, UK6, BI-9, CD041, CD042, CD046, CD 19, or R2029l.
  • the bacteriophages disclosed herein are used to treat an infection, a disease, or a condition, in the urinary tract of a subject.
  • the bacteriophages are used to modulate and/or kill target bacteria within the urinary tract flora of a subject.
  • the urinary tract flora includes, but is not limited, to Staphylococcus epidermidis, Enterococcus faecalis, and some alpha-hemolytic Streptococci.
  • the bacteriophages are used to selectively modulate and/or kill one or more target uropathogenic bacteria from a plurality of bacteria within the urinary tract flora of a subject.
  • the target bacterium is uropathogenic E. Coli (UPEC).
  • the bacteriophages disclosed herein are used to treat an infection, a disease, or a condition, on the skin of a subject. In some embodiments, the bacteriophages are used to modulate and/or kill target bacteria on the skin of a subject.
  • the bacteriophages disclosed herein are used to treat an infection, a disease, or a condition, on a mucosal membrane of a subject.
  • the bacteriophages disclosed herein are used to treat an infection, a disease, or a condition, on a mucosal membrane of a subject.
  • bacteriophages are used to modulate and/or kill target bacteria on the mucosal membrane of a subject.
  • the pathogenic bacteria are antibiotic resistant.
  • the pathogenic bacterium is methicillin-resistant Staphylococcus aureus (MRSA).
  • the one or more target bacteria present in the bacterial population form a biofilm.
  • the biofilm comprises pathogenic bacteria.
  • the bacteriophage disclosed herein is used to treat a biofilm.
  • non-limiting examples of target bacteria includes Escherichia spp., Salmonella spp., Bacillus spp. , Corynehacterium Clostridium spp., Clostridium spp., Psuedomonas spp., Clostridium spp., Lactococcus spp., Acinetoibacter spp., Mycobacterium spp., Myxococcus spp., Staphylococcus spp., Streptococcus spp., or cyanobacteria.
  • non limiting examples of bacteria include Escherichia coli, Salmonella enterica, Bacillus subtilis, Clostridium acetobutylicum, Clostridium ljungdahlii, Clostridium difficile, Acinetobacter baumannii, Mycobacterium tuberculosis, Myxococcus xanthus, Staphylococcus aureus,
  • non-limiting examples of bacteria include Staphylococcus aureus, methicillin resistant Staphylococcus aureus, Streptococcus pneumonia, carbapenem-resistant Enter oacteriaceae, Staphylococcus epidermidis, Staphylococcus salivarius, Corynehacterium minutissium, Corynehacterium pseudodiphtheriae, Corynehacterium stratium, Corynehacterium group Gl, Corynehacterium group G2, Streptococcus pneumonia, Streptococcus mitis, Streptococcus sanguis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Burkholderia cepacia, Serratia marcescens, Haemophilus influenzae, Moraxella sp., Neisser
  • bacteria include lactic acid bacteria including but not limited to Lactobacillus spp. and Bifidobacterium spp:, electrofuel bacterial strains including but not limited to Geobacter spp., Clostridium spp., or Ralstonia eutropha ; or bacteria pathogenic on, for example, plants and mammals.
  • the bacterium is Escherichia coli.
  • the bacterium is Clostridium difficile.
  • the bacteriophage treats acne and other related skin infections.
  • a target bacterium is a multiple drug resistant (MDR) bacteria strain.
  • An MDR strain is a bacteria strain that is resistant to at least one antibiotic.
  • a bacteria strain is resistant to an antibiotic class such as a cephalosporin, a fluoroquinolone, a carbapenem, a colistin, an aminoglycoside, vancomycin, streptomycin, and methicillin.
  • a bacteria strain is resistant to an antibiotic such as a Ceftobiprole, Ceftaroline, Clindamycin, Dalbavancin, Daptomycin, Linezolid, Mupirocin, Oritavancin, Tedizolid, Telavancin, Tigecy cline, Vancomycin, an Aminoglycoside, a Carbapenem, Ceftazidime, Cefepime,
  • MDR strains include: Vancomycin- Resistant Enterococci (VRE), Methicillin-Resistant Staphylococcus aureus (MRS A), Extended- spectrum b-lactamase (ESBLs) producing Gram-negative bacteria, Klebsiella pneumoniae carbapenemase (KPC) producing Gram-negatives, and Multidrug-Resistant gram negative rods (MDR GNR) MDRGN bacteria such as Enterobacter species E.coli, Klebsiella pneumoniae, Acinetobacter baumannii, or Pseudomonas aeruginosa.
  • VRE Vancomycin- Resistant Enterococci
  • MRS A Methicillin-Resistant Staphylococcus aureus
  • ESBLs Extended- spectrum b-lactamase
  • KPC Klebsiella pneumoniae carbapenemase
  • MDR GNR Multidrug-Resistant gram negative rods
  • the target bacterium is Klebsiella pneumoniae. In some embodiments, the target bacterium is Staphylococcus aureus. In some embodiments, the target bacterium is Enterococci . In some embodiments, the target bacterium is Acinetobacter. In some embodiments, the target bacterium is Pseudomonas. In some embodiments, the target bacterium is Enterobacter.
  • the target bacterium is Clostridium difficile. In some embodiments, the target bacterium is E.coli. In some embodiments, the target bacterium is Clostridium bolteae. In some embodiments, the methods and compositions disclosed herein are for use in veterinary and medical applications as well as research applications Microbiome
  • “Microbiome”,“microbiota”, and“microbial habitat” are used interchangeably hereinafter and refer to the ecological community of microorganisms that live on or in a subject’s bodily surfaces, cavities, and fluids.
  • Non-limiting examples of habitats of microbiome include: gut, colon, skin, skin surfaces, skin pores, vaginal cavity, umbilical regions, conjunctival regions, intestinal regions, stomach, nasal cavities and passages, gastrointestinal tract, urogenital tracts, saliva, mucus, and feces.
  • the microbiome comprises microbial material including, but not limited to, bacteria, archaea, protists, fungi, and viruses.
  • the microbial material comprises a gram-negative bacterium. In some embodiments, the microbial material comprises a gram-positive bacterium. In some embodiments, the microbial material comprises Proteobacteria, Actinobacteria, Bacteriodetes, or Firmicutes.
  • the bacteriophages as disclosed herein are used to modulate or kill target bacteria within the microbiome of a subject. In some embodiments, the bacteriophages are used to modulate and/or kill target bacteria within the microbiome by the CRISPR-Cas system, lytic activity, or a combination thereof. In some embodiments, the bacteriophages are used to modulate and/or kill target bacteria within the microbiome of a subject. In some embodiments, the bacteriophages are used to selectively modulate and/or kill one or more target bacteria from a plurality of bacteria within the microbiome of a subject.
  • the bacteriophages are used to selectively modulate and/or kill one or more target entereopathogenic bacteria from a plurality of bacteria within the microbiome of a subject.
  • the target entereopathogenic bacterium is entereopathogenic E. Coli (EPEC).
  • the bacteriophages are used to selectively modulate and/or kill one or more target diarreagenic bacteria from a plurality of bacteria within the microbiome of a subject.
  • the target diarreagenic bacterium is diarreagenic E.coli (DEC).
  • the bacteriophages are used to selectively modulate and/or kill one or more target Shiga-toxin producing bacteria from a plurality of bacteria within the microbiome of a subject.
  • the target Shiga- toxin producing bacterium is Shiga-toxin producing E.coli (STEC).
  • the bacteriophages are used to selectively modulate and/or kill one or more target enteropathogenic C. difficile bacteria strains within the microbiome of a subject including: CD043, CD05, CD073, CD093, CD180, CD106, CD128, CD199, CD111, CD108, CD25, CD148, CD154, FOBT195, CD03, CD038, CD112, CD196, CD105, UK1, UK6, BI-9, CD041, CD042, CD046, CD 19, or R2029l.
  • the bacteriophages are used to modulate or kill target single or plurality of bacteria within the microbiome or gut flora of the gastrointestinal tract of a subject. Modification (e.g., dysbiosis) of the microbiome or gut flora increases the risk for health conditions such as diabetes, mental disorders, ulcerative colitis, colorectal cancer, autoimmune disorders, obesity, diabetes, diseases of the central nervous system and inflammatory bowel disease.
  • An exemplary list of the bacteria associated with diseases and conditions of gastrointestinal tract and are being modulated or killed by the bacteriophages include strains, sub-strains, and enterotypes of enterobacteriaceae, pasteurellaceae, fusobacteriaceae, neisseriaceae, veillonellaceae, gemellaceae, bacteriodales, clostridiales, erysipelotrichaceae, bifidobacteriaceae bacteroides, faecalibacterium, roseburia, blautia, ruminococcus, coprococcus, streptococcus, dorea, blautia, ruminococcus, lactobacillus, enterococcus, streptococcus, escherichia coli, fusobacterium nucleatum, haemophilus parainfluenzae (pasteurellaceae), veillonella parvula, eikenella
  • a bacteriophage disclosed herein is administered to a subject to promote a healthy microbiome. In some embodiments, a bacteriophage disclosed herein is administered to a subject to restore a subject’s microbiome to a microbiome composition that promotes health. In some embodiments, a composition comprising a bacteriophage disclosed herein comprises a prebiotic or a third agent. In some embodiment, microbiome related disease or disorder is treated by a bacteriophage disclosed herein.
  • bacteriophages disclosed herein are further used for food and agriculture sanitation (including meats, fruits and vegetable sanitation), hospital sanitation, home sanitation, vehicle and equipment sanitation, industrial sanitation, etc.
  • bacteriophages disclosed herein are used for the removal of antibiotic-resistant or other undesirable pathogens from medical, veterinary, animal husbandry, or any additional environments bacteria are passed to humans or animals.
  • Environmental applications of phage in health care institutions are for equipment such as endoscopes and environments such as ICUs which are potential sources of nosocomial infection due to pathogens that are difficult or impossible to disinfect.
  • a phage disclosed herein is used to treat equipment or environments inhabited by bacterial genera such as Pseudomonas which become resistant to commonly used disinfectants.
  • phage compositions disclosed herein are used to disinfect inanimate objects.
  • an environment disclosed herein is sprayed, painted, or poured onto with aqueous solutions with phage titers.
  • a solution described herein comprises between 10 1 -10 20 plaque forming units (PFU)/ml.
  • a bacteriophage disclosed herein is applied by aerosolizing agents that include dry dispersants to facilitate distribution of the bacteriophage into the environment.
  • objects are immersed in a solution containing
  • bacteriophages disclosed herein are used as sanitation agents in a variety of fields.
  • phage or“bacteriophage” may be used, it should be noted that, where appropriate, this term should be broadly construed to include a single bacteriophage, multiple bacteriophages, such as a bacteriophage mixtures and mixtures of a bacteriophage with an agent, such as a disinfectant, a detergent, a surfactant, water, etc.
  • bacteriophages are used to sanitize hospital facilities, including operating rooms, patient rooms, waiting rooms, lab rooms, or other miscellaneous hospital equipment.
  • this equipment includes electrocardiographs, respirators, cardiovascular assist devices, intraaortic balloon pumps, infusion devices, other patient care devices, televisions, monitors, remote controls, telephones, beds, etc.
  • the bacteriophage is applied through an aerosol canister.
  • bacteriophage is applied by wiping the phage on the object with a transfer vehicle.
  • a bacteriophage described herein is used in conjunction with patient care devices.
  • bacteriophage is used in conjunction with a conventional ventilator or respiratory therapy device to clean the internal and external surfaces between patients.
  • ventilators include devices to support ventilation during surgery, devices to support ventilation of incapacitated patients, and similar equipment.
  • the ventilators include devices to support ventilation during surgery, devices to support ventilation of incapacitated patients, and similar equipment.
  • respiratory therapy includes automatic or motorized devices, or manual bag-type devices such as are commonly found in emergency rooms and ambulances.
  • respiratory therapy includes inhalers to introduce medications such as bronchodilators as commonly used with chronic obstructive pulmonary disease or asthma, or devices to maintain airway patency such as continuous positive airway pressure devices.
  • a bacteriophage described herein is used to cleanse surfaces and treat colonized people in an area where highly-contagious bacterial diseases, such as meningitis or enteric infections are present.
  • water supplies are treated with a composition disclosed herein.
  • bacteriophage disclosed herein is used to treat contaminated water, water found in cisterns, wells, reservoirs, holding tanks, aqueducts, conduits, and similar water distribution devices.
  • the bacteriophage is applied to industrial holding tanks where water, oil, cooling fluids, and other liquids accumulate in collection pools.
  • a bacteriophage disclosed herein is periodically introduced to the industrial holding tanks in order to reduce bacterial growth.
  • bacteriophages disclosed herein are used to sanitize a living area, such as a house, apartment, condominium, dormitory, or any living area.
  • the bacteriophage is used to sanitize public areas, such as theaters, concert halls, museums, train stations, airports, pet areas, such as pet beds, or litter boxes.
  • the bacteriophage is dispensed from conventional devices, including pump sprayers, aerosol containers, squirt bottles, pre-moistened towelettes, etc, applied directly to (e.g., sprayed onto) the area to be sanitized, or be transferred to the area via a transfer vehicle, such as a towel, sponge, etc.
  • a phage disclosed herein is applied to various rooms of a house, including the kitchen, bedrooms, bathrooms, garage, basement, etc. In some embodiments, a phage disclosed herein is in the same manner as conventional cleaners. In some embodiments, the phage is applied in conjunction with (before, after, or simultaneously with) conventional cleaners provided that the conventional cleaner is formulated so as to preserve adequate bacteriophage biologic activity.
  • a bacteriophage disclosed herein is added to a component of paper products, either during processing or after completion of processing of the paper products.
  • Paper products to which a bacteriophage disclosed herein is added include, but are not limited to, paper towels, toilet paper, moist paper wipes.
  • a bacteriophage described herein is used in any food product or nutritional supplement, for preventing contamination.
  • food or pharmaceuticals products are milk, yoghurt, curd, cheese, fermented milks, milk based fermented products, ice creams, fermented cereal based products, milk based powders, infant formulae or tablets, liquid suspensions, dried oral supplement, wet oral supplement, or dry-tube-feeding.
  • the broad concept of bacteriophage sanitation is applicable to other agricultural applications and organisms. Produce, including fruits and vegetables, dairy products, and other agricultural products. For example, freshly-cut produce frequently arrive at the processing plant contaminated with pathogenic bacteria. This has led to outbreaks of food-borne illness traceable to produce.
  • the application of bacteriophage preparations to agricultural produce substantially reduce or eliminate the possibility of food-borne illness through application of a single phage or phage mixture with specificity toward species of bacteria associated with food- borne illness.
  • bacteriophages are applied at various stages of production and processing to reduce bacterial contamination at that point or to protect against contamination at subsequent points.
  • specific bacteriophages are applied to produce in restaurants, grocery stores, produce distribution centers.
  • bacteriophages disclosed herein are periodically or continuously applied to the fruit and vegetable contents of a salad bar.
  • the application of bacteriophages to a salad bar or to sanitize the exterior of a food item is a misting or spraying process or a washing process.
  • a bacteriophage described herein is used in matrices or support media containing with packaging containing meat, produce, cut fruits and vegetables, and other foodstuffs.
  • polymers that are suitable for packaging are impregnated with a bacteriophage preparation.
  • a bacteriophage described herein is used in farm houses and livestock feed. In some embodiments, on a farm raising livestock, the livestock is provided with bacteriophage in their drinking water, food, or both. In some embodiments, a bacteriophage described herein is sprayed onto the carcasses and used to disinfect the slaughter area.
  • bacteriophages are natural, non-toxic products that will not disturb the ecological balance of the natural microflora in the way the common chemical sanitizers do, but will specifically lyse the targeted food-borne pathogens. Because bacteriophages, unlike chemical sanitizers, are natural products that evolve along with their host bacteria, new phages that are active against recently emerged, resistant bacteria are rapidly identified when required, whereas identification of a new effective sanitizer is a much longer process, several years.
  • the disclosure provides pharmaceutical compositions and methods of administering the same to treat bacterial, archaeal infections or to disinfect an area.
  • the pharmaceutical composition comprises any of the reagents discussed above in a pharmaceutically acceptable carrier.
  • a pharmaceutical composition or method disclosed herein treats Lung infections (CFP, NCFB, HAP/VAP) systemic infections (bacteremia, SSSI) GI microbiome dysbiosis (CDI) and/ or urinary tract infections (cUTI).
  • compositions disclosed herein comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.
  • the bacteriophages disclosed herein are formulated for
  • the manufacture of a pharmaceutical composition according to the disclosure the bacteriophage is admixed with, inter alia, an acceptable carrier.
  • the carrier is a solid (including a powder) or a liquid, or both, and is preferably formulated as a unit-dose composition.
  • one or more bacteriophages are incorporated in the
  • compositions disclosed herein which are prepared by any suitable method of a pharmacy.
  • a method of treating subject’s in-vivo comprising administering to a subject a pharmaceutical composition comprising a bacteriophage disclosed herein in a
  • the administration of the bacteriophage to a human subject or an animal in need thereof are by any means known in the art.
  • bacteriophages disclosed herein are for oral administration.
  • the bacteriophages are administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
  • compositions and methods suitable for buccal (sub-lingual) administration include lozenges comprising the bacteriophages in a flavored base, usually sucrose and acacia or tragacanth; and pastilles comprising the bacteriophages in an inert base such as gelatin and glycerin or sucrose and acacia.
  • methods and compositions of the present disclosure are suitable for parenteral administration comprising sterile aqueous and non-aqueous injection solutions of the bacteriophage.
  • these preparations are isotonic with the blood of the intended recipient.
  • these preparations comprise antioxidants, buffers, bacteriostals and solutes which render the composition isotonic with the blood of the intended recipient.
  • aqueous and non-aqueous sterile suspensions include suspending agents and thickening agents.
  • compositions disclosed herein are presented in unit ⁇ dose or multi-dose containers, for example sealed ampoules and vials, and are stored in a freeze- dried(lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, saline or water for injection on immediately prior to use.
  • sterile liquid carrier for example, saline or water for injection
  • methods and compositions suitable for rectal administration are presented as unit dose suppositories. In some embodiments, these are prepared by admixing the bacteriophage with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture. In some embodiments, methods and compositions suitable for topical application to the skin are in the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. In some embodiments, carriers which are used include petroleum jelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.
  • compositions suitable for transdermal administration are presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.
  • methods and compositions suitable for nasal administration or otherwise administered to the lungs of a subject include any suitable means, e.g., administered by an aerosol suspension of respirable particles comprising the bacteriophage compositions, which the subject inhales.
  • the respirable particles are liquid or solid.
  • aerosol includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages.
  • aerosols of liquid particles are produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer.
  • aerosols of solid particles comprising the composition is produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.
  • methods and compositions suitable for administering bacteriophages disclosed herein to a surface of an object or subject includes aqueous solutions.
  • such aqueous solutions are sprayed onto the surface of an object or subject.
  • the aqueous solutions are used to irrigate and clean a physical wound of a subject form foreign debris including bacteria.
  • the bacteriophages disclosed herein are administered to the subject in a therapeutically effective amount.
  • at least one bacteriophage composition disclosed herein is formulated as a pharmaceutical formulation.
  • a pharmaceutical formulation comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more bacteriophage disclosed herein.
  • a pharmaceutical formulation comprises a bacteriophage described herein and at least one of: an excipient, a diluent, or a carrier.
  • a pharmaceutical formulation comprises an excipient.
  • Excipients are described in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (1986) and includes but are not limited to solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, and lubricants.
  • Non-limiting examples of suitable excipients include but is not limited to a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a chelator, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, a coloring agent.
  • an excipient is a buffering agent.
  • suitable buffering agents include but is not limited to sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
  • a pharmaceutical formulation comprises any one or more buffering agent listed: sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium glucomate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium
  • polyphosphate potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide and other calcium salts.
  • an excipient is a preservative.
  • suitable preservatives include but is not limited to antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
  • antioxidants include but not limited to EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxy anisole (BHA), sodium sulfite, p-amino benzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol and N- acetyl cysteine.
  • preservatives include validamycin A, TL-3, sodium ortho vanadate, sodium fluoride, N-a-tosyl-Phe- chloromethylketone, N-a-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride,
  • diisopropylfluorophosphate diisopropylfluorophosphate, protease inhibitor, reducing agent, alkylating agent, antimicrobial agent, oxidase inhibitor, or other inhibitor.
  • a pharmaceutical formulation comprises a binder as an excipient.
  • suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyl oxoazoli done, polyvinylalcohols, C l2 -C l8 fatty acid alcohol,
  • polyethylene glycol polyols, saccharides, oligosaccharides, and combinations thereof.
  • the binders that are used in a pharmaceutical formulation are selected from starches such as potato starch, corn starch, wheat starch; sugars such as sucrose, glucose, dextrose, lactose, maltodextrin; natural and synthetic gums; gelatine; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinylpyrrolidone (povidone); polyethylene glycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols such as sorbitol, xylitol, mannitol and water or a combination thereof.
  • starches such as potato starch, corn starch, wheat starch
  • sugars such as sucrose, glucose, dextrose, lactose, maltodextrin
  • natural and synthetic gums such as cellulose derivatives such as microcrystalline cellulose,
  • a pharmaceutical formulation comprises a lubricant as an excipient.
  • suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.
  • lubricants that are in a pharmaceutical formulation are selected from metallic stearates (such as magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (such as sodium stearyl fumarate), fatty acids (such as stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffins, hydrogenated vegetable oils, leucine, polyethylene glycols (PEG), metallic lauryl sulphates (such as sodium lauryl sulphate, magnesium lauryl sulphate), sodium chloride, sodium benzoate, sodium acetate and talc or a combination thereof.
  • an excipient comprises a flavoring agent.
  • flavoring agents includes natural oils; extracts from plants, leaves, flowers, and fruits; and combinations thereof.
  • an excipient comprises a sweetener.
  • suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts such as a sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives of sucrose such as sucralose; and sugar alcohols such as sorbitol, mannitol, sylitol, and the like.
  • a pharmaceutical formulation comprises a coloring agent.
  • suitable color agents include food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), and external drug and cosmetic colors (Ext. D&C).
  • the pharmaceutical formulation disclosed herein comprises a chelator.
  • a chelator includes ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA); a disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium and
  • diammonium salt of EDTA a barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium, or zinc chelate of EDTA.
  • a pharmaceutical formulation comprises a diluent.
  • diluents include water, glycerol, methanol, ethanol, and other similar biocompatible diluents.
  • a diluent is an aqueous acid such as acetic acid, citric acid, maleic acid, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, or similar.
  • a pharmaceutical formulation comprises a surfactant.
  • surfactants are be selected from, but not limited to, polyoxyethylene sorbitan fatty acid esters (polysorbates), sodium lauryl sulphate, sodium stearyl fumarate, polyoxyethylene alkyl ethers, sorbitan fatty acid esters, polyethylene glycols (PEG), polyoxyethylene castor oil derivatives, docusate sodium, quaternary ammonium compounds, aminoacids such as L- leucine, sugar esters of fatty acids, glycerides of fatty acids or a combination thereof.
  • a pharmaceutical formulation comprises an additional pharmaceutical agent.
  • an additional pharmaceutical agent is an antibiotic agent.
  • an antibiotic agent is of the group consisting of aminoglycosides, ansamycins, carbacephem, carbapenems, cephalosporins (including first, second, third, fourth and fifth generation cephalosporins), lincosamides, macrolides, monobactams, nitrofurans, quinolones, penicillin, sulfonamides, polypeptides or tetracycline.
  • an antibiotic agent described herein is an aminoglycoside such as Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin or Paromomycin.
  • an antibiotic agent described herein is an Ansamycin such as Geldanamycin or Herbimycin
  • an antibiotic agent described herein is a carbacephem such as Loracarbef.
  • an antibiotic agent described herein is a carbapenem such as Ertapenem, Doripenem, Imipenem/Cilastatin or Meropenem.
  • an antibiotic agent described herein is a cephalosporins (first generation) such as Cefadroxil, Cefazolin, Cefalexin, Cefalotin or Cefalothin, or alternatively a Cephalosporins (second generation) such as Cefaclor, Cefamandole, Cefoxitin, Cefprozil or Cefuroxime.
  • an antibiotic agent is a Cephalosporins (third generation) such as Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftibuten,
  • Ceftizoxime and Ceftriaxone or a Cephalosporins (fourth generation) such as Cefepime or
  • an antibiotic agent described herein is a lincosamide such as Clindamycin and Azithromycin, or a macrolide such as Azithromycin, Clarithromycin,
  • an antibiotic agent described herein is a monobactams such as Aztreonam, or a nitrofuran such as Furazolidone or Nitrofurantoin.
  • an antibiotic agent described herein is a penicillin such as Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Nafcillin, Oxacillin, Penicillin G or V, Piperacillin, Temocillin and Ticarcillin.
  • an antibiotic agent described herein is a sulfonamide such as Mafenide, Sulfonamidochrysoidine, Sulfacetamide, Sulfadiazine, Silver sulfadiazine,
  • Sulfamethizole Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim, or Trimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP-SMX).
  • an antibiotic agent described herein is a quinolone such as
  • Ciprofloxacin Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin and Temafloxacin.
  • an antibiotic agent described herein is a polypeptide such as
  • an antibiotic agent described herein is a tetracycline such as
  • Demeclocycline Doxycycline, Minocycline or Oxytetracycline.
  • a composition disclosed herein is administered to patients by oral administration.
  • a dose of phage between 10 3 and l0 20 PFU is given.
  • the bacteriophage is present in a composition in an amount between 10 3 and 10 11 PFU.
  • the bacteriophage is present in a composition in an amount about 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , 10 19 , 10 20 , 10 21 , 10 22 , 10 23 , l0 24 PFU or more.
  • the bacteriophage is present in a composition in an amount of less thanlO 1 PFU. In some embodiments, the bacteriophage is present in a composition in an amount between l0 4 and 10 8 , l0 4 and 10 9 , l0 5 and 10 10 , or l0 7 and l0 u PFU. In some embodiments, a bacteriophage or a mixture is administered to a subject in need thereof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times a day. In some embodiments, a bacteriophage or a mixture is administered to a subject in need thereof at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times a week. In some
  • a bacteriophage or a mixture is administered to a subject in need thereof at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 times a month.
  • the compositions (bacteriophage) disclosed herein are administered before, during, or after the occurrence of a disease or condition. In some embodiment, the timing of administering the composition containing the bacteriophage varies. In some embodiments, the pharmaceutical compositions are used as a prophylactic and are administered continuously to subjects with a propensity to conditions or diseases in order to prevent the occurrence of the disease or condition. In some embodiments, pharmaceutical compositions are administered to a subject during or as soon as possible after the onset of the symptoms.
  • the administration of the compositions is initiated within the first 48 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, or within 3 hours of the onset of the symptoms.
  • the initial administration of the composition is via any route practical, such as by any route described herein using any formulation described herein.
  • the compositions is administered as soon as is practicable after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months. In some embodiments, the length of treatment will vary for each subject.
  • kits for use comprises the nucleic acid constructs for the CRISPR arrays, transcriptional activators, and/or anti-CRISPR polypeptides, as well as the bacteriophages and/or any other vectors/expression cassettes disclosed herein in a form suitable for introduction into a cell and/or administration to a subject.
  • the kit comprises other therapeutic agents, carriers, buffers, containers, devices for administration, and the like.
  • the kit comprises labels and/or instructions for repression of expression a target gene and/or modulation of repression of expression of a target gene.
  • labeling and/or instructions includes, for example, information concerning the amount, frequency and method of introduction and/or administration of the nucleic acid constructs for the CRISPR arrays, transcriptional activators, and anti-CRISPR polypeptides, as well as the bacteriophages and/or any other vectors/expression cassettes.
  • kits for the killing of one target bacterium comprising, consisting essentially of, consisting of nucleic acid constructs for the CRISPR arrays, transcriptional activators, and/or anti-CRISPR polypeptides, as well as the bacteriophages and/or any other vectors/expression cassettes necessary to achieve killing of the target bacteria by any embodiment disclosed herein.
  • the nucleic acid constructs for the CRISPR arrays, transcriptional activators, and/or anti-CRISPR polypeptides of said kits are comprised on a single vector or expression cassette or on separate vectors or expression cassettes or within a single bacteriophage or a plurality of bacteriophages.
  • a kit comprises one or more bacteriophage disclosed herein.
  • the kits comprise instructions for use.
  • the instructions for practicing the methods are recorded on a suitable recording medium.
  • the instructions are printed on a substrate, such as paper or plastic, etc.
  • the instructions are present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, flash drive, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g. via the Internet), are provided.
  • the kit includes a web address where the instructions are viewed and/or from which the instructions are downloaded.
  • Example 1 Overview for generating CRISPR enhanced bacteriophages
  • CRISPR-enhanced bacteriophages are phages that have been engineered to express CRISPR RNA constructs from a bacteriophage genome that maintains the essential genes for lytic lifestyle.
  • the steps involved are sourcing, isolating and identifying bacteriophages and cocktails of bacteriophages with broad host ranges against bacteria followed by engineering each phage to carry an expression construct (for example, crRNA) that targets the bacterium’s genome and validating optimized combinations of crPhages to be used as a clinical lead candidate.
  • the general processes are as schematically shown in steps 1-5 of Figure 1. Steps 1 - 5 are designed to identify a suitable number of wild-type bacteriophages such that they:
  • a cocktail described herein in comprises at least 2, 3,
  • each candidate bacteriophage to express a crRNA construct from the wild-type genome.
  • Each engineered crPhage is intended to retain lytic activity.
  • crPhages will then be subjected to in vitro analyses to assess host range and in vitro efficacy. These studies are intended to confirm that crPhages retain broad host range, if not expanded host range by ability to transduce lethal crRNA constructs in the absence of productive lytic infection, and improved lethality for each crPhage to the cognate wild-type bacteriophage.
  • Bacteriophage vectors were obtained from environmental sources in North Carolina. Phages were directly isolated from these samples by co-incubating with uropathgenic E. coli s ECOR 14, 62, 64 and 71. Each phage was then subjected to clonal purification by single plaque isolation across a total of three rounds of double agar overlays prior to amplification, filtration and long-term storage at 4 degrees Celsius. Each phage was amplified, filtered, subjected to cesium chloride gradient purification and dialyzed into lx tris-buffered saline.
  • Phage genomic DNA was extracted from purified phage stocks using a column-based phage DNA preparation kit (Norgen) and submitted for genome sequencing by MiSeq or PacBio sequencing, as appropriate, and assembly at a third party vendor (Genewiz).
  • a previously isolated phage, K1F was obtained that has been previously shown to infect uropathogenic E. coli isolates and have known genomes (K1F:
  • This library of wild-type bacteriophages was individually characterized against an E. coli panel for the ability to replicate and lyse each target. This process was expected to result in a wild- type bacteriophage panel that lyses approximately 90% of a panel of uropathogenic E. coli and displays minimal resistance during 24-hour challenge studies. From this master library, an abbreviated wild-type bacteriophage library was generated with retained predicted activity against approximately 90% of the uropathogenic E. coli panel.
  • phages were tested for lytic activity against an evolutionarily broad panel of E. coli. Briefly, phages were produced to high titer (10 9 - 10 11 PFU/mL), filtered and left suspended in growth media. Each target host was grown to mid-log phase and incorporated in soft agar overlays to create bacterial lawns. Phages were serially diluted down to approximately 10 3 - 10 5 PFU/mL and 5 microliters of each dilution was spot plated on each bacterial lawn. Host sensitivity to each phage was defined as any observable zone of clearance within each spot.
  • this analysis includes some phages that adsorb to a target host and cause a phenomenon termed lysis- from-without rather than ly si s-from -within caused by productive lytic infection.
  • lysis- from-without rather than ly si s-from -within caused by productive lytic infection.
  • phages were chosen that collectively infect 17 of 18 isolates tested (94%) in the panel, including 4 urinary pathogenic s isolated from human patients as shown in Table 2 below. Phages were isolated or tested against ECOR 14, 62, 64 or 71 as highlighted and then tested against the broader panel shown. Importantly, phage host range was considered as all productive lysis events and include events that resulted from ly si s-from -without. Table 2: Host range analysis of isolated bacteriophages against diverse set of E. coli stains.
  • Example 4 Primary resistance to isolate phages
  • Stable resistance phenotypes did not exhibit obvious lysis by inspection of growth curves during 24-hour cultures in media comprising a challenge bacteriophage. Bacteriophages that generate primary resistance in >50% of clones were excluded from further study. A further characterization step then measures the sensitivity of each clone to the other wild-type bacteriophages in the abbreviated library.
  • phage stocks were produced as crude lysates of the wild-type, wild-type bacteriophage in growth media by fermentation, filtration and validation of >l0 10 PFU/mL titers.
  • putative resistant clones were generated for specific host: phage pairings by incubating -108 colony forming units of an E. coli host with a high-titer lysate (>l0 8 PFU, MOI > 1.0) in a double agar overlay. After overnight culture, putative resistant clones were isolated by triple colony purification.
  • each clone was subjected to challenge with each original phage from the abbreviated lO-phage cocktail.
  • numerous clones with apparent resistance phenotypes defined as survival after initial challenge, apparently regained sensitivity after outgrowth in the absence of phage challenge as seen Table 3 below: Table 3: Resistance profile data for selected crPhages against E. coli ECOR71.
  • R Resistant
  • S Sensitive
  • T Transient (defined initially as being sensitive followed by emergences of a resistant phenotype)
  • X Not determined
  • Grey shaded boxes indicate results from clones which were re-challenged with the indicated bacteriophage used to isolate the original putatively resistant clone.
  • Example 5 Identification of lead bacteriophages for a cocktail
  • phages have collective activity against approximately 90% of the clinical isolate panel or greater; 2) phages result in infection of each by at least 2 phages within the cocktail, intended to ensure sensitivity to the cocktail in the event of resistance to any single bacteriophage; 3) phages do not generate primary resistance in more than 50% of clones isolated, with primary resistance defined as emergence of escape clones after challenge with an individual phage that retain resistance to that particular phage after clonal outgrowth and re-challenge, and lastly 4) phages do not generate cross-resistance to all other phages in the cocktail, defined as emergence of escape clones after initial challenge and clonal outgrowth that display sustained resistance to the initial challenge phage and novel resistance to other phages in the proposed cocktail to which that clone
  • Wild-type bacteriophage combinations shown in Table 2 (cumulative host range of approximately 94%) were compared to resistance profiles shown in Table 3. From these data, and based on the optimization parameters described above, five candidate bacteriophages have been identified with an approximately 94% host range coverage when tested against a panel of 18 genetically diverse E. coli isolates, with each having susceptibility to a minimum of 2 of the 5 phages. Each wild-type phage is predicted to result in less than 50% emergent clones displaying primary resistance and no clones that display resistance to all phages in the proposed cocktail. These data are summarized in Table 4, Table 5, and Table 6 and form the basis for preliminary development of a crPhage cocktail.
  • Each individual crRNA was validated in vitro by transformation into E. coli isolates to demonstrate activity of each individual crRNA against targeted E. coli sequences. These crRNAs are then assembled into arrays that are expressed from a bacteriophage genome and processed by endogenous CRISPR-Cas systems to target the host chromosome.
  • Table 4 Host range of 5 individual bacteriophages for proposed crPhage cocktail.
  • Table 5 Resistance profiles for 5 individual bacteriophages for a proposed crPhage cocktail. (Revised from Table 2 based on ECOR71 sensitivity to ⁇ E>KlF observed in Table 3)
  • Grey shaded boxes indicate results from clones w iich were re-challenged with the indicated bacteriophage used to isolate the original putatively resistant clone.
  • Table 6 Summary data for 5 individual bacteriophages for proposed crPhage cocktail.
  • Table 7 Summary of proposed preliminary crPhage cocktail.
  • Table 8 Summary of individual crRNA information.
  • Figure 2A shows a schematic diagram of the linear alignment of the identified CRISPR-Cas systems from within five strains of C. difficile with identification of the various CRISPR-Cas constituent components.
  • the CRISPR-Cas system operon structures for strains 630 and R20291 are further diagrammed in Figure 2B.
  • Initial crRNA arrays within the C. difficile targeting crPhages comprised a native leader sequence for a CRISPR array from C. difficile 630 or R20291 that were combined with the consensus repeat sequences from the native CRISPR arrays found in C. difficile.
  • the spacer sequence was defined by the consensus PAM sequence for the endogenous Type I-B systems in C. difficile and complementary to three selected C. difficile target host genes including: dmsB, phi f and phi2.
  • Two additional configurations were tested including: a crRNA array using an endogenous enolase promotor in lieu of the native leader sequence and a crRNA array wherein the second repeat sequences was changed to facilitate IDT synthesis (R2 A>G).
  • the configuration of the CRISPR-RNA for engineering the bacteriophage is thus leader-repeat-spacer-repeat.
  • the engineered bacteriophage was created through homologous recombination in a native bacterial host as a lysogen using democratized plasmids for genetic manipulation of Clostridia.
  • Clostridium difficile strains 630 and R20291 were grown over night in brain heart infusion (BHI) medium at 37° C in an anaerobic environment and then sub-cultured into 5 mL of fresh BHI at a 1% (vol/vol) inoculum. C. difficile strains were then incubated until an OD of 0.2 before beginning a CFU reduction assay. All preparation and handling of bacteriophages was performed as described previously. To each culture, a total MOI of 10 wild-type or CRISPR phage lysate was added with a final concentration of 10 mM MgCl 2 and 1 mM CaCl 2 . OD and CFU were then monitored over the course of 6 hours.
  • BHI brain heart infusion
  • CRISPR-enhanced bacteriophages against E. coli and C. difficile were developed from distinct obligate lytic bacteriophages that contain an identical DNA sequence encoding a functional self-targeting CRISPR RNA embedded in the wild-type phage genome.
  • treatment of an E. coli culture containing 10 10 bacterial cells with the native, unmodified phage resulted in a 5-log reduction of bacterial cells by lytic activity of the phage.
  • Treatment with the modified crPhage results in a further approximate 5-log improvement in killing activity of the E. coli.
  • the improvement in anti -microbial activity is independent of the innate lytic activity of the phage and is a result of the anti-microbial activity of the CRISPR array itself.
  • Treatment with the CRISPR array shows an approximate 7-log reduction in the bacterial cell population as seen in Figure 3B.
  • Example 8 crPhages have expanded host range and associated killing activity
  • Example 9 crPhages have enhanced killing activity over a wide range of C. difficile strains
  • the ability of a crPhage to be effective over a panel of different strains for C. difficile was determined.
  • the CRISPR-RNA was designed by incorporating a native leader sequence for a CRISPR array from Clostridium difficile R20291 and then combined with the consensus repeat sequences from the native CRISPR arrays found in C. difficile.
  • the spacer sequence was defined by the consensus PAM sequence for the endogenous Type I-B systems in C. difficile and the length was determined by the most represented spacer length found in endogenous CRISPR arrays in C. difficile.
  • the configuration of the CRISPR-RNA for engineering the bacteriophage is thus leader- repeat-spacer-repeat.
  • the engineered bacteriophage was created through homologous
  • Clostridium difficile strains were grown over night in brain heart infusion (BHI) medium at 37° C in an anaerobic environment and then sub-cultured into 5 mL of fresh BHI at a 1% (vol/vol) inoculum. C. difficile strains were then incubated until an OD of 0.1 for the OD reduction assay and OD of 0.2 for the CFU reduction assay. All preparation and handling of bacteriophages was performed as described previously. To each culture, a total MOI of 10 wild-type or CRISPR phage lysate was added with a final concentration of 10 mM MgCl 2 and 1 mM CaCl 2. OD and CFU were then monitored over the course of 6 hours.
  • BHI brain heart infusion
  • CFU colony forming unit
  • a CFU assay of crPhage fO ⁇ 146 and crPhage fO ⁇ 24-2 anti -bacterial activity was conducted for each crPhage individually as well as the when administered together. Co-administration showed improved killing efficacy as compared to treatment with a combination of both wild-type phages together. Although wild-type phage fO ⁇ 24-2 demonstrated the stronger anti -bacterial killing activity as compared to wild-type phage fO ⁇ 146, when administered together, the killing combined efficacy significantly diminished. This is suggestive of the wild-type fO ⁇ 146 phage potentially interfering with the activity of wild-type phage fO ⁇ 24-2. However, the combination of crPhage fO ⁇ 146 and crPhage fO ⁇ 24-2 together show equal to slightly improved bacterial killing ability as compared to that of wild-type phage fO ⁇ 24-2 by itself.
  • Example 10 CRISPR enhanced crPhages in silico design of CRISPR array for overcoming resistance rates
  • crRNA array targets include the following genes in order of highest to lowest percentage of strain coverage: 7.
  • nusG 99 % ,fusA’2 (99 %),fusA (98%), glyQ (98%), eno (95%), gapA’2 (91%), eno’2 (89%), and nusG’2 (73%).
  • crRNA targets include the following genes: acpP, csra, eno, fusA, gapA, glyQ, inf A, nusG, secY, trmD, and Tsf
  • crRNA targets include the following genes: acpP, csra, eno, fusA, gapA, glyQ, inf A, nusG, secY, trmD, and Tsf
  • Example 11 crPhages enhanced with LeuO transcriptional activator
  • CRISPR-enhanced bacteriophages against A. coli were developed as a cocktail of up to three distinct obligate lytic bacteriophages that contain an identical DNA sequence encoding a functional self-targeting CRISPR RNA (crRNA cassette) embedded in the wild-type phage genome.
  • Bacteriophages were engineered by one of two methods: (1) homologous recombination in E. coli cells with active phage infection or (2) by transformation of engineered phage DNA assembled outside of the cell to reconstitute active phages with engineered genomes. Each phage was engineered with a similar crRNA cassette that contained two elements: (1) a LeuO
  • crRNA expression cassette used in the engineered various crPhages contains a single crRNA as shown in Table 10 below: Table 10.
  • Bacteriophage crT4 was engineered by deleting the hoc gene and replacing with a crRNA cassette.
  • Bacteriophage crT7 was engineered by deleting gpO.7, gp4.3 , gp4.5 and gp4.7 and replacing with a crRNA cassette.
  • Bacteriophage crT7m was engineered by deleting gp0.6 , gp0.65 , gpO.7, gp4.3 , and gp4.5 and replacing with a crRNA cassette. Based on the observation that all phages were successfully engineered after these deletions, it was concluded that these early phage genes were non-essential for phage survival. Details of these engineered bacteriophages are summarized in Table 11 below:
  • LeuO Upon DNA transduction during infection, LeuO is expressed from the phage genome and subsequently upregulate expression of the endogenous Type I-E CRISPR-Cas3 operon in E. coli.
  • the synthetic //.sri -targeting crRNA is expressed from the phage genome that is recognized and processed by the endogenous Type I-E CRISPR-Cas3 protein complex. This crRNA is then loaded onto a CRISPR-Cas3 complex and thereby directs the targeting and degradation of target bacterial DNA.
  • Example 12 Prevalence and distribution of CRISPR-Cas systems in E. coli
  • CRISPR-Cas systems There are a diverse range of CRISPR-Cas systems types and subtypes, with the majority (>60%) of discovered systems belonging to the Type I group that shares the unique feature of having the Cas3 signature nuclease.
  • CRISPR-Cas3 systems are unique in that they generate single strand nicks, followed by processive exonucleolytic degradation of targeted DNA.
  • E. coli CRISPR- Cas systems belong to two distinct subtypes, Type I-E and Type I-F, that use this signature Cas3 nuclease for degradation.
  • E. coli uropathogenic E. coli
  • STEC Shiga toxin producing A. coli
  • DEC diarrheagenic . coli
  • EPEC enteropathogenic E. coli
  • Each genome was scanned for operons with similarity to canonical Type I-E or Type I-F E. coli CRISPR-Cas systems.
  • Figure 16A shows the relative amounts of each of these E. coli genomes.
  • Figure 16B shows that approximately 78% (487/625) of all strains already have the complete CRISPR-Cas3 system, either type I-E or type I-F.
  • the proposed product exploits the presence of CRISPR-Cas3 proteins in the majority of E. coli by delivering only the guides and accessories required to activate endogenous CRISPR-Cas3 systems to target the genome.
  • E.coli genomes were downloaded directly from NCBI with accession numbers, spanning a diversity of s including: uropathogenic E. coli (UPEC), Shiga toxin producing C. (STEC), various O-antigen: H-antigen serotype if coli , diarrheagenic . coli (DEC), and enteropathogenic . coli (EPEC). Genomes were then queried for genes annotated as“ CasB The CasB coding sequence and 5 kb flanking on either side were extracted for further annotation.
  • UPEC uropathogenic E. coli
  • STEC Shiga toxin producing C.
  • O-antigen H-antigen serotype if coli
  • DEC diarrheagenic . coli
  • EPEC enteropathogenic . coli
  • Example 14 Expression of LeuO is necessary to elicit CRISPR-Cas lethality
  • E. coli encode the necessary components for Type I CRISPR-Cas3 activity in their genome.
  • the E. coli Type I-E CRISPR-Cas3 operon is regulated by histone-like nucleoid structuring (H-NS) repression and is not expressed under normal culture conditions.
  • H-NS histone-like nucleoid structuring
  • LeuO acts in opposition to H-NS at overlapping promoter regions and activates gene expression.
  • the interplay between H-NS and LeuO activity has been studied in S. typhimurium and E. coli , by examining the global transcriptional changes related to LeuO overexpression or knockout. Under conventional culturing conditions, LeuO itself is not expressed but is upregulated during starvation and stationary phase.
  • a phagemid was designed encoding a LeuO expression cassette to overcome the wild-type repression of the endogenous CRISPR-Cas3 operon.
  • the designed phagemid was derived from the Ml 3
  • the phagemid also encodes a CRISPR array targeting the conserved E. coli ftsA gene, whereby expression of this array activates and direct self-targeting of Type I-E£ coli CRISPR- Cas3 systems to elicit cell death.
  • Figure 17 shows non-lytic Ml3-derived phagemid delivery of CRISPR constructs using the validated ftsA spacer sequence designed to test the dependence on LeuO expression for CRISPR- mediated lethality.
  • Phagemids were produced to titers of 10 9 transducing units per milliliter and maintained in growth media for in vitro studies.
  • the phagemid vector encodes the ftsA repeat- spacer array, LeuO expression cassette, and an Ml 3-compatible origin of replication. Lethality of phagemid vectors was tested via transduction of Ml 3 bacteriophages into a range of s including a parent EMG2 containing a wild-type H-NS repressed A.
  • BWACas BW25l l3-derivative lacking Cas3 genes
  • E. coli were infected with 10 9 transducing units per milliliter of each Ml 3 phagemid and plated on selective media to recover transduced cells and count surviving colony forming units (transductants).
  • Each was transduced with the following phagemids indicated in the legend: Control, generic Ml 3 transduction control; pCRISPR, phagemid that constitutively expresses non-targeting crRNA; LeuO, phagemid that constitutively expresses the E. coli LeuO gene;//&4, phagemid that constitutively expresses crRNA targeting conserved ftsA gene present in E.
  • coli;ftsA::LeuO phagemid constitutively expresses LeuO gene and crRNA targeting ftsA.
  • Co-delivery of LeuO and the ftsA- targeting spacer resulted in reductions in the range of 3.4- log ( ⁇ 0.04) to 4.3-log ( ⁇ 0.06) compared to control across each except the BWACas that lacks Cas3 activity, confirming that lethality is dependent on the constructs expressed from the phagemid genome.
  • CRISPR-Cas3 lethality by expression of a ftsA -targeting spacer alone was only observed in the BW+Cas cell line, demonstrating that removal of H-NS repression alone is not sufficient to rescue significant levels of endogenous CRISPR-Cas3 targeting.
  • both the ftsA spacer and LeuO are required for cell death in non-engineered, wild-type E. coli.
  • Example 15 LeuO enhanced crPhages have improved lethality kinetics
  • Each crPhage was systemically compared to wild-type phage to determine change in potency of CRISPR-enhanced phages compared to their respective wild-type bacteriophage.
  • crPhages and the corresponding wild-type bacteriophage were produced, filtered, and adjusted to the same titer in growth media. Based on existing publicly available sequencing data, the three wild-type phages have significantly different genomic architectures, but are obligate lytic phages (data not shown).
  • Target E. coli were incubated for 2 or 5 hours for crT7m, crT7 and crT4, respectively, in growth media at the indicated multiplicity-of-infection (ratio of phage to bacteria) for each phage. After incubation, cultures were immediately collected, serially diluted and plated to count surviving colonies.
  • Figure 19A- Figure 19E shows the dose-response in vitro kill curves for each crPhage.
  • Each crPhage was produced by standard lytic amplification; filtration and left suspended in the original growth media (LB broth). All experiments were conducted in LB broth. E. coli MG1655 was grown to mid-log phase and then mixed with the indicated multiplicity-of-infection (MO I) of each crPhage, crPhage cocktail or LB only negative control. Treated populations were grown under aerobic, shaking conditions at 37C for 24 hours in a plate reader to monitor growth of treated populations by optical density (OD 630 nm).
  • E. coli MG1655 was grown to mid-log phase and treated with multiplicity-of-infection (MOI; ratio of phage to bacteria) as follows: Figure 19A, crT7 was incubated at MOIs of 0.0001, 0.01, and 1.0; Figure 19B, crT7m was incubated at MOIs of 0.0009, 0.09, and 9.0; and Figure 19C, crT4 was incubated at MOIs of 0.0006, 0.06, and 6.0. Each phage was mixed in equal amounts to create a crPhage cocktail (‘Cocktail’) and was incubated at MOIs (for each crPhage) of 0.0006, 0.06, and 6.0 as seen in Figure 19D.
  • Figure 19E is a zoomed in graph from Figure 19D.
  • Time-to-lysis was defined as the time at which the first derivative of the growth curve reaches zero after the bacterial population crashes due to presumed lytic phage amplification.
  • Endonuclease degradation of host genomes is considered to be a non-lytic mechanism for killing bacteria, thus CRISPR-mediated lethality is not expected to be observed by growth curve analysis.
  • E. coli MG1655 was grown to mid-log phase and treated with multiplicity-of-infection (MOI; ratio of phage to bacteria) as indicated for each crPhage. Growth curves were smoothed and the first derivative of the smoothed lines were determined using the PRISM software suite. Time- to-lysis was calculated as the time where the first derivative reaches zero immediately following the initial observed population decline.
  • MOI multiplicity-of-infection
  • Example 17 LeuO enhanced crPhages in vivo tolerability
  • crT7 and crT7m were suspended in sterile, endotoxin-free 0.9% saline, while crT4 was suspended in sterile, endotoxin- free IX tris-buffered saline (pH 7.4) supplemented with lOmM of each CaCl 2 and MgCl 2. No overt toxicity was observed during veterinary observation and no measurable changes in body temperature or body weight were noted after dosing with each crPhage preparation as shown in Figure 21B- Figure 21G.
  • crPhages were prepared for the peritonitis model as described in Table 14 below:
  • Table 14 crPhages for an in vivo peritonitis model studies.
  • crT7 and crT7m were suspended in sterile, endotoxin-free 0.9% saline, while crT4 was suspended in sterile, endotoxin-free IX tris-buffered saline (pH 7.4) supplemented with lOmM each CaCl 2 and MgCl 2.
  • Single-dose administration of crPhage (2.0xl0 u PFU/dose of crT7, 3.7xl0 9 PFU/dose of crT7M or 6.0xl0 8 PFU/dose of crT4) resulted in significant protection in this acute, highly lethal bacterial challenge as seen in Figure 22B- Figure 22D. Control animals were treated with saline injections only.
  • Example 19 LeuO enhanced crPhages in vivo bioburden reduction in a thigh model
  • crPhages were prepared for an in vivo bioburden reduction in a thigh model as described in Table 15 below:
  • Table 15 crPhages for in vivo bioburden reduction in a thigh model studies.
  • mice were made neutropenic by intraperitoneal injection of 150 mg/kg cyclophosphamide into the left abdomen. Mice were inoculated with 10 5 CFU of E. coli MG1655 by intramuscular injection into the thigh 30 minutes prior to intramuscular injection with the indicated crPhage or IX tris-buffered saline (phage vehicle).
  • Each individual crPhage or cocktail of 3 crPhages were administered by intramuscular injection into the same thigh with 100 microliters of crPhage solution, corresponding to a dose of 4.0xl0 u PFU/dose of crT7, 2.0xl0 u PFU/dose of crT7M, 2.0xl0 10 PFU/dose of crT4 or the cocktail containing l.OxlO 10 PFU/dose of each phage.
  • whole thigh muscles were excised at the indicated time points, homogenized and immediately diluted and plated to count surviving bacterial colonies per gram of tissue.
  • Example 20 LeuO enhanced crPhages in vivo persistence and distribution studies measured by phage titration
  • crPhages were prepared for an in vivo persistence and distribution study by intraurethral administration as described in Table 16 below:
  • Table 16 crPhages for in vivo persistence and distribution studies.
  • Example 21 LeuO enhanced crPhages in vivo persistence and distribution studies measured by quantitative PCR
  • a quantitative PCR-based method was developed and validated for the detection of each crPhage within a given cocktail (data not shown) enabling detection levels down to 50 copies per ng of total DNA in complex samples (e.g. commingled mouse blood and whole DNA).
  • Quantitative PCR is a highly specific method to detect and quantify DNA and is theoretically able to measure the total amount of each engineered crPhage within samples as the primers are designed to recognize a specific phage genome containing an identical crRNA cassette insert.
  • crPhages were prepared for an in vivo persistence and distribution study by oral administration as described in Table 17 below:
  • Table 17 crPhages for in vivo persistence and distribution studies.
  • mice were gavaged with 0.2mL of 6% sodium bicarbonate to reduce stomach acid levels approximately 30 minutes prior to crPhage dosing.
  • a single dose of 2.7xl0 9 PFU total of each crT7, crT7m and crT4 in 200 pL IX tris-buffered saline (pH 7.4) was administered by oral gavage to N 3 mice per condition/time point.
  • a test article of crPhage cocktail containing crT7 and crT7m was administered either intravenously (l.OxlO 11 PFU/dose/phage) or by intracatheter instillation into the bladder (0.5xl0 u PFU/dose/phage) once daily for 7 consecutive days to female Crl:CD-l mice.
  • crPhages were prepared for an the 7-day toxicology study as described in Table 18 below:
  • Table 18 crPhages for 7-day toxicology study.
  • the crPhage preparation was prepared using sterile, endotoxin-free IX tris-buffered saline (pH 7.4).
  • Groups 1 and 2 (9 female mice/group) were dosed with 0.1 mL of vehicle (IX tris- buffered saline, pH 7.4) or test article in a tail vein.
  • Groups 3 and 4 (9 female mice/group) were dosed with 0.05 mL of vehicle or test article into the bladder using a catheter syringe.
  • Group assignment and dosage levels are described in Table 19 below:
  • Table 19 Group assignment and dose levels for non-GLP toxicology study.
  • Postmortem assessment included necropsy and measurement of selected organ weights. A full tissue list was collected at necropsy. Collected tissues were sectioned with one section frozen in liquid nitrogen (stored frozen at ⁇ -70 °C) and a second section was preserved in 10% neutral -buffered formalin.
  • the CRISPR-Cas operon in C. difficile is not regulated by LeuO and H-NS as seen in E. coli. Rather, regulation is regulated by glucose in ccpA-dependent manner in the absence of ccpA binding site.
  • the upregulation of CD2983 has been associated with the upregulation of the Cas operon during a nutrient shift. Further, the regulation of CD2983 appears to be controlled by CodY, a global stringent response regulator. The loss of proteins similar in sequence (>40% similarity) in a type I-D system has been shown to result in increased expression off the Cas operon.
  • CodY and CD2983 are analogous equivalents to HNS and LeuO in C. difficile.
  • Table 20 Summary of characteristics for Cas operon regulation in E. coli and C. difficile.
  • Example 24 Engineering and validation of a lysogeny module knockout bacteriophage
  • FIG. 26A exemplifies the number of surviving cells (CFU/mL) counted at various time points after the treatment. Surviving cells were further screened for the presence of lysogenized CD24-2.
  • Figure 26B exemplifies the % lysogens present at various time points after the treatment.
  • Example 25 Treatment of a microbiome-related disorder
  • a pharmaceutical composition comprising an engineered bacteriophage as described herein can be administered to the subject.
  • the pharmaceutical composition can modulate or kill singular or plural bacterial populations within the microbiome by CRISPR-Cas activity, lytic activity, or a combination thereof.
  • Example 26 UTI efficacy study illustrating reduction in E.coli in bladder and kidney following intraurethral (IU) or intravenous (IV) administration
  • mice colonized with NC101 were treated with saline, a phage cocktail (2.4xl0 10 PFU/mL); or ciprofloxacin.
  • Ciprofloxacin (l-cyclopropy]-6-fluoro-1.4dihydro-4-oxo-7-(l-piperazinyl)-3- quinolinecarhoxylic acid hydrochloride) is an antibiotic, while effective, use results in a myriad of side effect and bacterial resistance.
  • tissues were collected after a single dose or after 5 doses and analyzed for CFUs. Phage treatment reduces CFUs in both the bladder ( Figure 30A- Figure 30B) and kidneys ( Figure 30C - Figure 30D).
  • results exemplify that IU and IV delivery of phage reduces CFUs in the bladder. Further, the results exemplify that crPhage cocktail has 1.5- to 3.5-log improved kill over wtPhage cocktail at l20h in the bladder ( Figure 30B). The results also exemplify that regardless of delivery route, at l20h crPhage cocktail performs comparable with ciprofloxacin in the bladder.
  • the study further illustrates route-dependent penetration of phage into different tissues and fluids, such as urine (Figure 31A), kidney (Figure 31B), bladder (Figure 31C), and spleen (Figure 31D).
  • the results illustrate that there is measurable phage in the urine regardless of treatment route.
  • Example 27 UTI efficacy study comparison of research-grade material compared WT versus engineered cocktail via different administration routes
  • mice colonized with NC101 were treated with saline; a phage cocktail (2.4xl0 10 PFU/mL); or ciprofloxacin. Tissues were collected after a single dose or after 5 doses and analyzed for CFUs. Phage treatment reduces CFUs in both the bladder ( Figure 33A- Figure 33B) and kidneys ( Figure 33C - Figure 33D). The results exemplify that IU and IV delivery of phage reduces CFUs in the bladder.
  • Example 28 Clinical trial to assess the safety, pharmacokinetics and potential efficacy of crPhage in E.coli colonized adults
  • Phase lb - Safety, tolerability and PK study is conducted in patients with urinary tract colonization by maximum feasible dose BID via catheter instillation. Pharmacokinetics related to phage persistence time, distribution and elimination is confirmed in the bladder. Bacteriophages that are non-toxic and unable to infect human cells give high therapeutic index.
  • UTI urinary tract infections
  • MDR MDR
  • CR strains MDR and CR strains
  • IV administration and dosing regimen is evaluated.
  • Primary endpoint resolution of UTI symptoms at end of therapy and test of cure (TOC - 7 days after end of therapy) with demonstration of bacterial pathogen reductions ⁇ l0 3 CFU/mL on urine culture (Microbiological Success) measured at TOC.
  • Secondary endpoint Immunogenicity (presence of anti-phage antibodies), QoL measures (pain/discomfort, etc), durability of response.
  • Figure 34A is a schematic of an exemplary human study conducted in adults with reoccurring and asymptomatic E.coli colonization of the urinary tract.
  • Figure 34B is an exemplary study participant inclusion and exclusion criteria for the UTI Phase lb study.
  • Phase 1 Safety, tolerability and PK study is conducted in healthy volunteers (C. diff colonization naturally occurring). 10-14 day oral dosing regimen and 28 day follow-up is evaluated. Safety and tolerability is evaluated. PK analysis of phage and C. difficile in stool is evaluated over time.
  • Phase 2 Non-inferiority study, 2: 1 randomized, active vs. standard of care (i.e.
  • TTROD time to resolution of diarrhea
  • CD AD time to diarrhea
  • grade of diarrhea assessed up to 8 weeks following completion of treatment period
  • safety and tolerability assessment PK analysis
  • Phase 3 Efficacy study in larger patient population of first line and recurrent CDI compared to SOC (vancomycin) orally for 10-14 day dosing regimen (40 day study), sub-analysis of front line vs. recurrent patient treatment
  • Example 30 Engineered phages show increased killing against both Type IE and Type IF E.coli strains
  • strains and phages were prepared as follows. All experiments were performed in 96 well flat bottom clear plates with total final volume of 200uL in LB with salts (lOmM MgCl 2 and CaCl 2 ). Three strains were selected to compare WT and CRISPR phages. Each E. coli strain was placed in a microtiter plate either alone, with WTphage (p33s; p46), or with the crPhage (p33s-6; p46cr) at MOI 0.1. For host range experiments, the cultures were incubated at 37°C for 20 hours in a plate reader to monitor growth of populations by optical density (OD; 600 nm).
  • Example 31 Switching phage cocktails overcomes target bacterial resistance in E.coli
  • Example 32 Comparison of wild type phage PB1 and CRISPR-enhanced PB1 against Pseudomonas aeruginosa strains
  • a panel of 44 P. aeruginosa strains was mixed with either LB, PB1 or cr-PBl at an MOI of 0.01 ( ⁇ 5 x 10 5 bacteria and 5 x 10 3 phage or LB).
  • Strains + phage or LB were grown for 20 hours at 37 °C and the optical density at 600 nm (OD 60 o) was measured every hour to generate a growth curve.
  • the values in the table represent the ratio of the area under the curve (AUC) for PB1 or cr-PBl (PB. Engineered) compared to the uninfected control. A smaller number represents a larger decrease in optical density. Gray boxes indicate AUC ratios ⁇ 0.7. Hit percentage is the percent of strains with an AUC ratio ⁇ 0.7.
  • CFU Reductions Stationary phase cultures of LFP805 were diluted to an optical density of 1.0 and 10 pL of diluted cultures were added to 180 pL of LB (-10 5 bacteria). PB1 and cr-PBl were diluted to 10 8 PFU/ml and either 10 pL of phage (10 6 PFU) or 10 pL of LB were added. Cultures were grown with aeration at 37 °C and lO-fold dilutions were plated on LB agar at 4 hrs and 8 hours post-inoculation to determine the CFU/ml in each culture.
  • Example 33 Plasmid based killing of E.coli and P.aeruginosa by Type I CRISPR-Cas systems
  • E .coli Type I testing Five Type-I Cas systems were cloned from bacteria gDNA into pUCPl9. Corresponding E.coli (BL21) targeting spacers were cloned into a second compatible plasmid (pRSFlb). B121 electrocompetent cells were transformed with each Cas system plasmid and targeting spacer or pRSFlb control. The transformations were diluted and spot plated on Kan/Carb LB plates. CFUs were counted after overnight incubation at 37C. Results are exemplified in Figure 39A.
  • P. aeruginosa Type-I Testing Four Type-I Cas systems were cloned from bacteria gDNA into pUCPl9 along with a pseudomonas targeting spacer. Electrocompetent PA01 cells were made competent using the Locus lab protocol. The cells were transformed with the Cas + spacer plasmid or Cas plasmid. The transformations were diluted and spot plated on Carb300 LB plates. CFUs were counted after overnight incubation at 37C. Results are exemplified in Figure 39B. Results exemplify that 3 of 4 systems were able to successfully target PA01 with a minimum 3 -log reduction. 1 system showed no CFUs for either plasmid.

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JP2021522857A (ja) 2021-09-02
CN112424363A (zh) 2021-02-26
AU2019262200A1 (en) 2020-12-17
US20220002681A1 (en) 2022-01-06
US20220387531A1 (en) 2022-12-08
UY38215A (es) 2019-11-29
US20200354690A1 (en) 2020-11-12
US20230038106A1 (en) 2023-02-09
TW202014519A (zh) 2020-04-16
CA3099316A1 (en) 2019-11-07

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