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WO2022235799A2 - Phage compositions for staphylococcus comprising crispr-cas systems and methods of use thereof - Google Patents

Phage compositions for staphylococcus comprising crispr-cas systems and methods of use thereof Download PDF

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Publication number
WO2022235799A2
WO2022235799A2 PCT/US2022/027671 US2022027671W WO2022235799A2 WO 2022235799 A2 WO2022235799 A2 WO 2022235799A2 US 2022027671 W US2022027671 W US 2022027671W WO 2022235799 A2 WO2022235799 A2 WO 2022235799A2
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Prior art keywords
bacteriophage
crispr
composition
sequence
cas system
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WO2022235799A3 (en
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David G. Ousterout
Hannah Hewit TUSON
Lana MCMILLAN
Robert Mckee
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Locus Biosciences Inc
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Locus Biosciences Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10041—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • bacteriophage compositions comprising two or more bacteriophage.
  • the composition comprises a Phietavirus bacteriophage and Rosenblumvirus bacteriophage, Phietavirus bacteriophage and Kayvirus bacteriophage, Rosenblumvirus bacteriophage and Kayvirus bacteriophage, or Phietavirus bacteriophage, Rosenblumvirus bacteriophage, and Kayvirus bacteriophage.
  • the Phietavirus bacteriophage is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene.
  • composition comprises the Phietavirus bacteriophage and Rosenblumvirus bacteriophage. In some embodiments, the composition comprises the Phietavirus bacteriophage and Kayvirus bacteriophage. In some embodiments, the composition comprises the Rosenblumvirus bacteriophage and Kayvirus bacteriophage.
  • the composition comprises the Phietavirus bacteriophage, Rosenblumvirus, and Kayvirus bacteriophage. In some embodiments, the composition comprises the Kayvirus bacteriophage, wherein the Kayvirus bacteriophage comprises two or more Kayvirus bacteriophage. In some embodiments, the bacteriophage of the composition infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria.
  • the composition comprises a plurality of bacteriophage comprising a first bacteriophage and a second bacteriophage, wherein the plurality of bacteriophage infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria.
  • the Staphylococcus bacteria are selected from b004604, b004605,b004606,b004607,b004608,b004609,b004610,b004611,b004612,b004613, b004614,b004615,b004616,b004617,b004618,b004619,b004620,b004621,b004622, b004623,b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631 , b004632, b004633,b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641, b004642, b004643, b004644, b004645, b004646, b
  • the Staphylococcus bacteria comprises Staphylococcus aureus.
  • the infectivity is determined with a plaque assay or growth inhibition assay.
  • the at least about 90% is at least about 95%. In some embodiments, the at least about 90% is at least about 98%. In some embodiments, the at least about 90% is at least about 99%.
  • the at least about 30 Staphylococcus bacteria comprises b004604, b004605, b004606, b004607, b004608, b004609, b004610, b004611,b004612,b004613,b004614,b004615,b004616,b004617,b004618,b004619, b004620, b004621 , b004622, b004623, b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631 , b004632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641 , b004642, b004643, b004644,
  • the first bacteriophage and the second bacteriophage are of different genera.
  • the plurality of bacteriophage comprise a Phietavirus bacteriophage and Rosenblum virus bacteriophage, Phietavirus bacteriophage and Kayvirus bacteriophage, Rosenblumvirus bacteriophage and Kay virus bacteriophage, or Phietavirus bacteriophage, Rosenblumvirus bacteriophage, and Kayvirus bacteriophage.
  • the plurality of bacteriophage comprise a Phietavirus.
  • the Phietavirus is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene.
  • the lysogenic gene encodes for a repressor.
  • the composition comprises a plurality of bacteriophage comprising a first bacteriophage that is specific for a first receptor of a Staphylococcus bacteria, a second bacteriophage that is specific for a second receptor of the Staphylococcus bacteria, wherein the plurality of bacteriophage is more resilient to resistance by the Staphylococcus bacteria than the first or second bacteriophage alone.
  • the first receptor and the second receptor are different.
  • the first bacteriophage comprises a Phietavirus.
  • the first bacteriophage comprises a Phietavirus and a Rosenblumvirus.
  • the second bacteriophage comprises a Kayvirus.
  • the plurality of bacteriophage comprise: a Phietavirus bacteriophage and Rosenblumvirus bacteriophage, Phietavirus bacteriophage and Kayvirus bacteriophage, Rosenblumvirus bacteriophage andKay virus bacteriophage, or Phietavirus bacteriophage, Rosenblumvirus bacteriophage, andKayvirus bacteriophage.
  • the plurality of bacteriophage of the composition infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria.
  • the first bacteriophage and the second bacteriophage are of different genera. In some embodiments, the first bacteriophage and the second bacteriophage are capable of independently infecting at least 90% of the collection of Staphylococcus bacteria.
  • engineered bacteriophage In some embodiments, provided is a Phietavirus bacteriophage engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene. In some embodiments, the lysogenic gene encodes for a repressor. In some embodiments, the repressor comprises an amino acid sequence at least about 80% identical to SEQ ID NO: 47 or 48.
  • removal of the lysogenic gene comprises removing from about 1% to 100% of the lysogenic gene. In some embodiments, about 10 to about 1,200 base pairs of the lysogenic gene are removed. In some embodiments, the lysogenic gene is removed, replaced, or inactivated. In some embodiments, the lysogenic gene is removed. In some embodiments, the promoter of the lysogenic gene is removed, replaced, or inactivated.
  • bacteriophage compositions comprising the engineered bacteriophage.
  • the composition comprises the engineered Phietavirus.
  • the composition comprises a Rosenblumvirus.
  • the composition comprises aKayvirus.
  • the composition comprises a plurality of bacteriophage, wherein the plurality of bacteriophage infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria.
  • the composition and/or engineered bacteriophage provided herein comprises a nucleic acid encoding for an exogenous peptide selected from TreA, Lpi, DNAsel, RIP, FS3, PLNC8a, PLNC8P, LytM, LnqO, 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
  • the composition and/or engineered bacteriophage comprises one or more components of a CRISPR-Cas system.
  • the CRISPR-Cas system is a Type IB CRISPR- Cas system from Listeria monocytogenes (LMIB).
  • the composition and/or engineered bacteriophage provided herein comprises a spacer sequence or a crRNA transcribed therefrom, wherein the spacer sequence is complementary to a target nucleic acid sequence from a target gene in a target bacteria.
  • the target bacteria comprises a Staphylococcus bacteria.
  • a bacteriophage comprising a nucleic acid encoding an exogenous peptide selected from TreA, Lpi, DNAsel, RIP, FS3, PLNC8a, PLNC8P, LytM, LnqO, 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, peroxida
  • engineered bacteriophage comprising a CRISPR-Cas system, or one or more components thereof.
  • the engineered bacteriophage comprises a Type IB CRISPR-Cas system from Listeria monocytogenes (LMIB).
  • the engineered bacteriophage comprises one or more components of a CRISPR-Cas system.
  • the CRISPR-Cas system is a Type IB CRISPR-Cas system from Listeria monocytogenes (LMIB).
  • the LMIB encodes for a sequence at least 80% identical to any one of SEQ ID NOS: 25-29.
  • the engineered bacteriophage comprises a spacer sequence or a crRNA transcribed therefrom, wherein the spacer sequence is complementary to a target nucleic acid sequence from a target gene in a target bacteria.
  • the target bacteria comprises a Staphylococcus bacteria.
  • the bacteriophage is an engineered Phietavirus.
  • the bacteriophage is an engineered Rosenblumvirus.
  • the bacteriophage is an engineered Kay virus.
  • provided are bacteriophage compositions comprising the engineered bacteriophage.
  • the composition comprises the Phietavirus, and a Rosenblumvirus and/or a Kay virus. In some embodiments, the composition comprises the Rosenblumvirus, and a Phietavirus and/or a Kay virus. In some embodiments, the composition comprises the Kayvirus, and a Rosenblumvirus and/or a Phietavirus.
  • the composition and/or engineered bacteriophage provided herein are used in a method of treating a disease or condition related to Staphylococcus, the method comprising administering to a subject in need thereof the composition and/or engineered bacteriophage.
  • the Staphylococcus is causative of, and/or contributes to, the disease or condition.
  • Fig. 1 depicts the predicted lysogeny region of a Phietavirus bacteriophage pi 473.
  • Fig. 2 depicts the dilution series of wild-type (WT) pl473 and several variants plated on a lawn of Staphylococcus aureus using the double agar overlay method. WT phage and variants Var002 and Var006 produced small hazy plaques, while variants Var009, VarOlO and VarO 12 produced larger clearer plaques.
  • WT phage and variants Var002 and Var006 produced small hazy plaques
  • variants Var009, VarOlO and VarO 12 produced larger clearer plaques.
  • Fig. 3 depicts the larger plaque morphology for wild -type pi 473, VarOlO and VarO 12 in a close-up image, with red arrows indicating individual plaques.
  • Fig. 4 depicts the bacteriophage killing assay with strain b4063 (USA300 strain FPR3757) in LB challenged with Wild-type pi 473, Var012, or Var042 as measured by bacterial counts as a function of time.
  • Fig. 5 depicts the growth curves of three -S' aureus clinical isolates (b2991,b3022 and b3202) in LB challenged with pl473 WT or pl473 Var012.
  • Fig. 6 depicts the sequence of the VarOlO deletion (SEQ IDNO: 30).
  • Fig. 7 depicts the sequence of the VarO 12 deletion (SEQ IDNO: 31), which is also the first deletion in Var042.
  • Fig. 8 depicts the second deletion in Var042 (SEQ ID NO: 32).
  • Fig. 9 depicts PCR screening of a Kay virus phage engineered to contain a Type I
  • Fig. 10 depicts PCR screening of the InqO promoter into phage.
  • Fig. 11 depicts the concentration of b2655 inoculated with engineered and wildtype Kayvirus phage over time.
  • the engineered Kayvirus comprises three promotor variants used to drive expression of lacticin Q ( JnqQ ): a Peat -InqQ, PsarA -InqQ, or PtmpG- InqQ promoter.
  • Fig. 12 depicts an alignment of the genomes of parent Kayvirus phage with the engineered variant containing DNase I and the P cat promoter.
  • Fig. 13 depicts a Venn diagram showing the different bacteria strains targeted by Phietavirus, Rosenblumvirus, andKayvirus, and combinations of the bacteriophage.
  • bacteriophage engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene As a non limiting example, the lysogenic gene encodes for a repressor. In some embodiments, the bacteriophage is further engineered to comprise one or more components of a CRISPR-Cas system, and/or an antimicrobial peptide. In some embodiments, the lysogenic gene encodes for a repressor. In some embodiments, removal of the lysogenic gene comprises removing from about 1% to 100% of the lysogenic gene, or about 10 to about 1,200 base pairs of the lysogenic gene are removed.
  • the lysogenic gene encodes for an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47 or 48.
  • bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic.
  • the target bacterium is Staphylococcus spp.
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of the region annotated as “varOlO” (also referred to as a varOlO deletion) in Figure 6 (SEQ ID NO: 30).
  • varOlO also referred to as a varOlO deletion
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of the region annotated as “var012” (also referred to as a var012 deletion) in Figure 7 (SEQ ID NO: 31).
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of the region annotated as “var042” (also referred to as a var042 deletion) in Figure 8 (SEQ ID NO: 32).
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of the region annotated as “var009” (also referred to as a var009 deletion) (SEQ ID NO: 30).
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of at least a portion of a lysogenic gene, or a promoter of a lysogenic gene.
  • the portion is at least about 1% to 100% of the nucleotides of the lysogenic gene. In some embodiments, the portion is about 10 base pairs to all of the lysogenic gene.
  • the lysogenic gene encodes for an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47 or 48.
  • bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic.
  • the target bacterium is Staphylococcus spp.
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “varOlO” (also referred to as a varO!O deletion) in Figure 6 (SEQ ID NO: 30).
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “varO 12” (also referred to as a var012 deletion) in Figure 7 (SEQ ID NO: 31).
  • a peptide e.g., antimicrobial peptide
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “var042” (also referred to as a var042 deletion) in Figure 8 (SEQ ID NO: 32).
  • a peptide e.g., antimicrobial peptide
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “var009” (also referred to as a var009 deletion) (SEQ ID NO: 30). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of at least a portion of a lysogenic gene, or a promoter of a lysogenic gene.
  • the portion is at least about 1% to 100% of the nucleotides of the lysogenic gene. In some embodiments, the portion is about 10 base pairs to all of the lysogenic gene. In some embodiments, the lysogenic gene encodes for an amino acid sequence atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
  • bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic.
  • the target bacterium is Staphylococcus spp .
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “varOlO” (also referred to as a varO!O deletion) in Figure 6 (SEQ ID NO: 30).
  • varOlO also referred to as a varO!O deletion
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “var012” (also referred to as a var012 deletion) in Figure 7 (SEQ ID NO: 31).
  • var012 also referred to as a var012 deletion
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “var042” (also referred to as a var042 deletion) in Figure 8 (SEQ ID NO: 32).
  • var042 also referred to as a var042 deletion
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “var009” (also referred to as a var009 deletion) (SEQ ID NO: 30).
  • var009 also referred to as a var009 deletion
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of at least a portion of a lysogenic gene, or a promoter of a lysogenic gene.
  • the portion is at least about 1% to 100% of the nucleotides of the lysogenic gene.
  • the portion is about 10 base pairs to all of the lysogenic gene.
  • the lysogenic gene encodes for an amino acid sequence atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
  • compositions comprises a plurality of bacteriophage.
  • the plurality comprises one or more engineered bacteriophage, e.g., engineered to remove a lysogenic gene, and/or to include a payload such as a CRISPR-Cas component and/or antimicrobial peptide.
  • the plurality of bacteriophage target a wide host range. For instance, the composition targets at least about 90% of a collection of at least about 30 Staphylococcus bacteria.
  • the plurality of bacteriophage comprises a first bacteriophage that is specific for a first receptor of a Staphylococcus bacteria, a second bacteriophage that is specific fora second receptor of the Staphylococcus bacteria, wherein the plurality of bacteriophage is more resilient to resistance by the Staphylococcus bacteria than the first or second bacteriophage alone.
  • the bacteriophage is an obligate lytic bacteriophage. In some embodiments, the bacteriophage is a temperate bacteriophage with retained lysogeny genes. In some embodiments, the bacteriophage is a temperate bacteriophage with some lysogeny genes removed, replaced, or inactivated. In some embodiments, the bacteriophage is a temperate bacteriophage with a lysogeny gene removed, replaced, or inactivated, thereby rendering the bacteriophage lytic. In some embodiments, the bacteriophage is rendered lytic by removal of at least a portion of a lysogenic gene, or a promoter of a lysogenic gene.
  • the portion is at least about 1% to 100% of the nucleotides of the lysogenic gene. In some embodiments, the portion is less than about 1%. In some embodiments, the portion removed is a single base. In some embodiments, the portion is about 10 base pairs to all of the lysogenic gene.
  • the portion removed is about 10-1200, 10-1100, 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10- 200, 10-100, 50-1200, 50-1100, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700, 100- 600, 100-500, 100-400, 100-300, or 100-200 base pairs.
  • the lysogenic gene encodes for an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47 or 48.
  • the bacteriophage targets Staphylococcus spp. In some embodiments, the bacteriophage targets S. aureus. In some embodiments, the bacteriophage specifically targets Staphylococcus spp. over other bacterial species. In some embodiments, the bacteriophage targets Staphylococcus spp. in the absence of a CRISPR-Cas system.
  • the bacteriophage targets Staphylococcus spp.
  • the bacteriophage is a Kay virus, a Twortvirus, a Rosenblumvirus, a Phietavirus a Triavirus, a Dubowvirus, a Beceayunavirus, a Peeveelvirus, a Coventry virus, or a Rockefellervirus.
  • the bacteriophage is a Kayvirus.
  • the bacteriophage is a Twortvirus.
  • the bacteriophage is a Rosenblumvirus.
  • the bacteriophage is a Phietavirus.
  • the virus is a Triavirus. In some embodiments, the virus is a Dubowvirus. In some embodiments, the virus is a Beceayunavirus. In some embodiments, the virus is a Peeveelvirus. In some embodiments, the virus is a Coventryvirus. In some embodiments, the virus is a Rockefellervirus. In some embodiments, the bacteriophage encodes a CRISPR-Cas system. In some embodiments, the bacteriophage encodes a peptide.
  • the bacteriophage is a Phietavirus.
  • a non-limiting example Phietavirus is pi 473.
  • the Phietavirus comprises a nucleic acid encoding a CRISPR-Cas system.
  • the Phietavirus comprises a nucleic acid encoding a peptide.
  • the Phietavirus is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene. As anon - limiting example, the lysogenic gene encodes for a repressor.
  • the bacteriophage is further engineered to comprise one or more components of a CRISPR-Cas system, and/or an antimicrobial peptide.
  • the lysogenic gene encodes for a repressor.
  • removal of the lysogenic gene comprises removing from about 1 % to 100% of the lysogenic gene, or about 10 to about 1 ,200 base pairs of the lysogenic gene are removed.
  • the lysogenic gene encodes for an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ IDNO: 47 or 48.
  • the bacteriophage is a Rosenblumvirus.
  • the Rosenblumvirus comprises a nucleic acid encoding a CRISPR-Cas system. In some embodiments, the Rosenblumvirus comprises a nucleic acid encoding a peptide.
  • the bacteriophage is a Kayvirus. In some embodiments, the Kayvirus comprises a nucleic acid encoding a CRISPR-Cas system. In some embodiments, the Kayvirus comprises a nucleic acid encoding a peptide.
  • the bacteriophage is p 1473, which targets Staphyloccocus ssp. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with pi 473. In some embodiments, the bacteriophage is a pi 473 bacteriophage comprising a CRISPR-Cas system.
  • bacteriophages of interest are obtained from environmental sources or from 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 nucleic acid sequence is inserted into a bacteriophage, e.g., a nucleic acid sequence encoding one or more components of a CRISPR-Cas system, and/or a peptide.
  • the insertion of the nucleic acid sequence into a bacteriophage preserves the lytic activity of the bacteriophage.
  • the nucleic acid sequence is inserted into the bacteriophage genome.
  • the nucleic acid sequence is inserted into the bacteriophage genome at a transcription terminator site at the end of an operon of interest.
  • the nucleic acid sequence is inserted into the bacteriophage genome as a replacement for one or more removed non- essential genes. In some embodiments, the nucleic acid sequence is inserted into the bacteriophage genome as a replacement for one or more removed lysogenic genes. In some embodiments, the replacement of non-essential and/or lysogenic genes with the nucleic acid sequence 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 sequence preserves the lytic activity of the bacteriophage.
  • the replacement of non- essential and/or lysogenic genes with the nucleic acid sequence enhances the lytic activity of the bacteriophage. In some embodiments, the replacement of non-essential and/or lysogenic genes with the nucleic acid sequence renders a lysogenic bacteriophage lytic.
  • the nucleic acid sequence 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. In some embodiments, the nucleic acid sequence is introduced into the bacteriophage 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 multiple separate locations. In some embodiments, 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. In some embodiments, the removal of one or more non-essential and/or lysogenic genes renders a lysogenic bacteriophage into a lytic bacteriophage.
  • the bacteriophage is a temperate bacteriophage which has been rendered lytic by any of the aforementioned means.
  • 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.
  • 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 bacteriophage is rendered lytic by removal of the “varOlO” region annotated as “varOlO” deletion in Figure 6 (SEQ ID NO: 30).
  • the bacteriophage is rendered lytic by removal of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or 1200 nucleotides of the varOlO region.
  • the bacteriophage is rendered lytic by removal of the “varO 12” region annotated as “varO 12” deletion in Figure 7 (SEQ ID NO: 31).
  • the bacteriophage is rendered lytic by removal of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides of the varO 12 region. In some embodiments, the bacteriophage is rendered lytic by removal of at least In some embodiments, the bacteriophage is rendered lytic by removal of the “var042” region annotated as “var042” deletion in Figure 8 (SEQ ID NO: 32). In some embodiments, the bacteriophage is rendered lytic by removal of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides of the var042 region.
  • the bacteriophage is rendered lytic by removal of the “var009” deletion (SEQ ID NO: 30). In some embodiments, the bacteriophage is rendered lytic by removal of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides of the var009 region.
  • the bacteriophage is rendered lytic by removal of atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides encoding the repressor having an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47 or 48.
  • the bacteriophage is rendered lytic by the deletion of an antirepressor gene integrase, Pem-K like phage protein gene, or a combination thereof. In some embodiments, the bacteriophage is rendered lytic by the deletion of a gene depicted in Fig. 1. In some embodiments, the bacteriophage is rendered lytic by the deletion of an antirepressor gene, integrase, Pem-K like phage protein gene, or a combination thereof, wherein the gene is depicted in Fig. 1.
  • the bacteriophage is rendered lytic by deletion of atleast 10-2000, 20-2000, 30-2000, 40-2000, 50-2000, 60-2000, 70-2000, 80-2000, 90-2000, 100-2000, 200-2000, 300-2000, 400-2000, 500-2000, 600-2000, 700-2000, 80-2000, 900-2000, 1000-2000, 1100-2000, 1200-2000, 1300-2000, 1400-2000, 1500-2000, 1600-2000,1700-2000,1800-2000 or 1900-2000 basepairs of an antirepressor gene.
  • the bacteriophage is rendered lytic by deletion of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of an antirepressor 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 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 the self -targeting activity of the first introduced CRISPR array. In some embodiments, a temperate bacteriophage that is rendered lytic is prevented fromrevertingbackto lysogenic state by way of introducing an additional CRISPR array. In some embodiments, the bacteriophage does not confer any new properties onto the target bacterium beyond cellular death caused by lytic activity of the bacteriophage and/or the activity of the first or second 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.
  • temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of the region annotated as “varOlO” in Fig. 6, “var012” in Fig. 7, “var042” in Fig. 8, at least a portion of SEQ ID NO: 47 or 48, or combinations thereof.
  • the bacteriophage infects multiple bacterial strains.
  • 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 comprises at least a portion of an essential gene that is needed for survival of the target bacterium.
  • the essential gene is Tsf acpP, gapA, infA , secY, csrA, trmD,ftsA,fusA , glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fiis, adk, rpsK, rplR, ctrA,parC , tRNA-Ser, tRNA-Asn, or metK.
  • the target nucleotide sequence is in a non-essential gene or other genomic locus. In some embodiments, the target nucleotide sequence is in a non-essential gene. In some embodiments, the target nucleotide sequence is in a non-essential genomic locus. In some embodiments, the target nucleotide sequence is a noncoding sequence. In some embodiments, the noncoding sequence is an intergenic sequence. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a highly conserved sequence in a target bacterium. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a sequence present in the target bacterium.
  • the spacer sequence is complementary to a target nucleotide sequence that comprises all or a part of a promoter sequence of the essential gene.
  • the first nucleic acid sequence comprises a first CRISPR array further comprising at least one repeat sequence.
  • the at least one repeat sequence is operably linked to the first spacer sequence at either its 5 ’ end or its 3 ’ end.
  • the target bacterium is S. aureus.
  • a cocktail comprising two or more bacteriophage.
  • the two or more bacteriophage are selected from the lineage consisting of a Kay virus, a Twortvirus, a Rosenblumvirus, a Phietavirus or a Triavirus.
  • at least one bacteriophage of the cocktail comprises a CRISPR-Cas system.
  • at least two bacteriophages of the cocktail comprise a CRISPR-Cas system.
  • at least three bacteriophage of the cocktail comprise a CRISPR-Cas system.
  • at least four bacteriophage of the cocktail comprise a CRISPR-Cas system.
  • At least one bacteriophage of the cocktail does not comprise a CRISPR-Cas system. In some embodiments, at least two bacteriophages of the cocktail do not comprise a CRISPR-Cas system. In some embodiments, at least one bacteriophage of the cocktail comprises an antimicrobial peptide as described herein. In some embodiments, at least two bacteriophages of the cocktail comprise a nucleic acid encoding an antimicrobial peptide as described herein. In some embodiments, at least one bacteriophage comprises a nucleic acid encoding a CRISPR-Cas system and at least one bacteriophage comprises a nucleic acid encoding an antimicrobial peptide.
  • At least one bacteriophage of the cocktail comprises a nucleic acid encoding a CRISPR-Cas system. In some embodiments, the bacteriophage of the cocktail do not comprise a nucleic acid encoding a CRISPR-Cas system or an antimicrobial peptide.
  • the cocktail comprises a Phietavirus.
  • the Phietavirus is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene.
  • the cocktail comprises a Phietavirus and a Rosenblumvirus.
  • the cocktail comprises a Phietavirus and a Kayvirus.
  • the cocktail comprises a Phietavirus, Rosenblumvirus, and Kayvirus.
  • the Phietavirus comprises a nucleic acid encoding a CRISPR- Cas system.
  • the Phietavirus comprises a nucleic acid encoding an antimicrobial peptide. In some cases, the Phietavirus binds to a different bacteria receptor than another bacteriophage in the cocktail. In some cases, if the bacteria develops resistance and/or has a mutation that prevents infection with the another bacteriophage, the Phietavirus is capable of infecting the bacteria. In some such cases, the cocktail is more resilient against resistance formation by the bacteria than a single bacteriophage.
  • the cocktail comprises a Rosenblumvirus.
  • the cocktail comprises a Rosenblumvirus and a Phietavirus.
  • the Phietavirus is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene.
  • the cocktail comprises a Rosenblumvirus and a Kayvirus.
  • the cocktail comprises a Phietavirus, Rosenblumvirus, and Kayvirus.
  • the Rosenblumvirus comprises a nucleic acid encoding a CRISPR-Cas system.
  • the Rosenblumvirus comprises a nucleic acid encoding an antimicrobial peptide.
  • the Rosenblumvirus binds to a different bacteria receptor than another bacteriophage in the cocktail. In some cases, if the bacteria develops resistance and/or has a mutation that prevents infection with the another bacteriophage, the Rosenblumvirus is capable of infecting the bacteria. In some such cases, the cocktail is more resilient against resistance formation by the bacteria than a single bacteriophage.
  • the cocktail comprises a Kayvirus. In some example cocktails, the cocktail comprises a Kayvirus and a Rosenblum virus. In some example cocktails, the cocktail comprises a Kayvirus and a Phietavirus.
  • the Phietavirus is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene.
  • the cocktail comprises a Phietavirus, Rosenblumvirus, and Kayvirus.
  • the Kayvirus comprises a nucleic acid encoding a CRISPR-Cas system.
  • the Kayvirus comprises a nucleic acid encoding an antimicrobial peptide.
  • the Kayvirus binds to a different bacteria receptor than another bacteriophage in the cocktail.
  • the Kayvirus is capable of infecting the bacteria.
  • the cocktail is more resilient against resistance formation by the bacteria than a single 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.
  • a cocktail comprising a plurality of bacteriophages is used together.
  • the cocktail comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, ormorethan20 phages.
  • the cocktail comprises 2 phages.
  • the cocktail comprises 3 phages.
  • the cocktail comprises 4 phages.
  • the cocktail comprises 5 phages.
  • the cocktail comprises 6 phages.
  • At least one bacteriophage in the cocktail comprises a CRISPR array. In some embodiments, atleast2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, ormore than 20 bacteriophages present in the cocktail comprise a CRISPR array. In some embodiments, at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding a Cascade polypeptide. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bacteriophages present in the cocktail comprise a nucleic acid sequence encoding a Cascade polypeptide.
  • At least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding a Cas3 polypeptide.
  • atleast2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bacteriophages present in the cocktail comprise a nucleic acid sequence encoding a Cas3 polypeptide.
  • at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding an antimicrobial peptide.
  • the bacteriophage cocktail has a host range greater than that of an individual bacteriophage.
  • the increased host range may allow for targeting a large number of strains of S. aureus.
  • the bacteriophage cocktail targets at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more than 99% of strains of S. aureus.
  • aureus include: b004604,b004605,b004606, b004607,b004608,b004609,b004610,b004611,b004612,b004613,b004614,b004615, b004616,b004617,b004618,b004619,b004620,b004621,b004622,b004623,b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631 , b004632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641 , b004642, b004643,b004644, b004645, b004646, b004647, b004648,
  • the bacteriophage cocktail targets S. aureus specifically and does not target other species of bacteria. In some embodiments, the bacteriophage cocktail targets 5. aureus and does not target other Staphylococcus species. The increased host range may allow for targeting a large number of strains of Staphylococcus spp . In some embodiments, the bacteriophage cocktail targets at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more than 99% of strains of Staphylococcus spp. In some embodiments, the bacteriophage cocktail targets Staphylococcus spp. specifically and does not target other species of bacteria.
  • the bacteriophage cocktail targets Staphylococcus spp. and does not target non Staphylococcus spp. In some embodiments, the bacteriophage cocktail comprises two or more of: Phietavirus, Rosenblumvirus, and Kay virus.
  • the bacteriophage in the cocktail are selected to minimize the ability of the target bacteria to evolve resistance.
  • the cocktail comprises a first bacteriophage that is specific for a first receptor of a Staphylococcus bacteria, and a second bacteriophage that is specific fora second receptor of the Staphylococcus bacteria, wherein the plurality of bacteriophage is more resilient to resistance by the Staphylococcus bacteriathan the first or second bacteriophage alone. For instance, if the Staphylococcus bacteria develops resistance to the first bacteriophage, the bacteria is still susceptible to infection by the second bacteriophage, and vice versa.
  • the cocktail comprises at least two bacteriophage, wherein the first bacteriophage binds a first receptor on Staphylococcus and the second bacteriophage binds a second receptor on Staphylococcus.
  • the bacteriophage cocktail comprises two or more of: Phietavirus, Rosenblumvirus, and Kay virus.
  • the bacterium comprises one or more species of Staphylococcus . In some embodiments, the bacterium comprises one or more strains of Staphylococcus. In some embodiments, the target bacterium is Staphylococcus aureus. In some embodiments, the target bacterium is Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Staphylococcus salivarius, Staphylococcus argentis, Staphylococcus hemolyicus, Staphylococcus schweitzeri or any combination thereof.
  • MRSA methicillin-resistant Staphylococcus aureus
  • the target bacterium causes an infection or disease.
  • the infection or disease is acute or chronic.
  • the infection or disease is localized or systemic.
  • infection or disease is idiopathic.
  • the infection or disease is acquired through means including, but not limited to, respiratory inhalation, ingestion, skin and wound infections, bone 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.
  • the target bacterium causes urinary tract infection.
  • the target bacterium causes and/or exacerbates an inflammatory disease. In some embodiments, the target bacterium causes and/or exacerbates an autoimmune disease. In some embodiments, the target bacterium causes and/or exacerbates eczema. In some embodiments, the target bacterium causes and/or exacerbates Atopic Dermatitis. In some embodiments, the target bacterium causes and/or exacerbates inflammatory bowel disease (IBD). In some embodiments, the target bacterium causes and/or exacerbates psoriasis. In some embodiments, the target bacterium causes and/or exacerbates psoriatic arthritis (PA).
  • IBD inflammatory bowel disease
  • PA psoriatic arthritis
  • the target bacterium causes and/or exacerbates rheumatoid arthritis (RA). In some embodiments, the target bacterium causes and/or exacerbates systemic lupus erythematosus (SLE). In some embodiments, the target bacterium causes and/or exacerbates multiple sclerosis (MS). In some embodiments, the target bacterium causes and/or exacerbates Graves’ disease. In some embodiments, the target bacterium causes and/or exacerbates Hashimoto’s thyroiditis. In some embodiments, the target bacterium causes and/or exacerbates Myasthenia gravis. In some embodiments, the target bacterium causes and/or exacerbates vasculitis.
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • MS multiple sclerosis
  • the target bacterium causes and/or exacerbates Graves’ disease.
  • the target bacterium causes and/or exacerbates Hashimoto’s
  • the target bacterium causes and/or exacerbates cancer. In some embodiments, the target bacterium causes and/or exacerbates cancer progression. In some embodiments, the target bacterium causes and/or exacerbates cancer metastasis. In some embodiments, the target bacterium causes and/or exacerbates resistance to cancer therapy. In some embodiments, the therapy used to address cancer includes, but is not limited to, chemotherapy, immunotherapy, hormone therapy, targeted drug therapy, and/or radiation therapy.
  • the cancer develops in organs including, but not limited to the, anus, bladder, blood and blood components, bone, bone marrow, brain, breast, cervix uteri, colon and rectum, esophagus, kidney, larynx, lymphatic system, muscle (i.e., soft tissue), oral cavity and pharynx, ovary, pancreas, prostate, skin, small intestine, stomach, testis, thyroid, uterus, and/or vulva.
  • the target bacterium causes and/or exacerbates disorders of the central nervous system (CNS).
  • the target bacterium causes and/or exacerbates attention deficit/hyperactivity disorder (ADHD).
  • the target bacterium causes and/or exacerbates autism. In some embodiments, the target bacterium causes and/or exacerbates bipolar disorder. In some embodiments, the target bacterium causes and/or exacerbates major depressive disorder. In some embodiments, the target bacterium causes and/or exacerbates epilepsy. In some embodiments, the target bacterium causes and/or exacerbates neurodegenerative disorders including, but not limited to, Alzheimer’ s disease, Huntington’s disease, and/or Parkinson’s disease.
  • the target bacteria comprises a Staphylococcus selected from one or more of: b004604, b004605, b004606, b004607, b004608, b004609, b004610, b004611,b004612,b004613,b004614,b004615,b004616,b004617,b004618,b004619, b004620, b004621 , b004622, b004623, b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631 , b004632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641 , b004642, b
  • 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.
  • Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR-Cas system is used herein.
  • 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 CRISPR array encoding 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
  • CRISPR array encoding 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.
  • the proteins Cas5, Cas8c, and Cas7 form the Cascade effector complex.
  • Cas5 processes the pre-crRNA (which can take the form of a multi-spacer array, ora single spacer between two repeats) to produce individual crRNA(s)madeup of a hairpin structure formed from the remaining repeat sequence and a linear spacer.
  • the effector complex then binds to the processed crRNA and scans DNA to identify PAM sites.
  • the PAM is recognized by the Cas8c protein, which then acts to unwind the DNA duplex.
  • sequence 3 ’ of the PAM matches the crRNA spacer that is bound to effector complex, a conformational change in the complex occurs and Cas3 is recruited to the site. Cas3 then nicks the non-target strand and begins degrading the DNA.
  • the proteins Cas8bl, Cas7, and Cas5 form the Cascade effector complex.
  • Cas5 processes the pre-crRNA (which can take the form of a multi-spacer array, ora single spacer between two repeats) to produce individual crRNA(s)madeup of a hairpin structure formed from the remaining repeat sequence and a linear spacer.
  • the effector complex then binds to the processed crRNA and scans DNA to identify PAM sites.
  • the PAM is recognized by the Cas8bl protein, which then acts to unwind the DNA duplex.
  • the Type I-B system is from Listeria monocytogenes (LMIB) (SEQ ID NO: 22).
  • LMIB Listeria monocytogenes
  • the Type I-B system comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS: 25-29.
  • the CRISPR-Cas system is endogenous to the target bacterium.
  • the target bacterium comprises at least one gene encoding a Cas polypeptide.
  • the target bacterium comprises a nucleic acid encoding a Cas3 polypeptide.
  • the target bacterium comprises a nucleic acid encoding a CASCADE complex.
  • the target bacterium when a CRISPR-Cas system is endogenous to the target bacterium, the target bacterium comprises a nucleic acid sequence encoding a Cas3 polypeptide and a CASCADE complex. [0062] In some embodiments, the CRISPR-Cas system is exogenous to the target bacterium. In some embodiments, when a CRISPR-Cas system is exogenousto the target bacterium, the bacteriophage comprises at least one gene encoding a Cas polypeptide. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the bacteriophage comprises a nucleic acid encoding a Cas3 polypeptide.
  • the bacteriophage when a CRISPR-Cas system is exogenous to the target bacterium, the bacteriophage comprises a nucleic acid encoding a CASCADE complex. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the bacteriophage comprises a nucleic acid sequence encoding a Cas3 polypeptide and a CASCADE complex. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not comprise a nucleic acid encoding a Cas3 polypeptide.
  • the target bacterium when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not express a Cas3 polypeptide. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not comprise a nucleic acid encoding a CASCADE complex. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not express a CASCADE complex.
  • the target bacterium when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not comprise a nucleic acid sequence encoding a Cas3 polypeptide and a CASCADE complex. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not express a Cas3 polypeptide and a CASCADE complex.
  • the CRISPR-Cas system is a Type I CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type I-A CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type I-B CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type I-B CRISPR-Cas system derived from Listeria monocytogenes. In some embodiments, the CRISPR-Cas system is a Type I-C CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type I-D CRISPR- Cas system. In some embodiments, the CRISPR-Cas system is a Type I-E CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type I-F CRISPR-Cas system.
  • the CRISPR-Cas system is a Type I-U CRISPR-Cas system. 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.
  • processing of a CRISPR-array disclosed herein includes, but is not limited to, the following processes: 1) transcription of the nucleic acid encoding a pre-crRNA; 2) recognition of the pre-crRNAby Cascade and/ or specific members of Cascade, such as Cas6, and (3) processing of the pre-crRNA by Cascade or members of Cascade, such as Cas6, into mature crRNAs.
  • the mode of action for a Type I CRISPR system includes, but is not limited to, the following processes: 4) mature crRNA complexation with Cascade; 5) target recognition by the complexed mature crRNA/Cascade complex; and 6) nuclease activity at the target leading to DNA degradation.
  • the Type I CRISPR-Cas system is a Type I-A system
  • Type I-B system Type I-C system, Type I-D system, Type I-E system, Type I-F system, or Type I-U system.
  • the Type I CRISPR-Cas system is a Type I-A system.
  • the Type I CRISPR-Cas system is a Type I-B system.
  • the Type I CRISPR-Cas system is a Type I-C system.
  • the Type I CRISPR-Cas system is a Type I-D system.
  • Type I CRISPR-Cas system is a Type I-E system.
  • Type I CRISPR-Cas system is a Type I-F system.
  • the Type I CRISPR-Cas system is a Type I-U system. In some embodiments, the Type I CRISPR-Cas system comprises Cascade polypeptides. Type I Cascade polypeptides process CRISPR arrays to produce a processed RNA that is then used to bind the complex to a target sequence that is complementary to the spacer in the processed RNA.
  • 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 (Csh 1) polypeptide, a nucleotide sequence encoding a Cas7 (Csh2) polypeptide, and a nucleotide sequence encoding a Cas5 polypeptide (Type I-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 (CasB) poly
  • the Type I Cascade complex comprises a Cascade polypeptide disclosed herein [0067]
  • the Type I CRISPR-Cas system comprises Cascade polypeptides.
  • Type I Cascade polypeptides process CRISPR arrays to produce a processed RNA that is then used to bind the complex to a target sequence that is complementary to the spacer in the processed RNA.
  • 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 CRISPR-Cas system is a Type I-B CRISPR-Cas system from Listeria monocytogenes (LMIB).
  • the CRISPR-Cas system is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22. In some instances, the CRISPR- Cas system is encoded by a sequence comprising at least a portion having at least or about 3,
  • the CRISPR-Cas system is encodedby a sequence comprising at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
  • the CRISPR-Cas system is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23. In some instances, the CRISPR-Cas system is encoded by a sequence comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,
  • the CRISPR-Cas system is encoded by a sequence comprising at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200,205,210,
  • the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 25 (e.g., Cas6). In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 29 (e.g., Cas8).
  • the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 26 (e.g., Cas7). In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 27 (e.g., Cas5).
  • the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 28 (e.g., Cas3).
  • the CRISPR-Cas system comprises at least or about 95% homology to any one of SEQ ID NOS: 25 -29.
  • the CRISPR-Cas system comprises at least or about 97% homology to any one of SEQ ID NOS: 25-29.
  • the CRISPR-Cas system comprises at least or about 99% homology to any one of SEQ ID NOS: 25-29.
  • the CRISPR-Cas system comprises 100% homology to anyone of SEQ ID NOS: 25-29. In some instances, the CRISPR-Cas system comprises at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19,20, 21,22, 23,24, 25,26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
  • the CRISPR-Cas system comprises at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or more than 215 amino acidsof any one of SEQ ID NOS: 25-29.
  • the CRISPR-Cas system comprises a Cas6 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 33.
  • the CRISPR-Cas system comprises a Cas8 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34.
  • the CRISPR-Cas system comprises a Cas7 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 35.
  • the CRISPR-Cas system comprises a Cas5 polypeptide encodedby a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 36.
  • the CRISPR-Cas system comprises a Cas3 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 37.
  • the CRISPR-Cas system comprises a Cas6 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38.
  • the CRISPR-Cas system comprises a Cas8 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 39.
  • the CRISPR-Cas system comprises a Cas7 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 40.
  • the CRISPR-Cas system comprises a Cas5 polypeptide encodedby a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 41.
  • the CRISPR-Cas system comprises a Cas3 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 42.
  • the CRISPR array (crArray) disclosed herein comprises a spacer sequence and at least one repeat sequence.
  • the CRISPR array encodes a processed, mature crRNA.
  • the mature crRNA is introduced into a phage or a target bacterium described herein.
  • the phage comprises a nucleic acid that encodes a processed, mature crRNA.
  • an endogenous or exogenous Cas6 processes the CRISPR array into mature crRNA.
  • an exogenous Cas6 is introduced into the phage.
  • the phage comprises an exogenous Cas6.
  • an exogenous Cas6 is introduced into a target bacterium.
  • processing of a CRISPR-array disclosed herein includes, but is not limited to, the following processes: 1) transcription of the nucleic acid encoding a pre-crRNA; 2) recognition of the pre-crRNAby Cascade and/ or specific members of Cascade, such as Cas6, and (3) processing of the pre-crRNA by Cascade or members of Cascade, such as Cas6, into mature crRNAs.
  • the mode of action for a Type I CRISPR system includes, but is not limited to, the following processes: 4) mature crRNA complexation with Cascade; 5) target recognition by the complexed mature crRNA/Cascade complex; and 6) nuclease activity at the target leading to DNA degradation.
  • the CRISPR array comprises a spacer sequence. In some embodiments, the CRISPR array further comprises at least one repeat sequence. In some embodiments, the at least one repeat sequence is operably linked to the spacer sequence at either its 5 ’ end or its 3 ’ end. In some embodiments, 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 a target bacterium by targeting one or more target sequences.
  • the CRISPR array comprises, consists essentially of, or consists of 1 to about 100 spacer nucleotide sequences, each linked on its 5' end and its 3' end to a repeat nucleotide sequence.
  • the CRISPR array as disclosed herein comprises essentially of, or consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
  • the CRISPR array comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 24.
  • the repeat sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 43.
  • the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 44-46.
  • the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 44. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 45. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 46.
  • the spacer sequence is complementary to a target nucleotide sequence in a target bacterium.
  • the target nucleotide sequence is a coding region.
  • the coding region is an essential gene.
  • the coding region is a nonessential gene.
  • the target nucleotide sequence is a noncoding sequence.
  • the noncoding sequence is an intergenic sequence.
  • the spacer sequence is complementary to a target nucleotide sequence of a highly conserved sequence in a target bacterium.
  • the spacer sequence is complementary to a target nucleotide sequence of a sequence present in the target bacterium.
  • the spacer sequence is complementary to a target nucleotide sequence that comprises all or a part of a promoter sequence of the essential gene. In some embodiments, the spacer sequence comprises one, two, three, four, or five mismatches as compared to the target nucleotide sequence. In some embodiments, the mismatches are contiguous. In some embodiments, the mismatches are noncontiguous. In some embodiments, the spacer sequence has 70% complementarity to a target nucleotide sequence. In some embodiments, the spacer sequence has 80% complementarity to a target nucleotide sequence.
  • the spacer sequence is 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementarity to a target nucleotide sequence. In some embodiments, the spacer sequence has 100% complementarity to the target nucleotide sequence. In some embodiments, the 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 has 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.
  • the 5' region of the spacer sequence is 100% complementary to a target nucleotide sequence while the 3 ' region of the spacer is substantially complementary to the target nucleotide sequence and therefore the overall complementarity of the spacer sequence to the target nucleotide sequence 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 (seed region) is 100% complementary to the target nucleotide sequence, 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 nucleotide sequence.
  • the first 7 to 12 nucleotides of the 3' end of the spacer sequence is 100% complementary to the target nucleotide sequence, 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to the target nucleotide sequence.
  • 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%, 8
  • the first 7 to 10 nucleotides in the 3' end of the spacer sequence is 75%-99% complementary to the target nucleotide sequence, while the remaining nucleotides in the 5' region of the spacer sequence are at least about 50% to about 99% complementary to the target nucleotide sequence. In some embodiments, the first 7 to 10 nucleotides in the 3 ' end of the spacer sequence is 100% complementary to the target nucleotide sequence, 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 nucleotide sequence.
  • the first 10 nucleotides (within the seed region) of the spacer sequence is 100% complementary to the target nucleotide sequence, 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 nucleotide sequence.
  • 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) have about 75% complementarity or more (75% to about 100% complementarity) to the target nucleotide sequence, while the remainder of the spacer sequence have about 50% or more complementarity to the target nucleotide sequence.
  • the first 8 nucleotides at the 5' end of the spacer sequence have 100% complementarity to the target nucleotide sequence or have one or two mutations and therefore is about 88% complementary or about 75% complementary to the target nucleotide sequence, respectively, while the remainder of the spacer nucleotide sequence is at least about 50% or more complementary to the target nucleotide sequence.
  • the spacer sequence is about 15 nucleotides to about 150 nucleotides in length. In some embodiments, the 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, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99
  • the 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, at
  • the Listeria monocytogenes Type I-B Cas system has a spacer length of about30to 39 nucleotides, about31 to about38 nucleotides, about 32 to about 37 nucleotides, about 36 to about 37 nucleotides, or about 37 nucleotides. In some embodiments, the Listeria monocytogenes Type I-B system has a spacer length of about 37 nucleotides.
  • the Listeria monocytogenes Type I-B Cas system has a spacer length of at least about 10, at least about 15, atleast about 20, atleast about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 29, at least about 29, at least about 30, at least about 31, at least about32, at least about 33, at least about 34, at least about, at least about 35, atleast about 36, at least about 37, at least about 38, at least about 39, at least about 20, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, or more than about 45 nucleotides.
  • the identity of two or more spacer sequences of the CRISPR array is the same. In some embodiments, the identity of two or more spacer sequences of the CRISPR array is different. In some embodiments, the identity of two or more spacer sequences of the CRISPR array is different but are complementary to one or more target nucleotide sequences. In some embodiments, the identity of two or more spacer sequences of the 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 sequences of the CRISPR array is different and are complementary to one or more target nucleotide sequences that are not overlapping sequences.
  • the target nucleotide sequence 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. In some embodiments, a target nucleotide sequence is located adjacent to or flanked by a PAM (protospacer adjacent motif).
  • PAM protospacer adjacent motif
  • the PAM sequence 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 exact PAM sequence that is required varies between each different CRISPR-Cas system and is identified through established bioinformatics and experimental procedures.
  • Non-limiting examples of PAMs include CCA, CCT, CCG, TTC, AAG, AGG, ATG, GAG, and/or CC.
  • 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, and is directly recognized by Cascade.
  • Cascade generally recruits the endonuclease Cas3, which cleaves and degrades the target DNA.
  • the PAM is required for a Cas9/sgRNAto 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)
  • 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 the essential gene.
  • the spacer nucleotide sequence is complementary to a promoter, or a part thereof, of the essential gene.
  • the target nucleotide sequence comprises all or a part of a nucleotide sequence located on a coding or a non -coding strand of the essential gene. 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 the essential gene.
  • the 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.
  • 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. In some embodiments, the target nucleotide sequence comprises at least a portion of an essential gene that is needed for survival of the target bacterium.
  • the essential gene is Tsf acpP , gap A, infA , sec F, csrA, trmD,ftsA,fiisA , glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fus, adk, rpsK, rplR, ctrA,parC , tRNA-Ser, tRNA-Asn, or metK.
  • a non-essential gene is any gene of an organism that is not critical for survival. However, being non-
  • non-limiting examples of the target nucleotide sequence of interest includes a target nucleotide sequence 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.
  • the target nucleotide sequence is from a gene involved in cell-division, cell structure, metabolism, motility, pathogenicity, virulence, or antibiotic resistance.
  • the target nucleotide sequence is from a hypothetical gene whose function is not yet characterized. Thus, for example, these genes are any genes from any bacterium.
  • the appropriate spacer sequences for a full-construct phage maybe identified by locating a search set of representative genomes, searching the genomes with relevant parameters, and determining the quality of a spacer for use in a CRISPR engineered phage.
  • a suitable search set of representative genomes is located and acquired for the organism/species/target of interest.
  • the set of representative genomes may be found in a variety of databases, including without limitations the NCBI GenBank or the PATRIC database.
  • NCBI GenBank is one of the largest databases available and contains a mixture of reference and submitted genomes for nearly every organism sequenced to date.
  • the PATRIC (Pathosystems Resource Integration Center) database provides an additional comprehensive resource of genomes and provides a focus on clinically relevant strains and genomes relevant to a drug product. Both of the above databases allow for bulk downloading of genomes via FTP (File Transfer Protocol) servers, enabling rapid and programmatic dataset acquisition
  • Genomes are searched with relevant parameters to locate suitable spacer sequences. Genomes may be read from start to end, in both the forward and reverse complement orientations, to locate contiguous stretches of DNA that contain a PAM (Proto spacer Adjacent Motif) site.
  • the spacer sequence willbetheN-lengthDNA sequence 3' or 5’ adjacentto the PAM site (depending on the CRISPR system type), where N is specific to the Cas system of interest and is generally known ahead of time. Characterizing the PAM sequence and spacer sequences may be performed during the discovery and initial research of a Cas system. Every observed P AM-adjacent spacer may be saved to a file and/or database for downstream use. The exact PAM sequence that is required varies between each different CRISPR-Cas system and is identified through established bioinformatics and experimental procedures.
  • Each observed spacer may be evaluated to determine how many of the evaluated genomes they are present in.
  • the observed spacers may be evaluated to see how many times they may occur in each given genome. Spacers that occur in more than one location per genome may be advantageous because the Cas system may not be able to recognize the target site if a mutation occurs, and each additional "backup" site increases the likelihood that a suitable, non-mutated target location will be present.
  • the observed spacers may be evaluated to determine whether they occur in functionally annotated regions of the genome. If such information is available, the functional annotations maybe further evaluated to determine whether those regions of the genome are "essential" for the survival and function of the organism.
  • the spacer selection may be broadly applicable to many targeted genomes. Provided a large selection pool of conserved spacers exists, preference may be given to spacers that occur in regions of the genome that have known function, with higher preference given if those genomic regions are "essential" for survival and occur more than 1 time per genome.
  • the spacer comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 44-46. Repeat Nucleotide Sequences
  • a repeat nucleotide sequence of the 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).
  • the repeat nucleotide sequences are distinct from one another based on the known repeat nucleotide sequences of a CRISPR-Cas system.
  • the repeat nucleotide sequences are each composed of distinct secondary structures of a native repeat from a CRISPR-Cas system (e.g., an internal hairpin). In some embodiments, the repeat nucleotide sequences are a combination of distinct repeat nucleotide sequences operable with a CRISPR-Cas system.
  • the spacer sequence is linked at its 5' end to the 3’ end of a repeat sequence. In some embodiments, the spacer 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 sequence. In some embodiments, the about 1 to about 8, about 1 to about 10, about 1 to about 15 nucleotides of the repeat sequence are a portion of the 3 ’ end of a repeat sequence. In some embodiments, the spacer nucleotide sequence is linked at its 3' end to the 5’ end of a repeat sequence.
  • 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 sequence.
  • the about 1 to about 8, about 1 to about 10, about 1 to about 15 nucleotides of the repeat sequence are a portion of the 5 ’ end of a repeat sequence.
  • the spacer nucleotide sequence is linked at its 5' end to a first repeat sequence and linked at its 3' end to a second repeat sequence to forma repeat- spacer-repeat sequence.
  • the spacer sequence is linked at its 5' end to the 3 ’ end of a first repeat sequence and is linked at its 3 ' end to the 5 ’ of a second repeat sequence where the spacer sequence and the second repeat 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 comprises, consists essentially of, or consists of 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, 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,
  • spacer nucleotide sequences 100, or more, spacer nucleotide sequences.
  • the repeat sequence is identical to or substantially identical to a repeat sequence from a wild-type CRISPR loci.
  • the repeat sequence is a repeat sequence found in Table 3.
  • the repeat sequence is a sequence described herein.
  • the repeat sequence comprises a portion of a wild type repeat sequence (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous nucleotides of a wild type repeat sequence).
  • the repeat sequence comprises, consists essentially of, or consists of at least one nucleotide (e.g., 1, 2, 3,
  • the repeat sequence comprises, consists essentially of, or consists of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • the repeat sequence comprises about 20 to 40, 21 to 40, 22 to 4023 to 40, 24 to 40, 25 to 40,
  • the repeat sequence comprises about 20 to 35, 21 to 35, 22 to 35 23 to 35, 24 to 35, 25 to 35, 26 to 35, 27 to 35, 28 to 35, 29to 35, 30 to 30, 31 to 35, 32 to 35, 33 to 35, 34 to 35, 25 to 40, 25 to 39, 25 to 38, 25 to 37, 25 to 36, 25 to 35, 25 to 34, 25 to 33, 25 to 32, 25 to 31, 25 to 30, 25 to 29, 25 to 28, 25 to 26 nucleotides.
  • the system is a L monocytogenes Typel-B Cas system.
  • the L. monocytogenes Type I-B Cas system has a repeat length of about 25 to 38 nucleotides.
  • the repeat comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 43.
  • the nucleic acid sequence further comprises a transcriptional activator.
  • the transcriptional activator encoded regulates the expression of genes of interest within the Staphylococcus species.
  • the transcriptional activator activates the expression of genes of interest within the Staphylococcus species whether exogenous or endogenous.
  • the transcriptional activator activates the expression genes of interest within the Staphylococcus species by disrupting the activity of one or more inhibitory elements within the Staphylococcus species.
  • the inhibitory element comprises a transcriptional repressor. In some embodiments, the inhibitory element comprises a global transcriptional repressor.
  • the inhibitory element is a histone -like nucleoid-structuring (H-NS) protein orhomologue or functional fragment thereof.
  • the inhibitory dementis a leucine responsive regulatory protein (LRP).
  • the inhibitory dementis 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.
  • LRP leucine responsive regulatory protein
  • 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 leuO coding sequence, 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. Overexpression of LeuO upregulates the expression of the CRISPR-Cas system.
  • 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 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 sequence comprising a CRISPR array.
  • 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 sequence comprising a CRISPR array so as to increase the level of lethality of the CRISPR array against a bacterium.
  • transcriptional activator causes increase activity of a bacteriophage and/or the CRISPR-Cas system.
  • the nucleic acid sequences are operatively associated with a variety of promoters, terminators and other regulatory elements for expression in various organisms or cells.
  • the nucleic acid sequence further comprises a leader sequence.
  • the nucleic acid sequence further comprises a promoter sequence.
  • at least one promoter and/or terminator is operably linked the CRISPR array. 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 disclosed 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 the nucleic acid sequence is constitutive, inducible, temporally regulated, developmentally regulated, or chemically regulated.
  • the expression of the nucleic acid sequence is made constitutive, inducible, temporally regulated, developmentally regulated, or chemically regulated by operatively linking the nucleic acid sequence to a promoter functional in an organism of interest.
  • repression is made reversible by operatively linking the nucleic acid sequence to an inducible promoter that is functional in an organism of interest.
  • the choice of promoter disclosed herein varies 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, bacteriophages and compositions disclosed herein include promoters that are functional in bacteria.
  • L-arabinose inducible (araBAD , PBAD ) 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 , prol /, cst- 1, cadA, 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 -II.
  • araBAD L-arabinose
  • the promoter is a BBa_J23102 promoter.
  • the promoter works in a b road range of bacteria, such as BBa_J23104, BBa_J23109.
  • the promoter is derived from the target bacterium, such as endogenous CRISPR promoter, endogenous Cas operon promoter or the promoter from sarA, lipA, ptsH or cap l of S. aureus.
  • the promoter comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 16-20.
  • the promoter comprises at least a portion havingatleastor about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 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 or more than 50 nucleotides of any one of SEQ ID NOS: 16-20.
  • the promoter comprises at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or more than 215 nucleotides of any one of SEQ ID NOS: 16-20.
  • a bacteriophage disclosed herein is further genetically modified to express an antibacterial peptide, a functional fragment of an antibacterial peptide, and/or a lytic gene.
  • a bacteriophage disclosed herein express at least one antimicrobial agent or peptide disclosed herein.
  • the bacteriophage comprises a nucleic acid that encodes a peptide that prevents phage degradation or a peptide that assists in breaking down or degrading biofilm matrix.
  • a bacteriophage described herein comprises a nucleic acid that encodes a peptide that prevents phage degradation or enables escape of the phage from the host defenses.
  • 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 peptide comprises TreA (e.g., a sequence at least 80% identical to SEQ ID NO: 10).
  • the peptide comprises Lpi (e.g., a sequence at least 80% identical to SEQ ID NO: 11).
  • the bacteriophage comprises nucleic 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
  • 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 1,4-L-fucodise hydrolase, colanicacid, depolymerazing alginase, DNase I, or combinations thereof.
  • a bacteriophage disclosed herein secretes an enzyme disclosed herein.
  • the peptide disrupts quorum sensing and biofilm formation.
  • the peptide increases the sensitivity of abacterial cell to an antibiotic.
  • the enzyme comprises DNAse I (e.g., a sequence at least 80% identical to SEQ ID NO: 9). In some embodiments, the enzyme comprises RIP (e.g., a sequence at least 80% identical to SEQ ID NO: 14). In some embodiments, the enzyme comprises FS3 (e.g., a sequence at least 80% identical to SEQ ID NO: 12).
  • an antimicrobial agent or peptide is expressed and/or secreted by a bacteriophage disclosed herein.
  • the antimicrobial agent or peptide comprises PLNC8 a.
  • the antimicrobial agent or peptide comprises PLNC8p.
  • the antimicrobial agent or peptide comprises LytM.
  • the antimicrobial agent or peptide comprises an anti -restriction modification enzyme.
  • the antimicrobial agent or peptide comprises laciticin Q (LnqQ, e.g., a sequence at least 80% identical to SEQ ID NO: 15 or 50, e.g., SEQ ID NO: 50).
  • the LnqQ peptide is atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50 or 15. In some embodiments, the LnqQpeptideis atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50.
  • 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.
  • 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 the first and/or the second Type I CRISPR-Cas system.
  • the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 1.
  • the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 2.
  • the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 3. In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 4.
  • the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 5.
  • the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 8. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 9.
  • the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 10. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 11.
  • the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 12. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 14.
  • the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 15.
  • the nucleic acid sequence is an expression cassette or in an expression cassette.
  • the expression cassettes are designed to express the nucleic acid sequence disclosed herein.
  • the nucleic acid sequence is an expression cassette encoding components of a CRISPR-Cas system and/or peptide.
  • the nucleic acid sequence is an expression cassette encoding components of a Type I CRISPR-Cas system.
  • the nucleic acid sequence is an expression cassette encoding an operable CRISPR-Cas system.
  • the nucleic acid sequence is an expression cassette encoding the operable components of a Type I CRISPR-Cas system, including Cascade and Cas3.
  • the nucleic acid sequence is an expression cassette encoding the operable components of a Type I CRISPR- Cas system, including a crRNA, Cascade and Cas3.
  • the nucleic acid sequence is an expression cassette encoding a peptide (e.g., antimicrobial peptide).
  • an expression cassette comprising a nucleic acid 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 nucleic acid sequence of interest and for correct mRNA polyadenylation.
  • the termination region is native to the transcriptional initiation region, is native to the operably linked nucleic acid 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 nucleic acid sequence of interest, to the host, or any combination thereof).
  • terminators are operably linked to the nucleic acid sequence disclosed herein.
  • an expression cassette includes a nucleotide sequence for a selectable marker.
  • the nucleotide sequence encodes either a selectable or a screenable marker, depending on whether the marker confers a trait that is sel ected 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).
  • nucleic acid sequences disclosed herein e.g. nucleic acid sequence comprising a CRISPR array, CRISPR-Cas, peptide
  • 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 transforms prokaryotic or eukaryotic host either by integration into the cellular genome or existextrachromosomally (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.
  • the nucleic acid sequence encoding a payload is optimized for stable expression in a phage genome.
  • the insert is stable through at least 2, 3, 4, 5, 6, 7, 8,9, or 10 generations of passaging.
  • the nucleic acid sequence is optimized by optimizing the insertion site, modifying secondary structures, modifying DNA modification sites, modifying restriction enzyme motifs, codon optimization, GC% optimization, or a combination thereof.
  • the insertion site of the nucleic acid sequence is optimized.
  • the nucleic acid sequence is modified to remove secondary structures.
  • the bacteriophage comprises a nucleic acid insert modified from an exogenous nucleic acid described herein, wherein the nucleic acid comprises a first plurality of codons encoding for a first protein, and the nucleic acid insert comprises a second plurality of codons encoding for a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity, and wherein at least 50% of the second plurality of codons are high frequency codons in the bacteriophage genome.
  • described herein is a method of inserting an exogenous sequence comprising a plurality of codons encoding a first protein into a bacteriophage, the method comprising substituting one or more of the plurality of codons with a codon native to the bacteriophage to generate a nucleic acid insert encoding a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity.
  • nucleic acid insert modified from an exogenous nucleic acid, wherein the nucleic acid comprises a first plurality of codons encoding for a first protein, and the nucleic acid insert comprises a second plurality of codons encoding for a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity, and wherein at least 50% of the second plurality of codons are high frequency codons in the bacteriophage genome.
  • the first protein and the second protein have at least 95%, 97.5%, 99% or 99.5% sequence identity.
  • At least 50% ⁇ 60%, 70%, 80%, 90% or more than 90% of the second plurality of codons are high frequency codons in the bacteriophage genome. In some embodiments, the second plurality of codons match the profile of codons in the bacteriophage genome.
  • the nucleic acid sequence is modified to remove DNA modification sites.
  • the DNA modification sites comprise DNA methylation sites.
  • the nucleic acid sequence is modified to remove restriction enzyme motifs. In some embodiments, the nucleic acid sequence is modified to remove restriction enzyme motifs for a restriction enzyme derived from a bacterial species described herein. In some embodiments, the nucleic acid insert does not comprise, or comprises fewer than 10 sites recognized by abacterial enzyme.
  • the nucleic acid sequence 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%, andthe 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 nucleic acid sequences of this disclosure are codon optimized for expression in the organism/species of interest.
  • the nucleic acid sequence is modified to optimize the percent GC content.
  • the percent GC content is modified so that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90% or more than 90% of the nucleotides comprises guanine or cytosine.
  • the percent GC content is modified so thatno more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90% or more than 90% of the nucleotides comprises guanine or cytosine.
  • the exogenous nucleic acid is a bacterial nucleic acid.
  • the nucleic acid insert and the bacterial nucleic acid have less than 100%, 95%, 90%, 80%, 70%, 60%, or 50% sequence identity.
  • the first bacterial protein is a CRISPR-Cas protein as described herein. In some embodiments, the first bacterial protein is an antimicrobial agent and/or peptide as described herein.
  • the nucleic acid sequence, and/or expression cassettes disclosed herein are expressed transiently and/or stably incorporated into the genome of a host organism.
  • a the nucleic acid sequence and/or expression cassettes disclosed herein is introduced into a cell by any method known to those of skill in the art. Exemplary methods of transformation include transformation via electroporation of competent cells, passive uptake by competent cells, chemical transformation of competent cells, as well as any other electrical, chemical, physical (mechanical) and/or biological mechanism that results in the introduction of nucleic acid into a cell, including any combination thereof.
  • transformation of a cell comprises nuclear transformation.
  • transformation of a cell comprises plasmid transformation and conjugation.
  • nucleotide sequences when more than one nucleic acid 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 transformation event, or in separate transformation events.
  • a target bacterium comprising contacting or introducing into a target bacterium any of the bacteriophages disclosed herein.
  • a mixed population of bacterial cells having a first bacterial species that comprises a target nucleotide sequence in the essential gene and a second bacterial species that does not comprise a target nucleotide sequence in the essential gene, the method comprising introducing into the mixed population of bacterial cells any of the bacteriophages disclosed herein.
  • the target bacterium is killed solely by lytic activity of the bacteriophage. In some embodiments, the target bacterium is killed solely by activity of the CRISPR-Cas system. In some embodiments, the target bacterium is killed by the processing of the CRISPR array by a 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. In some embodiments, the target bacterium is killed solely by the antimicrobial peptide.
  • the target bacterium is killed by lytic activity of the bacteriophage in combination with activity of the Type I CRISPR-Cas system. In some embodiments, the target bacterium is killed by the activity of the Type I CRISPR-Cas system, independently of the lytic activity of the bacteriophage. In some embodiments, the activity of the Type I CRISPR-Cas system supplements or enhances the lytic activity of the bacteriophage. In some embodiments, the activity of the Type I CRISPR-Cas system and the lytic activity of the bacteriophage are additive.
  • the target bacterium is killed by lytic activity of the bacteriophage in combination with activity of the Type I CRISPR-Cas system and the anti microbial peptide. In some embodiments, the target bacterium is killed by the activity of the Type I CRISPR-Cas system, independently of the lytic activity of the bacteriophage and the anti-microbial peptide. In some embodiments, the activity of the Type I CRISPR-Cas system supplements or enhances the lytic activity of the bacteriophage and the anti-microbial peptide. In some embodiments, the activity of the Type I CRISPR-Cas system, the lytic activity of the bacteriophage, and the activity of the antimicrobial peptide are additive.
  • the lytic activity of the bacteriophage and the activity of the Type I CRISPR-Cas system is synergistic.
  • a synergistic activity is defined as an activity resulting in a greater level of phage kill than the additive combination of the lytic activity of the bacteriophage and the Type I CRISPR-Cas system.
  • the lytic activity of the bacteriophage is modulated by a concentration of the bacteriophage.
  • the activity of the Type I CRISPR-Cas system is modulated by a concentration of the bacteriophage.
  • the synergistic killing of the bacterium is modulated to favor killing by the lytic activity of the bacteriophage over the activity of the CRISPR-Cas system by increasing the concentration of bacteriophage administered to the bacterium. In some embodiments, the synergistic killing of the bacterium is modulated to disfavor killing by the lytic activity of the bacteriophage over the activity of the CRISPR-Cas system by decreasing the concentration of bacteriophage administered to the bacterium. In some embodiments, at low concentrations, lytic replication allows for amplification and killing of the target bacteria. In some embodiments, at high concentrations, amplification of a phage is not required.
  • the synergistic killing of the bacterium is modulated to favor killing by the activity of the CRISPR-Cas system 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-Cas system 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 lytic activity of the bacteriophage, the activity of the Type I CRISPR-Cas system, and the activity of the antimicrobial peptide is synergistic.
  • a synergistic activity is defined as an activity resulting in a greater level of phage kill than the additive combination of the lytic activity of the bacteriophage, the Type I CRISPR-Cas system, and the antimicrobial peptide.
  • the lytic activity of the bacteriophage is modulated by a concentration of the bacteriophage and the antimicrobial peptide.
  • the activity of the Type I CRISPR-Cas system is modulated by a concentration of the bacteriophage.
  • a bacteriophage disclosed herein is administered to patients intra-arterially, intravenously, intraurethrally, intramuscularly, orally, subcutaneously, by inhalation, or any combination thereof. In some embodiments, a bacteriophage disclosed herein is administered to patients by oral administration.
  • a bacteriophage disclosed herein is administered to patients by topical, cutaneous, transdermal, transmucosal, implantation, sublingual, buccal, rectal, vaginal, ocular, otic, or nasal administration. In some embodiments, a bacteriophage disclosed herein is administered to patients by any combination of the aforementioned routes of administration.
  • a dose of phage between 10 3 and 10 20 PFU is given. In some embodiments, a dose of phage between 10 3 and 10 10 PFU is given. In some embodiments, a dose of phage between 10 6 and 10 20 PFU is given. In some embodiments, a dose of phage between 10 6 and 10 10 PFU is given. For example, in some embodiments, the bacteriophage is presentin a composition in an amountbetween 10 3 and 10 U PFU.
  • the bacteriophage is presentin 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 , 10 24 PFU or more. In some embodiments, the bacteriophage is presentin a composition in an amount of less than 10 1 PFU.
  • the bacteriophage is presentin a composition in an amountbetween lCriand 10 8 , 10 4 and 10 9 , 10 5 and 10 10 , or 10 7 and 10 11 PFU.
  • a composition comprising two or more bacteriophage is administered to a subject, wherein each bacteriophage is administered 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 ,
  • a composition comprising two or more bacteriophage is administered to a subject, wherein each bacteriophage is administered in an amount of less than 10 1 PFU. In some embodiments, a composition comprising two or more bacteriophage is administered to a subject, wherein each bacteriophage is administered in an amountbetween 10 1 and 10 8 , 10 4 and 10 9 , 10 5 and 10 10 , or 10 7 and 10 U PFU.
  • a bacteriophage or a mixture is administered to a subject in need thereof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 times a day. In some embodiments, a bacteriophage or a mixture is administered to a subject in need thereof atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or21 times a week.
  • a bacteriophage or a mixture is administered to a subject in need thereof atleast 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, 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.
  • a bacteriophage or a mixture is administered to a subject in need thereof every 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, or 24 hours.
  • 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.
  • the bacteriophages disclosed herein treat or prevent diseases or conditions mediated or caused by bacteria as disclosed herein in a human or animal subject. In some embodiments, the bacteriophages disclosed herein treat or prevent diseases or conditions caused or exacerbated by bacteria as disclosed herein in a human or animal subject. 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. In some embodiments, a bacterial infection is treated by modulating the activity of the bacteria and/or by directly killing of the bacteria.
  • the bacterium is Staphylococcus spp. In some embodiments, the bacterium is S. aureus.
  • one or more Staphylococcus species present in a bacterial population are pathogenic.
  • 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 enteropathogenic bacteria from a plurality of bacteria within the microbiome or gut flora of a subject.
  • 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 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. In some embodiments, the bacteriophages are used to modulate and/or kill target bacteria on the mucosal membrane of a subject.
  • the pathogenic bacteria are antibiotic resistant. In some embodiments, the pathogenic bacteria is methicillin resistant. In some embodiments, the pathogenic bacteria is methicillin resistant Staphylococcus aureus.
  • 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.
  • the bacterium is includes Staphylococcus spp. In some embodiments, the bacterium is Staphylococcus aureus.
  • the bacteriophage treats acne and other related skin infections.
  • the Staphylococcus species 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.
  • the bacteria strain is Staphylococcus aureus.
  • the pathogenic bacteria is methicillin resistant Staphylococcus aureus.
  • the bacterium is S. aureus.
  • the methods and compositions disclosed herein are for use in veterinary and medical applications as well as research applications.
  • 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.
  • 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, Bacteroidetes, 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 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 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 S. aureus.
  • 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 ofEnterobacteriaceae,Pasteurellaceae, Fusobacteriaceae, Neisseriaceae, Veillonellaceae, Gemellaceae, Bacteriodales, Clostridiales, Erysipelotrichaceae, Bifidobacteriaceae, Bacteroides, Faecalibacterium, Roseburia,Blautia, Ruminococcus, Coprococcus, Streptococcus, Dorea, Blautia, Ruminococcus, Lactobacillus, Enterococcus, Streptococcus, Actinomyces, Lactococcus,Roseburia, Blautia, Dialister, Desulfovibrio, Escherichia, Lactobacillus, Coprococcus, Clostridium,
  • the bacteriophages are used to modulate or kill target single or plurality of bacteria within the microbiome or flora of the epidermis of a subject. Modification (e.g., dysbiosis) of the microbiome or skin flora increasesthe risk for health conditions such as eczema or Atopic Dermatitis.
  • 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 furtherused 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.
  • phage disclosed herein is used to treat equipment or environments inhabited by bacterial genera 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 lO O 20 plaque formingunits (PFU)/ml.
  • a bacteriophage disclosed herein is applied by aerosolizing agents that include dry dispersants to facilitate distribution of the bacteriophage into the environment.
  • aerosolizing agents that include dry dispersants to facilitate distribution of the bacteriophage into the environment.
  • objects are immersed in a solution containing bacteriophage disclosed herein.
  • 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. Examples of ventilators include devices to support ventilation during surgery, devices to support ventilation of incapacitated patients, and similar equipment.
  • the conventional 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-moistenedtowelettes, 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. [0163] In some embodiments, 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.
  • bacteriophage sanitation is applicable to other agricultural applications and organisms.
  • Produce including fruits and vegetables, dairy products, and other agricultural products.
  • freshly -cut produce frequently arrive at the processing plant contaminated with pathogenic bacteria. This has led to outbreaks of food- bome 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 micro flora 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.
  • compositions comprising (a) the nucleic acid sequences as disclosed herein; and (b) a pharmaceutically acceptable excipient. Also disclosed herein, in certain embodiments, are pharmaceutical compositions comprising (a) the bacteriophages as disclosed herein; and (b) a pharmaceutically acceptable excipient. Further disclosed herein, in certain embodiments, are pharmaceutical compositions comprising (a) the compositions as disclosed herein; and (b) a pharmaceutically acceptable excipient.
  • 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 bloodstream infections (BSI) and/or inflammatory diseases (e.g. atopic dermatitis (AD)).
  • a pharmaceutical composition or method disclosed herein treats eczema.
  • a pharmaceutical composition or method disclosed herein treats atopic dermatitis.
  • compositions disclosed herein comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.
  • the bacteriophages disclosed herein are formulated for administration in a pharmaceutical carrier in accordance with suitable methods.
  • 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 pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered in a therapeutically effective amount.
  • 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, transderm al 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 subj ect includes aqueous solutions.
  • 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 subj ect 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,
  • 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 Ethylenediaminetetraacetic acid (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.
  • EDTA Ethylenediaminetetraacetic acid
  • BHT butylated hydroxytoluene
  • BHA butylated hydroxy anisole
  • sodium sulfite sodium sulfite
  • glutathione propyl gallate
  • cysteine methionine
  • ethanol 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, 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, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 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, com starch, wheat starch; sugars such as sucrose, glucose, dextrose, lactose, maltodextrin; natural and synthetic gums; gelatine; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxy ethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinylpyrrolidone (povidone); polyethyleneglycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols such as sorbitol, xylitol, mannitol and water or a combination thereof.
  • starches such as potato starch, com starch, wheat starch
  • sugars such as sucrose, glucose, dextrose, lactose, maltodextrin
  • natural and synthetic gums such as cellulose derivatives such as micro
  • 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 (com 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; SteviaRebaudiana(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.
  • Non limiting examples of 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, amino acids 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 asErtapenem, Doripenem, Imipenem/CilastatinorMeropenem.
  • an antibiotic agent described herein is a cephalosporins (first generation) such as Cefadroxil, Cefazolin, Cef alexin, Cefalotin or Cefalothin, or alternatively a Cephalosporins (second generation) such as Cefaclor, Cefamandole, Cefoxitin, Cefprozil or Cefuroxime.
  • first generation such as Cefadroxil, Cefazolin, Cef alexin, Cefalotin or Cefalothin
  • Cephalosporins second generation
  • Cefaclor, Cefamandole, Cefoxitin, Cefprozil or Cefuroxime 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 Ceftobiprole.
  • an antibiotic agent described herein is alincosamide such as Clindamycin and Azithromycin, or a macrolide such as Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin and Spectinomycin.
  • 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.
  • 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 asMafenide, Sulfonamidochrysoidine, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim, or Trimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP-SMX).
  • a sulfonamide such asMafenide, Sulfonamidochrysoidine, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim, or Trimethoprim-Sul
  • 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 Bacitracin, Colistin or Polymyxin B.
  • an antibiotic agent described herein is a tetracycline such as Demeclocycline, Doxy cy cline, Minocycline or Oxy tetracycline.
  • a bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, wherein the bacteriophage has been rendered lytic.
  • the bacteriophage of any one of embodiments 1 -2 wherein the bacteriophage has been rendered lytic by the removal, alteration or replacement of a promoter of a lysogeny gene.
  • the bacteriophage of any one of embodiments 1 -2 wherein the bacteriophage has been rendered lytic by the removal of a functional element of a lysogeny gene.
  • the bacteriophage of any one of embodiments 1-12 wherein the bacteriophage infects multiple bacterial strains.
  • the essential bacterial gene is Tsf acpP, gapA, infA , secY, csrA, trmD,ftsA,fiisA , glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fus, adk, rpsK, rplR, ctrA,parC , tRNA-Ser, tRNA-Asn, or metK.
  • a bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, wherein the bacteriophage has been rendered lytic by removal of the var009 region, varO 10 region, varOl 2 region, var042 region, or a combination thereof from a temperate bacteriophage.
  • the bacteriophage of embodiment 34 wherein the essential bacterial gene is Tsf acpP, gapA, infA , secY, csrA, trmD,ftsA,fiisA , glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fus, adk, rpsK, rplR, ctrA,parC , tRNA-Ser, tRNA-Asn, or metK.
  • the essential bacterial gene is Tsf acpP, gapA, inf
  • the bacteriophage of embodiment 39 wherein the non-essential bacteriophage gene is a cl repressor gene, antirepressor gene, integrase, PemK-like phage protein gene, or any combination or genes found in varO 10, var012, orvar042.
  • the bacteriophage of any one of embodiments 39-40 wherein the target bacterium is S. aureus.
  • the bacteriophage of any one of embodiments 39-40, wherein the temperate bacteriophage is a Kayvirus, a Twortvirus, a Rosenblumvirus, a Phietavirus or a Triavirus.
  • a pharmaceutical composition comprising: a. a bacteriophage of any one of embodiments 1 -48; and b . a pharmaceutically acceptable excipient.
  • the pharmaceutical composition of embodiment 48, wherein the pharmaceutical composition comprises at least two bacteriophage selected from the bacteriophage of any one of embodiments 1-45.
  • the pharmaceutical composition of embodiment 49, wherein the bacteriophage are from the lineage consisting of a Kay virus, a Twortvirus, a Rosenblum virus, a Phietavirus or a Triavirus. 1.
  • composition of embodiment 50 wherein the pharmaceutical composition is in a form of a tablet, a capsule, a liquid, a syrup, an oral formulation, an intravenous formulation, an intranasal formulation, an ocular formulation, an otic formulation, a subcutaneous formulation, a topical formulation, atransdermal formulation, a transmucosal formulation, an inhalable respiratory formulation, a suppository, and any combination thereof.
  • a method for killing a target bacterium comprising introducing into the target bacterium a lytic bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in the target bacterium, thereby killing the target bacterium, wherein the bacteriophage is rendered lytic by removal of a var009 region, varOlO region, var012 region, var042 region, or a combination thereof from a temperate bacteriophage.3.
  • the bacteriophage infects multiple bacterial strains. .
  • the target nucleotide sequence comprises all or a part of a promoter sequence for the target gene. 5. The method of any one of embodiments 52-53, wherein 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 method of any one of embodiments 52-53, wherein the target nucleotide sequence comprises at least a portion of an essential bacterial gene that is needed for survival of the target bacterium.
  • the essential bacterial gene is Tsf acpP , gapA, inf A, secY, csrA, trmD,ftsA,fusA , glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB,pheT, injB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fus, adk, rpsK, rplR, ctrA,parC , tRNA-Ser, tRNA-Asn, or metK.
  • the non-essential bacteriophage gene is a cl repressor gene, antirepressor gene, integrase, PemK-like phage protein gene, or any combination or genes found in varOlO, var012, orvar042.
  • the method of any one of embodiments 52-63, wherein the bacteriophage is a Kayvirus, a Twortvirus, a Rosenblumvirus, a Phietavirus or a Triavirus.
  • the method of embodiment 64, wherein the bacteriophage comprises at least 80% sequence identity with pi 473.
  • any one of embodiments 52-65 wherein the target bacterium is killed by the lytic activity of the bacteriophage, by the activity of a CRISPR-Cas system using the first spacer sequence or the crRNA transcribed therefrom, or both.
  • the method of any one of embodiments 52-66 wherein the target bacterium is killed by the activity of the CRISPR-Cas system independently of the lytic activity of the bacteriophage.
  • activity of the CRISPR-Cas system supplements or enhances lytic activity of the bacteriophage.
  • 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 atleastone antibiotic comprises a cephalosporin, a fluoroquinolone, a carbapenem, a colistin, an aminoglycoside, vancomycin, daptomycin, streptomycin, methicillin or oxacillin. 3.
  • a bacteriophage comprising a nucleic acid sequence encoding a Type I CRISPR-Cas system comprising:
  • the target nucleotide sequence comprises a non-coding or intergenic sequence.
  • the bacteriophage of embodiment 90 wherein the essential gene is Tsf acpP, gap A, infA, secY, csrA, trmD, ftsA,fusA, glyQ, eno, nusG, dnaA, dnaS, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, glnS, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fus, adk, rpsK, rplR, ctrA,parC, tRNA-Ser, tRNA-Asn, or metK.
  • the essential gene is Tsf acpP,
  • the bacteriophage of embodiment 92, wherein the Cascade complex comprises: (i) a Cas7 polypeptide, a Cas8al polypeptide or a Cas8a2 polypeptide, a Cas5 polypeptide, a Csa5 polypeptide, a Cas6a polypeptide, a Cas3' polypeptide, and a Cas3” polypeptide having no nuclease activity (Typel-A CRISPR-Cas system);
  • the bacteriophage of any one of embodiments 84-94, wherein the nucleic acid sequence further comprises a promoter sequence.
  • the bacteriophage of embodiment 99 wherein the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic.
  • the bacteriophage of any one of embodiments 98-101 wherein 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 of any one of embodiments 84-103 wherein the bacteriophage is an obligate lytic bacteriophage. .
  • a pharmaceutical composition comprising:
  • a pharmaceutically acceptable excipient comprising: (b) a pharmaceutically acceptable excipient.
  • a method of killing an Staphylococcus species comprising introducing into the target bacterium a bacteriophage comprising a nucleic acid sequence encoding a Type I CRISPR-Cas system comprising:
  • a Cas3 polypeptide (c) a Cas3 polypeptide. .
  • the target nucleotide sequence comprises all or a part of a promoter sequence.
  • the target nucleotide sequence comprises all or a part of a nucleotide sequence located on a coding strand of a transcribed region of an essential gene. .
  • the Cascade complex comprises:
  • a Cas7 polypeptide, a Cas8al polypeptide or a Cas8a2 polypeptide a Cas5 polypeptide, a Csa5 polypeptide, a Cas6a polypeptide, a Cas3' polypeptide, and a Cas3” polypeptide having no nuclease activity (Type I-A CRISPR-Cas system);
  • a Csel polypeptide, a Cse2 polypeptide, a Cas7 polypeptide, a Cas5 polypeptide, and a Cas6e polypeptide (Type I-E CRISPR-Cas system); (vi) a Csy 1 polypeptide, a Csy2 polypeptide, a Csy3 polypeptide, and a Csy4 polypeptide (Typel-F CRISPR-Cas system); or
  • a Cas8u2 polypeptide, a Cas7 polypeptide, and a fused Cas5 -Cas6 polypeptide (Typel-U CRISPR-Cas system).
  • the Cascade complex comprises a Cas5d polypeptide, a Cas8c polypeptide, and a Cas7 polypeptide (Type I-C CRISPR- Cas system).
  • the nucleic acid sequence further comprises a promoter sequence.
  • the Staphylococcus species is killed solely by activity of the CRISPR-Cas system. .
  • any one of embodiments 122-123 wherein the Staphylococcus species is killed by lytic activity of the bacteriophage in combination with activity of the CRISPR-Cas system. .
  • the method of embodiment 121 wherein the Staphylococcus species is killed by the activity of the CRISPR-Cas system, independently of the lytic activity of the bacteriophage. .
  • the method of embodiment 121 wherein the activity of the CRISPR-Cas system supplements or enhancesthe lytic activity of the bacteriophage. .
  • the method of embodiment 121, wherein the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic. .
  • 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.”
  • the 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. Thus, 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 means 100% complementarity or identity with the comparator nucleotide sequence or it means less than 100% complementarity (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%, and the like, complementarity).
  • Complement or complementable may also be used in terms of a “complement” to or “complementing” a mutation.
  • complementarity refers 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, functional 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 nucleotide sequence” refers to the portion of a target gene (i.e., target region in the genome or the “protospacer sequence,” which is adjacent to a protospacer adjacent motif (PAM) sequence) 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.
  • a target gene 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
  • PAM protospacer adjacent motif
  • 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 exact PAM sequence that is required varies between each different CRISPR-Cas system. Non-limiting examples of PAMs include CCA, CCT, CCG, TTC, AAG, AGG, ATG, GAG, and/or CC.
  • the PAM in Type I systems, 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, and is directly recognized by Cascade.
  • the PAM for B. halodurans Type I-C systems, the PAM is YYC, where Y can be either T or C.
  • the PAM for the L. monocytogenes Type I-B system, the PAM is CCW, where W can be A or T.
  • 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 atthe C-terminusof Cas9).
  • type I Clustered Regularly Interspaced Short Palindromic Repeats CRISPRj-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.
  • polypeptides include, but are not limited to, the Cascade polypeptides of type I subtypes I-A, I-B, I-C, I-D, I-E, I-F, and I-U.
  • type I-A polypeptides include Cas7 (Csa2), Cas8al (Csxl3), Cas8a2 (Csx9), Cas5, Csa5, Cas6a, Cas3' and/or a Cas3".
  • type I-B polypeptides include Cas6b, Cas8b (Cshl), Cas7 (Csh2) and/or Cas5.
  • Non-limiting examples of type I-C polypeptides include Cas5d, Cas8c (Csdl), and/or Cas7 (Csd2).
  • Non-limiting examples of type I-D polypeptides include Casl0d(Csc3), Csc2, Cscl, and/or Cas6d.
  • Non -limiting examples of type I-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).
  • Non -limiting examples of type I-F polypeptides include Cas8u2, Cas7, and/or fused Cas5 -Cas6 polypeptide.
  • Non -limiting examples of type I-U polypeptides include Cas8al (Cstl), Cas7 (Cst2), Cas5 (Cst5t), and Cas3.
  • a recombinant nucleic acid described herein comprises, consists essentially of, or consists of, a nucleotide sequence encoding a subset of type-I 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 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).
  • 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.
  • CRISPR phage refers to a bacteriophage particle comprising bacteriophage DNA comprising at least one heterologous polynucleotide that encodes at least one component of a CRISPR-Cas system (e.g., CRISPR array, crRNA; e.g., PI bacteriophage comprising an insertion of a targeting crRNA).
  • the polynucleotide encodes at least one transcriptional activator of a CRISPR-Cas system.
  • the 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%, 9
  • substantial identity refer to two or more sequences or sub sequences that have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95, 96, 97, 98, or 99% identity.
  • 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. The 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 d etermined using BLASTX version 2.0 for translated nucleotide sequences andBLASTN version 2.0 for polynucleotide sequences.
  • the recombinant nucleic acid 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 nucleic 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% pure, or purer.
  • 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.
  • the terms with respect to 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 on a subject.
  • the bacterial population comprises one or more target bacterial species.
  • the one or more bacteria species in the bacterial population comprise one or more strains of one or more bacteria.
  • the target bacterial population causes an “infection”, “a disease”, or “a condition” that is acute or chronic.
  • the target bacterial population causes an “infection”, “a disease”, or “a condition” that is localized or systemic.
  • the target bacterial population causes an “infection”, “a disease”, or “a condition” that is idiopathic. In some embodiments, the target bacterial population causes an “infection”, “a disease”, or “a condition” that 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.
  • 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, as well as at liquid-air interfaces, and are medically important, accounting for over 80 percent of microbial infections in the body.
  • prevent refers 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).
  • prevent infection food, surfaces, medical tools and devices are treated with compositions and by methods disclosed herein.
  • subjects are mammals, avians, reptiles, amphibians, fish, crustaceans, and mollusks.
  • 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).
  • Fish subjects include but are not limited to species used in aquaculture (e.g., tuna, salmon, tilapia, catfish, carp, trout, cod, bass, perch, snapper, and the like).
  • Crustacean subjects include but are not limited to species used in aquaculture (e.g., shrimp, prawn, lobster, crayfish, crab).
  • Mollusk subjects include but are not limited to species used in aquaculture (e.g., abalone, mussel, oyster, clams, scallop).
  • 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.”
  • nucleic acid sequence in context of a nucleic acid sequence is a nucleic acid sequence that exists apart from its native environment.
  • pharmaceutically acceptable means a material that is not biologically or otherwise undesirable, i.e., the materials are administered to a subject without causing any undesirable biological effects such as toxicity.
  • in vivo is used to describe an event that takes place in a subject’s body.
  • in vitro is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained.
  • in vitro assays can encompass cell- based assays in which living or dead cells are employed.
  • In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
  • Example 1 Engineered phage used in this application
  • Recombinant bacteriophage were engineered to contain a genetic deletion in the predicted lysogeny module region.
  • Fig. 1 depicts the predicted lysogeny region of phage pi 473. Further, Fig. 1 depicts open reading frames encoding putative repressor and anti repressor proteins and an open reading frame encoding a putative integrase.
  • the first recombinant phage contains a genetic deletion that was introduced into the predicted lysogeny module region of the bacteriophage genome in the region including a putative anti repressor, and two clones were isolated and designated Var009 and VarOlO.
  • a second recombinant phage contains a genetic deletion that was introduced into the predicted lysogeny module region of the bacteriophage genome in the region including a putative repressor and a single clone was isolated and designated VarO 12.
  • a final recombinant phage (Var042) was isolated that contains the deletion from VarO 12 and a second deletion removing the predicted integrase gene.
  • Two additional control variants were generated with different deletions outside of the lysogeny region as controls (not shown), isolated and designated Var002 and Var006, respectively.
  • Fig. 6 depicts the sequence of the VarOlO deletion.
  • Fig. 7 depicts the sequence of the VarOl 2 deletion, which is also the first deletion in Var042.
  • Fig. 8 depicts the second deletion in Var042.
  • Fig. 2 depicts the dilution series of wild-type (WT) pi 473 and several variants plated on a lawn of S. aureus using the double agar overlay method.
  • WT wild-type
  • Var002 and Var006 produced small hazy plaques
  • variants Var009, VarOlO and VarO 12 produced larger clearer plaques.
  • Variants Var002 and Var006 contain mutations outside of the lysogeny region. The variants with mutants outside the lysogeny region do not show changes in plaque morphology indicating that these phage variants remain temperate.
  • Var009, VarOlO and VarO 12 with mutations in the lysogeny region show clearer plaques and higher efficiency of plaquing on the strain shown.
  • These data indicate that Var009, VarOlO and VarO 12 were successfully converted from lysogenic to lytic phenotype, as clear plaque morphology is a hallmark of lytic bacteriophages in S. aureus (References Mi tarai etal J. Bacteriol. 2016. doi: 10.1128/JB.00965-15, Garcia et al. Appl. Environ. Microbiol. 2009. doi: 10.1128/ AEM.01864-09) and is a known phenomenon in other examples of lysogenic phages that have been genetically converted to obligately lytic.
  • Fig. 3 depicts a close-up image of Fig. 2, showing the larger plaque morphology for wild-type pl473, VarOlO and Var012.
  • the arrows pointto individual phage plaques.
  • VarOlO and Var012 plaques display a clearer morphology compared to wild-type. Additionally, the zone of clearing with too many plaques to count is clearer in VarOlO and VarO 12 than the WT phage.
  • Example 3 Bacteriophage killing assay confirms lysogenic to lytic phenotype conversion
  • Fig. 4 depicts the bacteriophage killing assay with strain b4063 (USA300 strain FPR3757) in LB challenged with Wild-type pi 473, Var012, or Var042 as measured by bacterial counts as a function of time.
  • strain b4063 USA300 strain FPR3757
  • bacteria were inoculated into LB in triplicate. For cells only, nothing else was added.
  • Wild-type pi 473, VarO 12 or Var042 were added at an MOI of 1.
  • Bacterial CFUs were enumerated at 0, 2, 4, 6 and 24 h post inoculation. As depicted in Fig.
  • Fig. 5 depicts growth curves of three S. aureus clinical isolates (b2991, b3022 and b3202) in LB challenged with pi 473 WT or pi 473 Var012. Growth of the clinical isolates in LB was monitored by taking the OD at 600 nm periodically over a 24 hr time course. Control drowth curves are uninfected culture controls for each strain, while test growth curves are for cultures challenged with WT or VarO 12 at an MOI of 1. Arrows indicate regrowth of the clinical isolates in the WT challenged cultures while the VarO 12 challenged cultures remain suppressed out to 24 hours. Overall, the experimental comparison of the three S.
  • aureus clinical isolates (b2991, b3022 andb3202) in LB challenged with pi 473 WT or pi 473 VarO 12 shows more regrowth in strains challenged with WT phage, with b2991 showing the best recovery over a 24 hr time course.
  • Top agar overlays are prepared by mixing 100 pL of a saturated overnight culture of b4063 with 6 mL of 0.375% agar in LB containing 10 mMMgCh and 10 mMCaC ⁇ . After the top agar solidified, 2 pL drops of serial 10 -fold dilution series of pi 473 wt (wild type) and p 1473 -Cas (Cas system only) and p 1473 -crArray (targeting CRISPR Array + Cas system) are spotted onto the surface of the top agar. Plates are incubated at 37°C for ⁇ 18h, then imaged using a Keyence BZ-X800 microscope at 4X and 1 OX magnification.
  • Bacteriophage was engineered with a Type IB CRISPR-Cas system (LMIB). The engineered phage lysate was spotted onto a bacterial overlay in order to obtain isolated (clonal) plaques. Seven plaques were picked and screened by PCR for the presence of the desired insert. Each plaque was screened using two pairs of PCR primers, with one pair covering the upstream engineering junction (i.e., the site where the wild type phage genome meets the engineered insert) and a significant portion of the insert and one pair covering the downstream engineering junction and a significant portion of the insert. Since one primer from each primer pair binds within the insert, un-engineered phages will produce no PCR bands.
  • LMIB Type IB CRISPR-Cas system
  • L designates a DNA size ladder
  • the numbers 1 -7 indicate individual clonal plaques being screened
  • a and b indicate the two primer pairs. Both primer pairs produced bands of the expected sizes for all plaques, indicating that all plaques were successfully engineered with the LMIB insert (SEQ ID NO: 23).
  • Clinical isolate b2655 was inoculated with either wild -type (WT) phage (squares), c at -lnqQ, Ps arA -InqQ, or P TmpG -InqQ.
  • WT wild -type
  • Ps arA -InqQ wild -type phage
  • P TmpG P TmpG -InqQ.
  • three bacterial replicates were inoculated with phage at a multiplicity of infection of 1. Cultures were grown at 37°C in a shaking plate reader with optical density at 600 nm measured every 10 minutes for 18 hours. The results are depicted in FIG. 11. Phages with InqQ reduced the growth of the bacteria compared to WT phage starting at around 80 minutes and persisting throughout the experiment.
  • Example 7 An engineered bacteriophage comprising DNAse
  • a Kayvirusbacteriophage was engineered to contain DNAse I (SEQ NO: 1) and the Eca/ promoter (SEQ ID NO: 16).
  • the DNA sequence encoding mature human DNase was modified to optimize codon usage and remove sequences not tolerated by the phage while maintaining the amino acid identity. This sequence was placed downstream of the Peat promoter.
  • the Pcat-DNase I sequence was engineered into the phage and the sequence was confirmed by Illumina next generation sequencing of the engineered phage genome. The results are depicted in FIG. 12.
  • Example 8 Host range of a bacteriophage cocktail
  • a 308 strain panel of clinical isolates of S. aureus was used to test the host range of a bacteriophage cocktail.
  • the S. aureus strains used were b004604,b004605,b004606, b004607,b004608,b004609,b004610,b004611,b004612,b004613,b004614,b004615, b004616,b004617,b004618,b004619,b004620,b004621,b004622,b004623,b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631 , b004632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641 ,
  • Results are depicted in Table 1.
  • the ability of Kayviruses, aPhietavirus, and aRosenblumvirus to target the 308 strains of the panel was tested. As can be seen in FIG. 13, for a majority of strains tested, at least two phage infect a particular target bacteria, reducing the likelihood that a target bacteria would be able to evolve resistance against a particular phage and evade infection by another bacteriophage.
  • Table 1 Results of Host Range Assay

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Abstract

Disclosed here are phage compositions for Staphylococcus comprising CRISPR-Cas systems and methods of use thereof.

Description

PHAGE COMPOSITIONS FOR STAPHYLOCOCCUS COMPRISING CRISPR-CAS SYSTEMS AND METHODS OF USE THEREOF
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Application No.
63/184,696, filed May 5, 2021 , which application is incorporated herein by reference.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 53240-747_601_SL.TXT, created April 27, 2022, which is 70,586 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.
SUMMARY
[0003] In one aspect, provided herein is are bacteriophage compositions comprising two or more bacteriophage. In some embodiments, the composition comprises a Phietavirus bacteriophage and Rosenblumvirus bacteriophage, Phietavirus bacteriophage and Kayvirus bacteriophage, Rosenblumvirus bacteriophage and Kayvirus bacteriophage, or Phietavirus bacteriophage, Rosenblumvirus bacteriophage, and Kayvirus bacteriophage. In some embodiments, the Phietavirus bacteriophage is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene. In some embodiments, the lysogenic gene encodes for a repressor. In some embodiments, composition comprises the Phietavirus bacteriophage and Rosenblumvirus bacteriophage. In some embodiments, the composition comprises the Phietavirus bacteriophage and Kayvirus bacteriophage. In some embodiments, the composition comprises the Rosenblumvirus bacteriophage and Kayvirus bacteriophage.
In some embodiments, the composition comprises the Phietavirus bacteriophage, Rosenblumvirus, and Kayvirus bacteriophage. In some embodiments, the composition comprises the Kayvirus bacteriophage, wherein the Kayvirus bacteriophage comprises two or more Kayvirus bacteriophage. In some embodiments, the bacteriophage of the composition infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria.
[0004] In some embodiments, the composition comprises a plurality of bacteriophage comprising a first bacteriophage and a second bacteriophage, wherein the plurality of bacteriophage infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria. In some embodiments, the Staphylococcus bacteria are selected from b004604, b004605,b004606,b004607,b004608,b004609,b004610,b004611,b004612,b004613, b004614,b004615,b004616,b004617,b004618,b004619,b004620,b004621,b004622, b004623,b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631 , b004632, b004633,b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641, b004642, b004643, b004644, b004645, b004646, b004647, b004648, b004649, b004650, b004651, b004652, b004653, b004654, b004655, b004656, b004657, b004658, b004659, b004660, b004661 , b004662, b004663, b004664, b004665, b004666, b004667, b004668, b004669, b004670, b004671 , b004672, b004673, b004674, b004675, b004676, b004677, b004678, b004679, b004680, b004681 , b004682, b004683, b004684, b004685, b004686, b004687, b004688, b004689, b004690, b004691, b004692, b004693, b004694, b004695, b004696, b004697, b004698, b004699, b004700, b004701 , b004702, b004703, b004704, b004705,b004706, b004707, b004708, b004709, b004710, b004711 , b004712, b004713,b004714,b004715,b004716,b004717,b004718,b004719,b004720,b004721, b004722, b004723,b004724, b004725, b004726, b004727, b004728, b004729, b004730, b004731 , b004732, b004733, b004734, b004735, b004736, b004737, b004738, b004739, b004740, b004741 , b004742, b004743, b004744, b004745, b004746, b004747, b004748, b004749, b004750, b004751 , b004752, b004753, b004754, b004755, b004756, b004757, b004758, b004759, b004760, b004761 , b004762, b004763, b004764, b004765, b004766, b004767, b004768, b004769, b004770, b004771, b004772, b004773, b004774, b004775, b004776, b004777, b004778, b004779, b004780, b004781 , b004782, b004783, b004784, b004785,b004786, b004787, b004788, b004789, b004790, b004791 , b004792, b004793, b004794, b004795,b004796, b004797, b004798, b004799, b004800, b004801 , b004802, b004803,b004804, b004805, b004806, b004807, b004808, b004809, b004810, b004811 , b004812, b004813, b004814, b004815, b004816, b004817, b004818, b004819, b004820, b004821, b004822, b004823, b004824, b004825, b004826, b004827, b004828, b004829, b004830,b004831,b004832,b004833,b004834,b004835,b004836,b004837,b004838, b004839, b004840, b004841 , b004842, b004843, b004844, b004845, b004846, b004847, b004848,b004849,b004850,b004851,b004852,b004853,b004854,b004855,b004856, b004857, b004858, b004859, b004860, b004861 , b004862, b004863, b004864, b004865, b004866, b004867, b004868, b004869, b004870, b004871 , b004872, b004873, b004874, b004875,b004876, b004877, b004878, b004879, b004880, b004881 , b004882, b004883, b004884, b004885, b004886, b004887, b004888, b004889, b004890, b004891, b004892, b004893,b004894, b004895, b004896, b004897, b004898, b004899, b004900, b004901 , b004902, b004903,b004904, b004905, b004906, b004907, b004908, b004909, b004910, and b004911. In some embodiments, the Staphylococcus bacteria comprises Staphylococcus aureus. In some embodiments, the infectivity is determined with a plaque assay or growth inhibition assay. In some embodiments, the at least about 90% is at least about 95%. In some embodiments, the at least about 90% is at least about 98%. In some embodiments, the at least about 90% is at least about 99%. In some embodiments, the at least about 30 Staphylococcus bacteria comprises b004604, b004605, b004606, b004607, b004608, b004609, b004610, b004611,b004612,b004613,b004614,b004615,b004616,b004617,b004618,b004619, b004620, b004621 , b004622, b004623, b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631 , b004632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641 , b004642, b004643, b004644, b004645, b004646, b004647, b004648, b004649, b004650, b004651 , b004652, b004653, b004654, b004655, b004656, b004657, b004658, b004659, b004660, b004661 , b004662, b004663, b004664, b004665, b004666, b004667, b004668, b004669, b004670, b004671 , b004672, b004673, b004674, b004675, b004676, b004677, b004678, b004679, b004680, b004681 , b004682, b004683,b004684, b004685, b004686, b004687, b004688, b004689, b004690, b004691 , b004692, b004693,b004694, b004695, b004696, b004697, b004698, b004699, b004700, b004701, b004702, b004703, b004704, b004705, b004706, b004707, b004708, b004709, b004710,b004711,b004712,b004713,b004714,b004715,b004716,b004717,b004718, b004719, b004720, b004721 , b004722, b004723, b004724, b004725, b004726, b004727, b004728, b004729, b004730, b004731 , b004732, b004733, b004734, b004735, b004736, b004737, b004738, b004739, b004740, b004741 , b004742, b004743, b004744, b004745, b004746, b004747, b004748, b004749, b004750, b004751 , b004752, b004753, b004754, b004755,b004756, b004757, b004758, b004759, b004760, b004761 , b004762, b004763, b004764, b004765,b004766, b004767, b004768, b004769, b004770, b004771 , b004772, b004773,b004774, b004775, b004776, b004777, b004778, b004779, b004780, b004781 , b004782, b004783,b004784, b004785, b004786, b004787, b004788, b004789, b004790, b004791, b004792, b004793, b004794, b004795, b004796, b004797, b004798, b004799, b004800, b004801 , b004802, b004803, b004804, b004805, b004806, b004807, b004808, b004809,b004810,b004811,b004812,b004813,b004814,b004815,b004816,b004817, b004818, b004819, b004820, b004821 , b004822, b004823, b004824, b004825, b004826, b004827, b004828, b004829, b004830, b004831 , b004832, b004833, b004834, b004835, b004836, b004837, b004838, b004839, b004840, b004841 , b004842, b004843, b004844, b004845,b004846, b004847, b004848, b004849, b004850, b004851 , b004852, b004853, b004854,b004855,b004856,b004857,b004858,b004859,b004860,b004861,b004862, b004863,b004864, b004865, b004866, b004867, b004868, b004869, b004870, b004871 , b004872, b004873,b004874, b004875, b004876, b004877, b004878, b004879, b004880, b004881,b004882,b004883,b004884,b004885,b004886,b004887,b004888,b004889, b004890, b004891 , b004892, b004893, b004894, b004895, b004896, b004897, b004898, b004899, b004900, b004901 , b004902, b004903, b004904, b004905, b004906, b004907, b004908,b004909,b004910, and b004911. In some embodiments, the first bacteriophage and the second bacteriophage are of different genera. In some embodiments, the plurality of bacteriophage comprise a Phietavirus bacteriophage and Rosenblum virus bacteriophage, Phietavirus bacteriophage and Kayvirus bacteriophage, Rosenblumvirus bacteriophage and Kay virus bacteriophage, or Phietavirus bacteriophage, Rosenblumvirus bacteriophage, and Kayvirus bacteriophage. In some embodiments, the plurality of bacteriophage comprise a Phietavirus. In some embodiments, the Phietavirus is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene. In some embodiments, the lysogenic gene encodes for a repressor.
[0005] In some embodiments, the composition comprises a plurality of bacteriophage comprising a first bacteriophage that is specific for a first receptor of a Staphylococcus bacteria, a second bacteriophage that is specific for a second receptor of the Staphylococcus bacteria, wherein the plurality of bacteriophage is more resilient to resistance by the Staphylococcus bacteria than the first or second bacteriophage alone. In some embodiments, the first receptor and the second receptor are different. In some embodiments, the first bacteriophage comprises a Phietavirus. In some embodiments, the first bacteriophage comprises a Phietavirus and a Rosenblumvirus. In some embodiments, the second bacteriophage comprises a Kayvirus. In some embodiments, the plurality of bacteriophage comprise: a Phietavirus bacteriophage and Rosenblumvirus bacteriophage, Phietavirus bacteriophage and Kayvirus bacteriophage, Rosenblumvirus bacteriophage andKay virus bacteriophage, or Phietavirus bacteriophage, Rosenblumvirus bacteriophage, andKayvirus bacteriophage. In some embodiments, the plurality of bacteriophage of the composition infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria. In some embodiments, the first bacteriophage and the second bacteriophage are of different genera. In some embodiments, the first bacteriophage and the second bacteriophage are capable of independently infecting at least 90% of the collection of Staphylococcus bacteria. [0006] In another aspect, provided herein are engineered bacteriophage. In some embodiments, provided is a Phietavirus bacteriophage engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene. In some embodiments, the lysogenic gene encodes for a repressor. In some embodiments, the repressor comprises an amino acid sequence at least about 80% identical to SEQ ID NO: 47 or 48. In some embodiments, removal of the lysogenic gene comprises removing from about 1% to 100% of the lysogenic gene. In some embodiments, about 10 to about 1,200 base pairs of the lysogenic gene are removed. In some embodiments, the lysogenic gene is removed, replaced, or inactivated. In some embodiments, the lysogenic gene is removed. In some embodiments, the promoter of the lysogenic gene is removed, replaced, or inactivated.
[0007] In some embodiments, provided are bacteriophage compositions comprising the engineered bacteriophage. In some embodiments, the composition comprises the engineered Phietavirus. In some embodiments, the composition comprises a Rosenblumvirus. In some embodiments, the composition comprises aKayvirus. In some embodiments, the composition comprises a plurality of bacteriophage, wherein the plurality of bacteriophage infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria.
[0008] In some embodiments, the composition and/or engineered bacteriophage provided herein comprises a nucleic acid encoding for an exogenous peptide selected from TreA, Lpi, DNAsel, RIP, FS3, PLNC8a, PLNC8P, LytM, LnqO, 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, lyase, glycosyl hydroxylase, polyglucosamine (PGA) depolymerases, colonic acid depolymerase, 1,4-L-fucodise hydrolase, colanic acid, or depolymerazingalginase. In some embodiments, the composition and/or engineered bacteriophage comprises one or more components of a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type IB CRISPR- Cas system from Listeria monocytogenes (LMIB).
[0009] In some embodiments, the composition and/or engineered bacteriophage provided herein comprises a spacer sequence or a crRNA transcribed therefrom, wherein the spacer sequence is complementary to a target nucleic acid sequence from a target gene in a target bacteria. In some embodiments, the target bacteria comprises a Staphylococcus bacteria. [0010] A bacteriophage comprising a nucleic acid encoding an exogenous peptide selected from TreA, Lpi, DNAsel, RIP, FS3, PLNC8a, PLNC8P, LytM, LnqO, 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, lyase, glycosyl hydroxylase, polyglucosamine (PGA) depolymerases, colonic acid depolymerase, 1 ,4-L-fucodise hydrolase, colanic acid, or depolymerazing alginase.
[0011] In another aspect, provided herein are engineered bacteriophage comprising a CRISPR-Cas system, or one or more components thereof. In some embodiments, the engineered bacteriophage comprises a Type IB CRISPR-Cas system from Listeria monocytogenes (LMIB). In some embodiments, the engineered bacteriophage comprises one or more components of a CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type IB CRISPR-Cas system from Listeria monocytogenes (LMIB). In some embodiments, the LMIB encodes for a sequence at least 80% identical to any one of SEQ ID NOS: 25-29. In some embodiments, the engineered bacteriophage comprises a spacer sequence or a crRNA transcribed therefrom, wherein the spacer sequence is complementary to a target nucleic acid sequence from a target gene in a target bacteria. In some embodiments, the target bacteria comprises a Staphylococcus bacteria. In some embodiments, the bacteriophage is an engineered Phietavirus. In some embodiments, the bacteriophage is an engineered Rosenblumvirus. In some embodiments, the bacteriophage is an engineered Kay virus. In some embodiments, provided are bacteriophage compositions comprising the engineered bacteriophage. In some embodiments, the composition comprises the Phietavirus, and a Rosenblumvirus and/or a Kay virus. In some embodiments, the composition comprises the Rosenblumvirus, and a Phietavirus and/or a Kay virus. In some embodiments, the composition comprises the Kayvirus, and a Rosenblumvirus and/or a Phietavirus.
[0012] In some embodiments, the composition and/or engineered bacteriophage provided herein are used in a method of treating a disease or condition related to Staphylococcus, the method comprising administering to a subject in need thereof the composition and/or engineered bacteriophage. In some embodiments, the Staphylococcus is causative of, and/or contributes to, the disease or condition. INCORPORATION BY REFERENCE
[0013] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0015] Fig. 1 depicts the predicted lysogeny region of a Phietavirus bacteriophage pi 473. [0016] Fig. 2 depicts the dilution series of wild-type (WT) pl473 and several variants plated on a lawn of Staphylococcus aureus using the double agar overlay method. WT phage and variants Var002 and Var006 produced small hazy plaques, while variants Var009, VarOlO and VarO 12 produced larger clearer plaques.
[0017] Fig. 3 depicts the larger plaque morphology for wild -type pi 473, VarOlO and VarO 12 in a close-up image, with red arrows indicating individual plaques.
[0018] Fig. 4 depicts the bacteriophage killing assay with strain b4063 (USA300 strain FPR3757) in LB challenged with Wild-type pi 473, Var012, or Var042 as measured by bacterial counts as a function of time.
[0019] Fig. 5 depicts the growth curves of three -S' aureus clinical isolates (b2991,b3022 and b3202) in LB challenged with pl473 WT or pl473 Var012.
[0020] Fig. 6 depicts the sequence of the VarOlO deletion (SEQ IDNO: 30).
[0021] Fig. 7 depicts the sequence of the VarO 12 deletion (SEQ IDNO: 31), which is also the first deletion in Var042.
[0022] Fig. 8 depicts the second deletion in Var042 (SEQ ID NO: 32).
[0023] Fig. 9 depicts PCR screening of a Kay virus phage engineered to contain a Type I
CRISPR System IB from Listeria monocytogenes (LMIB).
[0024] Fig. 10 depicts PCR screening of the InqO promoter into phage.
[0025] Fig. 11 depicts the concentration of b2655 inoculated with engineered and wildtype Kayvirus phage over time. The engineered Kayvirus comprises three promotor variants used to drive expression of lacticin Q ( JnqQ ): a Peat -InqQ, PsarA -InqQ, or PtmpG- InqQ promoter.
[0026] Fig. 12 depicts an alignment of the genomes of parent Kayvirus phage with the engineered variant containing DNase I and the Pcat promoter.
[0027] Fig. 13 depicts a Venn diagram showing the different bacteria strains targeted by Phietavirus, Rosenblumvirus, andKayvirus, and combinations of the bacteriophage.
DETAILED DESCRIPTION
[0028] Disclosed herein, in certain embodiments, are bacteriophage engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene. As a non limiting example, the lysogenic gene encodes for a repressor. In some embodiments, the bacteriophage is further engineered to comprise one or more components of a CRISPR-Cas system, and/or an antimicrobial peptide. In some embodiments, the lysogenic gene encodes for a repressor. In some embodiments, removal of the lysogenic gene comprises removing from about 1% to 100% of the lysogenic gene, or about 10 to about 1,200 base pairs of the lysogenic gene are removed. In some embodiments, the lysogenic gene encodes for an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47 or 48.
[0029] Disclosed herein, in certain embodiments, are bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic. In one embodiment, the target bacterium is Staphylococcus spp. Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of the region annotated as “varOlO” (also referred to as a varOlO deletion) in Figure 6 (SEQ ID NO: 30). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of the region annotated as “var012” (also referred to as a var012 deletion) in Figure 7 (SEQ ID NO: 31). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of the region annotated as “var042” (also referred to as a var042 deletion) in Figure 8 (SEQ ID NO: 32). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of the region annotated as “var009” (also referred to as a var009 deletion) (SEQ ID NO: 30). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of at least a portion of a lysogenic gene, or a promoter of a lysogenic gene. In some embodiments, the portion is at least about 1% to 100% of the nucleotides of the lysogenic gene. In some embodiments, the portion is about 10 base pairs to all of the lysogenic gene. In some embodiments, the lysogenic gene encodes for an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47 or 48.
[0030] Disclosed herein, in certain embodiments, are bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic. In one embodiment, the target bacterium is Staphylococcus spp. Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “varOlO” (also referred to as a varO!O deletion) in Figure 6 (SEQ ID NO: 30). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “varO 12” (also referred to as a var012 deletion) in Figure 7 (SEQ ID NO: 31). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “var042” (also referred to as a var042 deletion) in Figure 8 (SEQ ID NO: 32). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “var009” (also referred to as a var009 deletion) (SEQ ID NO: 30). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of at least a portion of a lysogenic gene, or a promoter of a lysogenic gene. In some embodiments, the portion is at least about 1% to 100% of the nucleotides of the lysogenic gene. In some embodiments, the portion is about 10 base pairs to all of the lysogenic gene. In some embodiments, the lysogenic gene encodes for an amino acid sequence atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
47 or 48.
[0031] Disclosed herein, in certain embodiments, are bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic. In one embodiment, the target bacterium is Staphylococcus spp . Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “varOlO” (also referred to as a varO!O deletion) in Figure 6 (SEQ ID NO: 30). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “var012” (also referred to as a var012 deletion) in Figure 7 (SEQ ID NO: 31). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “var042” (also referred to as a var042 deletion) in Figure 8 (SEQ ID NO: 32). Further disclosed herein, in certain embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of the region annotated as “var009” (also referred to as a var009 deletion) (SEQ ID NO: 30). Further disclosed herein, in certain embodiments, temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, and a second nucleic acid sequence encoding a peptide (e.g., antimicrobial peptide), provided that the bacteriophage is rendered lytic by removal of at least a portion of a lysogenic gene, or a promoter of a lysogenic gene. In some embodiments, the portion is at least about 1% to 100% of the nucleotides of the lysogenic gene. In some embodiments, the portion is about 10 base pairs to all of the lysogenic gene. In some embodiments, the lysogenic gene encodes for an amino acid sequence atleast about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:
47 or 48.
[0032] Disclosed herein, in certain embodiments, are compositions comprises a plurality of bacteriophage. In some cases, the plurality comprises one or more engineered bacteriophage, e.g., engineered to remove a lysogenic gene, and/or to include a payload such as a CRISPR-Cas component and/or antimicrobial peptide. In some embodiments, the plurality of bacteriophage target a wide host range. For instance, the composition targets at least about 90% of a collection of at least about 30 Staphylococcus bacteria. In some embodiments, the plurality of bacteriophage comprises a first bacteriophage that is specific for a first receptor of a Staphylococcus bacteria, a second bacteriophage that is specific fora second receptor of the Staphylococcus bacteria, wherein the plurality of bacteriophage is more resilient to resistance by the Staphylococcus bacteria than the first or second bacteriophage alone. Bacteriophage
[0033] In some embodiments, the bacteriophage is an obligate lytic bacteriophage. In some embodiments, the bacteriophage is a temperate bacteriophage with retained lysogeny genes. In some embodiments, the bacteriophage is a temperate bacteriophage with some lysogeny genes removed, replaced, or inactivated. In some embodiments, the bacteriophage is a temperate bacteriophage with a lysogeny gene removed, replaced, or inactivated, thereby rendering the bacteriophage lytic. In some embodiments, the bacteriophage is rendered lytic by removal of at least a portion of a lysogenic gene, or a promoter of a lysogenic gene. In some embodiments, the portion is at least about 1% to 100% of the nucleotides of the lysogenic gene. In some embodiments, the portion is less than about 1%. In some embodiments, the portion removed is a single base. In some embodiments, the portion is about 10 base pairs to all of the lysogenic gene. For example, the portion removed is about 10-1200, 10-1100, 10-1000, 10-900, 10-800, 10-700, 10-600, 10-500, 10-400, 10-300, 10- 200, 10-100, 50-1200, 50-1100, 50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200, 50-100, 100-1200, 100-1100, 100-1000, 100-900, 100-800, 100-700, 100- 600, 100-500, 100-400, 100-300, or 100-200 base pairs. In some embodiments, the lysogenic gene encodes for an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47 or 48.
[0034] In some embodiments, the bacteriophage targets Staphylococcus spp. In some embodiments, the bacteriophage targets S. aureus. In some embodiments, the bacteriophage specifically targets Staphylococcus spp. over other bacterial species. In some embodiments, the bacteriophage targets Staphylococcus spp. in the absence of a CRISPR-Cas system.
[0035] In some embodiments, the bacteriophage targets Staphylococcus spp. In some embodiments, the bacteriophage is a Kay virus, a Twortvirus, a Rosenblumvirus, a Phietavirus a Triavirus, a Dubowvirus, a Beceayunavirus, a Peeveelvirus, a Coventry virus, or a Rockefellervirus. In some embodiments, the bacteriophage is a Kayvirus. In some embodiments, the bacteriophage is a Twortvirus. In some embodiments, the bacteriophage is a Rosenblumvirus. In some embodiments, the bacteriophage is a Phietavirus. In some embodiments, the virus is a Triavirus. In some embodiments, the virus is a Dubowvirus. In some embodiments, the virus is a Beceayunavirus. In some embodiments, the virus is a Peeveelvirus. In some embodiments, the virus is a Coventryvirus. In some embodiments, the virus is a Rockefellervirus. In some embodiments, the bacteriophage encodes a CRISPR-Cas system. In some embodiments, the bacteriophage encodes a peptide.
[0036] In some embodiments, the bacteriophage is a Phietavirus. A non-limiting example Phietavirus is pi 473. In some embodiments, the Phietavirus comprises a nucleic acid encoding a CRISPR-Cas system. In some embodiments, the Phietavirus comprises a nucleic acid encoding a peptide. In some embodiments, the Phietavirus is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene. As anon - limiting example, the lysogenic gene encodes for a repressor. In some embodiments, the bacteriophage is further engineered to comprise one or more components of a CRISPR-Cas system, and/or an antimicrobial peptide. In some embodiments, the lysogenic gene encodes for a repressor. In some embodiments, removal of the lysogenic gene comprises removing from about 1 % to 100% of the lysogenic gene, or about 10 to about 1 ,200 base pairs of the lysogenic gene are removed. In some embodiments, the lysogenic gene encodes for an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ IDNO: 47 or 48.
[0037] In some embodiments, the bacteriophage is a Rosenblumvirus. In some embodiments, the Rosenblumvirus comprises a nucleic acid encoding a CRISPR-Cas system. In some embodiments, the Rosenblumvirus comprises a nucleic acid encoding a peptide. [0038] In some embodiments, the bacteriophage is a Kayvirus. In some embodiments, the Kayvirus comprises a nucleic acid encoding a CRISPR-Cas system. In some embodiments, the Kayvirus comprises a nucleic acid encoding a peptide.
[0039] In some embodiments, the bacteriophage is p 1473, which targets Staphyloccocus ssp. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with pi 473. In some embodiments, the bacteriophage is a pi 473 bacteriophage comprising a CRISPR-Cas system.
[0040] In some embodiments, bacteriophages of interest are obtained from environmental sources or from 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.
[0041] In some embodiments, a nucleic acid sequence is inserted into a bacteriophage, e.g., a nucleic acid sequence encoding one or more components of a CRISPR-Cas system, and/or a peptide. In some embodiments, the insertion of the nucleic acid sequence into a bacteriophage preserves the lytic activity of the bacteriophage. In some embodiments, the nucleic acid sequence is inserted into the bacteriophage genome. In some embodiments, the nucleic acid sequence 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 sequence is inserted into the bacteriophage genome as a replacement for one or more removed non- essential genes. In some embodiments, the nucleic acid sequence is inserted into the bacteriophage genome as a replacement for one or more removed lysogenic genes. In some embodiments, the replacement of non-essential and/or lysogenic genes with the nucleic acid sequence 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 sequence preserves the lytic activity of the bacteriophage. In some embodiments, the replacement of non- essential and/or lysogenic genes with the nucleic acid sequence enhances the lytic activity of the bacteriophage. In some embodiments, the replacement of non-essential and/or lysogenic genes with the nucleic acid sequence renders a lysogenic bacteriophage lytic.
[0042] In some embodiments, the nucleic acid sequence 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. In some embodiments, the nucleic acid sequence is introduced into the bacteriophage 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 multiple separate locations. In some embodiments, the removal and/or inactivation of one or more non- essential and/or lysogenic genes does not affect the lytic activity of the bacteriophage. In some embodiments, the removal and/or inactivation of one or more non-essential and/or lysogenic genes preserves the lytic activity of the bacteriophage. In some embodiments, the removal of one or more non-essential and/or lysogenic genes renders a lysogenic bacteriophage into a lytic bacteriophage.
[0043] In some embodiments, 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. In some embodiments, the lytic activity of the bacteriophage is due to the removal, replacement, or inactivation of at least one lysogeny gene. In some embodiments, the lysogenic gene plays a role in the maintenance of lysogenic cycle in the bacteriophage. In some embodiments, the lysogenic gene plays a role in establishing the lysogenic cycle in the bacteriophage. In some embodiments, the lysogenic gene plays a role in both establishing the lysogenic cycle and in the maintenance of the lysogenic cycle in the bacteriophage.
[0044] In some embodiments, the lysogenic gene is a repressor gene. In some embodiments, the bacteriophage is rendered lytic by removal of the “varOlO” region annotated as “varOlO” deletion in Figure 6 (SEQ ID NO: 30). In some embodiments, the bacteriophage is rendered lytic by removal of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 or 1200 nucleotides of the varOlO region. In some embodiments, the bacteriophage is rendered lytic by removal of the “varO 12” region annotated as “varO 12” deletion in Figure 7 (SEQ ID NO: 31). In some embodiments, the bacteriophage is rendered lytic by removal of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides of the varO 12 region. In some embodiments, the bacteriophage is rendered lytic by removal of at least In some embodiments, the bacteriophage is rendered lytic by removal of the “var042” region annotated as “var042” deletion in Figure 8 (SEQ ID NO: 32). In some embodiments, the bacteriophage is rendered lytic by removal of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides of the var042 region. In some embodiments, the bacteriophage is rendered lytic by removal of the “var009” deletion (SEQ ID NO: 30). In some embodiments, the bacteriophage is rendered lytic by removal of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides of the var009 region. In some embodiments, the bacteriophage is rendered lytic by removal of atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides encoding the repressor having an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 47 or 48. In some embodiments, the bacteriophage is rendered lytic by the deletion of an antirepressor gene integrase, Pem-K like phage protein gene, or a combination thereof. In some embodiments, the bacteriophage is rendered lytic by the deletion of a gene depicted in Fig. 1. In some embodiments, the bacteriophage is rendered lytic by the deletion of an antirepressor gene, integrase, Pem-K like phage protein gene, or a combination thereof, wherein the gene is depicted in Fig. 1. In some embodiments, the bacteriophage is rendered lytic by deletion of atleast 10-2000, 20-2000, 30-2000, 40-2000, 50-2000, 60-2000, 70-2000, 80-2000, 90-2000, 100-2000, 200-2000, 300-2000, 400-2000, 500-2000, 600-2000, 700-2000, 80-2000, 900-2000, 1000-2000, 1100-2000, 1200-2000, 1300-2000, 1400-2000, 1500-2000, 1600-2000,1700-2000,1800-2000 or 1900-2000 basepairs of an antirepressor gene. In some embodiments, the bacteriophage is rendered lytic by deletion of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of an antirepressor gene. In some embodiments, the lysogenic gene is cl repressor gene. In some embodiments, the lysogenic gene is an activator gene. In some embodiments, the lysogenic gene is ell gene. In some embodiments, the lysogenic gene is int (integrase) gene. In some embodiments, 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. In some embodiments, a temperate bacteriophage is rendered lytic by the insertion of one or more lytic genes. 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 embodiments, a temperate bacteriophage is rendered lytic by altering the expression of one or more genes that contribute to the induction of a lytic cycle. In some embodiments, a temperate bacteriophage phenotypically changes from a lysogenic bacteriophage to a lytic bacteriophage. In some embodiments, a temperate bacteriophage is rendered lytic by environmental alterations. In some embodiments, 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). In some embodiments, 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 the self -targeting activity of the first introduced CRISPR array. In some embodiments, a temperate bacteriophage that is rendered lytic is prevented fromrevertingbackto lysogenic state by way of introducing an additional CRISPR array. In some embodiments, the bacteriophage does not confer any new properties onto the target bacterium beyond cellular death caused by lytic activity of the bacteriophage and/or the activity of the first or second CRISPR array.
[0045] In some embodiments, 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. In some embodiments, the insertion of one or more lytic genes is achieved by any suitable chemical, biochemical, and/or physical method by homologous recombination.
[0046] Further disclosed herein, in some embodiments, are temperate bacteriophages comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, provided that the bacteriophage is rendered lytic by removal of the region annotated as “varOlO” in Fig. 6, “var012” in Fig. 7, “var042” in Fig. 8, at least a portion of SEQ ID NO: 47 or 48, or combinations thereof. In some embodiments, the bacteriophage infects multiple bacterial strains. In some embodiments, 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 comprises at least a portion of an essential gene that is needed for survival of the target bacterium. In some embodiments, the essential gene is Tsf acpP, gapA, infA , secY, csrA, trmD,ftsA,fusA , glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fiis, adk, rpsK, rplR, ctrA,parC , tRNA-Ser, tRNA-Asn, or metK. In some embodiments, the target nucleotide sequence is in a non-essential gene or other genomic locus. In some embodiments, the target nucleotide sequence is in a non-essential gene. In some embodiments, the target nucleotide sequence is in a non-essential genomic locus. In some embodiments, the target nucleotide sequence is a noncoding sequence. In some embodiments, the noncoding sequence is an intergenic sequence. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a highly conserved sequence in a target bacterium. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a sequence present in the target bacterium. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence that comprises all or a part of a promoter sequence of the essential gene. In some embodiments, the first nucleic acid sequence comprises a first CRISPR array further comprising at least one repeat sequence. In some embodiments, the at least one repeat sequence is operably linked to the first spacer sequence at either its 5 ’ end or its 3 ’ end. In some embodiments, the target bacterium is S. aureus.
[0047] Also disclosed herein is a cocktail comprising two or more bacteriophage. In some embodiments, the two or more bacteriophage are selected from the lineage consisting of a Kay virus, a Twortvirus, a Rosenblumvirus, a Phietavirus or a Triavirus. In some embodiments, at least one bacteriophage of the cocktail comprises a CRISPR-Cas system. In some embodiments, at least two bacteriophages of the cocktail comprise a CRISPR-Cas system. In some embodiments, at least three bacteriophage of the cocktail comprise a CRISPR-Cas system. In some embodiments, at least four bacteriophage of the cocktail comprise a CRISPR-Cas system. In some embodiments, at least one bacteriophage of the cocktail does not comprise a CRISPR-Cas system. In some embodiments, at least two bacteriophages of the cocktail do not comprise a CRISPR-Cas system. In some embodiments, at least one bacteriophage of the cocktail comprises an antimicrobial peptide as described herein. In some embodiments, at least two bacteriophages of the cocktail comprise a nucleic acid encoding an antimicrobial peptide as described herein. In some embodiments, at least one bacteriophage comprises a nucleic acid encoding a CRISPR-Cas system and at least one bacteriophage comprises a nucleic acid encoding an antimicrobial peptide. In some embodiments, at least one bacteriophage of the cocktail comprises a nucleic acid encoding a CRISPR-Cas system. In some embodiments, the bacteriophage of the cocktail do not comprise a nucleic acid encoding a CRISPR-Cas system or an antimicrobial peptide.
[0048] In some embodiments, the cocktail comprises a Phietavirus. In some cases, the Phietavirus is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene. In some example cocktails, the cocktail comprises a Phietavirus and a Rosenblumvirus. In some example cocktails, the cocktail comprises a Phietavirus and a Kayvirus. In some example cocktails, the cocktail comprises a Phietavirus, Rosenblumvirus, and Kayvirus. In some cases, the Phietavirus comprises a nucleic acid encoding a CRISPR- Cas system. In some cases, the Phietavirus comprises a nucleic acid encoding an antimicrobial peptide. In some cases, the Phietavirus binds to a different bacteria receptor than another bacteriophage in the cocktail. In some cases, if the bacteria develops resistance and/or has a mutation that prevents infection with the another bacteriophage, the Phietavirus is capable of infecting the bacteria. In some such cases, the cocktail is more resilient against resistance formation by the bacteria than a single bacteriophage.
[0049] In some embodiments, the cocktail comprises a Rosenblumvirus. In some example cocktails, the cocktail comprises a Rosenblumvirus and a Phietavirus. In some cases, the Phietavirus is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene. In some example cocktails, the cocktail comprises a Rosenblumvirus and a Kayvirus. In some example cocktails, the cocktail comprises a Phietavirus, Rosenblumvirus, and Kayvirus. In some cases, the Rosenblumvirus comprises a nucleic acid encoding a CRISPR-Cas system. In some cases, the Rosenblumvirus comprises a nucleic acid encoding an antimicrobial peptide. In some cases, the Rosenblumvirus binds to a different bacteria receptor than another bacteriophage in the cocktail. In some cases, if the bacteria develops resistance and/or has a mutation that prevents infection with the another bacteriophage, the Rosenblumvirus is capable of infecting the bacteria. In some such cases, the cocktail is more resilient against resistance formation by the bacteria than a single bacteriophage. [0050] In some embodiments, the cocktail comprises a Kayvirus. In some example cocktails, the cocktail comprises a Kayvirus and a Rosenblum virus. In some example cocktails, the cocktail comprises a Kayvirus and a Phietavirus. In some cases, the Phietavirus is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene. In some example cocktails, the cocktail comprises a Phietavirus, Rosenblumvirus, and Kayvirus. In some cases, the Kayvirus comprises a nucleic acid encoding a CRISPR-Cas system. In some cases, the Kayvirus comprises a nucleic acid encoding an antimicrobial peptide. In some cases, the Kayvirus binds to a different bacteria receptor than another bacteriophage in the cocktail. In some cases, if the bacteria develops resistance and/or has a mutation that prevents infection with the another bacteriophage, the Kayvirus is capable of infecting the bacteria. In some such cases, the cocktail is more resilient against resistance formation by the bacteria than a single bacteriophage.
[0051] In some embodiments, a plurality of bacteriophages are used together. In some embodiments, the plurality of bacteriophages used together targets the same or different bacteria within a sample or subject. In some embodiments, a cocktail comprising a plurality of bacteriophages is used together. In some embodiments, the cocktail comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, ormorethan20 phages. In some embodiments, the cocktail comprises 2 phages. In some embodiments, the cocktail comprises 3 phages. In some embodiments, the cocktail comprises 4 phages. In some embodiments, the cocktail comprises 5 phages. In some embodiments, the cocktail comprises 6 phages. In some embodiments, at least one bacteriophage in the cocktail comprises a CRISPR array. In some embodiments, atleast2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, ormore than 20 bacteriophages present in the cocktail comprise a CRISPR array. In some embodiments, at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding a Cascade polypeptide. In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bacteriophages present in the cocktail comprise a nucleic acid sequence encoding a Cascade polypeptide. In some embodiments, at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding a Cas3 polypeptide. In some embodiments, atleast2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 bacteriophages present in the cocktail comprise a nucleic acid sequence encoding a Cas3 polypeptide. In some embodiments, at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding an antimicrobial peptide.
[0052] In some embodiments, the bacteriophage cocktail has a host range greater than that of an individual bacteriophage. The increased host range may allow for targeting a large number of strains of S. aureus. In some embodiments, the bacteriophage cocktail targets at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more than 99% of strains of S. aureus. In some embodiments, the strains of S. aureus include: b004604,b004605,b004606, b004607,b004608,b004609,b004610,b004611,b004612,b004613,b004614,b004615, b004616,b004617,b004618,b004619,b004620,b004621,b004622,b004623,b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631 , b004632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641 , b004642, b004643,b004644, b004645, b004646, b004647, b004648, b004649, b004650, b004651 , b004652, b004653,b004654, b004655, b004656, b004657, b004658, b004659, b004660, b004661, b004662, b004663, b004664, b004665, b004666, b004667, b004668, b004669, b004670, b004671 , b004672, b004673, b004674, b004675, b004676, b004677, b004678, b004679, b004680, b004681 , b004682, b004683, b004684, b004685, b004686, b004687, b004688, b004689, b004690, b004691 , b004692, b004693, b004694, b004695, b004696, b004697, b004698, b004699, b004700, b004701 , b004702, b004703, b004704, b004705, b004706, b004707, b004708, b004709, b004710, b004711 , b004712, b004713 , b004714, b004715,b004716,b004717,b004718,b004719,b004720,b004721,b004722,b004723, b004724, b004725,b004726, b004727, b004728, b004729, b004730, b004731 , b004732, b004733,b004734, b004735, b004736, b004737, b004738, b004739, b004740, b004741 , b004742, b004743,b004744, b004745, b004746, b004747, b004748, b004749, b004750, b004751 , b004752, b004753, b004754, b004755, b004756, b004757, b004758, b004759, b004760, b004761 , b004762, b004763, b004764, b004765, b004766, b004767, b004768, b004769, b004770, b004771 , b004772, b004773, b004774, b004775, b004776, b004777, b004778, b004779, b004780, b004781 , b004782, b004783, b004784, b004785, b004786, b004787, b004788, b004789, b004790, b004791 , b004792, b004793, b004794, b004795, b004796, b004797, b004798, b004799, b004800, b004801 , b004802, b004803, b004804, b004805,b004806, b004807, b004808, b004809, b004810, b004811 , b004812, b004813, b004814,b004815,b004816,b004817,b004818,b004819,b004820,b004821,b004822, b004823,b004824, b004825, b004826, b004827, b004828, b004829, b004830, b004831, b004832,b004833,b004834,b004835,b004836,b004837,b004838,b004839,b004840, b004841, b004842, b004843, b004844, b004845, b004846, b004847, b004848, b004849, b004850,b004851,b004852,b004853,b004854,b004855,b004856,b004857,b004858, b004859, b004860, b004861 , b004862, b004863, b004864, b004865, b004866, b004867, b004868, b004869, b004870, b004871 , b004872, b004873, b004874, b004875, b004876, b004877, b004878, b004879, b004880, b004881 , b004882, b004883, b004884, b004885, b004886, t>004887, b004888, b004889, b004890, b004891 , b004892, b004893, b004894, b004895,b004896, b004897, b004898, b004899, b004900, b004901 , b004902, b004903, b004904, b004905, b004906, b004907, b004908, b004909, b004910, and b004911. In some embodiments, the bacteriophage cocktail targets S. aureus specifically and does not target other species of bacteria. In some embodiments, the bacteriophage cocktail targets 5. aureus and does not target other Staphylococcus species. The increased host range may allow for targeting a large number of strains of Staphylococcus spp . In some embodiments, the bacteriophage cocktail targets at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more than 99% of strains of Staphylococcus spp. In some embodiments, the bacteriophage cocktail targets Staphylococcus spp. specifically and does not target other species of bacteria. In some embodiments, the bacteriophage cocktail targets Staphylococcus spp. and does not target non Staphylococcus spp. In some embodiments, the bacteriophage cocktail comprises two or more of: Phietavirus, Rosenblumvirus, and Kay virus.
[0053] In some embodiments, the bacteriophage in the cocktail are selected to minimize the ability of the target bacteria to evolve resistance. In some embodiments, the cocktail comprises a first bacteriophage that is specific for a first receptor of a Staphylococcus bacteria, and a second bacteriophage that is specific fora second receptor of the Staphylococcus bacteria, wherein the plurality of bacteriophage is more resilient to resistance by the Staphylococcus bacteriathan the first or second bacteriophage alone. For instance, if the Staphylococcus bacteria develops resistance to the first bacteriophage, the bacteria is still susceptible to infection by the second bacteriophage, and vice versa. In some embodiments, at least two bacteriophage from different genus target at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more than 99% of strains of S. aureus. In some embodiments, the cocktail comprises at least two bacteriophage, wherein the first bacteriophage binds a first receptor on Staphylococcus and the second bacteriophage binds a second receptor on Staphylococcus. In some embodiments, the bacteriophage cocktail comprises two or more of: Phietavirus, Rosenblumvirus, and Kay virus.
Staphylococcus
[0054] In some embodiments, the bacterium comprises one or more species of Staphylococcus . In some embodiments, the bacterium comprises one or more strains of Staphylococcus. In some embodiments, the target bacterium is Staphylococcus aureus. In some embodiments, the target bacterium is Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Staphylococcus salivarius, Staphylococcus argentis, Staphylococcus hemolyicus, Staphylococcus schweitzeri or any combination thereof.
[0055] In some embodiments, the target bacterium causes an infection or disease. In some embodiments, the infection or disease is acute or chronic. In some embodiments, the infection or disease is localized or systemic. In some embodiments, infection or disease is idiopathic.
In some embodiments, the infection or disease is acquired through means including, but not limited to, respiratory inhalation, ingestion, skin and wound infections, bone 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. In some embodiments, the target bacterium causes urinary tract infection. In some embodiments, the target bacterium causes and/or exacerbates an inflammatory disease. In some embodiments, the target bacterium causes and/or exacerbates an autoimmune disease. In some embodiments, the target bacterium causes and/or exacerbates eczema. In some embodiments, the target bacterium causes and/or exacerbates Atopic Dermatitis. In some embodiments, the target bacterium causes and/or exacerbates inflammatory bowel disease (IBD). In some embodiments, the target bacterium causes and/or exacerbates psoriasis. In some embodiments, the target bacterium causes and/or exacerbates psoriatic arthritis (PA). In some embodiments, the target bacterium causes and/or exacerbates rheumatoid arthritis (RA). In some embodiments, the target bacterium causes and/or exacerbates systemic lupus erythematosus (SLE). In some embodiments, the target bacterium causes and/or exacerbates multiple sclerosis (MS). In some embodiments, the target bacterium causes and/or exacerbates Graves’ disease. In some embodiments, the target bacterium causes and/or exacerbates Hashimoto’s thyroiditis. In some embodiments, the target bacterium causes and/or exacerbates Myasthenia gravis. In some embodiments, the target bacterium causes and/or exacerbates vasculitis. In some embodiments, the target bacterium causes and/or exacerbates cancer. In some embodiments, the target bacterium causes and/or exacerbates cancer progression. In some embodiments, the target bacterium causes and/or exacerbates cancer metastasis. In some embodiments, the target bacterium causes and/or exacerbates resistance to cancer therapy. In some embodiments, the therapy used to address cancer includes, but is not limited to, chemotherapy, immunotherapy, hormone therapy, targeted drug therapy, and/or radiation therapy. In some embodiments, the cancer develops in organs including, but not limited to the, anus, bladder, blood and blood components, bone, bone marrow, brain, breast, cervix uteri, colon and rectum, esophagus, kidney, larynx, lymphatic system, muscle (i.e., soft tissue), oral cavity and pharynx, ovary, pancreas, prostate, skin, small intestine, stomach, testis, thyroid, uterus, and/or vulva. In some embodiments, the target bacterium causes and/or exacerbates disorders of the central nervous system (CNS). In some embodiments, the target bacterium causes and/or exacerbates attention deficit/hyperactivity disorder (ADHD). In some embodiments, the target bacterium causes and/or exacerbates autism. In some embodiments, the target bacterium causes and/or exacerbates bipolar disorder. In some embodiments, the target bacterium causes and/or exacerbates major depressive disorder. In some embodiments, the target bacterium causes and/or exacerbates epilepsy. In some embodiments, the target bacterium causes and/or exacerbates neurodegenerative disorders including, but not limited to, Alzheimer’ s disease, Huntington’s disease, and/or Parkinson’s disease.
[0056] In some embodiments, the target bacteria comprises a Staphylococcus selected from one or more of: b004604, b004605, b004606, b004607, b004608, b004609, b004610, b004611,b004612,b004613,b004614,b004615,b004616,b004617,b004618,b004619, b004620, b004621 , b004622, b004623, b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631 , b004632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641 , b004642, b004643, b004644, b004645, b004646, b004647, b004648, b004649, b004650, b004651 , b004652, b004653, b004654, b004655, b004656, b004657, b004658, b004659, b004660, b004661 , b004662, b004663, b004664, b004665, b004666, b004667, b004668, b004669, b004670, b004671 , b004672, b004673, b004674, b004675, b004676, b004677, b004678, b004679, b004680, b004681 , b004682, b004683,b004684, b004685, b004686, b004687, b004688, b004689, b004690, b004691 , b004692, b004693,b004694, b004695, b004696, b004697, b004698, b004699, b004700, b004701, b004702, b004703, b004704, b004705, b004706, b004707, b004708, b004709, b004710,b004711,b004712,b004713,b004714,b004715,b004716,b004717,b004718, b004719, b004720, b004721 , b004722, b004723, b004724, b004725, b004726, b004727, b004728, b004729, b004730, b004731 , b004732, b004733, b004734, b004735, b004736, b004737, b004738, b004739, b004740, b004741 , b004742, b004743, b004744, b004745, b004746, b004747, b004748, b004749, b004750, b004751 , b004752, b004753, b004754, b004755,b004756, b004757, b004758, b004759, b004760, b004761 , b004762, b004763, b004764, b004765,b004766, b004767, b004768, b004769, b004770, b004771 , b004772, b004773,b004774, b004775, b004776, b004777, b004778, b004779, b004780, b004781 , b004782, b004783,b004784, b004785, b004786, b004787, b004788, b004789, b004790, b004791, b004792, b004793, b004794, b004795, b004796, b004797, b004798, b004799, b004800, b004801 , b004802, b004803, b004804, b004805, b004806, b004807, b004808, b004809, b004810, b004811 , b004812, b004813, b004814, b004815, b004816, b004817, b004818, b004819, b004820, b004821 , b004822, b004823, b004824, b004825, b004826, b004827, b004828, b004829, b004830, b004831 , b004832, b004833, b004834, b004835, b004836,b004837,b004838,b004839,b004840,b004841,b004842,b004843,b004844, b004845,b004846, b004847, b004848, b004849, b004850, b004851 , b004852, b004853, b004854,b004855,b004856,b004857,b004858,b004859,b004860,b004861,b004862, b004863,b004864, b004865, b004866, b004867, b004868, b004869, b004870, b004871 , b004872, b004873, b004874, b004875, b004876, b004877, b004878, b004879, b004880, b004881,b004882,b004883,b004884,b004885,b004886,b004887,b004888,b004889, b004890, b004891 , b004892, b004893, b004894, b004895, b004896, b004897, b004898, b004899, b004900, b004901 , b004902, b004903, b004904, b004905, b004906, b004907, b004908, b004909, b004910, and b004911.
CRISPR/CAS Systems
[0057] 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. There is a diversity of CRISPR-Cas systems based on the set of cas genes and their phylogenetic relationship. There are at least six different types (I through VI) where Type I represents over 50% of all identified systems in both bacteria and archaea. In some embodiments, a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR-Cas system is used herein.
[0058] 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 CRISPR array encoding 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. In the Type I-E system, 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.
[0059] In the Type I-C system, the proteins Cas5, Cas8c, and Cas7 form the Cascade effector complex. Cas5 processes the pre-crRNA (which can take the form of a multi-spacer array, ora single spacer between two repeats) to produce individual crRNA(s)madeup of a hairpin structure formed from the remaining repeat sequence and a linear spacer. The effector complex then binds to the processed crRNA and scans DNA to identify PAM sites. In the Type I-C system, the PAM is recognized by the Cas8c protein, which then acts to unwind the DNA duplex. If the sequence 3 ’ of the PAM matches the crRNA spacer that is bound to effector complex, a conformational change in the complex occurs and Cas3 is recruited to the site. Cas3 then nicks the non-target strand and begins degrading the DNA.
[0060] In the Type I-B system, the proteins Cas8bl, Cas7, and Cas5 form the Cascade effector complex. Cas5 processes the pre-crRNA (which can take the form of a multi-spacer array, ora single spacer between two repeats) to produce individual crRNA(s)madeup of a hairpin structure formed from the remaining repeat sequence and a linear spacer. The effector complex then binds to the processed crRNA and scans DNA to identify PAM sites. In the Type I-B system, the PAM is recognized by the Cas8bl protein, which then acts to unwind the DNA duplex. If the sequence 3 ’ of the PAM matches the crRNA spacer that is bound to effector complex, a conformational change in the complex occurs and Cas3 is recruited to the site. Cas3 then nicks the non-target strand and begins degrading the DNA. In some embodiments, the Type I-B system is from Listeria monocytogenes (LMIB) (SEQ ID NO: 22). In some embodiments, the Type I-B system comprises a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOS: 25-29.
[0061] In some embodiments, the CRISPR-Cas system is endogenous to the target bacterium. In some embodiments, when a CRISPR-Cas system is endogenous to the target bacterium, the target bacterium comprises at least one gene encoding a Cas polypeptide. In some embodiments, when a CRISPR-Cas system is endogenous to the target bacterium, the target bacterium comprises a nucleic acid encoding a Cas3 polypeptide. In some embodiments, when a CRISPR-Cas system is endogenous to the target bacterium, the target bacterium comprises a nucleic acid encoding a CASCADE complex. In some embodiments, when a CRISPR-Cas system is endogenous to the target bacterium, the target bacterium comprises a nucleic acid sequence encoding a Cas3 polypeptide and a CASCADE complex. [0062] In some embodiments, the CRISPR-Cas system is exogenous to the target bacterium. In some embodiments, when a CRISPR-Cas system is exogenousto the target bacterium, the bacteriophage comprises at least one gene encoding a Cas polypeptide. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the bacteriophage comprises a nucleic acid encoding a Cas3 polypeptide. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the bacteriophage comprises a nucleic acid encoding a CASCADE complex. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the bacteriophage comprises a nucleic acid sequence encoding a Cas3 polypeptide and a CASCADE complex. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not comprise a nucleic acid encoding a Cas3 polypeptide. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not express a Cas3 polypeptide. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not comprise a nucleic acid encoding a CASCADE complex. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not express a CASCADE complex. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not comprise a nucleic acid sequence encoding a Cas3 polypeptide and a CASCADE complex. In some embodiments, when a CRISPR-Cas system is exogenous to the target bacterium, the target bacterium does not express a Cas3 polypeptide and a CASCADE complex.
[0063] In some embodiments, the CRISPR-Cas system is a Type I CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type I-A CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type I-B CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type I-B CRISPR-Cas system derived from Listeria monocytogenes. In some embodiments, the CRISPR-Cas system is a Type I-C CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type I-D CRISPR- Cas system. In some embodiments, the CRISPR-Cas system is a Type I-E CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a Type I-F CRISPR-Cas system.
In some embodiments, the CRISPR-Cas system is a Type I-U CRISPR-Cas system. 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.
[0064] In some embodiments, processing of a CRISPR-array disclosed herein includes, but is not limited to, the following processes: 1) transcription of the nucleic acid encoding a pre-crRNA; 2) recognition of the pre-crRNAby Cascade and/ or specific members of Cascade, such as Cas6, and (3) processing of the pre-crRNA by Cascade or members of Cascade, such as Cas6, into mature crRNAs. In some embodiments, the mode of action for a Type I CRISPR system includes, but is not limited to, the following processes: 4) mature crRNA complexation with Cascade; 5) target recognition by the complexed mature crRNA/Cascade complex; and 6) nuclease activity at the target leading to DNA degradation. [0065] In some embodiments, the Type I CRISPR-Cas system is a Type I-A system,
Type I-B system, Type I-C system, Type I-D system, Type I-E system, Type I-F system, or Type I-U system. In some embodiments, the Type I CRISPR-Cas system is a Type I-A system. In some embodiments, the Type I CRISPR-Cas system is a Type I-B system. In some embodiments, the Type I CRISPR-Cas system is a Type I-C system. In some embodiments, the Type I CRISPR-Cas system is a Type I-D system. In some embodiments, the Type I CRISPR-Cas system is a Type I-E system. In some embodiments, the Type I CRISPR-Cas system is a Type I-F system. In some embodiments, the Type I CRISPR-Cas system is a Type I-U system. In some embodiments, the Type I CRISPR-Cas system comprises Cascade polypeptides. Type I Cascade polypeptides process CRISPR arrays to produce a processed RNA that is then used to bind the complex to a target sequence that is complementary to the spacer in the processed RNA. In some embodiments, 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.
[0066] In some embodiments, the Type I Cascade complex comprises: (a) a nucleotide sequence encoding a Cas6b polypeptide, a nucleotide sequence encoding a Cas8b (Csh 1) polypeptide, a nucleotide sequence encoding a Cas7 (Csh2) polypeptide, and a nucleotide sequence encoding a Cas5 polypeptide (Type I-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 (CasB) polypeptide, a nucleotide sequence encoding a Cas7 (CasC) polypeptide, a nucleotide sequence encoding a Cas5 (CasD) polypeptide, and a nucleotide sequence encoding a Cas6e (CasE) polypeptide (Type I-E); (d) a nucleotide sequence encoding a Cysl polypeptide, a nucleotide sequence encoding a Cys2 polypeptide, a nucleotide sequence encoding a Cas7 (Cys3) polypeptide, and a nucleotide sequence encoding a Cas6f polypeptide (Type I-F); (e) a nucleotide sequence encoding a Cas7 (Csa2) polypeptide, a nucleotide sequence encoding a Cas8al (Csxl3) polypeptide or a Cas8a2 (Csx9) polypeptide, a nucleotide sequence encoding a Cas5 polypeptide, a nucleotide sequence encoding a Csa5 polypeptide, a nucleotide sequence encoding a Cas6a polypeptide, a nucleotide sequence encoding a Cas3' polypeptide, and a nucleotide sequence encoding a Cas3 " polypeptide having no nuclease activity (Type I- A); (f) a nucleotide sequence encoding a Casl Od (Csc3) polypeptide, a nucleotide sequence encoding a Csc2 polypeptide, a nucleotide sequence encoding a Cscl polypeptide, and a nucleotide sequence encoding a Cas6d polypeptide (Type I-D); and/or (g) Cas8u2 polypeptide, a Cas7 polypeptide, and a fused Cas5 -Cas6 polypeptide (Type I-U);. In some embodiments, the Type I Cascade complex comprises a Cascade polypeptide disclosed herein [0067] In some embodiments, the Type I CRISPR-Cas system comprises Cascade polypeptides. Type I Cascade polypeptides process CRISPR arrays to produce a processed RNA that is then used to bind the complex to a target sequence that is complementary to the spacer in the processed RNA. In some embodiments, 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. In some embodiments, the CRISPR-Cas system is a Type I-B CRISPR-Cas system from Listeria monocytogenes (LMIB).
[0068] In some embodiments, the CRISPR-Cas system is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 22. In some instances, the CRISPR- Cas system is encoded by a sequence comprising at least a portion having at least or about 3,
4, 5, 6, 7, 8, 9, 10, 12, 14, 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 or more than 50 nucleotides of SEQ ID NO. 22. In some instances, the CRISPR-Cas system is encodedby a sequence comprising at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 105, 110, 115 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or more than 215 nucleotides of SEQ ID NO. 22. In some embodiments, the CRISPR-Cas system is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23. In some instances, the CRISPR-Cas system is encoded by a sequence comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12,
14, 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 or more than 50 nucleotides of SEQ ID NO. 23. In some instances, the CRISPR-Cas system is encoded by a sequence comprising at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200,205,210,
215, or more than 215 nucleotides of SEQ ID NO. 23.
[0069] In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 25 (e.g., Cas6). In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 29 (e.g., Cas8). In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 26 (e.g., Cas7). In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 27 (e.g., Cas5). In some embodiments, the CRISPR-Cas system comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 28 (e.g., Cas3). In some instances, the CRISPR-Cas system comprises at least or about 95% homology to any one of SEQ ID NOS: 25 -29. In some instances, the CRISPR-Cas system comprises at least or about 97% homology to any one of SEQ ID NOS: 25-29. In some instances, the CRISPR-Cas system comprises at least or about 99% homology to any one of SEQ ID NOS: 25-29. In some instances, the CRISPR-Cas system comprises 100% homology to anyone of SEQ ID NOS: 25-29. In some instances, the CRISPR-Cas system comprises at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 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 ormorethan 50 amino acidsof anyone of SEQ ID NOS: 25-29. In some instances, the CRISPR-Cas system comprises at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or more than 215 amino acidsof any one of SEQ ID NOS: 25-29.
[0070] In some embodiments, the CRISPR-Cas system comprises a Cas6 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 33. In some embodiments, the CRISPR-Cas system comprises a Cas8 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34. In some embodiments, the CRISPR-Cas system comprises a Cas7 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 35. In some embodiments, the CRISPR-Cas system comprises a Cas5 polypeptide encodedby a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 36. In some embodiments, the CRISPR-Cas system comprises a Cas3 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 37.
[0071] In some embodiments, the CRISPR-Cas system comprises a Cas6 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38. In some embodiments, the CRISPR-Cas system comprises a Cas8 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 39. In some embodiments, the CRISPR-Cas system comprises a Cas7 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 40. In some embodiments, the CRISPR-Cas system comprises a Cas5 polypeptide encodedby a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 41. In some embodiments, the CRISPR-Cas system comprises a Cas3 polypeptide encoded by a sequence with at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ IDNO: 42.
CRISPR Array
[0072] In some embodiments, the CRISPR array (crArray) disclosed herein comprises a spacer sequence and at least one repeat sequence. In some embodiments, the CRISPR array encodes a processed, mature crRNA. In some embodiments, the mature crRNA is introduced into a phage or a target bacterium described herein. In some embodiments, the phage comprises a nucleic acid that encodes a processed, mature crRNA. In some embodiments, an endogenous or exogenous Cas6 processes the CRISPR array into mature crRNA. In some embodiments, an exogenous Cas6 is introduced into the phage. In some embodiments, the phage comprises an exogenous Cas6. In some embodiments, an exogenous Cas6 is introduced into a target bacterium.
[0073] In some embodiments, processing of a CRISPR-array disclosed herein includes, but is not limited to, the following processes: 1) transcription of the nucleic acid encoding a pre-crRNA; 2) recognition of the pre-crRNAby Cascade and/ or specific members of Cascade, such as Cas6, and (3) processing of the pre-crRNA by Cascade or members of Cascade, such as Cas6, into mature crRNAs. In some embodiments, the mode of action for a Type I CRISPR system includes, but is not limited to, the following processes: 4) mature crRNA complexation with Cascade; 5) target recognition by the complexed mature crRNA/Cascade complex; and 6) nuclease activity at the target leading to DNA degradation. [0074] In some embodiments, the CRISPR array comprises a spacer sequence. In some embodiments, the CRISPR array further comprises at least one repeat sequence. In some embodiments, the at least one repeat sequence is operably linked to the spacer sequence at either its 5 ’ end or its 3 ’ end. In some embodiments, 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 a target bacterium by targeting one or more target sequences. In some embodiments, the CRISPR array comprises, consists essentially of, or consists of 1 to about 100 spacer nucleotide sequences, each linked on its 5' end and its 3' end to a repeat nucleotide sequence. In some embodiments, the CRISPR array as disclosed herein, comprises essentially of, or consists of 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, 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, ormore, spacer nucleotide sequences. In some embodiments, the CRISPR array comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 24. In some embodiments, the repeat sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 43. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 44-46. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 44. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 45. In some embodiments, the spacer sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 46.
Spacer Sequence
[0075] In some embodiments, the spacer sequence is complementary to a target nucleotide sequence in a target bacterium. In some embodiments, the target nucleotide sequence is a coding region. In some embodiments, the coding region is an essential gene. In some embodiments, the coding region is a nonessential gene. In some embodiments, the target nucleotide sequence is a noncoding sequence. In some embodiments, the noncoding sequence is an intergenic sequence. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a highly conserved sequence in a target bacterium. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a sequence present in the target bacterium. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence that comprises all or a part of a promoter sequence of the essential gene. In some embodiments, the spacer sequence comprises one, two, three, four, or five mismatches as compared to the target nucleotide sequence. In some embodiments, the mismatches are contiguous. In some embodiments, the mismatches are noncontiguous. In some embodiments, the spacer sequence has 70% complementarity to a target nucleotide sequence. In some embodiments, the spacer sequence has 80% complementarity to a target nucleotide sequence. In some embodiments, the spacer sequence is 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementarity to a target nucleotide sequence. In some embodiments, the spacer sequence has 100% complementarity to the target nucleotide sequence. In some embodiments, the 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 has 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 the spacer sequence is 100% complementary to a target nucleotide sequence while the 3 ' region of the spacer is substantially complementary to the target nucleotide sequence and therefore the overall complementarity of the spacer sequence to the target nucleotide sequence is less than 100%. For example, in some embodiments, the first 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides in the 3' region of a 20 nucleotide spacer sequence (seed region) is 100% complementary to the target nucleotide sequence, 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 nucleotide sequence. In some embodiments, the first 7 to 12 nucleotides of the 3' end of the spacer sequence is 100% complementary to the target nucleotide sequence, 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to the target nucleotide sequence. In some embodiments, the first 7 to 10 nucleotides in the 3' end of the spacer sequence is 75%-99% complementary to the target nucleotide sequence, while the remaining nucleotides in the 5' region of the spacer sequence are at least about 50% to about 99% complementary to the target nucleotide sequence. In some embodiments, the first 7 to 10 nucleotides in the 3 ' end of the spacer sequence is 100% complementary to the target nucleotide sequence, 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 nucleotide sequence. In some embodiments, the first 10 nucleotides (within the seed region) of the spacer sequence is 100% complementary to the target nucleotide sequence, 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 nucleotide sequence. In some embodiment, 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) have about 75% complementarity or more (75% to about 100% complementarity) to the target nucleotide sequence, while the remainder of the spacer sequence have about 50% or more complementarity to the target nucleotide sequence. In some embodiments, the first 8 nucleotides at the 5' end of the spacer sequence have 100% complementarity to the target nucleotide sequence or have one or two mutations and therefore is about 88% complementary or about 75% complementary to the target nucleotide sequence, respectively, while the remainder of the spacer nucleotide sequence is at least about 50% or more complementary to the target nucleotide sequence.
[0076] In some embodiments, the spacer sequence is about 15 nucleotides to about 150 nucleotides in length. In some embodiments, the 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, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 nucleotides or more). In some embodiments, the 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, at least about 10, at least about 15, at least about 20, at least about 25, at least about 30, at least about 32, at least about35, at least about 40, at least about 44, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, atleast about 130, atleast about 140, atleast about 150 nucleotides in length, or more, and any value or range therein. In some embodiments, the Listeria monocytogenes Type I-B Cas system has a spacer length of about30to 39 nucleotides, about31 to about38 nucleotides, about 32 to about 37 nucleotides, about 36 to about 37 nucleotides, or about 37 nucleotides. In some embodiments, the Listeria monocytogenes Type I-B system has a spacer length of about 37 nucleotides. In some embodiments, the Listeria monocytogenes Type I-B Cas system has a spacer length of at least about 10, at least about 15, atleast about 20, atleast about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 29, at least about 29, at least about 30, at least about 31, at least about32, at least about 33, at least about 34, at least about, at least about 35, atleast about 36, at least about 37, at least about 38, at least about 39, at least about 20, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, or more than about 45 nucleotides.
[0077] In some embodiments, the identity of two or more spacer sequences of the CRISPR array is the same. In some embodiments, the identity of two or more spacer sequences of the CRISPR array is different. In some embodiments, the identity of two or more spacer sequences of the CRISPR array is different but are complementary to one or more target nucleotide sequences. In some embodiments, the identity of two or more spacer sequences of the 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 sequences of the CRISPR array is different and are complementary to one or more target nucleotide sequences that are not overlapping sequences. In some embodiments, the target nucleotide sequence 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. In some embodiments, a target nucleotide sequence is located adjacent to or flanked by a PAM (protospacer adjacent motif).
[0078] The PAM sequence 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 exact PAM sequence that is required varies between each different CRISPR-Cas system and is identified through established bioinformatics and experimental procedures. Non-limiting examples of PAMs include CCA, CCT, CCG, TTC, AAG, AGG, ATG, GAG, and/or CC. For Type I systems, 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, and is directly recognized by Cascade. Once a protospacer is recognized, Cascade generally recruits the endonuclease Cas3, which cleaves and degrades the target DNA. For Type II systems, the PAM is required for a Cas9/sgRNAto 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)
[0079] In some embodiments, the target nucleotide sequence in the bacterium to be killed is any essential target nucleotide sequence of interest. In some embodiments, the target nucleotide sequence is a non-essential sequence. In some embodiments, 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 the essential gene. In some embodiments, the spacer nucleotide sequence is complementary to a promoter, or a part thereof, of the essential gene. In some embodiments, the target nucleotide sequence comprises all or a part of a nucleotide sequence located on a coding or a non -coding strand of the essential gene. 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 the essential gene. [0080] In some embodiments, the 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 embodiments, 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 comprises at least a portion of an essential gene that is needed for survival of the target bacterium. In some embodiments, the essential gene is Tsf acpP , gap A, infA , sec F, csrA, trmD,ftsA,fiisA , glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fus, adk, rpsK, rplR, ctrA,parC , tRNA-Ser, tRNA-Asn, or metK. In some embodiments, 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.
[0081] In some embodiments, non-limiting examples of the target nucleotide sequence of interest includes a target nucleotide sequence 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. In some embodiments, the target nucleotide sequence is from a gene involved in cell-division, cell structure, metabolism, motility, pathogenicity, virulence, or antibiotic resistance. In some embodiments, the target nucleotide sequence is from a hypothetical gene whose function is not yet characterized. Thus, for example, these genes are any genes from any bacterium.
[0082] The appropriate spacer sequences for a full-construct phage maybe identified by locating a search set of representative genomes, searching the genomes with relevant parameters, and determining the quality of a spacer for use in a CRISPR engineered phage. [0083] First, a suitable search set of representative genomes is located and acquired for the organism/species/target of interest. The set of representative genomes may be found in a variety of databases, including without limitations the NCBI GenBank or the PATRIC database. NCBI GenBank is one of the largest databases available and contains a mixture of reference and submitted genomes for nearly every organism sequenced to date. Specifically, for pathogenic prokaryotes, the PATRIC (Pathosystems Resource Integration Center) database provides an additional comprehensive resource of genomes and provides a focus on clinically relevant strains and genomes relevant to a drug product. Both of the above databases allow for bulk downloading of genomes via FTP (File Transfer Protocol) servers, enabling rapid and programmatic dataset acquisition
[0084] Next, the genomes are searched with relevant parameters to locate suitable spacer sequences. Genomes may be read from start to end, in both the forward and reverse complement orientations, to locate contiguous stretches of DNA that contain a PAM (Proto spacer Adjacent Motif) site. The spacer sequence willbetheN-lengthDNA sequence 3' or 5’ adjacentto the PAM site (depending on the CRISPR system type), where N is specific to the Cas system of interest and is generally known ahead of time. Characterizing the PAM sequence and spacer sequences may be performed during the discovery and initial research of a Cas system. Every observed P AM-adjacent spacer may be saved to a file and/or database for downstream use. The exact PAM sequence that is required varies between each different CRISPR-Cas system and is identified through established bioinformatics and experimental procedures.
[0085] Next, the quality of a spacer for use in a CRISPR engineered phage is determined.
Each observed spacer may be evaluated to determine how many of the evaluated genomes they are present in. The observed spacers may be evaluated to see how many times they may occur in each given genome. Spacers that occur in more than one location per genome may be advantageous because the Cas system may not be able to recognize the target site if a mutation occurs, and each additional "backup" site increases the likelihood that a suitable, non-mutated target location will be present. The observed spacers may be evaluated to determine whether they occur in functionally annotated regions of the genome. If such information is available, the functional annotations maybe further evaluated to determine whether those regions of the genome are "essential" for the survival and function of the organism. By focusing on spacers that occur in all, or nearly all, evaluated genomes of interest (>= 99%), the spacer selection may be broadly applicable to many targeted genomes. Provided a large selection pool of conserved spacers exists, preference may be given to spacers that occur in regions of the genome that have known function, with higher preference given if those genomic regions are "essential" for survival and occur more than 1 time per genome.
[0086] In some embodiments, the spacer comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 44-46. Repeat Nucleotide Sequences
[0087] In some embodiments, a repeat nucleotide sequence of the CRISPR array comprises a nucleotide sequence of any known repeat nucleotide sequence of a CRISPR-Cas system. In some embodiment, the CRISPR-Cas system is a Type I CRISPR-Cas system. In some embodiment, 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). In some embodiments, the repeat nucleotide sequences are distinct from one another based on the known repeat nucleotide sequences of a CRISPR-Cas system. In some embodiments, the repeat nucleotide sequences are each composed of distinct secondary structures of a native repeat from a CRISPR-Cas system (e.g., an internal hairpin). In some embodiments, the repeat nucleotide sequences are a combination of distinct repeat nucleotide sequences operable with a CRISPR-Cas system.
[0088] In some embodiments, the spacer sequence is linked at its 5' end to the 3’ end of a repeat sequence. In some embodiments, the spacer 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 sequence. In some embodiments, the about 1 to about 8, about 1 to about 10, about 1 to about 15 nucleotides of the repeat sequence are a portion of the 3 ’ end of a repeat sequence. In some embodiments, the spacer nucleotide sequence is linked at its 3' end to the 5’ end of a repeat 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 sequence. In some embodiments, the about 1 to about 8, about 1 to about 10, about 1 to about 15 nucleotides of the repeat sequence are a portion of the 5 ’ end of a repeat sequence.
[0089] In some embodiments, the spacer nucleotide sequence is linked at its 5' end to a first repeat sequence and linked at its 3' end to a second repeat sequence to forma repeat- spacer-repeat sequence. In some embodiments, the spacer sequence is linked at its 5' end to the 3 ’ end of a first repeat sequence and is linked at its 3 ' end to the 5 ’ of a second repeat sequence where the spacer sequence and the second repeat sequence are repeated to form a repeat-(spacer-repeat)n sequence such that n is any integer from 1 to 100. In some embodiments, a repeat-(spacer-repeat)n sequence comprises, consists essentially of, or consists of 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, 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.
[0090] In some embodiments, the repeat sequence is identical to or substantially identical to a repeat sequence from a wild-type CRISPR loci. In some embodiments, the repeat sequence is a repeat sequence found in Table 3. In some embodiments, the repeat sequence is a sequence described herein. In some embodiments, the repeat sequence comprises a portion of a wild type repeat sequence (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous nucleotides of a wild type repeat sequence). In some embodiments, the 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, 100, or more nucleotides, or any range therein). In some embodiments, the repeat sequence comprises, consists essentially of, or consists of no more than 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, 26, 27,2 - 8, 29, 30, 31, 3 · 2, 33, 34, 35, 36, 37, 38
39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides. In some embodiments, the repeat sequence comprises about 20 to 40, 21 to 40, 22 to 4023 to 40, 24 to 40, 25 to 40,
26 to 40, 27 to 40, 28 to 40, 29 to 40, 30 to 30, 31 to 40, 32 to 40, 33 to 40, 34 to 40, 35 to 40, 36 to 40, 37 to 40, 38 to 40, 39 to 40, 20 to 39, 20 to 38, 20 to 37, 20 to 36, 20 to 35, 20 to 34, 20 to 33, 20 to 32, 20 to 31, 20 to 30, 20 to 29, 20 to 28, 20 to 26, 20 to 25, 20to 24, 20 to 23,
20 to 22, or 20 to 21 nucleotides. In some embodiments, the repeat sequence comprises about 20 to 35, 21 to 35, 22 to 35 23 to 35, 24 to 35, 25 to 35, 26 to 35, 27 to 35, 28 to 35, 29to 35, 30 to 30, 31 to 35, 32 to 35, 33 to 35, 34 to 35, 25 to 40, 25 to 39, 25 to 38, 25 to 37, 25 to 36, 25 to 35, 25 to 34, 25 to 33, 25 to 32, 25 to 31, 25 to 30, 25 to 29, 25 to 28, 25 to 26 nucleotides. In some embodiments, the system is a L monocytogenes Typel-B Cas system. In some embodiments, the L. monocytogenes Type I-B Cas system has a repeat length of about 25 to 38 nucleotides.
[0091] In some embodiments, the repeat comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 43.
Transcriptional Activators
[0092] In some embodiments, the nucleic acid sequence further comprises a transcriptional activator. In some embodiments, the transcriptional activator encoded regulates the expression of genes of interest within the Staphylococcus species. In some embodiments, the transcriptional activator activates the expression of genes of interest within the Staphylococcus species whether exogenous or endogenous. In some embodiments, the transcriptional activator activates the expression genes of interest within the Staphylococcus species by disrupting the activity of one or more inhibitory elements within the Staphylococcus species. In some embodiments, the inhibitory element comprises a transcriptional repressor. In some embodiments, the inhibitory element comprises a global transcriptional repressor. In some embodiments the inhibitory element is a histone -like nucleoid-structuring (H-NS) protein orhomologue or functional fragment thereof. In some embodiments, the inhibitory dementis a leucine responsive regulatory protein (LRP). In some embodiments, the inhibitory dementis a CodY protein.
[0093] In some bacteria, the CRISPR-Cas system is poorly expressed and considered silent under most environmental conditions. In these bacteria, 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.
[0094] Similarly, in some bacteria, the repression of the CRISPR-Cas system is controlled by an inhibitory element, for example the leucine responsive regulatory protein (LRP). LRP has been implicated in binding to upstream and downstream regions of the transcriptional start sites. Notably, the 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.
[0095] In some instances, the repression of CRISPR-Cas system is also controlled by inhibitory element CodY. 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. [0096] In some embodiments, the transcriptional activator is a LeuO polypeptide, any homolog or functional fragment thereof, a leuO coding sequence, or an agent that upregulates LeuO. In some embodiments, the transcriptional activator comprises any ortholog or functional equivalent of LeuO. In some bacteria, 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. Overexpression of LeuO upregulates the expression of the CRISPR-Cas system.
[0097] In some embodiments, the expression of LeuO leads to disruption of an inhibitory element. In some embodiments, the disruption of an inhibitory element due to expression of LeuO removes the transcriptional repression of a CRISPR-Cas system. In some embodiments, the expression of LeuO removes transcriptional repression of a CRISPR-Cas system due to activity of H-NS. In some embodiments, the disruption of an inhibitory element due to the expression of LeuO causes an increase in the expression of a CRISPR-Cas system. In some embodiments, 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 sequence comprising 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 sequence comprising a CRISPR array so as to increase the level of lethality of the CRISPR array against a bacterium. In some embodiments, transcriptional activator causes increase activity of a bacteriophage and/or the CRISPR-Cas system.
Regulatory Elements
[0098] In some embodiments, the nucleic acid sequences are operatively associated with a variety of promoters, terminators and other regulatory elements for expression in various organisms or cells. In some embodiments, the nucleic acid sequence further comprises a leader sequence. In some embodiments, the nucleic acid sequence further comprises a promoter sequence. In some embodiments, at least one promoter and/or terminator is operably linked the CRISPR array. 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 disclosed herein. A regulatory element as used herein is endogenous or heterologous. In some embodiments, 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.
[0099] In some embodiments, expression of the nucleic acid sequence is constitutive, inducible, temporally regulated, developmentally regulated, or chemically regulated. In some embodiments, the expression of the nucleic acid sequence is made constitutive, inducible, temporally regulated, developmentally regulated, or chemically regulated by operatively linking the nucleic acid sequence to a promoter functional in an organism of interest. In some embodiments, repression is made reversible by operatively linking the nucleic acid sequence to an inducible promoter that is functional in an organism of interest. The choice of promoter disclosed herein varies depending on the quantitative, temporal and spatial requirements for expression, and also depending on the host cell to be transformed.
[0100] Exemplary promoters for use with the methods, bacteriophages and compositions disclosed herein include promoters that are functional in bacteria. For example, L-arabinose inducible ( araBAD , PBAD ) promoter, any lac promoter, L-rhamnose inducible (rhaPBAD) promoter, T7 RNA polymerase promoter, trc promoter, tac promoter, lambda phage promoter (PLPL-9G-50), anhydrotetracycline-inducible (let A) promoter, trp , Ipp, phoA , recA , prol /, cst- 1, cadA, 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 -II. coll 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. In some embodiments, the promoter is a BBa_J23102 promoter. In some embodiments, the promoter works in a b road range of bacteria, such as BBa_J23104, BBa_J23109. In some embodiments the promoter is derived from the target bacterium, such as endogenous CRISPR promoter, endogenous Cas operon promoter or the promoter from sarA, lipA, ptsH or cap l of S. aureus.
[0101] In some embodiments, the promoter comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 16-20. In some instances, the promoter comprises at least a portion havingatleastor about3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 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 or more than 50 nucleotides of any one of SEQ ID NOS: 16-20. In some instances, the promoter comprises at least a portion having at least or about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or more than 215 nucleotides of any one of SEQ ID NOS: 16-20.
Antimicrobial Agents and Peptides
[0102] In some embodiments, a bacteriophage disclosed herein is further genetically modified to express an antibacterial peptide, a functional fragment of an antibacterial peptide, and/or a lytic gene. In some embodiments, a bacteriophage disclosed herein express at least one antimicrobial agent or peptide disclosed herein. In some embodiments, the bacteriophage comprises a nucleic acid that encodes a peptide that prevents phage degradation or a peptide that assists in breaking down or degrading biofilm matrix.
[0103] In some embodiments, a bacteriophage described herein comprises a nucleic acid that encodes a peptide that prevents phage degradation or enables escape of the phage from the host defenses. In some embodiments, 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. In some embodiments, the peptide comprises TreA (e.g., a sequence at least 80% identical to SEQ ID NO: 10). In some embodiments, the peptide comprises Lpi (e.g., a sequence at least 80% identical to SEQ ID NO: 11).
[0104] In some embodiments, the bacteriophage comprises nucleic acids which encode enzymes which assist in breaking down or degrading biofilm matrix. In some embodiments, 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 lyase. In some embodiments, 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 1,4-L-fucodise hydrolase, colanicacid, depolymerazing alginase, DNase I, or combinations thereof. In some embodiments, a bacteriophage disclosed herein secretes an enzyme disclosed herein. In some embodiments, the peptide disrupts quorum sensing and biofilm formation. In some embodiments, the peptide increases the sensitivity of abacterial cell to an antibiotic. In some embodiments, the enzyme comprises DNAse I (e.g., a sequence at least 80% identical to SEQ ID NO: 9). In some embodiments, the enzyme comprises RIP (e.g., a sequence at least 80% identical to SEQ ID NO: 14). In some embodiments, the enzyme comprises FS3 (e.g., a sequence at least 80% identical to SEQ ID NO: 12).
[0105] In some embodiments, an antimicrobial agent or peptide is expressed and/or secreted by a bacteriophage disclosed herein. In some embodiments, the antimicrobial agent or peptide comprises PLNC8 a. In some embodiments, the antimicrobial agent or peptide comprises PLNC8p. In some embodiments, the antimicrobial agent or peptide comprises LytM. In some embodiments, the antimicrobial agent or peptide comprises an anti -restriction modification enzyme. In some embodiments, the antimicrobial agent or peptide comprises laciticin Q (LnqQ, e.g., a sequence at least 80% identical to SEQ ID NO: 15 or 50, e.g., SEQ ID NO: 50). In some embodiments, the LnqQ peptide is atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50 or 15. In some embodiments, the LnqQpeptideis atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 50.
[0106] In some embodiments, an antimicrobial agent or peptide is expressed and/or secreted by a bacteriophage disclosed herein. In some embodiments, 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. In some embodiments, 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 the first and/or the second Type I CRISPR-Cas system.
[0107] In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 1. In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 2. In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 3. In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 4. In some embodiments, the antimicrobial agent or peptide is encoded by a sequence comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 5.
[0108] In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 8. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 9. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 10. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 11. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 12. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 14. In some embodiments, the antimicrobial agent or peptide comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO. 15.
Expression Cassette
[0109] In some embodiments, the nucleic acid sequence is an expression cassette or in an expression cassette. In some embodiments, the expression cassettes are designed to express the nucleic acid sequence disclosed herein. In some embodiments, the nucleic acid sequence is an expression cassette encoding components of a CRISPR-Cas system and/or peptide. In some embodiments, the nucleic acid sequence is an expression cassette encoding components of a Type I CRISPR-Cas system. In some embodiments, the nucleic acid sequence is an expression cassette encoding an operable CRISPR-Cas system. In some embodiments, the nucleic acid sequence is an expression cassette encoding the operable components of a Type I CRISPR-Cas system, including Cascade and Cas3. In some embodiments, the nucleic acid sequence is an expression cassette encoding the operable components of a Type I CRISPR- Cas system, including a crRNA, Cascade and Cas3. In some embodiments, the nucleic acid sequence is an expression cassette encoding a peptide (e.g., antimicrobial peptide).
[0110] In some embodiments, an expression cassette comprising a nucleic acid 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. In some embodiments, an expression cassette is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
[0111] In some embodiments, an expression cassette includes a transcriptional and/or translational termination region (i.e. termination region) that is functional in the selected host cell. In some embodiments, termination regions are responsible for the termination of transcription beyond the heterologous nucleic acid sequence of interest and for correct mRNA polyadenylation. In some embodiments, the termination region is native to the transcriptional initiation region, is native to the operably linked nucleic acid 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 nucleic acid sequence of interest, to the host, or any combination thereof). In some embodiments, terminators are operably linked to the nucleic acid sequence disclosed herein.
[0112] In some embodiments, an expression cassette includes a nucleotide sequence for a selectable marker. In some embodiments, the nucleotide sequence encodes either a selectable or a screenable marker, depending on whether the marker confers a trait that is sel ected 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).
Vectors
[0113] In addition to expression cassettes, the nucleic acid sequences disclosed herein (e.g. nucleic acid sequence comprising a CRISPR array, CRISPR-Cas, peptide) are used in connection with vectors. 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 transforms prokaryotic or eukaryotic host either by integration into the cellular genome or existextrachromosomally (e.g. autonomous replicating plasmid with an origin of replication). Additionally, included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms. In some embodiments, a shuttle vector replicates in actinomycetes and bacteria and/or eukaryotes. In some embodiments, 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. In some embodiments, the vector is a bi-functional expression vector which functions in multiple hosts.
Sequence Optimization
[0114] In some embodiments, the nucleic acid sequence encoding a payload (e.g. the nucleic acid insert) is optimized for stable expression in a phage genome. In some embodiments, the insert is stable through at least 2, 3, 4, 5, 6, 7, 8,9, or 10 generations of passaging. In some embodiments, the nucleic acid sequence is optimized by optimizing the insertion site, modifying secondary structures, modifying DNA modification sites, modifying restriction enzyme motifs, codon optimization, GC% optimization, or a combination thereof. In some embodiments, the insertion site of the nucleic acid sequence is optimized. In some embodiments, the nucleic acid sequence is modified to remove secondary structures.
[0115] In certain embodiments, the bacteriophage comprises a nucleic acid insert modified from an exogenous nucleic acid described herein, wherein the nucleic acid comprises a first plurality of codons encoding for a first protein, and the nucleic acid insert comprises a second plurality of codons encoding for a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity, and wherein at least 50% of the second plurality of codons are high frequency codons in the bacteriophage genome. In certain aspects, described herein is a method of inserting an exogenous sequence comprising a plurality of codons encoding a first protein into a bacteriophage, the method comprising substituting one or more of the plurality of codons with a codon native to the bacteriophage to generate a nucleic acid insert encoding a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity. In certain aspects, described herein is a nucleic acid insert modified from an exogenous nucleic acid, wherein the nucleic acid comprises a first plurality of codons encoding for a first protein, and the nucleic acid insert comprises a second plurality of codons encoding for a second protein, wherein the first protein and the second protein have at least 90% amino acid sequence identity, and wherein at least 50% of the second plurality of codons are high frequency codons in the bacteriophage genome. In some embodiments, the first protein and the second protein have at least 95%, 97.5%, 99% or 99.5% sequence identity. In some embodiments, at least 50%< 60%, 70%, 80%, 90% or more than 90% of the second plurality of codons are high frequency codons in the bacteriophage genome. In some embodiments, the second plurality of codons match the profile of codons in the bacteriophage genome.
[0116] In some embodiments, the nucleic acid sequence is modified to remove DNA modification sites. In some embodiments, the DNA modification sites comprise DNA methylation sites.
[0117] In some embodiments, the nucleic acid sequence is modified to remove restriction enzyme motifs. In some embodiments, the nucleic acid sequence is modified to remove restriction enzyme motifs for a restriction enzyme derived from a bacterial species described herein. In some embodiments, the nucleic acid insert does not comprise, or comprises fewer than 10 sites recognized by abacterial enzyme.
[0118] In some embodiments, the nucleic acid sequence 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. When the nucleotide sequences are to be expressed in the nucleus, 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%, andthe like) to the native nucleotide sequence but which still encodes a polypeptide having the same function as that encoded by the original nucleotide sequence. In some embodiments, the nucleic acid sequences of this disclosure are codon optimized for expression in the organism/species of interest.
[0119] In some embodiments, the nucleic acid sequence is modified to optimize the percent GC content. In some embodiments, the percent GC content is modified so that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90% or more than 90% of the nucleotides comprises guanine or cytosine. In some embodiments, the percent GC content is modified so thatno more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% 90% or more than 90% of the nucleotides comprises guanine or cytosine.
[0120] In some embodiments, the exogenous nucleic acid is a bacterial nucleic acid. In some embodiments, the nucleic acid insert and the bacterial nucleic acid have less than 100%, 95%, 90%, 80%, 70%, 60%, or 50% sequence identity. In some embodiments, the first bacterial protein is a CRISPR-Cas protein as described herein. In some embodiments, the first bacterial protein is an antimicrobial agent and/or peptide as described herein.
Transformation
[0121] In some embodiments, the nucleic acid sequence, and/or expression cassettes disclosed herein are expressed transiently and/or stably incorporated into the genome of a host organism. In some embodiments, a the nucleic acid sequence and/or expression cassettes disclosed herein is introduced into a cell by any method known to those of skill in the art. Exemplary methods of transformation include transformation via electroporation of competent cells, passive uptake by competent cells, chemical transformation of competent cells, as well as any other electrical, chemical, physical (mechanical) and/or biological mechanism that results in the introduction of nucleic acid into a cell, including any combination thereof. In some embodiments, transformation of a cell comprises nuclear transformation. In some embodiments, transformation of a cell comprises plasmid transformation and conjugation.
[0122] In some embodiments, when more than one nucleic acid sequence is introduced, the nucleotide sequences 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 transformation event, or in separate transformation events.
METHODS OF USE
[0123] Disclosed herein, in certain embodiments, are methods of killing a target bacterium comprising contacting or introducing into a target bacterium any of the bacteriophages disclosed herein.
[0124] Further disclosed herein, in certain embodiments, are methods of modifying a mixed population of bacterial cells having a first bacterial species that comprises a target nucleotide sequence in the essential gene and a second bacterial species that does not comprise a target nucleotide sequence in the essential gene, the method comprising introducing into the mixed population of bacterial cells any of the bacteriophages disclosed herein.
[0125] Also disclosed herein, in certain embodiments, are methods of treating a disease in an individual in need thereof, the method comprising administering to the individual any of the bacteriophages disclosed herein.
[0126] In some embodiments, the target bacterium is killed solely by lytic activity of the bacteriophage. In some embodiments, the target bacterium is killed solely by activity of the CRISPR-Cas system. In some embodiments, the target bacterium is killed by the processing of the CRISPR array by a 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. In some embodiments, the target bacterium is killed solely by the antimicrobial peptide.
[0127] In some embodiments, the target bacterium is killed by lytic activity of the bacteriophage in combination with activity of the Type I CRISPR-Cas system. In some embodiments, the target bacterium is killed by the activity of the Type I CRISPR-Cas system, independently of the lytic activity of the bacteriophage. In some embodiments, the activity of the Type I CRISPR-Cas system supplements or enhances the lytic activity of the bacteriophage. In some embodiments, the activity of the Type I CRISPR-Cas system and the lytic activity of the bacteriophage are additive.
[0128] In some embodiments, the target bacterium is killed by lytic activity of the bacteriophage in combination with activity of the Type I CRISPR-Cas system and the anti microbial peptide. In some embodiments, the target bacterium is killed by the activity of the Type I CRISPR-Cas system, independently of the lytic activity of the bacteriophage and the anti-microbial peptide. In some embodiments, the activity of the Type I CRISPR-Cas system supplements or enhances the lytic activity of the bacteriophage and the anti-microbial peptide. In some embodiments, the activity of the Type I CRISPR-Cas system, the lytic activity of the bacteriophage, and the activity of the antimicrobial peptide are additive.
[0129] In some embodiments, the lytic activity of the bacteriophage and the activity of the Type I CRISPR-Cas system is synergistic. In some embodiments, a synergistic activity is defined as an activity resulting in a greater level of phage kill than the additive combination of the lytic activity of the bacteriophage and the Type I CRISPR-Cas system. In some embodiments, the lytic activity of the bacteriophage is modulated by a concentration of the bacteriophage. In some embodiments, the activity of the Type I CRISPR-Cas system is modulated by a concentration of the bacteriophage. [0130] 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-Cas system by increasing the concentration of bacteriophage administered to the bacterium. In some embodiments, the synergistic killing of the bacterium is modulated to disfavor killing by the lytic activity of the bacteriophage over the activity of the CRISPR-Cas system by decreasing the concentration of bacteriophage administered to the bacterium. In some embodiments, at low concentrations, lytic replication allows for amplification and killing of the target bacteria. In some embodiments, at high concentrations, amplification of a phage is not required. In some embodiments, the synergistic killing of the bacterium is modulated to favor killing by the activity of the CRISPR-Cas system 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-Cas system 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.
[0131] In some embodiments, the lytic activity of the bacteriophage, the activity of the Type I CRISPR-Cas system, and the activity of the antimicrobial peptide is synergistic. In some embodiments, a synergistic activity is defined as an activity resulting in a greater level of phage kill than the additive combination of the lytic activity of the bacteriophage, the Type I CRISPR-Cas system, and the antimicrobial peptide. In some embodiments, the lytic activity of the bacteriophage is modulated by a concentration of the bacteriophage and the antimicrobial peptide. In some embodiments, the activity of the Type I CRISPR-Cas system is modulated by a concentration of the bacteriophage.
Administration Routes and Dosage
[0132] Dose and duration of the administration of a composition disclosed herein will depend on a variety of factors, including the subject’s age, subject’s weight, and tolerance of the phage. In some embodiments, a bacteriophage disclosed herein is administered to patients intra-arterially, intravenously, intraurethrally, intramuscularly, orally, subcutaneously, by inhalation, or any combination thereof. In some embodiments, a bacteriophage disclosed herein is administered to patients by oral administration. In some embodiments, a bacteriophage disclosed herein is administered to patients by topical, cutaneous, transdermal, transmucosal, implantation, sublingual, buccal, rectal, vaginal, ocular, otic, or nasal administration. In some embodiments, a bacteriophage disclosed herein is administered to patients by any combination of the aforementioned routes of administration.
[0133] In some embodiments, a dose of phage between 103and 1020PFU is given. In some embodiments, a dose of phage between 103 and 1010PFU is given. In some embodiments, a dose of phage between 106and 1020 PFU is given. In some embodiments, a dose of phage between 106and 1010PFU is given. For example, in some embodiments, the bacteriophage is presentin a composition in an amountbetween 103and 10UPFU. In some embodiments, the bacteriophage is presentin a composition in an amount about 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024PFU or more. In some embodiments, the bacteriophage is presentin a composition in an amount of less than 101 PFU. In some embodiments, the bacteriophage is presentin a composition in an amountbetween lCriand 108, 104and 109, 105and 1010, or 107and 1011 PFU. In some embodiments, a composition comprising two or more bacteriophage is administered to a subject, wherein each bacteriophage is administered in an amount about 103, 104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020,
1021, 1022, 1023, 1024PFU or more. In some embodiments, a composition comprising two or more bacteriophage is administered to a subject, wherein each bacteriophage is administered in an amount of less than 101 PFU. In some embodiments, a composition comprising two or more bacteriophage is administered to a subject, wherein each bacteriophage is administered in an amountbetween 101 and 108, 104and 109, 105 and 1010, or 107and 10UPFU.
[0134] 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, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24 times a day. In some embodiments, a bacteriophage or a mixture is administered to a subject in need thereof atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or21 times a week. In some embodiments, a bacteriophage or a mixture is administered to a subject in need thereof atleast 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, 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. In some embodiments, a bacteriophage or a mixture is administered to a subject in need thereof every 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, or 24 hours.
[0135] In some embodiments, 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. In some embodiments, 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. In some embodiments, the initial administration of the composition is via any route practical, such as by any route described herein using any formulation described herein. In some embodiments, 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.
Bacterial Infections
[0136] Disclosed herein, in certain embodiments, are methods of treating bacterial infections. In some embodiments, the bacteriophages disclosed herein treat or prevent diseases or conditions mediated or caused by bacteria as disclosed herein in a human or animal subject. In some embodiments, the bacteriophages disclosed herein treat or prevent diseases or conditions caused or exacerbated by bacteria as disclosed herein in a human or animal subject. 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. In some embodiments, a bacterial infection is treated by modulating the activity of the bacteria and/or by directly killing of the bacteria.
[0137] In some embodiments, the bacterium is Staphylococcus spp. In some embodiments, the bacterium is S. aureus.
[0138] In some embodiments, one or more Staphylococcus species present in a bacterial population are pathogenic.
[0139] In some embodiments, 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 enteropathogenic bacteria from a plurality of bacteria within the microbiome or gut flora of a subject.
[0140] In some embodiments, the bacteriophages disclosed herein are used to treat an infection, a disease, or a condition, in the urinary tract of a subject. In some embodiments, 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. In some embodiments, 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.
[0141] In some embodiments, 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.
[0142] In some embodiments, the bacteriophages disclosed herein are used to treat an infection, a disease, or a condition, on a mucosal membrane of a subject. In some embodiments, the bacteriophages are used to modulate and/or kill target bacteria on the mucosal membrane of a subject.
[0143] In some embodiments, the pathogenic bacteria are antibiotic resistant. In some embodiments, the pathogenic bacteria is methicillin resistant. In some embodiments, the pathogenic bacteria is methicillin resistant Staphylococcus aureus.
[0144] In some embodiments, the one or more target bacteria present in the bacterial population form a biofilm. In some embodiments, the biofilm comprises pathogenic bacteria. In some embodiments, the bacteriophage disclosed herein is used to treat a biofilm.
[0145] In some embodiments, the bacterium is includes Staphylococcus spp. In some embodiments, the bacterium is Staphylococcus aureus.
[0146] In some embodiments, the bacteriophage treats acne and other related skin infections.
[0147] In some embodiments, the Staphylococcus species is a multiple drug resistant (MDR) bacteria strain. An MDR strain is a bacteria strain that is resistant to at least one antibiotic. In some embodiments, 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. In some embodiments, the bacteria strain is Staphylococcus aureus. In some embodiments, the pathogenic bacteria is methicillin resistant Staphylococcus aureus. [0148] In some embodiments, the bacterium is S. aureus. In some embodiments, the methods and compositions disclosed herein are for use in veterinary and medical applications as well as research applications.
Microbiome
[0149] “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. In some embodiments, the microbiome comprises microbial material including, but not limited to, bacteria, archaea, protists, fungi, and viruses. In some embodiments, 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, Bacteroidetes, or Firmicutes.
[0150] In some embodiments, 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. [0151] In some embodiments, 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 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 S. aureus.
[0152] In some embodiments, 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 ofEnterobacteriaceae,Pasteurellaceae, Fusobacteriaceae, Neisseriaceae, Veillonellaceae, Gemellaceae, Bacteriodales, Clostridiales, Erysipelotrichaceae, Bifidobacteriaceae, Bacteroides, Faecalibacterium, Roseburia,Blautia, Ruminococcus, Coprococcus, Streptococcus, Dorea, Blautia, Ruminococcus, Lactobacillus, Enterococcus, Streptococcus, Actinomyces, Lactococcus,Roseburia, Blautia, Dialister, Desulfovibrio, Escherichia, Lactobacillus, Coprococcus, Clostridium, Bifidobacterium, Klebsiella, Granulicatella, Eubacterium, Anaerostipes, Parabacteroides, Coprobacillus, Gordonibacter, Collinsella, Bacteroides, Faecalibacterium, Anaerotruncus, Alistipes, Haemophilus, Anaerococcus, Veillonella, Arevotella, Akkermansia, Bilophila, Sutterella, Eggerthella, Holdemania, Gemella, Peptoniphilus, Rothia, Pediococcus, Citrobacter, Odoribacter, Enterobacteria, Fusobacterium, Proteus, Escherichia coli , Fusobacterium nucleatum, Haemophilus parainfluenzae (Pasteur ellaceae), Veillonella parvula, Eikenella corrodens (Neisseriaceae), Gemella moribillum, Bacteroides vulgatus, Bacteroides caccae, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium dentum, Blautia hansenii, Ruminococcus gnavus, Clostridium nexile, Faecalibacterium prausnitzii, Ruminoccus torques, Clostridium bolteae, Eubacterium rectale, Roseburia intestinalis, and Coprococcus iomes.
[0153] In some embodiments, the bacteriophages are used to modulate or kill target single or plurality of bacteria within the microbiome or flora of the epidermis of a subject. Modification (e.g., dysbiosis) of the microbiome or skin flora increasesthe risk for health conditions such as eczema or Atopic Dermatitis.
[0154] In some embodiments, 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. Environmental Therapy
[0155] In some embodiments, bacteriophages disclosed herein are furtherused for food and agriculture sanitation (including meats, fruits and vegetable sanitation), hospital sanitation, home sanitation, vehicle and equipment sanitation, industrial sanitation, etc. In some embodiments, 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.
[0156] 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. In some embodiments, a phage disclosed herein is used to treat equipment or environments inhabited by bacterial genera which become resistant to commonly used disinfectants. In some embodiments, phage compositions disclosed herein are used to disinfect inanimate objects. In some embodiments, an environment disclosed herein is sprayed, painted, or poured onto with aqueous solutions with phage titers. In some embodiment a solution described herein comprises between lO O20 plaque formingunits (PFU)/ml. In some embodiments, a bacteriophage disclosed herein is applied by aerosolizing agents that include dry dispersants to facilitate distribution of the bacteriophage into the environment. In some embodiments, objects are immersed in a solution containing bacteriophage disclosed herein.
Sanitation
[0157] In some embodiments, bacteriophages disclosed herein are used as sanitation agents in a variety of fields. Although the terms “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. [0158] In some embodiments, bacteriophages are used to sanitize hospital facilities, including operating rooms, patient rooms, waiting rooms, lab rooms, or other miscellaneous hospital equipment. In some embodiments, this equipment includes electrocardiographs, respirators, cardiovascular assist devices, intraaortic balloon pumps, infusion devices, other patient care devices, televisions, monitors, remote controls, telephones, beds, etc. In some situations, the bacteriophage is applied through an aerosol canister. In some embodiments, bacteriophage is applied by wiping the phage on the object with a transfer vehicle. [0159] In some embodiments, a bacteriophage described herein is used in conjunction with patient care devices. In some embodiment, bacteriophage is used in conjunction with a conventional ventilator or respiratory therapy device to clean the internal and external surfaces between patients. Examples of ventilators include devices to support ventilation during surgery, devices to support ventilation of incapacitated patients, and similar equipment. In some embodiments, the conventional therapy includes automatic or motorized devices, or manual bag-type devices such as are commonly found in emergency rooms and ambulances. In some embodiments, 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.
[0160] In some embodiment, 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.
[0161] In some embodiments, water supplies are treated with a composition disclosed herein. In some embodiments, bacteriophage disclosed herein is used to treat contaminated water, water found in cisterns, wells, reservoirs, holding tanks, aqueducts, conduits, and similar water distribution devices. In some embodiments, the bacteriophage is applied to industrial holding tanks where water, oil, cooling fluids, and other liquids accumulate in collection pools. In some embodiments, a bacteriophage disclosed herein is periodically introduced to the industrial holding tanks in order to reduce bacterial growth.
[0162] In some embodiments, bacteriophages disclosed herein are used to sanitize a living area, such as a house, apartment, condominium, dormitory, or any living area. In some embodiments, 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. In this capacity, the bacteriophage is dispensed from conventional devices, including pump sprayers, aerosol containers, squirt bottles, pre-moistenedtowelettes, 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. In some embodiments, 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. [0163] In some embodiments, 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.
Food Safety
[0164] In some embodiments, a bacteriophage described herein is used in any food product or nutritional supplement, for preventing contamination. Examples for 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.
[0165] 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- bome illness traceable to produce. In some embodiments, 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. In some embodiments, 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.
[0166] In some embodiments, specific bacteriophages are applied to produce in restaurants, grocery stores, produce distribution centers. In some embodiments, bacteriophages disclosed herein are periodically or continuously applied to the fruit and vegetable contents of a salad bar. In some embodiments, 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.
[0167] In some embodiments, a bacteriophage described herein is used in matrices or support media containing with packaging containing meat, produce, cut fruits and vegetables, and other foodstuffs. In some embodiments, polymers that are suitable for packaging are impregnated with a bacteriophage preparation.
[0168] In some embodiments, 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.
[0169] The use of specific bacteriophages as biocontrol agents on produce provides many advantages. For example, bacteriophages are natural, non-toxic products that will not disturb the ecological balance of the natural micro flora 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.
PHARMACEUTICAL COMPOSITIONS
[0170] Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising (a) the nucleic acid sequences as disclosed herein; and (b) a pharmaceutically acceptable excipient. Also disclosed herein, in certain embodiments, are pharmaceutical compositions comprising (a) the bacteriophages as disclosed herein; and (b) a pharmaceutically acceptable excipient. Further disclosed herein, in certain embodiments, are pharmaceutical compositions comprising (a) the compositions as disclosed herein; and (b) a pharmaceutically acceptable excipient.
[0171] In some embodiments, the disclosure provides pharmaceutical compositions and methods of administering the same to treat bacterial, archaeal infections or to disinfect an area. In some embodiments, the pharmaceutical composition comprises any of the reagents discussed above in a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition or method disclosed herein treats bloodstream infections (BSI) and/or inflammatory diseases (e.g. atopic dermatitis (AD)). In some embodiments, a pharmaceutical composition or method disclosed herein treats eczema. In some embodiments, a pharmaceutical composition or method disclosed herein treats atopic dermatitis.
[0172] In some embodiments, compositions disclosed herein comprise medicinal agents, pharmaceutical agents, carriers, adjuvants, dispersing agents, diluents, and the like.
[0173] In some embodiments, the bacteriophages disclosed herein are formulated for administration in a pharmaceutical carrier in accordance with suitable methods. In some embodiments, the manufacture of a pharmaceutical composition according to the disclosure, the bacteriophage is admixed with, inter alia, an acceptable carrier. In some embodiments, the carrier is a solid (including a powder) or a liquid, or both, and is preferably formulated as a unit-dose composition. In some embodiments, one or more bacteriophages are incorporated in the compositions disclosed herein, which are prepared by any suitable method of a pharmacy.
[0174] In some embodiment, a method of treating subject’s in-vivo , comprising administering to a subject a pharmaceutical composition comprising a bacteriophage disclosed herein in a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is administered in a therapeutically effective amount. In some embodiments, the administration of the bacteriophage to a human subject or an animal in need thereof are by any means known in the art.
[0175] In some embodiments, bacteriophages disclosed herein are for oral administration. In some embodiments, 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. In some embodiments, 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.
[0176] In some embodiments, methods and compositions of the present disclosure are suitable for parenteral administration comprising sterile aqueous and non-aqueous injection solutions of the bacteriophage. In some embodiments, these preparations are isotonic with the blood of the intended recipient. In some embodiments, these preparations comprise antioxidants, buffers, bacteriostals and solutes which render the composition isotonic with the blood of the intended recipient. In some embodiments, aqueous and non-aqueous sterile suspensions include suspending agents and thickening agents. In some embodiments, 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.
[0177] In some embodiment, 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, transderm al enhancers, and combinations of two or more thereof.
[0178] In some embodiments, methods and 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.
[0179] In some embodiments, 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. In some embodiments, the respirable particles are liquid or solid. As used herein, “aerosol” includes any gas-borne suspended phase, which is capable of being inhaled into the bronchioles or nasal passages. In some embodiments, aerosols of liquid particles are produced by any suitable means, such as with a pressure - driven aerosol nebulizer or an ultrasonic nebulizer. In some embodiments, aerosols of solid particles comprising the composition is produced with any solid particulate medicament aerosol generator, by techniques known in the pharmaceutical art.
[0180] In some embodiment, methods and compositions suitable for administering bacteriophages disclosed herein to a surface of an object or subj ect includes aqueous solutions. In some embodiments, such aqueous solutions are sprayed onto the surface of an object or subject. In some embodiment, the aqueous solutions are used to irrigate and clean a physical wound of a subj ect form foreign debris including bacteria.
[0181] In some embodiments, the bacteriophages disclosed herein are administered to the subject in a therapeutically effective amount. In some embodiments, at least one bacteriophage composition disclosed herein is formulated as a pharmaceutical formulation. In some embodiments, 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. In some instances, a pharmaceutical formulation comprises a bacteriophage described herein and at least one of: an excipient, a diluent, or a carrier.
[0182] In some embodiments, 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. [0183] 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.
[0184] In some embodiments, an excipient is a buffering agent. Non-limiting examples of suitable buffering agents include but is not limited to sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. In some embodiments, 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.
[0185] In some embodiments an excipient is a preservative. Non -limiting examples of suitable preservatives include but is not limited to antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol. In some embodiments, antioxidants include but not limited to Ethylenediaminetetraacetic acid (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. In some embodiments, 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, protease inhibitor, reducing agent, alkylating agent, antimicrobial agent, oxidase inhibitor, or other inhibitor.
[0186] In some embodiments, a pharmaceutical formulation comprises a binder as an excipient. Non-limiting examples of suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
[0187] In some embodiments, the binders that are used in a pharmaceutical formulation are selected from starches such as potato starch, com starch, wheat starch; sugars such as sucrose, glucose, dextrose, lactose, maltodextrin; natural and synthetic gums; gelatine; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxy ethyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinylpyrrolidone (povidone); polyethyleneglycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols such as sorbitol, xylitol, mannitol and water or a combination thereof.
[0188] In some embodiments, a pharmaceutical formulation comprises a lubricant as an excipient. Non-limiting examples of 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. In some embodiments, 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.
[0189] In some embodiments, an excipient comprises a flavoring agent. In some embodiments, flavoring agents includes natural oils; extracts from plants, leaves, flowers, and fruits; and combinations thereof.
[0190] In some embodiments, an excipient comprises a sweetener. Non-limiting examples of suitable sweeteners include glucose (com 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; SteviaRebaudiana(Stevioside); chloro derivatives of sucrose such as sucralose; and sugar alcohols such as sorbitol, mannitol, sylitol, and the like.
[0191] In some instances, a pharmaceutical formulation comprises a coloring agent. Non limiting examples of 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).
[0192] In some embodiments, the pharmaceutical formulation disclosed herein comprises a chelator. In some embodiments, 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. [0193] In some instances, a pharmaceutical formulation comprises a diluent. Non limiting examples of diluents include water, glycerol, methanol, ethanol, and other similar biocompatible diluents. In some embodiments, a diluent is an aqueous acid such as acetic acid, citric acid, maleic acid, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, or similar.
[0194] In some embodiments, a pharmaceutical formulation comprises a surfactant. In some embodiments, 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, amino acids such as L- leucine, sugar esters of fatty acids, glycerides of fatty acids or a combination thereof.
[0195] In some instances, a pharmaceutical formulation comprises an additional pharmaceutical agent. In some embodiments, an additional pharmaceutical agent is an antibiotic agent. In some embodiments, 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. [0196] In some embodiments, an antibiotic agent described herein is an aminoglycoside such as Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin or Paromomycin. In some embodiments, an antibiotic agent described herein is an Ansamycin such as Geldanamycin or Herbimycin.
[0197] In some embodiments, an antibiotic agent described herein is a carbacephem such as Loracarbef. In some embodiments, an antibiotic agent described herein is a carbapenem such asErtapenem, Doripenem, Imipenem/CilastatinorMeropenem.
[0198] In some embodiments, an antibiotic agent described herein is a cephalosporins (first generation) such as Cefadroxil, Cefazolin, Cef alexin, Cefalotin or Cefalothin, or alternatively a Cephalosporins (second generation) such as Cefaclor, Cefamandole, Cefoxitin, Cefprozil or Cefuroxime. In some embodiments, 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 Ceftobiprole.
[0199] In some embodiments, an antibiotic agent described herein is alincosamide such as Clindamycin and Azithromycin, or a macrolide such as Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin and Spectinomycin.
[0200] In some embodiments, an antibiotic agent described herein is a monobactams such as Aztreonam, or a nitrofuran such as Furazolidone or Nitrofurantoin.
[0201] In some embodiments, 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. [0202] In some embodiments, an antibiotic agent described herein is a sulfonamide such asMafenide, Sulfonamidochrysoidine, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim, or Trimethoprim-Sulfamethoxazole (Co-trimoxazole) (TMP-SMX).
[0203] In some embodiments, 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. [0204] In some embodiments, an antibiotic agent described herein is a polypeptide such as Bacitracin, Colistin or Polymyxin B.
[0205] In some embodiments, an antibiotic agent described herein is a tetracycline such as Demeclocycline, Doxy cy cline, Minocycline or Oxy tetracycline.
EMBODIMENTS
1. A bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, wherein the bacteriophage has been rendered lytic.
2. The bacteriophage of embodiment 1, wherein the bacteriophage is derived from a temperate bacteriophage.
3. The bacteriophage of any one of embodiments 1 -2, wherein the bacteriophage has been rendered lytic by removal, replacement, or inactivation of a lysogenic gene.
4. The bacteriophage of any one of embodiments 1 -2, wherein the bacteriophage has been rendered lytic by removal of a var009 region.
5. The bacteriophage of any one of embodiments 1 -2, wherein the bacteriophage has been rendered lytic by removal of a varO 10 region.
6. The bacteriophage of any one of embodiments 1 -2, wherein the bacteriophage has been rendered lytic by the removal of a var012 region. The bacteriophage of any one of embodiments 1 -2, wherein the bacteriophage has been rendered lytic by the removal of a var042 region. The bacteriophage of any one of embodiments 1 -2, wherein the bacteriophage has been rendered lytic by the removal of a e002 region. The bacteriophage of any one of embodiments 1 -2, wherein the bacteriophage has been rendered lytic by the removal of a regulatory element of a lysogeny gene. The bacteriophage of any one of embodiments 1 -2, wherein the bacteriophage has been rendered lytic by the removal, alteration or replacement of a promoter of a lysogeny gene. The bacteriophage of any one of embodiments 1 -2, wherein the bacteriophage has been rendered lytic by the removal of a functional element of a lysogeny gene. The bacteriophage of any one of embodiments 1-11, wherein the bacteriophage has been rendered lytic via a second CRISPR array comprising a second spacer directed to a lysogenic gene. The bacteriophage of any one of embodiments 1-12, wherein the bacteriophage infects multiple bacterial strains. The bacteriophage of any one of embodiments 1-13, wherein the target nucleotide sequence comprises all or a part of a promoter sequence for the target gene. The bacteriophage of any one of embodiments 1-14, wherein 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 bacteriophage of any one of embodiments 1-15, wherein the target nucleotide sequence comprises at least a portion of an essential bacterial gene that is needed for survival of the target bacterium. The bacteriophage of embodiment 16, wherein the essential bacterial gene is Tsf acpP, gapA, infA , secY, csrA, trmD,ftsA,fiisA , glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fus, adk, rpsK, rplR, ctrA,parC , tRNA-Ser, tRNA-Asn, or metK. The bacteriophage of any one of embodiments 1-17, wherein the target nucleotide sequence is in a non-essential bacterial gene or genomic locus. The bacteriophage of any one of embodiments 1-18, wherein the first nucleic acid sequence is a first CRISPR array further comprising at least one repeat sequence. The bacteriophage of embodimentl9, wherein the at least one repeat sequence is operably linked to the first spacer sequence at either its 5 ’ end or its 3 ’ end. The bacteriophage of any one of embodiments 1 -20, wherein the first nucleic acid is inserted into a non-essential bacteriophage gene or other genomic locus. The bacteriophage of embodiment 21, wherein the non-essential gene is a cl repressor gene, antirepressor gene, integrase, PemK -like phage protein gene, or any combination or genes found in varO 10, or varO 12. The bacteriophage of any one of embodiments 1 -22, wherein the target bacterium is S. aureus. The bacteriophage of any one of embodiments 1 -23, wherein the bacteriophage is a Kayvirus, a Twortvirus, a Rosenblumvirus, a Phietavirus or a Triavirus. The bacteriophage of embodiment 24, wherein the bacteriophage comprises at least 80% sequence identity to pi 473. The bacteriophage of embodiment 25, wherein the CRISPR-Cas system is endogenous to the target bacterium. The bacteriophage of embodiment 26, wherein the CRISPR-Cas system is exogenous to the target bacterium. The bacteriophage of any one of embodiments 1-27, wherein the CRISPR-Cas system is a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR- Cas system, a Type IV CRISPR-Cas system, or a Type V CRISPR-Cas system. The bacteriophage of any one of embodiments 1 -28, wherein the CRISPR-Cas system is a Type I CRISPR-Cas system. A bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a target bacterium, wherein the bacteriophage has been rendered lytic by removal of the var009 region, varO 10 region, varOl 2 region, var042 region, or a combination thereof from a temperate bacteriophage. The bacteriophage of embodiment 30, wherein the bacteriophage infects multiple bacterial strains. The bacteriophage of any one of embodiments 1 -31, wherein the target nucleotide sequence comprises all or a part of a promoter sequence for the target gene. The bacteriophage of any one of embodiments 1-32, wherein 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 bacteriophage of any one of embodiments 1-33, wherein the target nucleotide sequence comprises at least a portion of an essential bacterial gene that is needed for survival of the target bacterium. The bacteriophage of embodiment 34, wherein the essential bacterial gene is Tsf acpP, gapA, infA , secY, csrA, trmD,ftsA,fiisA , glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fus, adk, rpsK, rplR, ctrA,parC , tRNA-Ser, tRNA-Asn, or metK. The bacteriophage of any one of embodiments 1-35, wherein the target nucleotide sequence is in a non-essential bacterial gene or genomic locus. The bacteriophage of any one of embodiments 1-36, wherein the first nucleic acid sequence is a first CRISPR array further comprising at least one repeat sequence. The bacteriophage of embodiment 37, wherein the at least one repeat sequence is operably linked to the first spacer sequence at either its 5 ’ end or its 3 ’ end. The bacteriophage of any one of embodiments 1-38, wherein the first nucleic acid is inserted into a non-essential bacteriophage gene or other genomic locus. The bacteriophage of embodiment 39, wherein the non-essential bacteriophage gene is a cl repressor gene, antirepressor gene, integrase, PemK-like phage protein gene, or any combination or genes found in varO 10, var012, orvar042. The bacteriophage of any one of embodiments 39-40, wherein the target bacterium is S. aureus. The bacteriophage of any one of embodiments 39-40, wherein the temperate bacteriophage is a Kayvirus, a Twortvirus, a Rosenblumvirus, a Phietavirus or a Triavirus. The bacteriophage of embodiment 42, wherein the bacteriophage comprises at least 80% sequence identity with pi 473. The bacteriophage of embodiment 43, wherein the CRISPR-Cas system is endogenous to the target bacterium. The bacteriophage of embodiment 43 , wherein the CRISPR-Cas system is exogenous to the target bacterium. 6. The bacteriophage of any one of embodiments 44-45, wherein the CRISPR-Cas system is a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a Type IV or a Type V system. 7. The temperate bacteriophage of any one of embodiments44-46, wherein the CRISPR- Cas system is a Type I CRISPR-Cas system. 8. A pharmaceutical composition comprising: a. a bacteriophage of any one of embodiments 1 -48; and b . a pharmaceutically acceptable excipient. 9. The pharmaceutical composition of embodiment 48, wherein the pharmaceutical composition comprises at least two bacteriophage selected from the bacteriophage of any one of embodiments 1-45. 0. The pharmaceutical composition of embodiment 49, wherein the bacteriophage are from the lineage consisting of a Kay virus, a Twortvirus, a Rosenblum virus, a Phietavirus or a Triavirus. 1. The pharmaceutical composition of embodiment 50, wherein the pharmaceutical composition is in a form of a tablet, a capsule, a liquid, a syrup, an oral formulation, an intravenous formulation, an intranasal formulation, an ocular formulation, an otic formulation, a subcutaneous formulation, a topical formulation, atransdermal formulation, a transmucosal formulation, an inhalable respiratory formulation, a suppository, and any combination thereof. . A method for killing a target bacterium, the method comprising introducing into the target bacterium a lytic bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or a crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in the target bacterium, thereby killing the target bacterium, wherein the bacteriophage is rendered lytic by removal of a var009 region, varOlO region, var012 region, var042 region, or a combination thereof from a temperate bacteriophage.3. The method of embodiment 52, wherein the bacteriophage infects multiple bacterial strains. . The method of any one of embodiments 52-53, wherein the target nucleotide sequence comprises all or a part of a promoter sequence for the target gene. 5. The method of any one of embodiments 52-53, wherein 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 method of any one of embodiments 52-53, wherein the target nucleotide sequence comprises at least a portion of an essential bacterial gene that is needed for survival of the target bacterium. The method of embodiment 56, wherein the essential bacterial gene is Tsf acpP , gapA, inf A, secY, csrA, trmD,ftsA,fusA , glyQ, eno, nusG, dnaA, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, dnaE, rpoA, rpoB,pheT, injB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fus, adk, rpsK, rplR, ctrA,parC , tRNA-Ser, tRNA-Asn, or metK. The method of any one of embodiments 52-57, wherein the target nucleotide sequence is in a non-essential bacterial gene or genomic locus. The method of any one of embodiments 52-58, wherein the firstnucleic acid sequence is a first CRISPR array further comprising at least one repeat sequence. The method of embodiment 59, wherein the at least one repeat sequence is operably linked to the first spacer sequence at either its 5 ’ end or its 3 ’ end. The method of any one of embodiments 52-60, wherein the firstnucleic acid is inserted into a non-essential bacteriophage gene. The method of embodiment 61, wherein the non-essential bacteriophage gene is a cl repressor gene, antirepressor gene, integrase, PemK-like phage protein gene, or any combination or genes found in varOlO, var012, orvar042. The method of any one of embodiments 52-62, wherein the target bacterium is S. aureus. The method of any one of embodiments 52-63, wherein the bacteriophage is a Kayvirus, a Twortvirus, a Rosenblumvirus, a Phietavirus or a Triavirus. The method of embodiment 64, wherein the bacteriophage comprises at least 80% sequence identity with pi 473. The method of any one of embodiments 52-65, wherein the target bacterium is killed by the lytic activity of the bacteriophage, by the activity of a CRISPR-Cas system using the first spacer sequence or the crRNA transcribed therefrom, or both. The method of any one of embodiments 52-66, wherein the target bacterium is killed by the activity of the CRISPR-Cas system independently of the lytic activity of the bacteriophage. The method of any one of embodiments 52-66, wherein activity of the CRISPR-Cas system supplements or enhances lytic activity of the bacteriophage. The method of any one of embodiments 52-66, wherein lytic activity of the temperate bacteriophage and activity of the CRISPR-Cas system are synergistic. 0. The method of any one of embodiments 52-69, wherein lytic activity of the temperate bacteriophage, activity of the CRISPR-Cas system, or both is modulated by a concentration of the bacteriophage. 1. The method of any one of embodiments 52-70, wherein the CRISPR-Cas system is endogenous to the target bacterium. . The method of any one of embodiments 52-70, wherein the CRISPR-Cas system is exogenous to the target bacterium. 3. The method of any one of embodiments 52-72, wherein the CRISPR-Cas system is a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a Type IV, or a Type V system. . The method of any one of embodiments 52-73, wherein the CRISPR-Cas system is a Type I CRISPR-Cas system. 5. The method of any one of embodiments 52-74, wherein the temperate bacteriophage does not confer any new properties onto the target bacterium beyond cellular death caused by the lytic activity of the temperate bacteriophage, beyond the activity of the CRISPR-Cas array, or both. 6. A method of treating a disease in an individual in need thereof, the method comprising administering the pharmaceutical composition of any one of embodiments 48-51. 7. The method of embodiment 76, wherein the individual is a mammal. 8. The method of any one of embodiments 76-77, wherein the disease is abacterial infection. 9. The method of embodiment 78, wherein a bacterium causingthe bacterial infection is an Staphylococcus aureus, methicillin resistant Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus salivarius, Stapylococcus argentis, Staphylococcus hemolyicus, Staphylocuccus schweitzeri or any combination thereof. 0. The method of embodiment 78, wherein the bacterium is a drug resistant bacterium that is resistant to at least one antibiotic. 1. The method of embodiment 80, wherein the bacterium is a multi -drug resistant bacterium that is resistant to at least one antibiotic. . The method of any one of embodiments 80-81, wherein the atleastone antibiotic comprises a cephalosporin, a fluoroquinolone, a carbapenem, a colistin, an aminoglycoside, vancomycin, daptomycin, streptomycin, methicillin or oxacillin. 3. The method of any one of embodiments 76-82, wherein the administering is intra arterial, intravenous, intraurethral, intramuscular, oral, subcutaneous, inhalation, topical, cutaneous, transdermal, transmucosal, implantation, sublingual, buccal, rectal, vaginal, ocular, otic, or nasal administration or any combination thereof. . A bacteriophage comprising a nucleic acid sequence encoding a Type I CRISPR-Cas system comprising:
(a) a CRISPR array comprising a spacer sequence complementary to target nucleotide sequence in a Staphylococcus species;
(b) a Cascade polypeptide; and
(c) a Cas3 polypeptide. 5. The bacteriophage of embodiment 84, wherein the CRISPR array further comprises at least one repeat sequence. 6. The bacteriophage of embodiment 85, wherein the at least one repeat sequence is operably linked to the spacer sequence at either its 5’ end or its 3 ’ end. 7. The bacteriophage of any one of embodiments 84-86 wherein the target nucleotide sequence comprises a coding sequence. 8. The bacteriophage of any one of embodiments 84-86, wherein the target nucleotide sequence comprises a non-coding or intergenic sequence. 9. The bacteriophage of any one of embodiments 84-86, wherein the target nucleotide sequence comprises all or a part of a promoter sequence. 0. The bacteriophage of embodiment 89, wherein the target nucleotide sequence comprises all or a part of a nucleotide sequence located on a coding strand of a transcribed region of an essential gene. 1. The bacteriophage of embodiment 90, wherein the essential gene is Tsf acpP, gap A, infA, secY, csrA, trmD, ftsA,fusA, glyQ, eno, nusG, dnaA, dnaS, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, glnS, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fus, adk, rpsK, rplR, ctrA,parC, tRNA-Ser, tRNA-Asn, or metK. . The bacteriophage of any one of embodiments 84-91, wherein the Cascade polypeptide forms a Cascade complex of a Type I-A CRISPR-Cas system, a Type I-B CRISPR-Cas system, a Type I-C CRISPR-Cas system, a Type I-D CRISPR-Cas system, a Type I-E CRISPR-Cas system, or a Type I-F CRISPR-Cas system. 3. The bacteriophage of embodiment 92, wherein the Cascade complex comprises: (i) a Cas7 polypeptide, a Cas8al polypeptide or a Cas8a2 polypeptide, a Cas5 polypeptide, a Csa5 polypeptide, a Cas6a polypeptide, a Cas3' polypeptide, and a Cas3” polypeptide having no nuclease activity (Typel-A CRISPR-Cas system);
(ii) a Cas6b polypeptide, a Cas8b polypeptide, a Cas7 polypeptide, and a Cas5 polypeptide (Typel-B CRISPR-Cas system);
(iii) a Cas5d polypeptide, a Cas8c polypeptide, and a Cas7 polypeptide (Typel-C CRISPR-Cas system);
(iv) a CaslOd polypeptide, a Csc2 polypeptide, a Cscl polypeptide, a Cas6d polypeptide (Typel-D CRISPR-Cas system);
(v) a Csel polypeptide, a Cse2 polypeptide, a Cas7 polypeptide, a Cas5 polypeptide, and a Cas6e polypeptide (Type I-E CRISPR-Cas system);
(vi) a Csyl polypeptide, a Csy2 polypeptide, a Csy3 polypeptide, and a Csy4 polypeptide (Typel-F CRISPR-Cas system); or
(vii) Cas8u2 polypeptide, a Cas7 polypeptide, and a fused Cas5 -Cas6 polypeptide (Type I-U CRISPR-Cas system). The bacteriophage of embodiment 93, wherein the Cascade complex comprises a Cas5d polypeptide, a Cas8c polypeptide, and a Cas7 polypeptide (Type I-C CRISPR- Cas system). The bacteriophage of any one of embodiments 84-94, wherein the nucleic acid sequence further comprises a promoter sequence. The bacteriophage of any one of embodiments 84-95, wherein the Staphylococcus species is killed solely by lytic activity of the bacteriophage. The bacteriophage of any one of embodiments 84-95, wherein the Staphylococcus species is killed solely by activity of the CRISPR-Cas system. The bacteriophage of any one of embodiments 84-95, wherein the Staphylococcus species is killed by lytic activity of the bacteriophage in combination with activity of the CRISPR-Cas system. The bacteriophage of embodiment 98, wherein the Staphylococcus species is killed by the activity of the CRISPR-Cas system, independently of the lytic activity of the bacteriophage. . The bacteriophage of embodiment 99, wherein the activity of the CRISPR-Cas system supplements or enhances the lytic activity of the bacteriophage. . The bacteriophage of embodiment 99, wherein the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic. . The bacteriophage of any one of embodiments 98-101, wherein 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 of any one of embodiments 84-102, wherein the bacteriophage infects multiple bacterial strains. . The bacteriophage of any one of embodiments 84-103, wherein the bacteriophage is an obligate lytic bacteriophage. . The bacteriophage of any one of embodiments 84-104, wherein the bacteriophage is a temperate bacteriophage that is rendered lytic. . The bacteriophage of embodiment 105, wherein the temperate bacteriophage is rendered lytic by the removal, replacement, or inactivation of a lysogeny gene.. The bacteriophage of any one of embodiments 84-106, wherein the nucleic acid sequence is inserted into a non-essential bacteriophage gene. . A pharmaceutical composition comprising:
(a) the bacteriophage of any one of embodiments 84-107; and
(b) a pharmaceutically acceptable excipient. . The pharmaceutical composition of embodiment 108, wherein the pharmaceutical composition comprises at least two bacteriophage. . The pharmaceutical composition of embodiment 108 or 109, wherein the pharmaceutical composition is in the form of a tablet, a capsule, a liquid, a syrup, an oral formulation, an intravenous formulation, an intranasal formulation, an ocular formulation, an otic formulation, a subcutaneous formulation, a topical formulation, a transdermal formulation, a transmucosal formulation, an inhalable respiratory formulation, a suppository, and any combination thereof. . A method of killing an Staphylococcus species comprising introducing into the target bacterium a bacteriophage comprising a nucleic acid sequence encoding a Type I CRISPR-Cas system comprising:
(a) a CRISPR array comprising a spacer sequence complementary to target nucleotide sequence in the Staphylococcus species;
(b) a Cascade polypeptide; and
(c) a Cas3 polypeptide. . The method of any one of embodiments 111, wherein the CRISPR array further comprises at least one repeat sequence. . The method of embodiment 112, wherein the at least one repeat sequence is operably linked to the spacer sequence at either its 5’ end or its 3 ’ end. . The method of any one of embodiments 111-112, wherein the CRISPR array comprises at least about 90% sequence identity to a sequence as set forth in Fig. 1.. The method of any one of embodiments 111-114, wherein the target nucleotide sequence comprises a coding sequence. . The method of any one of embodiments 111-114, wherein the target nucleotide sequence comprises a non-coding or intergenic sequence. . The method of any one of embodiments 111-114, wherein the target nucleotide sequence comprises all or a part of a promoter sequence. . The method of any one of embodiments 111-114, wherein the target nucleotide sequence comprises all or a part of a nucleotide sequence located on a coding strand of a transcribed region of an essential gene. . The method of embodiment 118, wherein the essential gene is t sf, acpP, gap A, infA, secY, csrA, trmD, ftsA, fiisA, glyQ, eno, nusG, dnaA, dnaS, pheS, rplB, gltX, hisS, rplC, aspS, gyrB, glnS, dnaE, rpoA, rpoB, pheT, infB, rpsC, rplF, alaS, leuS, serS, rplD, gyrA, glmS,fus, adk, rpsK, rplR, ctrA,parC, tRNA-Ser, tRNA-Asn, or metK. . The method of any one of embodiments 111-119, wherein the Cascade polypeptide forms a Cascade complex of a Type I-A CRISPR-Cas system, a Type I-B CRISPR-Cas system, a Type I-C CRISPR-Cas system, a Type I-D CRISPR-Cas system, a Type I-E CRISPR-Cas system, or a Type I-F CRISPR-Cas system. . The method of embodiment 120, wherein the Cascade complex comprises:
(i) a Cas7 polypeptide, a Cas8al polypeptide or a Cas8a2 polypeptide, a Cas5 polypeptide, a Csa5 polypeptide, a Cas6a polypeptide, a Cas3' polypeptide, and a Cas3” polypeptide having no nuclease activity (Type I-A CRISPR-Cas system);
(ii) a Cas6b polypeptide, a Cas8b polypeptide, a Cas7 polypeptide, and a Cas5 polypeptide (Type I-B CRISPR-Cas system);
(iii) a Cas5d polypeptide, a Cas8c polypeptide, and a Cas7 polypeptide (Type I-C CRISPR-Cas system);
(iv) a CaslOd polypeptide, a Csc2 polypeptide, a Cscl polypeptide, a Cas6d polypeptide (Type I-D CRISPR-Cas system);
(v) a Csel polypeptide, a Cse2 polypeptide, a Cas7 polypeptide, a Cas5 polypeptide, and a Cas6e polypeptide (Type I-E CRISPR-Cas system); (vi) a Csy 1 polypeptide, a Csy2 polypeptide, a Csy3 polypeptide, and a Csy4 polypeptide (Typel-F CRISPR-Cas system); or
(vii) a Cas8u2 polypeptide, a Cas7 polypeptide, and a fused Cas5 -Cas6 polypeptide (Typel-U CRISPR-Cas system). . The method of embodiment 121, wherein the Cascade complex comprises a Cas5d polypeptide, a Cas8c polypeptide, and a Cas7 polypeptide (Type I-C CRISPR- Cas system). . The method of any one of embodiments 111-122, wherein the nucleic acid sequence further comprises a promoter sequence. . The method of any one of embodiments 122-123 , wherein the Staphylococcus species is killed solely by activity of the CRISPR-Cas system. . The method of any one of embodiments 122-123, wherein the Staphylococcus species is killed by lytic activity of the bacteriophage in combination with activity of the CRISPR-Cas system. . The method of embodiment 121, wherein the Staphylococcus species is killed by the activity of the CRISPR-Cas system, independently of the lytic activity of the bacteriophage. . The method of embodiment 121, wherein the activity of the CRISPR-Cas system supplements or enhancesthe lytic activity of the bacteriophage. . The method of embodiment 121, wherein the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic. . The method of any one of embodiments 126-128, wherein the lytic activity of the bacteriophage, the activity of the CRISPR-Cas system, or both, is modulated by a concentration of the bacteriophage. . The method of any one of embodiments 111-129, wherein the bacteriophage infects multiple bacterial strains. . The method of any one of embodiments 111-130, wherein the bacteriophage is an obligate lytic bacteriophage. . The method of any one of embodiments 111-130, wherein the bacteriophage is a temperate bacteriophage that is rendered lytic. . The method of embodiment 132, wherein the temperate bacteriophage is rendered lytic by the removal, replacement, or inactivation of a lysogeny gene. . The method of any one of embodiments 111-134, wherein the nucleic acid sequence is inserted in pace of or adjacent to a non-essential bacteriophage gene. 135. The method of any one of embodiments 111-135, wherein a mixed population of bacterial cells comprises the Staphylococcus species.
136. A bacteriophage derived from a temperate bacteriophage that has been rendered lytic, wherein the bacteriophage has been rendered lytic by removal, replacement, or inactivation of a lysogenic gene.
137. The bacteriophage of embodiment 136, wherein the bacteriophage has been rendered lytic by removal of a var009 region.
138. The bacteriophage of embodiment 136, wherein the bacteriophage has been rendered lytic by removal of a varOlO region.
139. The bacteriophage of embodiment 136, wherein the bacteriophage has been rendered lytic by the removal of a varO 12 region.
140. The bacteriophage of embodiment 136, wherein the bacteriophage has been rendered lytic by the removal of a var042 region.
141. The bacteriophage of embodiment 136, wherein the bacteriophage has been rendered lytic by the removal of a regulatory element of a lysogeny gene.
142. The bacteriophage of embodiment 136, wherein the bacteriophage has been rendered lytic by the removal, alteration or replacement of a promoter of a lysogeny gene.
143. The bacteriophage of embodiment 136, wherein the bacteriophage has been rendered lytic by the removal of a functional element of a lysogeny gene.
DEFINITIONS
[0206] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description of the disclosure herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure.
[0207] Unless the context indicates otherwise, it is specifically intended that the various features of the disclosure described herein are able of being used in any combination. Moreover, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein are excluded or omitted. To illustrate, if the specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, are omitted and disclaimed singularly or in any combination. [0208] One of skill in the art will understand the interchangeability of terms designating the various CRISPR-Cas systems and their components due to a lack of consistency in the literature and an ongoing effort in the art to unify such terminology. Likewise, one of skill in the art will also understand the interchangeability of terms designating the various anti- CRISPR proteins due to a lack of consistency in the literature and an ongoing effort in the art to unify such terminology.
[0209] As used in the description and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").
[0210] The term “about” as used herein when referring to a measurable value such as a dosage or time period and the like refers to variations of ± 20%, ± 10%, ± 5%, ± 1%, + 0.5%, or even ± 0.1% of the specified amount. As used herein, phrases such as “between X and Y” and “between about X and Y” should be interpreted to include X and Y. As used herein, 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.”
[0211] The term “comprise”, “comprises”, and “comprising”, “includes”, “including”, “have” and “having”, as used herein, specify the presence of the stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof.
[0212] As used herein, the 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. Thus, the term "consisting essentially of’ when used in a claim of this disclosure is not intended to be interpreted to be equivalent to “comprising.”
[0213] The term “consists of’ and “consisting of’, as used herein, excludes any features, steps, operations, elements, and/or components not otherwise directly stated. The use of "consisting of" limits only the features, steps, operations, elements, and/or components set forth in that clause and does exclude other features, steps, operations, elements, and/or components from the claim as a whole.
[0214] As used herein, “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). [0215] “Complement” as used herein means 100% complementarity or identity with the comparator nucleotide sequence or it means less than 100% complementarity (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%, and the like, complementarity). Complement or complementable may also be used in terms of a “complement” to or “complementing” a mutation.
[0216] The terms “complementary” or “complementarity”, as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base- pairing. For example, the 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.
[0217] As used herein, 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, functional 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.
[0218] As used herein, a “target nucleotide sequence” refers to the portion of a target gene that is complementary to the spacer sequence of the recombinant CRISPR array.
[0219] As used herein, a “target nucleotide sequence” refers to the portion of a target gene (i.e., target region in the genome or the “protospacer sequence,” which is adjacent to a protospacer adjacent motif (PAM) sequence) 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. [0220] As used herein, the term “protospacer adjacent motif’ or “PAM” refers to a DNA sequence present on the target DNA molecule adjacent to the nucleotide sequence matching the spacer sequence. 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 exact PAM sequence that is required varies between each different CRISPR-Cas system. Non-limiting examples of PAMs include CCA, CCT, CCG, TTC, AAG, AGG, ATG, GAG, and/or CC. In some instances, in Type I systems, 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, and is directly recognized by Cascade. In some instances, for B. halodurans Type I-C systems, the PAM is YYC, where Y can be either T or C. In some instances, for the L. monocytogenes Type I-B system, the PAM is CCW, where W can be A or T. Once a cognate protospacer and PAM are recognized, Cas3 is recruited, which then cleaves and degrades the target DNA. For Type II systems, 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 atthe C-terminusof Cas9). [0221] As used herein, type I Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRj-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. These polypeptides include, but are not limited to, the Cascade polypeptides of type I subtypes I-A, I-B, I-C, I-D, I-E, I-F, and I-U. 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 I-B polypeptides include Cas6b, Cas8b (Cshl), Cas7 (Csh2) and/or Cas5. Non-limiting examples of type I-C polypeptides include Cas5d, Cas8c (Csdl), and/or Cas7 (Csd2). Non-limiting examples of type I-D polypeptides include Casl0d(Csc3), Csc2, Cscl, and/or Cas6d. Non -limiting examples of type I-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). Non -limiting examples of type I-F polypeptides include Cas8u2, Cas7, and/or fused Cas5 -Cas6 polypeptide. Non -limiting examples of type I-U polypeptides include Cas8al (Cstl), Cas7 (Cst2), Cas5 (Cst5t), and Cas3. In some embodiments, a recombinant nucleic acid described herein comprises, consists essentially of, or consists of, a nucleotide sequence encoding a subset of type-I 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.
[0222] 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. In a recombinant CRISPR array, the combination of repeat sequences and spacer sequences is synthetic, made by man and not found in nature. In some embodiments, 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.
[0223] As used herein, “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). 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.
[0224] 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.
[0225] As used herein, the term “CRISPR phage”, “CRISPR enhanced phage”, and “crPhage” refers to a bacteriophage particle comprising bacteriophage DNA comprising at least one heterologous polynucleotide that encodes at least one component of a CRISPR-Cas system (e.g., CRISPR array, crRNA; e.g., PI bacteriophage comprising an insertion of a targeting crRNA). In some embodiments, the polynucleotide encodes at least one transcriptional activator of a CRISPR-Cas system. In some embodiments, the polynucleotide encodes at least one component of an anti-CRISPR polypeptide of a CRISPR-Cas system. [0226] As used herein, 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 algorithms or by visual inspection. In some embodiments, substantial identity refer to two or more sequences or sub sequences that have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95, 96, 97, 98, or 99% identity. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The 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.
[0227] 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. In some instances, “Percent identity” is d etermined using BLASTX version 2.0 for translated nucleotide sequences andBLASTN version 2.0 for polynucleotide sequences. [0228] In some embodiments, the recombinant nucleic acid 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. In some instances, 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. In representative embodiments, the isolated nucleic 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% pure, or purer.
[0229] 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. With respect to an infection, a disease or a condition, the term refers to a decrease in the symptoms or other manifestations of the infection, disease or condition. In some embodiments, 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.
[0230] The terms with respect to an “infection”, “a disease”, or “a condition”, used herein, refer to any adverse, negative, or harmful physiological condition in a subject. In some embodiments, the source of an “infection”, “a disease”, or “a condition”, is the presence of a target bacterial population in and/or on a subject. In some embodiments, the bacterial population comprises one or more target bacterial species. In some embodiments, the one or more bacteria species in the bacterial population comprise one or more strains of one or more bacteria. In some embodiments, the target bacterial population causes an “infection”, “a disease”, or “a condition” that is acute or chronic. In some embodiments, the target bacterial population causes an “infection”, “a disease”, or “a condition” that is localized or systemic.
In some embodiments, the target bacterial population causes an “infection”, “a disease”, or “a condition” that is idiopathic. In some embodiments, the target bacterial population causes an “infection”, “a disease”, or “a condition” that 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.
[0231] As used herein the term “biofilm” means an accumulation of microorganisms embedded in a matrix of polysaccharide. Biofilms form on solid biological or non -biological surfaces, as well as at liquid-air interfaces, and are medically important, accounting for over 80 percent of microbial infections in the body.
[0232] 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). Thus, in some embodiments, to prevent infection, food, surfaces, medical tools and devices are treated with compositions and by methods disclosed herein.
[0233] The terms “individual”, or “subject” as used herein includes any animal that has or is susceptible to an infection, disease or condition involving bacteria. Thus, in some embodiments, subjects are mammals, avians, reptiles, amphibians, fish, crustaceans, and mollusks. 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). Fish subjects include but are not limited to species used in aquaculture (e.g., tuna, salmon, tilapia, catfish, carp, trout, cod, bass, perch, snapper, and the like). Crustacean subjects include but are not limited to species used in aquaculture (e.g., shrimp, prawn, lobster, crayfish, crab). Mollusk subjects include but are not limited to species used in aquaculture (e.g., abalone, mussel, oyster, clams, scallop). In some embodiments, 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. In some embodiments, a subject is a human.”
[0234] As used herein, the term “isolated: in context of a nucleic acid sequence is a nucleic acid sequence that exists apart from its native environment. [0235] As used herein, “pharmaceutically acceptable” means a material that is not biologically or otherwise undesirable, i.e., the materials are administered to a subject without causing any undesirable biological effects such as toxicity.
[0236] As used herein, the term “in vivo” is used to describe an event that takes place in a subject’s body.
[0237] As used herein, the term “in vitro” is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell- based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.
EXAMPLES
[0238] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1: Engineered phage used in this application
[0239] Recombinant bacteriophage were engineered to contain a genetic deletion in the predicted lysogeny module region. Fig. 1 depicts the predicted lysogeny region of phage pi 473. Further, Fig. 1 depicts open reading frames encoding putative repressor and anti repressor proteins and an open reading frame encoding a putative integrase. The first recombinant phage contains a genetic deletion that was introduced into the predicted lysogeny module region of the bacteriophage genome in the region including a putative anti repressor, and two clones were isolated and designated Var009 and VarOlO. A second recombinant phage contains a genetic deletion that was introduced into the predicted lysogeny module region of the bacteriophage genome in the region including a putative repressor and a single clone was isolated and designated VarO 12. A final recombinant phage (Var042) was isolated that contains the deletion from VarO 12 and a second deletion removing the predicted integrase gene. Two additional control variants were generated with different deletions outside of the lysogeny region as controls (not shown), isolated and designated Var002 and Var006, respectively. Fig. 6 depicts the sequence of the VarOlO deletion. Fig. 7 depicts the sequence of the VarOl 2 deletion, which is also the first deletion in Var042. Fig. 8 depicts the second deletion in Var042. Example 2: Engineered phage variants exhibit lytic phenotype in S. aureus
[0240] Fig. 2 depicts the dilution series of wild-type (WT) pi 473 and several variants plated on a lawn of S. aureus using the double agar overlay method. As depicted, the WT phage and variants Var002 and Var006 produced small hazy plaques, while variants Var009, VarOlO and VarO 12 produced larger clearer plaques. Variants Var002 and Var006 contain mutations outside of the lysogeny region. The variants with mutants outside the lysogeny region do not show changes in plaque morphology indicating that these phage variants remain temperate. Var009, VarOlO and VarO 12, with mutations in the lysogeny region show clearer plaques and higher efficiency of plaquing on the strain shown. These data indicate that Var009, VarOlO and VarO 12 were successfully converted from lysogenic to lytic phenotype, as clear plaque morphology is a hallmark of lytic bacteriophages in S. aureus (References Mi tarai etal J. Bacteriol. 2016. doi: 10.1128/JB.00965-15, Garcia et al. Appl. Environ. Microbiol. 2009. doi: 10.1128/ AEM.01864-09) and is a known phenomenon in other examples of lysogenic phages that have been genetically converted to obligately lytic. The ability of VarOlO and VarO 12 to plaque at higher efficiency (i.e. lower down the plate, when the phage is more dilute) may indicate that the modified phage is no longer susceptible to interference from the endogenous prophage present in the bacterial strain, as previously observed for other phages following conversion from temperate to obligate lytic (Reference: Zhang etal. Microbiol. 2013. doi: 10.1099/mic.0.067116-0). Fig. 3 depicts a close-up image of Fig. 2, showing the larger plaque morphology for wild-type pl473, VarOlO and Var012. In Fig. 3, the arrows pointto individual phage plaques. As depicted, VarOlO and Var012 plaques display a clearer morphology compared to wild-type. Additionally, the zone of clearing with too many plaques to count is clearer in VarOlO and VarO 12 than the WT phage.
Example 3: Bacteriophage killing assay confirms lysogenic to lytic phenotype conversion
[0241] Fig. 4 depicts the bacteriophage killing assay with strain b4063 (USA300 strain FPR3757) in LB challenged with Wild-type pi 473, Var012, or Var042 as measured by bacterial counts as a function of time. For the bacteriophage killing assay with strain b4063 (USA300 strain FPR3757), bacteria were inoculated into LB in triplicate. For cells only, nothing else was added. For the other phage, Wild-type pi 473, VarO 12 or Var042 were added at an MOI of 1. Bacterial CFUs were enumerated at 0, 2, 4, 6 and 24 h post inoculation. As depicted in Fig. 4, at the 24 hr time point, the bacterial counts had recovered in the pi 473 WT treated bacteria, but the two variants showed sustained killing to limit of detection (LOD). Temperate phages often show quick rebound due to the outgrowth of lysogens which are resistant to infection by the phage. The lack of rebound in Var012 and Var042 is consistent with the inability of these variants to form lysogens.
Example 4: Comparison of rebound on S. aureus clinical isolates [0242] Fig. 5 depicts growth curves of three S. aureus clinical isolates (b2991, b3022 and b3202) in LB challenged with pi 473 WT or pi 473 Var012. Growth of the clinical isolates in LB was monitored by taking the OD at 600 nm periodically over a 24 hr time course. Control drowth curves are uninfected culture controls for each strain, while test growth curves are for cultures challenged with WT or VarO 12 at an MOI of 1. Arrows indicate regrowth of the clinical isolates in the WT challenged cultures while the VarO 12 challenged cultures remain suppressed out to 24 hours. Overall, the experimental comparison of the three S. aureus clinical isolates (b2991, b3022 andb3202) in LB challenged with pi 473 WT or pi 473 VarO 12 shows more regrowth in strains challenged with WT phage, with b2991 showing the best recovery over a 24 hr time course.
Example 5. Phage lytic activity when engineered with CRISPR-Cas full construct
[0243] Top agar overlays are prepared by mixing 100 pL of a saturated overnight culture of b4063 with 6 mL of 0.375% agar in LB containing 10 mMMgCh and 10 mMCaC^. After the top agar solidified, 2 pL drops of serial 10 -fold dilution series of pi 473 wt (wild type) and p 1473 -Cas (Cas system only) and p 1473 -crArray (targeting CRISPR Array + Cas system) are spotted onto the surface of the top agar. Plates are incubated at 37°C for ~18h, then imaged using a Keyence BZ-X800 microscope at 4X and 1 OX magnification.
Example 6: Engineering of a lytic bacteriophage
[0244] Bacteriophage was engineered with a Type IB CRISPR-Cas system (LMIB). The engineered phage lysate was spotted onto a bacterial overlay in order to obtain isolated (clonal) plaques. Seven plaques were picked and screened by PCR for the presence of the desired insert. Each plaque was screened using two pairs of PCR primers, with one pair covering the upstream engineering junction (i.e., the site where the wild type phage genome meets the engineered insert) and a significant portion of the insert and one pair covering the downstream engineering junction and a significant portion of the insert. Since one primer from each primer pair binds within the insert, un-engineered phages will produce no PCR bands. In FIG. 9, L designates a DNA size ladder, the numbers 1 -7 indicate individual clonal plaques being screened, and a and b indicate the two primer pairs. Both primer pairs produced bands of the expected sizes for all plaques, indicating that all plaques were successfully engineered with the LMIB insert (SEQ ID NO: 23).
[0245] After engineering of the three promoter variants of InqQ (SEQ NO: 49), P cat-lnqQ (SEQ ID NOS: 16, 49), PSarA -InqQ (SEQ ID NOS: 17, 49), or P TmpG-lnqQ (SEQ ID NOS: 18, 49), into Kay virus phage, polymerase chain reaction (PCR) was used to confirm insertion of the promoter + InqQ into the phages. This PCR reaction used one primer upstream of the insertion site and one primer in the inserted sequence. The results are depicted in FIG. 10. Sequencing of the phage DNA confirmed insertion of the variants in the phage.
[0246] Clinical isolate b2655 was inoculated with either wild -type (WT) phage (squares), cat-lnqQ, PsarA -InqQ, or PTmpG -InqQ. For each sample, three bacterial replicates were inoculated with phage at a multiplicity of infection of 1. Cultures were grown at 37°C in a shaking plate reader with optical density at 600 nm measured every 10 minutes for 18 hours. The results are depicted in FIG. 11. Phages with InqQ reduced the growth of the bacteria compared to WT phage starting at around 80 minutes and persisting throughout the experiment.
Example 7: An engineered bacteriophage comprising DNAse
Figure imgf000090_0001
[0247] A Kayvirusbacteriophage was engineered to contain DNAse I (SEQ NO: 1) and the Eca/ promoter (SEQ ID NO: 16). The DNA sequence encoding mature human DNase was modified to optimize codon usage and remove sequences not tolerated by the phage while maintaining the amino acid identity. This sequence was placed downstream of the Peat promoter. The Pcat-DNase I sequence was engineered into the phage and the sequence was confirmed by Illumina next generation sequencing of the engineered phage genome. The results are depicted in FIG. 12.
Example 8: Host range of a bacteriophage cocktail
[0248] A 308 strain panel of clinical isolates of S. aureus was used to test the host range of a bacteriophage cocktail. The S. aureus strains used were b004604,b004605,b004606, b004607,b004608,b004609,b004610,b004611,b004612,b004613,b004614,b004615, b004616,b004617,b004618,b004619,b004620,b004621,b004622,b004623,b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631 , b004632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641 , b004642, b004643,b004644, b004645, b004646, b004647, b004648, b004649, b004650, b004651 , b004652, b004653,t>004654, b004655, b004656, b004657, b004658, b004659, b004660, b004661, b004662, b004663, b004664, b004665, b004666, b004667, b004668, b004669, b004670, b004671 , b004672, b004673, b004674, b004675, b004676, b004677, b004678, b004679, b004680, b004681 , b004682, b004683, b004684, b004685, b004686, b004687, b004688, b004689, b004690, b004691 , b004692, b004693, b004694, b004695, b004696, b004697, b004698, b004699, b004700, b004701 , b004702, b004703, b004704, b004705, b004706, b004707, b004708, b004709, b004710, b004711 , b004712, b004713 , b004714, b004715,b004716,b004717,b004718,b004719,b004720,b004721,b004722,b004723, b004724, b004725,b004726, b004727, b004728, b004729, b004730, b004731 , b004732, b004733,b004734, b004735, b004736, b004737, b004738, b004739, b004740, b004741 , b004742, b004743,b004744, b004745, b004746, b004747, b004748, b004749, b004750, b004751 , b004752, b004753, b004754, b004755, b004756, b004757, b004758, b004759, b004760, b004761 , b004762, b004763, b004764, b004765, b004766, b004767, b004768, b004769, b004770, b004771 , b004772, b004773, b004774, b004775, b004776, b004777, b004778, b004779, b004780, b004781 , b004782, b004783, b004784, b004785, b004786, b004787, b004788, b004789, b004790, b004791 , b004792, b004793, b004794, b004795, b004796, b004797, b004798, b004799, b004800, b004801 , b004802, b004803, b004804, b004805,b004806, b004807, b004808, b004809, b004810, b004811 , b004812, b004813, b004814,b004815,b004816,b004817,b004818,b004819,b004820,b004821,b004822, b004823,b004824, b004825, b004826, b004827, b004828, b004829, b004830, b004831, b004832,b004833,b004834,b004835,b004836,b004837,b004838,b004839,b004840, b004841, b004842, b004843, b004844, b004845, b004846, b004847, b004848, b004849, b004850,b004851,b004852,b004853,b004854,b004855,b004856,b004857,b004858, b004859, b004860, b004861 , b004862, b004863, b004864, b004865, b004866, b004867, b004868, b004869, b004870, b004871 , b004872, b004873, b004874, b004875, b004876, b004877, b004878, b004879, b004880, b004881 , b004882, b004883, b004884, b004885, b004886, b004887, b004888, b004889, b004890, b004891 , b004892, b004893, b004894, b004895,b004896, b004897, b004898, b004899, b004900, b004901 , b004902, b004903, b004904, b004905, b004906, b004907, b004908, b004909, b004910, b004911 . Results are depicted in Table 1. The ability of Kayviruses, aPhietavirus, and aRosenblumvirus to target the 308 strains of the panel was tested. As can be seen in FIG. 13, for a majority of strains tested, at least two phage infect a particular target bacteria, reducing the likelihood that a target bacteria would be able to evolve resistance against a particular phage and evade infection by another bacteriophage. Table 1: Results of Host Range Assay
Figure imgf000092_0001
[0249] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicingthe invention. It is intended thatthe following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.
SEQUENCES
Figure imgf000093_0001
Figure imgf000093_0002
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001

Claims

1. A composition comprising a Phietavirus bacteriophage and Rosenblumvirus bacteriophage, Phietavirus bacteriophage and Kay virus bacteriophage, Rosenblumvirus bacteriophage and Kayvirus bacteriophage, or Phietavirus bacteriophage, Rosenblumvirus bacteriophage, and Kayvirus bacteriophage.
2. The composition of claim 1, comprisingthe Phietavirus bacteriophage, wherein the Phietavirus bacteriophage is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene.
3. The composition of claim 2, wherein the lysogenic gene encodes for a repressor.
4. The composition of any one of claims 1 -3, comprisingthe Phietavirus bacteriophage and Rosenblumvirus bacteriophage.
5. The composition of any one of claims 1-3, comprisingthe Phietavirus bacteriophage and Kayvirus bacteriophage.
6. The composition of any one of claims 1 -3, comprisingthe Rosenblumvirus bacteriophage and Kayvirus bacteriophage.
7. The composition of any one of claims 1 -3, comprisingthe Phietavirus bacteriophage, Rosenblumvirus, and Kayvirus bacteriophage.
8. The composition of any one of claims 1 -7, comprisingthe Kayvirus bacteriophage, wherein the Kayvirus bacteriophage comprises two or more Kayvirus bacteriophage.
9. The composition of any one of claims 1 -8, wherein the bacteriophage of the composition infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria.
10. A composition comprising a plurality of bacteriophage comprising a first bacteriophage and a second bacteriophage, wherein the plurality of bacteriophage infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria.
11. The composition of claim 10, wherein the Staphylococcus bacteria are selected from b004604, b004605, b004606, b004607, b004608, b004609, b004610, b004611 , b004612,b004613,b004614,b004615,b004616,b004617,b004618,b004619, b004620, b004621,b004622, b004623, b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631, b004632, b004633, b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641 , b004642, b004643, b004644, b004645, b004646, b004647, b004648, b004649, b004650, b004651 , b004652, b004653, b004654, b004655, b004656, b004657, b004658, b004659, b004660, b004661,b004662, b004663, b004664, b004665, b004666, b004667, b004668, t>004669, b004670, b004671, b004672, b004673, b004674, b004675, b004676, b004677, b004678, b004679, b004680, b004681 , b004682, b004683, b004684, b004685, b004686, b004687, b004688, b004689, b004690, b004691 , b004692, b004693, b004694, b004695, b004696, b004697, b004698, b004699, b004700, b004701,b004702, b004703, b004704, b004705, b004706, b004707, b004708,b004709,b004710,b004711,b004712,b004713,b004714,b004715, b004716, b004717, b004718, b004719, b004720, b004721 , b004722, b004723, b004724, b004725, b004726, b004727, b004728, b004729, b004730, b004731 , b004732, b004733, b004734, b004735, b004736, b004737, b004738, b004739, b004740, b004741,b004742, b004743, b004744, b004745, b004746, b004747, b004748, b004749, b004750, b004751, b004752, b004753, b004754, b004755, b004756, b004757, b004758, b004759, b004760, b004761 , b004762, b004763, b004764, b004765, b004766, b004767, b004768, b004769, b004770, b004771 , b004772, b004773, b004774, b004775, b004776, b004777, b004778, b004779, b004780, b004781,b004782, b004783, b004784, b004785, b004786, b004787, b004788, b004789, b004790, b004791, b004792, b004793, b004794, b004795, b004796, b004797, b004798, b004799, b004800, b004801 , b004802, b004803, b004804, b004805, b004806, b004807, b004808, b004809, b004810, b004811 , b004812,b004813,b004814,b004815,b004816,b004817,b004818,b004819, b004820, b004821,b004822, b004823, b004824, b004825, b004826, b004827, b004828, b004829, b004830, b004831, b004832, b004833, b004834, b004835, b004836, b004837, b004838, b004839, b004840, b004841 , b004842, b004843, b004844, b004845, b004846, b004847, b004848, b004849, b004850, b004851 , b004852,b004853,b004854,b004855,b004856,b004857,b004858,b004859, b004860, b004861,b004862, b004863, b004864, b004865, b004866, b004867, b004868, b004869, b004870, b004871, b004872, b004873, b004874, b004875, b004876, b004877, b004878, b004879, b004880, b004881 , b004882, b004883, b004884,b004885,b004886,b004887,b004888,b004889,b004890,b004891, b004892, b004893, b004894, b004895, b004896, b004897, b004898, b004899, b004900, b004901,b004902, b004903, b004904, b004905, b004906, b004907, b004908, b004909, b004910, and b004911.
12. The composition of claim 10 or claim 11, wherein the Staphylococcus bacteria comprises Staphylococcus aureus.
13. The composition of any one of claims 10-12, wherein the infectivity is determined with a plaque assay or growth inhibition assay.
14. The composition of any one of claims 10-13, wherein the at least about 90% is at least about 95%.
15. The composition of any one of claims 10-13, wherein the at least about 90% is at least about 98%.
16. The composition of any one of claims 10-13, wherein the at least about 90% is at least about 99%.
17. The composition of any oneof claims 10-16, wherein the at least about 30 Staphylococcus bacteria comprises b004604, b004605, b004606, b004607, b004608, b004609,b004610,b004611,b004612,b004613,b004614,b004615,b004616, b004617, b004618, b004619, b004620, b004621, b004622, b004623, b004624, b004625, b004626, b004627, b004628, b004629, b004630, b004631 , b004632, b004633,b004634, b004635, b004636, b004637, b004638, b004639, b004640, b004641, b004642, b004643, b004644, b004645, b004646, b004647, b004648, b004649, b004650, b004651 , b004652, b004653, b004654, b004655, b004656, b004657, b004658, b004659, b004660, b004661, b004662, b004663, b004664, b004665, b004666, b004667, b004668, b004669, b004670, b004671 , b004672, b004673, b004674, b004675, b004676, b004677, b004678, b004679, b004680, b004681, b004682, b004683, b004684, b004685, b004686, b004687, b004688, b004689, b004690, b004691, b004692, b004693, b004694, b004695, b004696, b004697, b004698, b004699, b004700, b004701, b004702, b004703, b004704, b004705, b004706, b004707, b004708, b004709, b004710, b004711 , b004712, b004713,b004714,b004715,b004716,b004717,b004718,b004719,b004720, b004721, b004722, b004723, b004724, b004725, b004726, b004727, b004728, b004729, b004730, b004731, b004732, b004733, b004734, b004735, b004736, b004737, b004738, b004739, b004740, b004741, b004742, b004743, b004744, b004745, b004746, b004747, b004748, b004749, b004750, b004751 , b004752, b004753,b004754, b004755, b004756, b004757, b004758, b004759, b004760, b004761, b004762, b004763, b004764, b004765, b004766, b004767, b004768, b004769, b004770, b004771, b004772, b004773, b004774, b004775, b004776, b004777, b004778, b004779, b004780, b004781, b004782, b004783, b004784, b004785, b004786, b004787, b004788, b004789, b004790, b004791 , b004792, b004793,b004794, b004795, b004796, b004797, b004798, b004799, b004800, b004801, b004802, b004803, b004804, b004805, b004806, b004807, b004808, b004809,b004810,b004811,b004812,b004813,b004814,b004815,b004816, b004817, b004818, b004819, b004820, b004821, b004822, b004823, b004824, b004825, b004826, b004827, b004828, b004829, b004830, b004831 , b004832, b004833,b004834,b004835,b004836,b004837,b004838,b004839,b004840, b004841, b004842, b004843, b004844, b004845, b004846, b004847, b004848, b004849, b004850,b004851,b004852,b004853,b004854,b004855,b004856, b004857,b004858,b004859,b004860,b004861,b004862,b004863,b004864, b004865, b004866, b004867, b004868, b004869, b004870, b004871 , b004872, b004873,b004874, b004875, b004876, b004877, b004878, b004879, b004880, b004881,b004882, b004883, b004884, b004885, b004886, b004887, b004888, b004889, b004890, b004891, b004892, b004893, b004894, b004895, b004896, b004897, b004898, b004899, b004900, b004901, b004902, b004903, b004904, b004905, b004906, b004907, b004908, b004909, b004910, and b004911.
18. The composition of any oneof claims 10-17, wherein the first bacteriophage and the second bacteriophage are of different genera.
19. The composition of any oneof claims 10-18, wherein the plurality of bacteriophage comprise a Phietavirus bacteriophage and Rosenblum virus bacteriophage, Phietavirus bacteriophage and Kayvirus bacteriophage, Rosenblum virus bacteriophage and Kayvirus bacteriophage, or Phietavirus bacteriophage, Rosenblumvirus bacteriophage, and Kayvirus bacteriophage.
20. The composition of any oneof claims 10-19, wherein the plurality of bacteriophage comprise a Phietavirus.
21. The composition of claim 20, wherein the Phietavirus is engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene.
22. The composition of claim 21 , wherein the lysogenic gene encodes for a repressor.
23. A composition comprising a plurality of bacteriophage comprising a first bacteriophage that is specific for a first receptor of a Staphylococcus bacteria, a second bacteriophage that is specific for a second receptor of the Staphylococcus bacteria, wherein the plurality of bacteriophage is more resilient to resistance by the Staphylococcus bacteria than the first or second bacteriophage alone.
24. The composition of claim 23, wherein the first receptor and the second receptor are different.
25. The composition of claim 23 or claim 24, wherein the first bacteriophage comprises a Phietavirus.
26. The composition of claim 23 or claim 24, wherein the first bacteriophage comprises a Phietavirus and aRosenblumvirus.
27. The composition of any one of claims23-26, wherein the second bacteriophage comprises a Kayvirus.
28. The composition of claim 23 or claim 24, wherein the plurality of bacteriophage comprise: a Phietavirus bacteriophage andRosenblumvirus bacteriophage, Phietavirus bacteriophage and Kayvirus bacteriophage, Rosenblum virus bacteriophage and Kayvirus bacteriophage, or Phietavirus bacteriophage, Rosenblumvirus bacteriophage, and Kayvirus bacteriophage.
29. The composition of any one of claims23 -28, wherein the plurality of bacteriophage of the composition infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria.
30. The composition of any one of claims23-29, wherein the first bacteriophage and the second bacteriophage are of different genera.
31. The composition of claim 30, wherein the first bacteriophage and the second bacteriophage are capable of independently infecting at least 90% of the collection of Staphylococcus bacteria.
32. A Phietavirus bacteriophage engineered to remove, replace, or inactivate a lysogenic gene or a promoter of a lysogenic gene.
33. The bacteriophage of claim 32, wherein the lysogenic gene encodes for a repressor.
34. The bacteriophage of claim 33, wherein the repressor comprises an amino acid sequence at least about 80% identical to SEQ ID NO: 47 or 48.
35. The bacteriophage of any one of claims 32-34, wherein removal of the lysogenic gene comprises removing from about 1% to 100% of the lysogenic gene.
36. The bacteriophage of claim 35, wherein about 10 to about 1,200 base pairs of the lysogenic gene are removed.
37. The bacteriophage of any one of claims 32-36, wherein the lysogenic gene is removed, replaced, or inactivated.
38. The bacteriophage of claim 37, wherein the lysogenic gene is removed.
39. The bacteriophage of any one of claims 32-38, wherein the promoter of the lysogenic gene is removed, replaced, or inactivated.
40. The bacteriophage of any one of claims 32-39, further comprising a Rosenblumvirus.
41. The bacteriophage of any one of claims 32-40, further comprising a Kay virus.
42. The bacteriophage of any one of claims 32-41, comprising a plurality of bacteriophage comprising the Phietavirus bacteriophage, wherein the plurality of bacteriophage infect at least about 90% of a collection of at least about 30 Staphylococcus bacteria.
43. The composition of any one of claims 1 -31 , or the bacteriophage of any one of claims 32-42, comprising a nucleic acid encoding for an exogenous peptide selected from TreA, Lpi, DNAsel, RIP, FS3, PLNC8a, PLNC8p, LytM, LnqO, 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, lyase, glycosyl hydroxylase, polyglucosamine (PGA) depolymerases, colonic acid depolymerase, 1,4- L-fucodise hydrolase, colanic acid, or depolymerazing alginase .
44. The composition of any one of claims 1 -31, or the bacteriophage of any one of claims 32-42, comprising one or more components of a CRISPR-Cas system.
45. The composition or bacteriophage of claim 44, wherein the CRISPR-Cas system is a Type IB CRISPR-Cas system from Listeria monocytogenes (LMIB).
46. The composition of any one of claims 1 -31, or the bacteriophage of any one of claims 32-42, comprising a spacer sequence or a crRNA transcribed therefrom, wherein the spacer sequence is complementary to a target nucleic acid sequence from a target gene in a target bacteria.
47. The composition or bacteriophage of claim 46, wherein the target bacteria comprises a Staphylococcus bacteria.
48. A bacteriophage comprising a nucleic acid encoding an exogenous peptide selected from TreA, Lpi, DNAsel, RIP, FS3, PLNC8a, PLNC8p, LytM, LnqO, 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, lyase, glycosyl hydroxylase, polyglucosamine (PGA) depolymerases, colonic acid depolymerase, 1,4- L-fucodise hydrolase, colanic acid, or depolymerazing alginase .
49. A bacteriophage comprising a Type IB CRISPR-Cas system from Listeria monocytogenes (LMIB).
50. The bacteriophage of claim 48, wherein the bacteriophage further comprises one or more components of a CRISPR-Cas system.
51. The bacteriophage of claim 50, wherein the CRISPR-Cas system is a Type IB CRISPR-Cas system from Listeria monocytogenes (LMIB).
52. The bacteriophage of claim 49 or claim 51 , wherein the LMIB encodes for a sequence at least 80% identical to any one of SEQ ID NOS: 25-29.
53. The bacteriophage of any one of claims 48-52, comprising a spacer sequence or a crRNA transcribed therefrom, wherein the spacer sequence is complementary to a target nucleic acid sequence from a target gene in a target bacteria.
54. The bacteriophage of claim 53, wherein the target bacteria comprises a Staphylococcus bacteria.
55. The bacteriophage of any one of claims 48-54, wherein the bacteriophage is an engineered Phietavirus.
56. The bacteriophage of any one of claims 48-54, wherein the bacteriophage is an engineered Rosenblum virus.
57. The bacteriophage of any one of claims 48-54, wherein the bacteriophage is an engineered Kay virus.
58. A composition comprising the bacteriophage of claim 55, further comprising a Rosenblumvirus and/or a Kay virus.
59. A composition comprising the bacteriophage of claim 56, further comprising a Phietavirus and/or a Kayvirus.
60. A composition comprising the bacteriophage of claim 57, further comprising a Rosenblumvirus and/or a Phietavirus.
61. A method of treating a disease or condition related to Staphylococcus, the method comprising administering to a subject in need thereof the bacteriophage of any one of claims 32-57, or the composition of any one of claims 1 -31, 43-47, or 58-60.
62. The method of claim 61, wherein Staphylococcus is causative of, and/or contributes to, the disease or condition.
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