CN117729853A

CN117729853A - Phage compositions for pseudomonas comprising a CRISPR-CAS system and methods of use thereof

Info

Publication number: CN117729853A
Application number: CN202180089302.9A
Authority: CN
Inventors: 保罗·加罗福洛; 大卫·G·奥斯特劳特; 库尔特·塞利; 汉纳·休伊特·图森; 拉娜·麦克米兰; 罗伯特·麦基; 泰勒·彭克; 肖恩·斯蒂尔; 克里斯多夫·拉德纳; 加布里埃尔·奥-哈利斯
Original assignee: Rocos Biosciences
Current assignee: Rocos Biosciences
Priority date: 2020-11-05
Filing date: 2021-11-04
Publication date: 2024-03-19
Also published as: TW202305126A

Abstract

Phage compositions comprising CRISPR-Cas systems targeting pseudomonas species and methods of use thereof are disclosed herein.

Description

Phage compositions for pseudomonas comprising a CRISPR-CAS system and methods of use thereof

Cross reference

The present application claims the benefit of U.S. provisional application No. 63/110,288, filed on 5 months 11 in 2020, and U.S. provisional application No. 63/184,728, filed on 5 months 5 in 2021, both of which are incorporated herein by reference in their entirety.

Sequence listing

The present application contains a sequence listing submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created at 2021, 10 months, 27 days, and named 53240-743_601_sl.txt, of size 71,774 bytes.

Disclosure of Invention

In certain embodiments, disclosed herein are phage (phage) compositions comprising a CRISPR-Cas system and methods of use thereof.

In certain embodiments, disclosed herein is a bacteriophage (bacterial) comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising: a CRISPR array comprising one or more spacer sequences complementary to a target nucleotide sequence in a pseudomonas species; a cascades polypeptide; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. 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 one or more spacer sequences at its 5 'end or its 3' end. In some embodiments, the repeat sequence comprises at least about 90% sequence identity to any one of SEQ ID NOS.26-30. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the target nucleotide sequence comprises a coding sequence. In some embodiments, the target nucleotide sequence comprises a non-coding sequence or an intergenic sequence. In some embodiments, the target nucleotide sequence comprises all or part of a promoter sequence. In some embodiments, the promoter sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 1-11. In some embodiments, the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding strand of the transcribed region of the essential gene. In some embodiments, the essential gene is Tsf, acpP, gapA, 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, tRNAasn or metK. In some embodiments, the cascades polypeptide forms a cascades complex of an I-A type CRISPR-Cas system, an I-B type CRISPR-Cas system, an I-C type CRISPR-Cas system, an I-D type CRISPR-Cas system, an I-E type CRISPR-Cas system, or an I-F type CRISPR-Cas system. In some embodiments, the cascades complex comprises: (i) A Cas7 polypeptide, a Cas8a1 polypeptide or a Cas8a2 polypeptide, a Cas5 polypeptide, a Csa5 polypeptide, and a Cas6a polypeptide, wherein the Cas3 polypeptide comprises a Cas3' polypeptide and a Cas3 "polypeptide having no nuclease activity (type I-a CRISPR-Cas system); (ii) Cas6B polypeptides, cas8B polypeptides, cas7 polypeptides, and Cas5 polypeptides (type I-B CRISPR-Cas system); (iii) Cas5d polypeptides, cas8C polypeptides, and Cas7 polypeptides (type I-C CRISPR-Cas system); (iv) Cas10D polypeptide, csc2 polypeptide, csc1 polypeptide, cas6D polypeptide (I-D CRISPR-Cas system); (v) Cse1, cse2, cas7, cas5, and Cas6E polypeptides (I-E CRISPR-Cas system); (vi) Csy1 polypeptides, csy2 polypeptides, csy3 polypeptides, and Csy4 polypeptides (type I-F CRISPR-Cas system). In some embodiments, the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81), and a Cas7 polypeptide (optionally SEQ ID NO: 82) (type I-C CRISPR-Cas system). In some embodiments, the nucleic acid sequence further comprises a promoter sequence. In some embodiments, the pseudomonas species is killed only by the lytic activity of the bacteriophage. In some embodiments, the pseudomonas species is killed by only the activity of the CRISPR-Cas system. In some embodiments, the pseudomonas species is killed by a combination of lytic activity of the bacteriophage and activity of the CRISPR-Cas system. In some embodiments, the killing of the pseudomonas species by the activity of the CRISPR-Cas system is independent of the lytic activity of the bacteriophage. In some embodiments, the activity of the CRISPR-Cas system complements or enhances the lytic activity of the bacteriophage. In some embodiments, the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic. In some embodiments, the lytic activity of the bacteriophage, the activity of the CRISPR-Cas system, or both, is modulated by the concentration of the bacteriophage. In some embodiments, the bacteriophage infects a plurality of bacterial strains. In some embodiments, the bacteriophage is an obligate lytic bacteriophage. In some embodiments, the bacteriophage is a temperate bacteriophage that is conferred lytic. In some embodiments, the temperate bacteriophage is rendered lytic by removal, replacement or inactivation of a lysogenic gene. In some embodiments, the bacteriophage comprises Φkz virus (PhiKZvirus), Φkmv virus (PhiKMV virus), bruney virus (brunejosis virus), sa Mu Na virus (Samunavirus), south China virus (Nankokuvirus), abilet virus (abijan virus), begarter virus (baikal virus), bei Telei virus (Beetrevirus), kadaban virus (Casadabanvirus), cetex virus (cittex virus), vesicular virus (cyvista virus), de-trary virus (detrev virus), eltex virus (eltex virus), holohol Wei Bingdu (hollyvirus), small hat virus (kochia suruvirus), li Tu virus (livala virus), lu Saipu Ma Bingdu (luzsepiim virus), niperum virus (qikali virus), pizebra virus (pizebra virus), pizebra virus (pizoprairius), pizebra virus (pizebra virus), pizoma virus (pizebra virus), pizebra virus (pizebra virus), and other viruses (pizebra virus). In some embodiments, the bacteriophage is engineered from a bacteriophage that infects pseudomonas. In some embodiments, the bacteriophage that infects pseudomonas comprises a wild-type pna phage subtype listed in table 5A, wherein the phage infects pseudomonas target, such as with a positive (+) tag (e.g., phage p1106 infects b 002548). In some embodiments, the bacterial phage that infects pseudomonas comprises an engineered pna phage subtype listed in table 5A, wherein the phage infects a target pseudomonas, such as with a positive (+) tag (e.g., p1106e003 infects b 002548). In some embodiments, the bacterial phage that infects pseudomonas includes a wild-type sal Mu Na virus phage subtype, an engineered sal Mu Na virus phage subtype, a wild-type Φkz virus, a wild-type Φkmv virus, or a wild-type brunesoid virus (Bruynoghevirus), e.g., as set forth in table 5B, wherein the phage infects a target pseudomonas, e.g., labeled with a plus (+) sign. As listed in table 5A, the wild-type pna phage subtypes may be p1106, p1587, p1835, p2037, p2363, p2421 and/or pb1, while the engineered pna phage subtypes may be p1106e003, p1587e002, p1835e002, p2037e002, p2363e003 and/or p2421e002. As listed in table 5B, the wild-type salsa Mu Na viral phage subtypes may be p1772, p2131, p2132, and/or p2973, the engineered salsa Mu Na viral phage subtypes may be pb1e002, p1772e005, p2131e002, p2132e002, and/or p2973e002, the wild-type Φkz viral phage subtypes may be p1194 and/or p4430, the wild-type Φkmv viral phage subtypes may be p2167, and the wild-type brunesoid viral phage subtypes may be p1695 and p3278. In some embodiments, the bacteriophage that infects pseudomonas is a southern national virus. In some embodiments, the bacteriophage that infects pseudomonas kills pseudomonas. In some embodiments, the bacteriophage that infects pseudomonas does not infect staphylococcus aureus. In some embodiments, the bacteriophage that infects pseudomonas does not kill staphylococcus aureus. In some embodiments, the bacteriophage that kills pseudomonas does not infect staphylococcus aureus. In some embodiments, the bacteriophage that kills pseudomonas does not kill staphylococcus aureus. In some embodiments, the bacteriophage that infects pseudomonas does not infect klebsiella pneumoniae. In some embodiments, the bacteriophage that infects pseudomonas does not kill klebsiella pneumoniae. In some embodiments, the bacteriophage that kills pseudomonas does not infect klebsiella pneumoniae. In some embodiments, the bacteriophage that kills pseudomonas does not kill klebsiella pneumoniae. In some embodiments, the bacteriophage that infects pseudomonas does not infect enterococcus faecium. In some embodiments, the bacteriophage that infects pseudomonas does not kill enterococcus faecium. In some embodiments, the bacteriophage that kills pseudomonas does not infect enterococcus faecium. In some embodiments, the bacteriophage that kills pseudomonas does not kill enterococcus faecium. In some embodiments, the bacteriophage that infects pseudomonas does not infect enterobacter cloacae. In some embodiments, the bacteriophage that infects pseudomonas does not kill enterobacter cloacae. In some embodiments, the bacteriophage that kills pseudomonas does not infect enterobacter cloacae. In some embodiments, the bacteriophage that kills pseudomonas does not kill enterobacter cloacae. In some embodiments, the bacteriophage that infects pseudomonas does not infect acinetobacter baumannii. In some embodiments, the bacteriophage that infects pseudomonas does not kill acinetobacter baumannii. In some embodiments, the bacteriophage that kills pseudomonas does not infect acinetobacter baumannii. In some embodiments, the bacteriophage that kills pseudomonas does not kill acinetobacter baumannii. In some embodiments, the bacteriophage that infects pseudomonas does not infect staphylococcus epidermidis. In some embodiments, the bacteriophage that infects pseudomonas does not kill staphylococcus epidermidis. In some embodiments, the bacteriophage that kills pseudomonas does not infect staphylococcus epidermidis. In some embodiments, the bacteriophage that kills pseudomonas does not kill staphylococcus epidermidis. In some embodiments, the combination of bacteriophages infects pseudomonas. As one non-limiting example, the combination infects at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of pseudomonas in table 5A. As one non-limiting example, the combination infects at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of pseudomonas in table 5B. As one non-limiting example, the combination infects at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of pseudomonas in table 6B. In some embodiments, the combination of bacteriophages kills pseudomonas. As one non-limiting example, the combination kills at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the pseudomonas in table 5A. As one non-limiting example, the combination kills at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the pseudomonas in table 5B. As one non-limiting example, the combination kills at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the pseudomonas in table 6B.

In some embodiments, a cocktail system is provided that comprises one or more bacteriophage. In some embodiments, the bacteriophage contained in the cocktail system comprises any one of p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB1, or two or more phages thereof. In some embodiments, the bacteriophage cocktail system used herein comprises one, two, three, four, five, six, or more selected from the group consisting of p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, and PB 1. In some embodiments, the bacteriophage is a bacteriophage having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a bacteriophage selected from p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 and PB 1. In some embodiments, the bacteriophage in the bacteriophage cocktail system as used herein includes any one, two, three, four, five, six, or more from p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002, or PB1 wt. In some embodiments, the bacteriophage in the bacteriophage cocktail system used herein includes a bacteriophage having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132e002, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002, or PB1wt, or two or more thereof. In some embodiments, the bacteriophage in the bacteriophage cocktail system as used herein includes any one or more of p1106e003, p1835e002, p1772e005, p2131e002, p1194, p4430 and p 1695. In some embodiments, the nucleic acid sequence is inserted at or near the location of a non-essential bacteriophage gene. In some embodiments, the bacteriophage is selected from the group consisting of p1106e003, p1835e002, p1772e005, and p2131e002. In some embodiments, the bacteriophage in the bacteriophage cocktail system as used herein includes any one or more of p1106e003, p1835e002, p1772e005, p2131e002, and p 1194. In some embodiments, the bacteriophage in the bacteriophage cocktail system as used herein includes any one or more of p1106e003, p1835e002, p1772e005, p2131e002, and p 4430. In some embodiments, the bacteriophage in the bacteriophage cocktail system as used herein includes any one or more of p1106e003, p1835e002, p1772e005, p2131e002 and p 1695. In some embodiments, the bacteriophage in the bacteriophage cocktail system as used herein includes any one or more of e p e003, p1835e002, p1772e005, p2131e002, p1194, and p 1695. In some embodiments, the bacteriophage in the bacteriophage cocktail system as used herein includes any one or more of p1106e003, p1835e002, p1772e005, p2131e002, p4430 and p 1695. In some embodiments, the bacteriophage cocktail system comprises a bacteriophage of CK000512 (p 1106e003, p1835e002, p1772e005, p2131e002, p4430, and p 1695).

In certain embodiments, disclosed herein are pharmaceutical compositions comprising: (a) a bacteriophage as described herein; and (b) a pharmaceutically acceptable excipient. In some embodiments, the bacteriophage is selected from the group consisting of Φkz virus, Φkmv virus, brunesoid virus, sal Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cetuximab virus, vesicular virus, detrile virus, ehrlichia virus, holo Wei Bingdu, chikungunya virus, livina virus, lu Saipu pedicle Ma Bingdu, nipena virus, parkenna virus, pameke virus, boenmer virus, filteirus, puli Mo Liji virus, sapenjang virus, starbuchner virus, takiki virus, johne virus, atameria virus, and prana virus. In some embodiments, the pharmaceutical composition comprises at least six bacteriophage, wherein the bacteriophage is selected from the group consisting of p1106e003, p1835e002, p1772e005, p2131e002, p1194 and p1695 bacteriophage. In some embodiments, the pharmaceutical composition comprises at least six bacteriophage, wherein the bacteriophage in the bacteriophage cocktail system used herein comprises any one or more of p1106e003, p1835e002, p1772e005, p2131e002, p4430, and p1695. In some embodiments, the pharmaceutical composition is in the form of a tablet, liquid, syrup, oral formulation (formulation), intravenous formulation, intranasal formulation, ophthalmic formulation, otic formulation, subcutaneous formulation, inhalable respiratory formulation, suppository, lyophilized formulation, nebulizable formulation, and any combination thereof. In one embodiment, the pharmaceutical composition comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430 and p1695 in an nebulizable formulation for pulmonary delivery.

In certain aspects, disclosed herein is a method of killing a pseudomonas species comprising introducing into the target bacterium a nucleic acid sequence encoding a type I CRISPR-Cas system from a bacteriophage, the nucleic acid comprising: a CRISPR array comprising one or more spacer sequences complementary to a target nucleotide sequence in said pseudomonas species; a cascades polypeptide; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. 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 one or more spacer sequences at its 5 'end or its 3' end. In some embodiments, the repeat sequence comprises at least about 90% sequence identity to any one of SEQ ID NOS.26-30. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the target nucleotide sequence comprises a coding sequence. In some embodiments, the target nucleotide sequence comprises a non-coding sequence or an intergenic sequence. In some embodiments, the target nucleotide sequence comprises all or part of a promoter sequence. In some embodiments, the promoter sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 1-11. In some embodiments, the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding strand of the transcribed region of the essential gene. In some embodiments, the essential gene is Tsf, acpP, gapA, 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, tRNAasn or metK. In some embodiments, the cascades polypeptide forms a cascades complex of an I-A type CRISPR-Cas system, an I-B type CRISPR-Cas system, an I-C type CRISPR-Cas system, an I-D type CRISPR-Cas system, an I-E type CRISPR-Cas system, or an I-F type CRISPR-Cas system. In some embodiments, the cascades complex comprises: (i) A Cas7 polypeptide, a Cas8a1 polypeptide or a Cas8a2 polypeptide, a Cas5 polypeptide, a Csa5 polypeptide, and a Cas6a polypeptide, wherein the Cas3 polypeptide comprises a Cas3' polypeptide and a Cas3 "polypeptide having no nuclease activity (type I-a CRISPR-Cas system); (ii) Cas6B polypeptides, cas8B polypeptides, cas7 polypeptides, and Cas5 polypeptides (type I-B CRISPR-Cas system); (iii) Cas5d polypeptides, cas8C polypeptides, and Cas7 polypeptides (type I-C CRISPR-Cas system); (iv) Cas10D polypeptide, csc2 polypeptide, csc1 polypeptide, cas6D polypeptide (I-D CRISPR-Cas system); (v) Cse1, cse2, cas7, cas5, and Cas6E polypeptides (I-E CRISPR-Cas system); (vi) Csy1 polypeptides, csy2 polypeptides, csy3 polypeptides, and Csy4 polypeptides (type I-F CRISPR-Cas system). In some embodiments, the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81), and a Cas7 polypeptide (optionally SEQ ID NO: 82) (type I-C CRISPR-Cas system). In some embodiments, the nucleic acid sequence further comprises a promoter sequence. In some embodiments, the pseudomonas species is killed by only the activity of the CRISPR-Cas system. In some embodiments, the pseudomonas species is killed by a combination of lytic activity of the bacteriophage and activity of the CRISPR-Cas system. In some embodiments, the killing of the pseudomonas species by the activity of the CRISPR-Cas system is independent of the lytic activity of the bacteriophage. In some embodiments, the activity of the CRISPR-Cas system complements or enhances the lytic activity of the bacteriophage. In some embodiments, the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic. In some embodiments, the lytic activity of the bacteriophage, the activity of the CRISPR-Cas system, or both, is modulated by the concentration of the bacteriophage. In some embodiments, the bacteriophage infects a plurality of bacterial strains. In some embodiments, the bacteriophage is an obligate lytic bacteriophage. In some embodiments, the bacteriophage is a temperate bacteriophage that is conferred lytic. In some embodiments, the temperate bacteriophage is rendered lytic by removal, replacement or inactivation of a lysogenic gene. In some embodiments, the bacteriophage comprises Φkz virus, Φkmv virus, brunesoid virus, sal Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cetuximab virus, vesicular virus, detrilavirus, ehrlift virus, holo Wei Bingdu, chikungunya virus, livina virus, lu Saipu pedicle Ma Bingdu, niprina virus, parkrina virus, pamek's virus, boenjim virus, filteirus, puli Mo Liji virus, sapetrex virus, starbuchner virus, takiki virus, johne virus, atameria virus, or prina virus bacteriophages. In some embodiments, the bacteriophage is part of a bacteriophage cocktail. In some embodiments, the bacteriophage or bacteriophage cocktail comprises p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB1, or two or more phages thereof. In some embodiments, the bacteriophage is a bacteriophage having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a bacteriophage selected from p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 and PB 1. In some embodiments, the bacteriophage or bacteriophage cocktail comprises p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132e002, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more thereof. In some embodiments, the bacteriophage or bacteriophage cocktail comprises a bacteriophage having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132e002, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more thereof. In some embodiments, the nucleic acid sequence is inserted at or near the location of a non-essential bacteriophage gene. In some embodiments, the mixed population of bacterial cells comprises the pseudomonas species. In some embodiments, the bacteriophage cocktail comprises p1106e003, p1835e002, p1772e005, p2131e002, p1194, and p1695. In some embodiments, the bacteriophage cocktail comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430, and p1695.

In certain aspects, disclosed herein is a method of treating a disease or condition in an individual in need thereof, the method comprising administering to the individual a bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising: a CRISPR array; a cascades polypeptide comprising one or more spacer sequences complementary to a target nucleotide sequence in a pseudomonas species; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. 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 one or more spacer sequences at its 5 'end or its 3' end. In some embodiments, the repeat sequence comprises at least about 90% sequence identity to any one of SEQ ID NOS.26-30. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the target nucleotide sequence comprises a coding sequence. In some embodiments, the target nucleotide sequence comprises a non-coding sequence or an intergenic sequence. In some embodiments, the target nucleic acid sequence comprises all or part of a promoter sequence. In some embodiments, the promoter sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 1-11. In some embodiments, the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding strand of the transcribed region of the essential gene. In some embodiments, the essential gene is Tsf, acpP, gapA, 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, tRNAasn or metK. In some embodiments, the cascades polypeptide forms a cascades complex of an I-A type CRISPR-Cas system, an I-B type CRISPR-Cas system, an I-C type CRISPR-Cas system, an I-D type CRISPR-Cas system, an I-E type CRISPR-Cas system, or an I-F type CRISPR-Cas system. In some embodiments, the cascades complex comprises: (i) A Cas7 polypeptide, a Cas8a1 polypeptide or a Cas8a2 polypeptide, a Cas5 polypeptide, a Csa5 polypeptide, and a Cas6a polypeptide, wherein the Cas3 polypeptide comprises a Cas3' polypeptide and a Cas3 "polypeptide having no nuclease activity (type I-a CRISPR-Cas system); (ii) Cas6B polypeptides, cas8B polypeptides, cas7 polypeptides, and Cas5 polypeptides (type I-B CRISPR-Cas system); (iii) Cas5d polypeptides, cas8C polypeptides, and Cas7 polypeptides (type I-C CRISPR-Cas system); (iv) Cas10D polypeptide, csc2 polypeptide, csc1 polypeptide, cas6D polypeptide (I-D CRISPR-Cas system); (v) Cse1, cse2, cas7, cas5, and Cas6E polypeptides (I-E CRISPR-Cas system); (vi) Csy1 polypeptides, csy2 polypeptides, csy3 polypeptides, and Csy4 polypeptides (type I-F CRISPR-Cas system). In some embodiments, the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81), and a Cas7 polypeptide (optionally SEQ ID NO: 82) (type I-C CRISPR-Cas system). In some embodiments, the nucleic acid sequence further comprises a promoter sequence. In some embodiments, the pseudomonas species is killed by only the activity of the CRISPR-Cas system. In some embodiments, the pseudomonas species is killed by a combination of lytic activity of the bacteriophage and activity of the CRISPR-Cas system. In some embodiments, the killing of the pseudomonas species by the activity of the CRISPR-Cas system is independent of the lytic activity of the bacteriophage. In some embodiments, the activity of the CRISPR-Cas system complements or enhances the lytic activity of the bacteriophage. In some embodiments, the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic. In some embodiments, the lytic activity of the bacteriophage, the activity of the CRISPR-Cas system, or both, is modulated by the concentration of the bacteriophage. In some embodiments, the bacteriophage infects a plurality of bacterial strains. In some embodiments, the bacteriophage is an obligate lytic bacteriophage. In some embodiments, the bacteriophage is a temperate bacteriophage that is conferred lytic. In some embodiments, the temperate bacteriophage is rendered lytic by removal, replacement or inactivation of a lysogenic gene. In some embodiments, the bacteriophage comprises Φkz virus, Φkmv virus, brunesoid virus, sal Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cetuximab virus, vesicular virus, detrilavirus, ehrlift virus, holo Wei Bingdu, chikungunya virus, livina virus, lu Saipu pedicle Ma Bingdu, niprina virus, parkrina virus, pameke virus, boendeum virus, filteirus, puli Mo Liji virus, sapetrex virus, starbuch virus, takiwiki virus, jojohne virus, atameria virus, or prana virus. In some embodiments, the bacteriophage is part of a bacteriophage cocktail. In some embodiments, the bacteriophage or bacteriophage cocktail comprises p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB1, or two or more phages thereof. In some embodiments, the bacteriophage is a bacteriophage having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a bacteriophage selected from p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 and PB 1. In some embodiments, the bacteriophage or bacteriophage cocktail comprises p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132e002, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more thereof. In some embodiments, the bacteriophage or bacteriophage cocktail comprises a bacteriophage having at least 80%, at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132e002, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more thereof. In some embodiments, the nucleic acid sequence is inserted at or near the location of a non-essential bacteriophage gene. In some embodiments, the method further comprises administering at least one additional bacteriophage type. In some embodiments, the method further comprises administering 2, 3, 4, 5, 6, 7, 8, 9, or 10 different bacteriophage. In some embodiments, the method further comprises administering at least six different bacteriophage. In some embodiments, the method further comprises administering at least six different bacteriophage, wherein the bacteriophage in the bacteriophage cocktail system used herein comprises any one or more of p1106e003, p1835e002, p1772e005, p2131e002, p1194, and p1695. In some embodiments, the method further comprises administering at least six different bacteriophage, wherein the bacteriophage comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430, and p1695. In some embodiments, the disease is a bacterial infection. In some embodiments, the bacterial infection is a pseudomonas bacterial infection. In some embodiments, the bacterial infection is associated with cystic fibrosis. In some embodiments, the bacterial infection is associated with non-cystic fibrosis bronchiectasis. In some embodiments, the disease or condition is cystic fibrosis. In some embodiments, the disease or condition is non-cystic fibrosis bronchiectasis. In some embodiments, the bacterial infection is a pseudomonas bacterial infection associated with cystic fibrosis. In some embodiments, the bacterial infection is a pseudomonas bacterial infection associated with non-cystic fibrosis bronchiectasis. In some embodiments, the disease or condition is pneumonia. For example, the pneumonia is hospital-acquired pneumonia, ventilator-acquired pneumonia, community-acquired pneumonia, or healthcare-acquired pneumonia. In some embodiments, the disease or condition is a Blood System Infection (BSI). In some embodiments, the pseudomonas species responsible for the disease or condition is a drug resistant pseudomonas species. In some embodiments, the drug-resistant pseudomonas species is resistant to at least one antibiotic. In some embodiments, the pseudomonas species responsible for the disease or condition is a multidrug resistant pseudomonas species. In some embodiments, the multi-drug resistant pseudomonas species is resistant to at least one antibiotic. In some embodiments, the antibiotic comprises cephalosporin, fluoroquinolone, carbapenem, colistin, aminoglycoside, vancomycin, streptomycin, or methicillin. In some embodiments, the pseudomonas species is pseudomonas aeruginosa. In some embodiments, the administration is intrA-Arterial, intravenous, intra-urethral, intramuscular, oral, subcutaneous, inhaled, topical, dermal, transdermal, transmucosal, implanted, sublingual, buccal, rectal, vaginal, ocular, otic, or nasal administration, or any combination thereof. In some embodiments, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent comprises tobramycin. In some embodiments, the subject is a mammal. In some embodiments, the additional therapeutic agent comprises a drug for improving airway function. In some embodiments, the additional therapeutic agent comprises a drug for reducing airway responsiveness. In some embodiments, the additional therapeutic agent comprises a drug for reducing airway inflammation. In some embodiments, the additional therapeutic agent comprises a bronchodilator. In some embodiments, the additional therapeutic agent comprises a drug for increasing oxygen utilization. In some embodiments, the additional therapeutic agent comprises a drug for reducing airway mucus production. In some embodiments, the additional therapeutic agent comprises dnase. In some embodiments, the additional therapeutic agent is saline. In some embodiments, the additional therapeutic agent is a therapeutic method comprising cough exercises, e.g., for treating cystic fibrosis.

In certain aspects, disclosed herein is a bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising: a CRISPR array comprising one or more spacer sequences complementary to a target nucleotide sequence in a pseudomonas species; a cascades polypeptide comprising Cas5, cas8c, and Cas 7; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or at least 90% sequence identity to any one of SEQ ID NOS 12-23, 31-74, or 88-120. 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 one or more spacer sequences at its 5 'end or its 3' end. In some embodiments, the repeat sequence comprises at least about 90% sequence identity to any one of SEQ ID NOS.26-30. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the target nucleotide sequence comprises a coding sequence. In some embodiments, the target nucleotide sequence comprises a non-coding sequence or an intergenic sequence. In some embodiments, the target nucleotide sequence comprises all or part of a promoter sequence. In some embodiments, the promoter sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 1-11. In some embodiments, the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding strand of the transcribed region of the essential gene. In some embodiments, the essential gene is Tsf, acpP, gapA, 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, tRNAasn or metK. In some embodiments, the nucleic acid sequence further comprises a promoter sequence. In some embodiments, the pseudomonas species is killed only by the lytic activity of the bacteriophage. In some embodiments, the pseudomonas species kills only the activity of the CRISPR-Cas system. In some embodiments, the pseudomonas species is killed by a combination of lytic activity of the bacteriophage and activity of the CRISPR-Cas system. In some embodiments, the killing of the pseudomonas species by the activity of the CRISPR-Cas system is independent of the lytic activity of the bacteriophage. In some embodiments, the activity of the CRISPR-Cas system complements or enhances the lytic activity of the bacteriophage. In some embodiments, the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic. In some embodiments, the lytic activity of the bacteriophage, the activity of the CRISPR-Cas system, or both, is modulated by the concentration of the bacteriophage. In some embodiments, the bacteriophage infects a plurality of bacterial strains. In some embodiments, the bacteriophage is an obligate lytic bacteriophage. In some embodiments, the bacteriophage is a temperate bacteriophage that is conferred lytic. In some embodiments, the temperate bacteriophage is rendered lytic by removal, replacement or inactivation of a lysogenic gene. In some embodiments, the bacteriophage is from one of the following: Φkz virus, Φkmv virus, brunejo virus, sal Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cet-ter-chi virus, vesicular virus, detrilavirus, hallo Wei Bingdu, eriosema virus, lirana virus, lu Saipu pedicle Ma Bingdu, nipena virus, parkenna virus, pamex virus, boende-mer virus, filtei virus, pri Mo Liji virus, sapendamate virus, starbuch virus, teltei virus, jojo-subunit virus, ataegre-trie virus, or prana virus. In some embodiments, the bacteriophage as used herein includes any one or more of p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB1, or two or more thereof. In some embodiments, the bacteriophage is a bacteriophage having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a bacteriophage selected from p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 and PB 1. In some embodiments, the bacteriophage in use herein includes any one or more of p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132e002, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more phages thereof. In some embodiments, the bacteriophage as used herein includes any one or more of a bacteriophage having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more thereof. In some embodiments, the bacteriophage used herein is selected from the group consisting of p1106e003, p1835e002, p1772e005, p2131e002, p1194, and p1695. In some embodiments, the bacteriophage in the bacteriophage cocktail system as used herein includes any one or more of p1106e003, p1835e002, p1772e005, p2131e002, p4430 and p1695. In some embodiments, the nucleic acid sequence is inserted into a non-essential bacteriophage gene. In some embodiments, disclosed herein is a pharmaceutical composition comprising: (a) a bacteriophage as disclosed herein; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises at least two different bacteriophage. In some embodiments, the pharmaceutical composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different bacteriophage. In some embodiments, the pharmaceutical composition comprises at least six different bacteriophage. In some embodiments, the pharmaceutical composition comprises at least six different bacteriophage, wherein the bacteriophage as used herein comprises any one or more of p1106e003, p1835e002, p1772e005, p2131e002, p1194, and p1695. In some embodiments, the pharmaceutical composition comprises at least six different bacteriophage, wherein the bacteriophage as used herein comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430 and p1695. In some embodiments, the pharmaceutical composition is in the form of a tablet, capsule, liquid, syrup, oral formulation, intravenous formulation, intranasal formulation, ophthalmic formulation, otic formulation, subcutaneous formulation, topical formulation, transdermal formulation, transmucosal formulation, inhalable respiratory formulation, suppository, lyophilized formulation, nebulizable formulation, and any combination thereof. In some embodiments, the pharmaceutical composition comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430 and p1695 in an aerosolizable formulation for pulmonary delivery.

In certain aspects, disclosed herein is a method of sterilizing a surface in need thereof, the method comprising administering to the surface a bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising: a CRISPR array; a cascades polypeptide comprising one or more spacer sequences complementary to a target nucleotide sequence in a pseudomonas species; and Cas3 polypeptides. In some embodiments, the surface is a hospital surface, a vehicle surface, a device surface, or an industrial surface.

In certain aspects, disclosed herein is a method of preventing contamination in a food product or nutritional supplement, the method comprising contacting the food product or nutritional supplement with a bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising: a CRISPR array; a cascades polypeptide comprising one or more spacer sequences complementary to a target nucleotide sequence in a pseudomonas species; and Cas3 polypeptides. In some embodiments, the food product or nutritional supplement comprises milk, yogurt, curd, cheese, fermented milks, milk based fermented products, ice cream, fermented cereal based products, milk based powders, infant formulas or tablets, liquid suspensions, dry oral supplements, wet oral supplements, or dry tube feeds.

In certain aspects, disclosed herein is a bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising: a CRISPR array comprising a spacer sequence complementary to a target nucleotide sequence in a pseudomonas species, wherein said spacer sequence comprises SEQ ID NOs 12, 16 and 20; a cascades polypeptide; and Cas3 polypeptides.

In certain aspects, disclosed herein is a bacteriophage comprising at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a bacteriophage selected from p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the bacteriophage as used herein includes any one or more of bacteriophage having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more thereof. In some embodiments, a bacteriophage cocktail system is provided that comprises two additional bacteriophage. In some embodiments, the bacteriophage cocktail system comprises a bacteriophage of CK000512 (p 1106e003, p1835e002, p1772e005, p2131e002, p4430, and p 1695). In some embodiments, the bacteriophage further comprises a CRISPR array; a cascades polypeptide comprising one or more spacer sequences complementary to a target nucleotide sequence in a pseudomonas species; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. 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 one or more spacer sequences at its 5 'end or its 3' end. In some embodiments, the repeat sequence comprises at least about 90% sequence identity to any one of SEQ ID NOS.26-30. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the target nucleotide sequence comprises a coding sequence. In some embodiments, the target nucleotide sequence comprises a non-coding sequence or an intergenic sequence. In some embodiments, the target nucleotide sequence comprises all or part of a promoter sequence. In some embodiments, the promoter sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 1-11.

In certain aspects, disclosed herein is a composition comprising at least four bacteriophage comprising: a first bacteriophage comprising p1106e003 or having at least 80% sequence identity to p1106e 003; a second bacteriophage comprising p1835e002 or having at least 80% sequence identity to p1835e 002; a third bacteriophage comprising p1772e005 or having at least 80% sequence identity to p1772e 005; and a fourth bacteriophage comprising p2131e002 or having at least 80% sequence identity to p2131e 002. In some embodiments, the composition further comprises a fifth bacteriophage comprising p1194 or having at least 80% sequence identity to p 1194. In some embodiments, the composition further comprises a fifth bacteriophage comprising p1695 or having at least 80% sequence identity to p 1695. In some embodiments, the composition further comprises a fifth bacteriophage comprising p4430 or having at least 80% sequence identity to p 4430. In some embodiments, the composition further comprises a sixth bacteriophage comprising p1695 or having at least 80% sequence identity to p 1695. In some embodiments, the composition comprises a bacteriophage of CK000512 (p 1106e003, p1835e002, p1772e005, p2131e002, p4430 and p 1695).

In any of the method embodiments herein, the bacteriophage of the method is engineered from a pseudomonas-infected bacteriophage. In some embodiments, the bacteriophage that infects pseudomonas comprises a wild-type pna phage subtype listed in table 5A, wherein the phage infects pseudomonas target, such as with a positive (+) tag (e.g., phage p1106 infects b 002548). In some embodiments, the bacterial phage that infects pseudomonas comprises an engineered pna phage subtype listed in table 5A, wherein the phage infects a target pseudomonas, such as with a positive (+) tag (e.g., p1106e003 infects b 002548). In some embodiments, the bacterial phage infected with pseudomonas comprises a wild-type salsa Mu Na virus phage subtype, an engineered salsa Mu Na virus phage subtype, a wild-type Φkz virus, a wild-type Φkmv virus, or a wild-type brunesabout virus, e.g., as set forth in table 5B, wherein the phage infects a target pseudomonas, e.g., marked with a plus (+) sign. As listed in table 5A, the wild-type pna phage subtypes may be p1106, p1587, p1835, p2037, p2363, p2421 and/or pb1, while the engineered pna phage subtypes may be p1106e003, p1587e002, p1835e002, p2037e002, p2363e003 and/or p2421e002. As listed in table 5B, the wild-type salsa Mu Na viral phage subtype may be p1772, p2131, p2132 and/or p2973, the engineered salsa Mu Na viral phage subtype may be pb1e002, p1772e005, p2131e002, p2132e002 and/or p2973e002, the wild-type Φkz viral phage subtype may be p1194 and/or p4430, the wild-type Φkmv viral phage subtype may be p2167, and the wild-type brunesoid viral phage subtype may be p1695 and/or p3278. In some embodiments, the bacteriophage that infects pseudomonas is a south China virus, Φkz virus, Φkmv virus, bruneshop virus, sal Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cetex virus, vesicular virus, de-tei virus, aether virus, holo Wei Bingdu, xima virus, livina virus, lu Saipu ti Ma Bingdu, nipena virus, parkona virus, pamex virus, boenmer virus, filtei virus, pri Mo Liji virus, saprotamate virus, starbucus virus, taerty virus, jojoba virus, ataxia virus, or prana virus. In some embodiments, the bacteriophage that infects pseudomonas kills pseudomonas. In some embodiments, the bacteriophage that infects pseudomonas does not infect staphylococcus aureus. In some embodiments, the bacteriophage that infects pseudomonas does not kill staphylococcus aureus. In some embodiments, the bacteriophage that kills pseudomonas does not infect staphylococcus aureus. In some embodiments, the bacteriophage that kills pseudomonas does not kill staphylococcus aureus. In some embodiments, the bacteriophage that infects pseudomonas does not infect klebsiella pneumoniae. In some embodiments, the bacteriophage that infects pseudomonas does not kill klebsiella pneumoniae. In some embodiments, the bacteriophage that kills pseudomonas does not infect klebsiella pneumoniae. In some embodiments, the bacteriophage that kills pseudomonas does not kill klebsiella pneumoniae. In some embodiments, the bacteriophage that infects pseudomonas does not infect enterococcus faecium. In some embodiments, the bacteriophage that infects pseudomonas does not kill enterococcus faecium. In some embodiments, the bacteriophage that kills pseudomonas does not infect enterococcus faecium. In some embodiments, the bacteriophage that kills pseudomonas does not kill enterococcus faecium. In some embodiments, the bacteriophage that infects pseudomonas does not infect enterobacter cloacae. In some embodiments, the bacteriophage that infects pseudomonas does not kill enterobacter cloacae. In some embodiments, the bacteriophage that kills pseudomonas does not infect enterobacter cloacae. In some embodiments, the bacteriophage that kills pseudomonas does not kill enterobacter cloacae. In some embodiments, the bacteriophage that infects pseudomonas does not infect acinetobacter baumannii. In some embodiments, the bacteriophage that infects pseudomonas does not kill acinetobacter baumannii. In some embodiments, the bacteriophage that kills pseudomonas does not infect acinetobacter baumannii. In some embodiments, the bacteriophage that kills pseudomonas does not kill acinetobacter baumannii. In some embodiments, the bacteriophage that infects pseudomonas does not infect staphylococcus epidermidis. In some embodiments, the bacteriophage that infects pseudomonas does not kill staphylococcus epidermidis. In some embodiments, the bacteriophage that kills pseudomonas does not infect staphylococcus epidermidis. In some embodiments, the bacteriophage that kills pseudomonas does not kill staphylococcus epidermidis. In some embodiments, the combination of bacteriophages infects pseudomonas. As one non-limiting example, the combination infects at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of pseudomonas in table 5A. As one non-limiting example, the combination infects at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of pseudomonas in table 5B. As one non-limiting example, the combination infects at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of pseudomonas in table 6B. In some embodiments, the combination of bacteriophages kills pseudomonas. As one non-limiting example, the combination kills at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the pseudomonas in table 5A. As one non-limiting example, the combination kills at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the pseudomonas in table 5B. As one non-limiting example, the combination kills at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the pseudomonas in table 6B.

Drawings

The novel features believed characteristic of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

FIG. 1A depicts the sequence and arrangement of crArray1 (SEQ ID NO: 83). FIG. 1B depicts the sequence and arrangement of crArray2 (SEQ ID NO: 84). FIG. 1C depicts the sequence and arrangement of crArray3 (SEQ ID NO: 85). FIG. 1D depicts the sequence and arrangement of crArray4 (SEQ ID NO: 86). FIG. 1E depicts the sequence and arrangement of crArray 5 (SEQ ID NO: 87). FIGS. 1F-1K depict the arrangement of crArray1 and PaIC insertions (SEQ ID NO: 25). FIGS. 1L-1Q depict the arrangement of crArray3 and PaIC insertions (SEQ ID NO: 24).

Figure 2A (upper panel) depicts the effect of transforming two pseudomonas aeruginosa strains with crRNA-containing plasmids using the endogenous CRISPR-Cas3 system, as measured by the number of transformants in Colony Forming Units (CFU). The lower panel shows the effect of transforming pseudomonas aeruginosa with plasmids containing crRNA (containing 3 spacers) and exogenous I-C Cas operon, which resulted in transformants with less than the detection limit. Figure 2B depicts the number of bacterial transformants obtained per mL of Cas operon null mutant transformed into pseudomonas aeruginosa strain B1121. Array1 targets the bacterial genome, while array2 is a non-targeted control. The different plasmids were normalized to the empty vector control plasmid with respect to molar concentration. Fig. 2C depicts the effect of transforming individual spacer or 3-spacer arrays targeting rpoB or ftsA (i.e., array3 or array 4) into pseudomonas aeruginosa strains with (b 1121) or without (b 1121 Cas KO) endogenous I-C type Cas operon.

FIG. 3A depicts a schematic representation of the genome of wild-type phage p1772 and its engineered variants. Bars below the genome axis indicate the region of the genome that was removed and replaced. The schematic below the phage genome shows the DNA used to replace the WT phage gene in the deleted region. Arrays 1, 3, and 4 target the bacterial genome and kill bacteria in the presence of the I-C Cas operon. The spacers in array 2 are non-targeted, but the array is structurally identical to the three targeted arrays. Figure 3B compares the sequences of p1772e005 (targeting the crarray1+cas system) after passage 5 or 10 times at 37 degrees celsius. No difference in insertion was observed at the nucleotide level, indicating the stability of engineered phage expressing CRISPR-Cas 3.

Figure 4 illustrates that phages engineered with CRISPR-Cas3 show no structural changes. There was no apparent morphological difference between p1772wt (wild type), p1772e004 (Cas system only) and p1772e005 (targeting crarray+cas system) when imaged by TEM.

Fig. 5A-5C illustrate that full construct phages amplify similarly to wild-type parent phages in variants of different Cas types and retain similar host ranges. Figure 5A depicts in vitro amplification titers of p1772wt (wild type), p1772e004 (Cas system) and p1772e005 (targeting crarray1+cas system) in pseudomonas aeruginosa strains containing an I-F type Cas system. Figure 5B depicts in vitro amplification titers of p1772wt and p1772e005 in pseudomonas aeruginosa strains containing an I-C Cas system. FIG. 5C depicts host ranges for p1772wt, p1772e004 and p1772e005 on 44 P.aeruginosa strains. If (AUC in the presence of phage)/(AUC in the absence of phage) is less than 0.65, then the phage is considered to infect the given strain.

FIGS. 6A-6E illustrate efficient expression of exogenous CRISPR-Cas3 systems from phage genomes. Fig. 6A depicts a schematic of a spacer array and Cas operon inserted into an engineered variant of p 1772. Figures 6B-6D depict the expression of crArray, cas3 and Cas8 in pseudomonas aeruginosa strain B1121 infected with p1772wt (wild-type) or p1772e005 (targeting crArray1+ Cas system) within 1600 minutes. Figure 6E depicts Cas3 expression 1 and 24 hours after infection with p1772E005, p2131E002 (targeting crarray1+cas system) and p2132E002 (targeting crarray1+cas system).

Figure 7 depicts plaques generated by the spreading of p1772wt (wild-type) or p1772e005 (targeting the crarray1+cas system) onto pseudomonas aeruginosa.

Fig. 8A illustrates the results of a plate-based kill assay. p1772wt (wild type), p1772e004 (Cas system only), p1772e006 (crArray 1 only) and p1772e005 (crArray 1+cas system targeted) were mixed with pseudomonas aeruginosa at a multiplicity of infection (MOI) of 100 to 0.0000954. Fig. 8B depicts a portion of a plate as set forth in fig. 8A at a greater magnification. FIG. 8C shows quantification of relative fluorescence units of P.aeruginosa infected with p1772wt, p1772e008 (non-targeted crArray2+Cas system), p1772e006 and p1772e005 at MOI of 1.5

Fig. 9A depicts the growth of p1772wt (wild type), p1772e004 (Cas system only), p1772e005 (targeted to crarray1+cas system) and p1772e006 (targeted to crArray1 only) in pseudomonas aeruginosa strain b1121 over 24 hours when inoculated at an MOI of 1. FIG. 9B depicts the growth of p1772wt, p1772e004, p1772e005 and p1772e006 in P.aeruginosa strain B1121 over 24 hours when inoculated at MOI of 10. FIG. 9C depicts the growth of p1772wt, p1772e004, p1772e005 and p1772e006 in P.aeruginosa strain b1121 over 24 hours when inoculated at MOI of 100.

FIG. 10A depicts the growth of P.aeruginosa cultures mixed with p1772wt (wild type), p1772e008 (non-targeted crArray2+Cas system), p1772e006 (targeted crArray1 only) and pArray3 (targeted crArray3+Cas system) on agar plates at MOI of 100 to 0.0001. FIG. 10B depicts the growth of P.aeruginosa cultures mixed with p1772wt, p1772e008, p1772e006 and pArray4 (targeting the crArray4+Cas system) on agar plates at MOI of 100 to 0.0001. FIG. 10C is an inset of FIG. 10A, depicting an enlarged view of p1772e006 compared to pArray3 at MOI of 0.0244 (top row) and 0.00610 (bottom row). FIG. 10D is a quantification of fluorescent signals from bacteria after infection with phage at an MOI of about 1.5 in FIG. 10A. FIG. 10E is a quantification of fluorescent signals from bacteria after infection with phage at an MOI of about 1.5 in FIG. 10B.

FIG. 11 depicts the growth of P.aeruginosa cultures mixed with p1772 variants with different promoters on agar plates. FIG. 11A shows the growth of P.aeruginosa cultures mixed with p1772wt (wild type) and variants containing the same crArray1 and Cas system driven by different promoters on agar plates at MOI of 100 to 0.00001. FIG. 11B shows quantification of cellular fluorescence from FIG. 11A at an MOI of 1.5.

FIG. 12A depicts quantification of fluorescent growth of P.aeruginosa cultures mixed with p2132wt (wild type) and p2132e002 (targeting crArray1+Cas system) on agar plates at MOI of 1.5. FIG. 12B depicts quantification of fluorescence from growth of P.aeruginosa cultures mixed with p2973wt (wild type) and p2973e002 (targeting crArray1+Cas system) on agar plates at MOI of 1.5.

FIG. 13 depicts the growth of different strains of Pseudomonas aeruginosa cultures mixed with different phage variants on agar plates. P4209wt (wild type) and p4209E002 (targeting crArray1+Cas system) were mixed with the b2550 (type I-E Cas), b2631 (type I-F Cas), b2816 (type I-E/I-F Cas) and b2825 (inactive type I Cas) strains of P.aeruginosa and plated after 0, 3 or 24 hour incubation.

Figure 14 depicts the efficacy of crArray/Cas insertion in multiple pseudomonas aeruginosa strains. P4209wt (wild type), p4209E001 (Cas system only) and p4209E002 (crArray1+cas system targeted) were plaque on the b2550 (type I-E Cas), b2631 (type I-F Cas), b2816 (type I-E/I-F Cas) and b2825 (inactive type I Cas) strains of P.aeruginosa.

Figures 15A-15D illustrate in vivo efficacy results comparing p1772wt (wild type) and p1772e005 (targeting crarray1+cas system). Fig. 15A is a schematic diagram depicting the experimental setup of fig. 15B-15D. Figure 15B depicts the efficacy of phage when injected into the thigh muscle of mice. The left panel depicts the number of Colony Forming Units (CFU) recovered 6 hours after infection. The right panel depicts the number of Plaque Forming Units (PFU) recovered 6 hours after infection. Figure 15C depicts the efficacy of phage when injected into the thigh muscle of mice and depicts the number of CFUs (upper panel) and PFUs (lower panel) recovered 8 and 24 hours after infection. Figure 15D depicts the efficacy of phage when administered intravenously and depicts the number of CFUs (upper panel) and PFUs (lower panel) recovered at 9, 12, 15 and 24 hours post-infection. Fig. 15E depicts the experimental setup of fig. 15F. Figure 15F depicts dose response with p1772wt and p1772e005 treatment and depicts amounts of CFU (upper panel) and PFU (lower panel) recovered 8 and 24 hours post infection. Data are shown as mean ± SEM. * p <0.05, < p <0.01, < p <0.001, < p <0.0001. One-way ANOVA and multiple comparisons or two-way ANOVA and base tests.

Figure 16 illustrates that CRISPR-Cas3 engineered reference phage PB1e002 (crarray1+cas system) cooperates with p1772e005 (crarray1+cas system).

FIG. 17 depicts the number of transformants produced after transfection of Pseudomonas with inserts containing different spacer sequences.

Figure 18A depicts an assay for testing efficiency in CRISPR engineered phage alone or in cocktails. Fig. 18B depicts the effect of cocktail treatment in mice infected with pseudomonas aeruginosa B2631. Fig. 18C depicts the effect of cocktail treatment in mice infected with pseudomonas aeruginosa b 1121. Fig. 18D depicts the effect of cocktail treatment in mice infected with pseudomonas aeruginosa b 3144. Figure 18E depicts the effect of phage treatment with CRISPR engineering compared to wild type phage. Figure 18F depicts a graphical representation of an assay for testing cocktail efficacy compared to treatment with phage alone. Fig. 18G depicts the effect of treatment with phage alone or phage cocktails (as shown in the figure) in mice infected with pseudomonas aeruginosa b 2631. Figure 18H depicts the effect of treatment with cocktails or phage alone in mice infected with pseudomonas aeruginosa b 3144. FIG. 18I depicts the effect of treatment with cocktails or phage alone in mice infected with P.aeruginosa.

Fig. 19A depicts an assay for testing cocktail efficacy. Fig. 19B depicts the effect of treatment with phage cocktail CK125 in mice infected with pseudomonas aeruginosa B2631. Fig. 19C depicts the effect of cocktail treatment in mice infected with pseudomonas aeruginosa b 1121. Fig. 19D depicts the effect of cocktail treatment in mice infected with pseudomonas aeruginosa b 3144. TOB = tobramycin.

Fig. 20A shows a graphical representation of an experimental setup for testing phage cocktail CK125 for anti-biofilm activity against preformed biofilms from key pseudomonas aeruginosa strains b1121 and b2631 using a Minimum Biofilm Eradication Concentration (MBEC) assay. Left side of fig. 20B, inhibition for 24h, middle, inhibition for 48 h. On the right, a table depicting comparison data of MBEC IC50 with phage cocktails or ciprofloxacin is shown.

Fig. 21 (upper panel) shows experimental setup and results of testing phage cocktail PACK512 for anti-biofilm activity against biofilms from respiratory/clinical isolates of pseudomonas aeruginosa strains b1121, b2631 and b2631 using Minimum Biofilm Eradication Concentration (MBEC) method. Inhibition in% at 24h, right, 48h on the left side of fig. 22 (bottom).

Fig. 22A shows a test set-up for testing the bactericidal activity of a cocktail in the presence of human mucin. FIG. 22B shows the results from the assay, bacterial load in different treatments, left side, inhibition by Pseudomonas aeruginosa strain B1121 from respiratory isolates; on the right, the results of inhibition by Pseudomonas aeruginosa strain b2631 from the CF isolate.

Fig. 23A shows a test setup for testing the bactericidal efficacy of cocktails in the presence of mucins and a comparison with tobramycin. Figure 23B shows bacterial load in the presence of cocktail or tobramycin treatment compared to no treatment control.

Figure 24 shows data from phage level testing after bacterial removal in the lower respiratory tract infection model of pseudomonas aeruginosa.

Detailed Description

In certain embodiments, disclosed herein are bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising: (a) CRISPR arrays (also known as "crArray"); (b) a cascades polypeptide; and (c) a Cas3 polypeptide. In certain embodiments, also disclosed herein are pharmaceutical compositions comprising the bacteriophage disclosed herein. In certain embodiments, further disclosed herein are methods of killing a pseudomonas species comprising introducing into the pseudomonas species a bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising: (a) a CRISPR array; (b) a cascades polypeptide; and (c) a Cas3 polypeptide. In certain embodiments, further disclosed herein are methods of treating a disease or condition in an individual in need thereof, the method comprising administering to the individual a nucleic acid sequence encoding a type I CRISPR-Cas system comprising: (a) a CRISPR array; (b) a cascades polypeptide; and (c) Cas3 polypeptide

Certain terms

Unless defined otherwise, 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.

Unless the context indicates otherwise, it is expressly indicated that the various features of the disclosure described herein can be used in any combination. Furthermore, the present disclosure also contemplates that, in some embodiments, any feature or combination of features set forth herein is excluded or omitted. For example, if the specification states that a composition comprises components A, B and C, then it is expressly indicated that either or a combination of A, B or C is omitted and discarded, either alone or in any combination.

Those skilled in the art will understand the interchangeability of terms for individual CRISPR-Cas systems and their components due to lack of consistency in the literature and efforts are underway in the art to unify these terms.

As used in the specification 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. Furthermore, as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, and includes no combinations when interpreted in the alternative conjunctive ("or").

As used herein, the term "about" when referring to a measurable value such as a dose or period of time, refers to a change of a particular amount of ±20%, ±10%, ±5%, ±1%, +0.5% or even ±0.1%. As used herein, phrases such as "between X and Y" and "between about X and Y" should be construed 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".

As used herein, the terms "comprises," "comprising," "includes," "including," and "having" specify the presence of 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.

As used herein, the transitional phrase "consisting essentially of … …" means that the scope of the claims should be interpreted to encompass the specific materials or steps recited in the claims, as well as those materials or steps that do not materially affect the basic and novel characteristics of the claimed disclosure. Accordingly, the term "consisting essentially of … …" is not intended to be interpreted as equivalent to "comprising" when used in the claims of the present disclosure.

As used herein, the term "consisting of … …" excludes any feature, step, operation, element, and/or component not directly recited. The use of "consisting of … …" merely limits the features, steps, operations, elements, and/or components set forth in the clause and excludes other features, steps, operations, elements, and/or components from the claims in general.

As used herein, the term "complementary" or "complementarity" refers to the natural binding of polynucleotides by base pairing under the conditions of salt and temperature allowed. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A". Complementarity between two single-stranded molecules is "partial" in that only some nucleotides bind, or complete when complete complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

As used herein, "complement" means 100% complementarity or identity to a comparative nucleotide sequence, or means less than 100% complementarity (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% etc. complementarity). Complement or complementation may also be used in the "complement" or "complement" aspects of the mutation.

As used herein, the terms "CRISPR phage," "CRISPR-enhanced phage," and "crPhage" refer to a bacteriophage particle comprising bacteriophage DNA comprising at least one heterologous polynucleotide encoding at least one component of a CRISPR-Cas system (e.g., a CRISPR array, crRNA; e.g., a P1 bacteriophage comprising an insertion that targets the 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.

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 refers to two or more sequences or subsequences having 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 is used as a reference sequence for comparison with the test sequence. When using a sequence comparison algorithm, test and reference sequences are input 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 of the test sequence relative to the reference sequence based on the specified program parameters.

The optimal alignment of sequences for the alignment window is performed by means such as the local homology algorithms of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and optionally by computerized embodiments of these algorithms, as may beWisconsin/>A portion of (Accelrys inc., san Diego, CA) obtained GAP, BESTFIT, FASTA and TFASTA. The "identity score" of an aligned segment of a test sequence and a reference sequence is the number of identical components that are common to both aligned sequences divided by the total number of components in the reference sequence segment (i.e., the entire reference sequence or a smaller defined portion of the reference sequence). Percent sequence identity is expressed as the identity score multiplied by 100. One or more polynucleotide sequences are compared to a full length polynucleotide sequence or a portion or longer polynucleotide sequence thereof. In some cases, BLASTX version 2.0 is used for translated nucleotide sequences and BLASTN version 2.0 is used for polynucleotide sequences to determine "percent identity".

As used herein, a "target nucleotide sequence" refers to a portion of a target gene (i.e., a target region in a genome or "protospacer sequence" that 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 greater ratio)) to a spacer sequence in a CRISPR array.

As used herein, the term "protospacer adjacent motif" or "PAM" refers to a DNA sequence present on a target DNA molecule adjacent to a nucleotide sequence that matches a spacer sequence. This motif is present in the target gene in the vicinity of the region to which the spacer sequence binds, because the spacer sequence is complementary to the region; and identifies the initiation point for base pairing with the spacer nucleotide sequence. The exact PAM sequence required varies between each different CRISPR-Cas system. Non-limiting examples of PAM include CCA, CCT, CCG, TTC, AAG, AGG, ATG, GAG and/or CC. In some cases, in type I systems, PAM is located directly 5 'of the sequence that matches the spacer, and thus 3' of the sequence that base pairs with the spacer nucleotide sequence, and is directly recognized by cascades. In some cases, PAM is yc for a bacillus salt tolerant I-C system, where Y may be T or C. In some cases, PAM is TTC for pseudomonas aeruginosa type I-C systems. Once homologous protospacer sequences and PAMs are identified, cas3 is recruited, which then cleaves and degrades the target DNA. For type II systems, cas 9/sgrnas require PAM to form an R loop to interrogate specific DNA sequences through their watson-crick pairing of the guide RNA with the genome. PAM specificity is a function of the DNA binding specificity of the Cas9 protein (e.g., the protospacer adjacent motif recognition domain at the C-terminus of Cas 9).

As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, tRNA, rRNA, miRNA, anti-micrornas, regulatory RNAs, and the like. The gene may or may not be capable of being used to produce a functional protein or gene product. Genes comprise coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and/or 5 'and 3' non-translated regions). A gene is "isolated," which refers to a nucleic acid that is substantially or essentially free of components that normally exist in association with the nucleic acid in its natural state. Such components include other cellular material, media from recombinant production, and/or various chemicals for chemical synthesis of nucleic acids.

The term "treatment" refers to a reduction in the severity of, or at least a partial improvement or amelioration of, a condition in a subject, and some degree of alleviation, alleviation or reduction of at least one clinical symptom, and/or delay of progression of a disease or condition, and/or delay of onset of a disease or condition. With respect to an infection, disease or condition, the term refers to a reduction in symptoms or other manifestations of the infection, disease or condition. In some embodiments, treatment reduces 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 term "preventing" (and grammatical variations thereof) refers to preventing and/or delaying the onset of an infection, disease, condition, and/or clinical symptom in a subject, and/or a reduction in the severity of the infection, disease, condition, and/or clinical symptom onset relative to what would occur without the methods disclosed herein prior to the onset of the disease, condition, and/or clinical symptom. Thus, in some embodiments, to prevent infection, foods, surfaces, medical tools, and devices are treated with the compositions and methods disclosed herein.

The terms used herein with respect to "infection", "disease" or "condition" refer to any adverse, negative or deleterious physiological condition in a subject that results from the presence of a target bacterium in the subject. The terms are used interchangeably.

As used herein, the term "individual" or "subject" includes any animal suffering from or susceptible to an infection, disease or condition involving bacteria. Thus, in some embodiments, the subject is a mammal, bird, reptile, amphibian, fish, crustacean, or mollusc. Mammalian subjects include, but are not limited to, humans, non-human primates (e.g., gorilla, monkey, baboon, chimpanzee, etc.), dogs, cats, goats, horses, pigs, cattle, sheep, etc., and laboratory animals (e.g., rats, guinea pigs, mice, gerbils, hamsters, etc.). Bird subjects include, but are not limited to, chickens, ducks, turkeys, geese, quails, pheasants, and birds raised as pets (e.g., parrots, budgerigars, meracil parrots, canaries, etc.). Fish subjects include, but are not limited to, species used in aquaculture (e.g., tuna, salmon, tilapia, catfish, carp, trout, cod, weever, jetstrefoil, sea bream, etc.). Crustacean subjects include, but are not limited to, species used in aquaculture (e.g., shrimp, prawn, lobster, crayfish, crab, etc.). Mollusc subjects include, but are not limited to, species used in aquaculture (e.g., abalone, mussel, oyster, clams, scallops, etc.). In some embodiments, suitable subjects include male and female, as well as subjects of any age, including embryonic (e.g., intrauterine or intraegg), infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a human.

As used herein, the term "isolated" in the context of a nucleic acid sequence is a nucleic acid sequence that exists away from its natural environment.

As used herein, an "expression cassette" means a recombinant nucleic acid molecule (e.g., a recombinant nucleic acid molecule and CRISPR array disclosed herein) comprising a nucleotide sequence of interest, wherein the nucleotide sequence is operably associated with at least a control sequence (e.g., a promoter).

As used herein, "chimeric" refers to a nucleic acid molecule or polypeptide in which at least two components are derived from different sources (e.g., different organisms, different coding regions).

As used herein, a "selectable marker" means a nucleotide sequence that, when expressed, confers a unique phenotype on host cells expressing the marker, thereby allowing such transformed cells to be distinguished from those cells without the marker.

As used herein, a "vector" refers to a composition for transferring, delivering, or introducing a nucleic acid (or nucleic acids) into a cell.

As used herein, "pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing any undesirable biological effects such as toxicity.

As used herein, the term "biofilm" means the accumulation of microorganisms embedded in a polysaccharide matrix. The biofilm is formed on solid biological or non-biological surfaces, has important medical significance and accounts for more than 80% of in-vivo microbial infection.

As used herein, the term "in vivo" is used to describe events that occur within a subject.

As used herein, the term "in vitro" is used to describe an event that occurs in a container for holding laboratory reagents such that it is separated from the biological source from which the material was obtained. In vitro assays may encompass cell-based assays in which living or dead cells are employed. In vitro assays may also encompass cell-free assays that do not employ intact cells.

CRISPR/CAS system

CRISPR-Cas systems are the natural adaptive immune system found in bacteria and archaea. The CRISPR system is a nuclease system involved in the defense against invading phages and plasmids, which provides some form of acquired immunity. Based on the collection of Cas genes and their phylogenetic relationship, the CRISPR-Cas system has diversity. There are at least six different types (I to VI), where type I represents more than 50% of all identification systems in bacteria and archaea. In some embodiments, a type I, type II, type IV, type V, or type VI CRISPR-Cas system is used herein.

Type I systems fall into seven subtypes, including: I-A, I-B, I-C, I-D, I-E, I-F and I-U. Type I CRISPR-Cas systems include a multi-subunit complex called cascades (a complex associated with antiviral defenses), cas3 (a protein with nuclease, helicase and exonuclease activity responsible for degrading target DNA) and CRISPR arrays encoding crrnas (stabilizing cascades and directing cascades and Cas3 to DNA targets). Cascade forms a complex with crRNA, and protein-RNA pairs recognize their genomic targets by complementary base pairing between the 5' end of the crRNA sequence and a predefined protospacer. Such complexes are directed to homologous loci of pathogen DNA via crRNA within the pathogen genome and regions encoded within Protospacer Adjacent Motifs (PAMs). Base pairing occurs between crRNA and target DNA sequences, resulting in conformational changes. In type I-E systems, PAM is recognized by CasA proteins within cascades, which then unwind flanking DNA to assess the extent of base pairing between the target and spacer portions of crRNA. Sufficient recognition results in cascades recruiting and activating Cas3.Cas3 then cleaves the non-target strand and begins to degrade the strand in the 3 'to 5' direction.

In type I-C systems, proteins Cas5, cas8C, and Cas7 form cascades effector complexes. Cas5 processes the pre-crRNA (which may take the form of a multi-spacer array or a single spacer between two repeats) to produce a single crRNA composed of hairpin structures formed of the remaining repeat sequences and linear spacers. The effector complex then binds to the processed crRNA and the DNA is scanned to identify PAM sites. In type I-C systems, PAM is recognized by Cas8C protein and then functions to break down the DNA duplex. If the sequence 3' of PAM matches the crRNA spacer bound to the effector complex, the complex undergoes a conformational change and Cas3 is recruited to the site. Cas3 then cleaves the non-target strand and begins to degrade DNA. In some cases, cas5 includes Cas5, cas5c, cas5d, and a sequence having at least 90% identity to SEQ ID No. 76. In some cases, cas7 includes Cas7, cas7c, and a sequence having at least 90% identity to SEQ ID No. 78. In some cases, cas8 includes Cas8, cas8c, and a sequence having at least 90% identity to SEQ ID No. 77.

In some embodiments, the CRISPR-Cas system is internally derived from a pseudomonas species. In some embodiments, the CRISPR-Cas system is externally derived from a pseudomonas species. 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-C CRISPR-Cas system. In some embodiments, the CRISPR-Cas system is a type I-C CRISPR-Cas system derived from pseudomonas aeruginosa. In some embodiments, the CRISPR-Cas system is an I-D type 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 an I-U type 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.

In some embodiments, processing of the CRISPR arrays disclosed herein includes, but is not limited to, the following processes: 1) Transcription of nucleic acid encoding pre-crRNA; 2) The pre-crRNA is recognized by cascades and/or specific components of cascades (e.g., cas 6), and 3) the pre-crRNA is processed into mature crRNA by cascades or components of cascades (e.g., cas 6). In some embodiments, modes of action of a type I CRISPR system include, but are not limited to, the following processes: 4) Compounding mature crRNA with Cascade; 5) Target recognition by the complexed mature crRNA/cascades complex; and 6) nuclease activity at the target that results in DNA degradation.

In some embodiments, provided herein are components of a CRISPR-Cas system and bacteriophage comprising the same. As one example, provided herein is a nucleic acid sequence comprising at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to any one of SEQ ID NOs 83-87. In some cases, the nucleic acid sequence comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO 83. In some cases, the nucleic acid sequence comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO 84. In some cases, the nucleic acid sequence comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 85. In some cases, the nucleic acid sequence comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO 86. In some cases, the nucleic acid sequence comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO 87. In some cases, the nucleic acid sequence comprises at least about 90% identity to SEQ ID NO. 83. In some cases, the nucleic acid sequence comprises at least about 90% identity to SEQ ID NO. 84. In some cases, the nucleic acid sequence comprises at least about 90% identity to SEQ ID NO. 85. In some cases, the nucleic acid sequence comprises at least about 90% identity to SEQ ID NO. 86. In some cases, the nucleic acid sequence comprises at least about 90% identity to SEQ ID NO. 87. In any of these embodiments, the nucleic acid sequence may comprise a sequence having at least 90% identity to any of SEQ ID NOs 12-23, 31-74 or 88-120. In one non-limiting example, the nucleic acid sequence comprises one or more of the following: SEQ ID NO. 12, SEQ ID NO. 16 and SEQ ID NO. 20.

Further provided herein are CRISPR-Cas system components comprising a nucleic acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 24. In some cases, the nucleic acid sequence comprises at least about 90% identity to SEQ ID NO. 24. In any of these embodiments, the nucleic acid sequence may comprise a sequence having at least 90% identity to any of SEQ ID NOs 12-23, 31-74 or 88-120. In one non-limiting example, the nucleic acid sequence comprises one or more of the following: SEQ ID NO. 12, SEQ ID NO. 16 and SEQ ID NO. 20.

Provided herein are CRISPR-Cas system components comprising at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 25. In some cases, the nucleic acid sequence comprises at least about 90% identity to SEQ ID NO. 25. In any of these embodiments, the nucleic acid sequence may comprise a sequence having at least 90% identity to any of SEQ ID NOs 12-23, 31-74 or 88-120. In one non-limiting example, the nucleic acid sequence comprises one or more of the following: SEQ ID NO. 12, SEQ ID NO. 16 and SEQ ID NO. 20.

CRISPR phage

In certain embodiments, disclosed herein are bacteriophage compositions comprising a CRISPR-Cas system and methods of use thereof.

Bacteriophages or "phages" represent a group of bacterial viruses and are engineered or derived from environmental sources. The individual bacteriophage host range is usually narrow, which means that the bacteriophage has a high degree of specificity for one strain or a small number of strains of a certain bacterial species, and this specificity makes them uniquely antibacterial. Bacteriophages are bacterial viruses that rely on the cellular machinery of the host for replication. Depending on their lifestyle, bacteriophages are generally classified as virulent phages or temperate phages. Toxic bacteriophages, also known as lytic bacteriophages, can only undergo lytic replication. Lytic bacteriophages infect host cells, make multiple rounds of replication, and trigger cell lysis to release newly produced bacteriophage particles. In some embodiments, the lytic bacteriophages disclosed herein retain their replication ability. In some embodiments, the lytic bacteriophage disclosed herein retain their ability to trigger cell lysis. In some embodiments, lytic bacteriophages disclosed herein retain their ability to replicate and trigger cell lysis. In some embodiments, the bacteriophage disclosed herein comprises a CRISPR array. In some embodiments, the CRISPR array does not affect the ability of the bacteriophage to replicate and/or trigger cell lysis. Mild or lysogenic bacteriophages can undergo lysogens in which the phage ceases replication and is stably present in the host cell, either integrating into the bacterial genome or being maintained as an extrachromosomal plasmid. Mild phages can also undergo lytic replication, similar to their lytic bacteriophage counterparts. Whether temperate phages replicate or undergo lysogens at the time of infection depends on a number of factors including growth conditions and the physiological state of the cells. Bacterial cells having lysogenic phages integrated into their genome are known as lysogenic bacteria or lysogens. Exposure to adverse conditions may trigger reactivation of lysogenic phages, termination of the lysogenic state, and restoration of phage lytic replication. This process is called induction. Adverse conditions that favor termination of the lysogenic state include drying, exposure to UV or ionizing radiation, and exposure to mutagenic substances. This results in phage gene expression, reversal of the integration process, and lytic proliferation. In some embodiments, the temperate bacteriophage disclosed herein is conferred lytic. The term "lysogenic gene" refers to any gene whose gene product promotes the lysogen of a temperate phage. As in the case where the integrase protein aids in the integration of the bacteriophage into the host genome, the lysogenic genes may contribute directly thereto. Lysogenic genes can also indirectly promote lysogenicity, as in the case where CI transcriptional regulators prevent transcription of genes required for lytic replication, thereby facilitating maintenance of lysogenicity.

Three general methods are used for packaging and delivering synthetic DNA by bacteriophages. In the first approach, the synthetic DNA is recombined into the phage genome in a targeted manner, which typically involves selectable markers. In the second method, restriction sites within phage are used to introduce synthetic DNA in vitro. In a third approach, a plasmid, which typically encodes a phage packaging site and a lytic origin of replication, is packaged as an assembled part of a phage particle. The resulting plasmid is called a "phagemid".

Phage are limited to a given bacterial strain for evolutionary reasons. In some cases, injection of their genetic material into incompatible strains may be counterproductive. Thus, phages have evolved to specifically infect a limited typical type of bacterial strain. However, some phages have been found to inject their genetic material into a variety of bacteria. A classical example is the P1 phage, which has been demonstrated to be capable of DNA injection in a variety of gram-negative bacteria.

Phage capsids have a limited capacity, which means that their genome size must be kept within a small range in order to be packaged correctly. Since the DNA encoding the Cas operon + CRISPR array is quite large (6000 bp total), other DNA must be removed from the phage genome in order to make room for the Cas system. Exemplary phages engineered herein comprise Cas operons and CRISPR arrays inserted into phages such that the phages remain viable.

In some embodiments, disclosed herein are bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a pseudomonas species. In some embodiments, the bacteriophage comprises a first nucleic acid sequence encoding a first spacer sequence or crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in pseudomonas aeruginosa, provided that the bacteriophage is rendered lytic. In some embodiments, the bacteriophage is a temperate bacteriophage. In some embodiments, the bacteriophage is rendered lytic by removal, replacement, or inactivation of a lysogenic gene. In some embodiments, the lysogenic genes play a role in maintaining the lysogenic cycle in the bacteriophage. In some embodiments, the lysogenic genes play a role in establishing the lysogenic cycle in the bacteriophage. In some embodiments, lysogenic genes play a role in establishing and maintaining lysogenic cycles in the bacteriophage. In some embodiments, the lysogenic gene is a repressor gene. In some embodiments, the lysogenic gene is a cI repressor gene. In some embodiments, the bacteriophage is rendered lytic by removal of a regulatory element of the lysogen. In some embodiments, the bacteriophage is rendered lytic by removal of a promoter of the lysogen. In some embodiments, the bacteriophage is rendered lytic by removal of a functional element of the lysogen. In some embodiments, the lysogenic gene is an activator gene. In some embodiments, the lysogenic gene is a cII gene. In some embodiments, the lysogenic gene is the lexA gene. In some embodiments, the lysogenic gene is the int (integrase) gene. In some embodiments, two or more lysogenic genes are removed, replaced, or inactivated to cause arrest of the bacteriophage lysogenic cycle and/or induction of the lytic cycle. In some embodiments, the bacteriophage is conferred lytic via a second CRISPR array comprising a second spacer directed to a lysogen. In some embodiments, the bacteriophage is rendered lytic by insertion of one or more lytic genes. In some embodiments, the bacteriophage is rendered lytic by insertion of one or more genes that help induce a lytic cycle. In some embodiments, the bacteriophage is rendered lytic by altering the expression of one or more genes that help induce a lytic cycle. In some embodiments, the bacteriophage phenotypically changes from lysogenic bacteriophage to lytic bacteriophage. In some embodiments, the phenotypic change is via self-targeting of the CRISPR-Cas system to render the bacteriophage lytic because it is insoluble. In some embodiments, the self-targeted CRISPR-Cas comprises a self-targeted crRNA from a prophage genome and kills lysogens. In some embodiments, the bacteriophage is rendered lytic by an environmental change. In some embodiments, environmental changes include, but are not limited to, changes in temperature, pH, or nutrients; exposure to antibiotics, hydrogen peroxide, foreign DNA or DNA damaging agents; the presence of organic carbon; and the presence of heavy metals, for example in the form of chromium (VI). In some embodiments, the bacteriophage that is rendered lytic is prevented from reverting to a lysogenic state. In some embodiments, bacteriophages that are rendered lytic are prevented from reverting to lysogenic state by the introduction of additional CRIPSR arrays. In some embodiments, the bacteriophage does not confer any new properties to the pseudomonas species other than cell death caused by the lytic activity of the bacteriophage and/or the activity of the CRISPR array.

In some embodiments, further disclosed herein are temperate bacteriophage comprising a first nucleic acid sequence encoding a first spacer sequence or crRNA transcribed therefrom, wherein the first spacer sequence is complementary to a target nucleotide sequence from a target gene in a pseudomonas species, provided that the bacteriophage is rendered lytic. In some embodiments, the bacteriophage infects a plurality of bacterial strains. In some embodiments, the target nucleotide sequence comprises all or part of a promoter sequence of the target gene. In some embodiments, the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding strand of the transcribed region of the target gene. In some embodiments, the target nucleotide sequence comprises at least a portion of an essential gene required for the survival of a pseudomonas species. In some embodiments, the target nucleotide sequence comprises a highly conserved non-coding sequence or intergenic sequence. In some embodiments, the target sequence is an intergenic sequence located between the essential gene rpmF and the conserved hypothetical protein. In some embodiments, the essential gene is Tsf, acpP, gapA, 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, tRNAasn or metK. In some embodiments, the essential gene is dnaA, ftsA, gyrB, dnaN, glnS or rpoB. In some embodiments, the target sequence is the boundary between PA4325 (hypothetical protein), PA1310 (phnW, pyruvate aminotransferase), or PA2970 (rpmF, 50S ribosomal protein L32) and PA2971 (conserved hypothetical protein). In some embodiments, the target nucleotide sequence is in a non-essential gene. In some embodiments, the target nucleotide sequence is a non-coding sequence. In some embodiments, the non-coding 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 pseudomonas species. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a sequence present in a pseudomonas species. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence that comprises all or part of a promoter sequence of an essential gene. In some embodiments, the first nucleic acid sequence comprises a first CRISPR array comprising at least one repeat sequence. In some embodiments, the at least one repeat sequence is operably linked to the first spacer sequence at its 5 'end or its 3' end.

In some embodiments, the bacteriophage DNA is from a lysogenic or temperate bacteriophage. In some embodiments, the bacteriophage includes, but is not limited to, p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4209, p4430, or PB1, or two or more phages thereof.

In some embodiments, the bacteriophage of interest is obtained from an environmental source or commercial research provider. In some embodiments, the obtained bacteriophage is screened for lytic activity against a library of bacteria and related strains. In some embodiments, bacterial phage are screened for their ability to develop primary resistance in the bacteria being screened for a library of bacteria and their related strains.

In some embodiments, the nucleic acid is inserted into the bacteriophage genome. In some embodiments, the nucleic acid comprises a crArray, cas system, or a combination thereof. In some embodiments, the nucleic acid is inserted into the bacteriophage genome at a transcription terminator site at the end of the operon of interest. In some embodiments, the nucleic acid is inserted into the bacteriophage genome as a replacement for one or more removed non-essential genes. In some embodiments, the nucleic acid is inserted into the bacteriophage genome as a replacement for one or more removed lysogens. In some embodiments, replacement of non-essential and/or lysogenic genes with nucleic acids enhances the lytic activity of the bacteriophage. In some embodiments, replacement of non-essential and/or lysogenic genes with nucleic acids confers lytic properties to the lysogenic bacteriophage.

In some embodiments, the nucleic acid is introduced into the bacteriophage genome at a first location, and one or more non-essential and/or lysogenic genes are removed and/or inactivated separately from the bacteriophage genome at different locations. In some embodiments, removal of one or more non-essential and/or lysogenic genes renders the lysogenic bacteriophage a lytic bacteriophage. Similarly, in some embodiments, one or more lytic genes are introduced into the bacteriophage to render the non-lytic, lysogenic bacteriophage a lytic bacteriophage.

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, insertion of one or more cleavage genes is achieved by homologous recombination by any suitable chemical, biochemical and/or physical means.

In some embodiments, the non-essential gene to be removed and/or replaced from the bacteriophage is a gene that is not essential for the survival of the bacteriophage. In some embodiments, the non-essential genes to be removed and/or replaced from the bacteriophage are genes that are not essential for the induction and/or maintenance of the lytic cycle.

In certain embodiments, disclosed herein are bacteriophage comprising an intact exogenous CRISPR system. In some embodiments, 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, a type V CRISPR-Cas system, or a type VI CRISPR-Cas system. In certain embodiments, disclosed herein are bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising: (a) a CRISPR array; (b) a cascades polypeptide; and (c) a Cas3 polypeptide. In some embodiments, the CRISPR-Cas system comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 24. In some embodiments, the CRISPR-Cas system comprises at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID No. 25.

In some embodiments, the bacteriophage is p1106 (ATCC deposit number PTA-127024), wherein the bacteriophage comprises a type I CRISPR-Cas system. In some embodiments, the bacteriophage is p1587 (ATCC deposit No. PTA-127027), wherein the bacteriophage comprises a type I CRISPR-Cas system. In some embodiments, the bacteriophage is p1772 (ATCC deposit No. PTA-127030), wherein the bacteriophage comprises a type I CRISPR-Cas system. In some embodiments, the bacteriophage is p1835 (ATCC accession number PTA-127032), wherein the bacteriophage comprises a type I CRISPR-Cas system. In some embodiments, the bacteriophage is p2037 (ATCC accession number PTA-127034), wherein the bacteriophage comprises a type I CRISPR-Cas system. In some embodiments, the bacteriophage is p2131 (ATCC accession number PTA-127036), wherein the bacteriophage comprises a type I CRISPR-Cas system. In some embodiments, the bacteriophage is p2132 (ATCC accession number PTA-127038), wherein the bacteriophage comprises a type I CRISPR-Cas system. In some embodiments, the bacteriophage is p2363 (ATCC deposit No. PTA-127041), wherein the bacteriophage comprises a type I CRISPR-Cas system. In some embodiments, the bacteriophage is p2421 (ATCC accession number PTA-127043), wherein the bacteriophage comprises a type I CRISPR-Cas system. In some embodiments, the bacteriophage is p2973 (ATCC accession number PTA-127045), wherein the bacteriophage comprises a type I CRISPR-Cas system. In some embodiments, the bacteriophage is PB1 (ATCC deposit number PTA-127049), wherein the bacteriophage comprises a type I CRISPR-Cas system. In some embodiments, the bacteriophage comprises a bacteriophage listed in table 1A.

In some embodiments, the bacteriophage comprises a CRISPR system having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 24. In some embodiments, the bacteriophage comprises a CRISPR system having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 25.

In some embodiments, the bacteriophage is p1106e003 (ATCC deposit No. PTA-127023) or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to p1106e 003. In some embodiments, the bacteriophage is p1587e002 (ATCC deposit No. PTA-127026) or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to p1587e 002. In some embodiments, the bacteriophage is p1772e005 (ATCC deposit No. PTA-127029) or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to p1772e 005. In some embodiments, the bacteriophage is p1835e002 (ATCC deposit No. PTA-127031) or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to p1835e 002. In some embodiments, the bacteriophage is p2037e002 (ATCC deposit No. PTA-127033) or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to p2037e 002. In some embodiments, the bacteriophage is p2131e002 (ATCC deposit No. PTA-127035) or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to p2131e 002. In some embodiments, the bacteriophage is p2132e002 (ATCC deposit No. PTA-127037) or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to p2132e 002. In some embodiments, the bacteriophage is p2363e003 (ATCC deposit No. PTA-127040) or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to p2363e 003. In some embodiments, the bacteriophage is p2421e002 (ATCC accession No. PTA-127042) or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to p2421e 002. In some embodiments, the bacteriophage is p2973e002 (ATCC deposit No. PTA-127044) or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to p2973e 002. In some embodiments, the bacteriophage is PB1e002 (ATCC deposit No. PTA-127048) or has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to PB1e 002. In some embodiments, the bacteriophage comprises a bacteriophage listed in table 1A. In some embodiments, a bacteriophage cocktail system is provided that includes one or more engineered bacteriophage and a wild-type bacteriophage. In some embodiments, the wild-type phage is a phage of table 5A, table 5B, or table 6A. In some embodiments, the wild-type phage is a wild-type pluravirus. Non-limiting exemplary wild-type pluronic viruses include p1106, p1587, p1835, p2037, p2363, p2421 and pb1. In some embodiments, the wild-type phage is wild-type sal Mu Na virus. Non-limiting exemplary wild-type salsa Mu Na viruses include p1772, p2121, p2132 and p2973. In some embodiments, the wild-type phage is a wild-type southern virus. In some embodiments, the wild-type phage is a wild-type Φkz virus. Non-limiting examples of wild-type Φkz viruses include p1194p.b008 and p4430. In some embodiments, the wild-type phage is a wild-type Φkmv virus. One non-limiting example of a wild-type Φkmv virus is p2167. In some embodiments, the wild-type phage is wild-type brunesoid virus. Non-limiting examples of wild-type bruneshop viruses include p1695 and p3278. In some embodiments, the wild-type phage is p1194. In some embodiments, the wild-type phage is p4430. In some embodiments, the wild-type phage is p1695. In some embodiments, the wild-type phage is Φkz virus, Φkmv virus, brunesoid virus, sal Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cetuximab virus, vesicular virus, detrilavirus, ehrlift virus, holo Wei Bingdu, chikumi virus, livina virus, lu Saipu ti Ma Bingdu, nipenv, parkrina virus, pamex virus, boenmer virus, filteirus, pri Mo Liji virus, sapetrex virus, starbuchner virus, takii virus, johne virus, atameria virus, or prina virus.

In some embodiments, the bacteriophage cocktail system comprises CK000512 (p 1106e003, p1835e002, p1772e005, p2131e002, p4430, and p 1695).

In some embodiments, multiple bacteriophage are used together. In some embodiments, the plurality of bacteriophage used together target the same or different bacteria within a sample or subject. In some embodiments, cocktails comprising multiple bacteriophages are used together. In some embodiments, the cocktail comprises at least 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, or 27 phages selected from table 1A. In some embodiments, the cocktail comprises 2 phages selected from table 1A. In some embodiments, the cocktail comprises 3 phages selected from table 1A. In some embodiments, the cocktail comprises 3 phages selected from table 1A. In some embodiments, the cocktail comprises 4 phages selected from table 1A. In some embodiments, the cocktail comprises 5 phages selected from table 1A. In some embodiments, the cocktail comprises 6 phages selected from table 1A. In some embodiments, the cocktail comprises a cocktail selected from table 6A. In some embodiments, at least one bacteriophage in the cocktail comprises a CRISPR array. In some embodiments, at least 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, or 27 bacteriophage present in the cocktail comprises a CRISPR array. In some embodiments, at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding a cascades 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, 21, 22, 23, 24, 25, 26, or 27 bacteriophage present in the cocktail comprises a nucleic acid sequence encoding a cascades polypeptide. In some embodiments, at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding a Cas3 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, 21, 22, 23, 24, 25, 26, or 27 bacteriophage present in the cocktail comprises a nucleic acid sequence encoding a Cas3 polypeptide. In some embodiments, the cocktail comprises p1106e003, p1835e002, p1772e005, and p2131e002. In some embodiments, the cocktail further comprises p1194. In some embodiments, the cocktail further comprises p1695. In some embodiments, the cocktail further comprises p4430. In some embodiments, the cocktail comprises p1106e003, p1835e002, p1772e005, p2131e002, p1194, and p1695. In some embodiments, the cocktail comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430, and p1695.

In some embodiments, provided herein is a Φkz viral bacteriophage comprising a type I CRISPR-Cas system. In some embodiments, the Φkz virus is p1194 or p4430. In some embodiments, the CRISPR-Cas system comprises one or more spacer sequences that are complementary to a target nucleotide sequence in a pseudomonas species; a cascades polypeptide; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. In some embodiments, the CRISPR array comprises at least one repeat sequence comprising at least about 90% sequence identity to any one of SEQ ID NOS: 26-30. In some embodiments, the nucleic acid sequence further comprises a promoter sequence, for example selected from SEQ ID NOs 1-11. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81), and a Cas7 polypeptide (optionally SEQ ID NO: 82) (type I-C CRISPR-Cas system). In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID NO. 24. In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID No. 25.

In some embodiments, provided herein is a Φkmv viral bacteriophage comprising a type I CRISPR-Cas system. In some embodiments, the Φkmv virus is p2167. In some embodiments, the CRISPR-Cas system comprises one or more spacer sequences that are complementary to a target nucleotide sequence in a pseudomonas species; a cascades polypeptide; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. In some embodiments, the CRISPR array comprises at least one repeat sequence comprising at least about 90% sequence identity to any one of SEQ ID NOS: 26-30. In some embodiments, the nucleic acid sequence further comprises a promoter sequence, for example selected from SEQ ID NOs 1-11. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81), and a Cas7 polypeptide (optionally SEQ ID NO: 82) (type I-C CRISPR-Cas system). In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID NO. 24. In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID No. 25.

In some embodiments, provided herein is a brunesoid virus bacteriophage comprising a type I CRISPR-Cas system. In some embodiments, the brunesoid virus is p1695 or p3278. In some embodiments, the CRISPR-Cas system comprises one or more spacer sequences that are complementary to a target nucleotide sequence in a pseudomonas species; a cascades polypeptide; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. In some embodiments, the CRISPR array comprises at least one repeat sequence comprising at least about 90% sequence identity to any one of SEQ ID NOS: 26-30. In some embodiments, the nucleic acid sequence further comprises a promoter sequence, for example selected from SEQ ID NOs 1-11. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81), and a Cas7 polypeptide (optionally SEQ ID NO: 82) (type I-C CRISPR-Cas system). In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID NO. 24. In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID No. 25.

In some embodiments, provided herein is a salsa Mu Na viral bacteriophage comprising a type I CRISPR-Cas system. In some embodiments, the salsa Mu Na virus is p1772, p2131, p2132 or p2973. In some embodiments, the CRISPR-Cas system comprises one or more spacer sequences that are complementary to a target nucleotide sequence in a pseudomonas species; a cascades polypeptide; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. In some embodiments, the CRISPR array comprises at least one repeat sequence comprising at least about 90% sequence identity to any one of SEQ ID NOS: 26-30. In some embodiments, the nucleic acid sequence further comprises a promoter sequence, for example selected from SEQ ID NOs 1-11. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81), and a Cas7 polypeptide (optionally SEQ ID NO: 82) (type I-C CRISPR-Cas system). In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID NO. 24. In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID No. 25.

In some embodiments, provided herein is a proviral bacteriophage comprising a type I CRISPR-Cas system. In some embodiments, the pluravirus is p1106, p1587, p1835, p2037, p2363, p2421 or pb1. In some embodiments, the CRISPR-Cas system comprises one or more spacer sequences that are complementary to a target nucleotide sequence in a pseudomonas species; a cascades polypeptide; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. In some embodiments, the CRISPR array comprises at least one repeat sequence comprising at least about 90% sequence identity to any one of SEQ ID NOS: 26-30. In some embodiments, the nucleic acid sequence further comprises a promoter sequence, for example selected from SEQ ID NOs 1-11. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81), and a Cas7 polypeptide (optionally SEQ ID NO: 82) (type I-C CRISPR-Cas system). In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID NO. 24. In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID No. 25.

In some embodiments, provided herein is a southern viral bacteriophage comprising a type I CRISPR-Cas system. In some embodiments, the CRISPR-Cas system comprises one or more spacer sequences that are complementary to a target nucleotide sequence in a pseudomonas species; a cascades polypeptide; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. In some embodiments, the CRISPR array comprises at least one repeat sequence comprising at least about 90% sequence identity to any one of SEQ ID NOS: 26-30. In some embodiments, the nucleic acid sequence further comprises a promoter sequence, for example selected from SEQ ID NOs 1-11. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81), and a Cas7 polypeptide (optionally SEQ ID NO: 82) (type I-C CRISPR-Cas system). In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID NO. 24. In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID No. 25.

In some embodiments, provided herein is an arbwhat virus, begalvirus, bei Telei virus, casadaban virus, cetrimide virus, vesicular virus, detrilavirus, ehrlift virus, holo Wei Bingdu, xihat virus, liimage virus, lu Saipu pedicle Ma Bingdu, niprina virus, parkona virus, pamez virus, boidelmv virus, filteirus, pri Mo Liji virus, saprotamate virus, starbuchner virus, tatty virus, jojoba virus, or ataxia virus bacteriophage comprising a CRISPR-Cas system type I. In some embodiments, the CRISPR-Cas system comprises one or more spacer sequences that are complementary to a target nucleotide sequence in a pseudomonas species; a cascades polypeptide; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. In some embodiments, the CRISPR array comprises at least one repeat sequence comprising at least about 90% sequence identity to any one of SEQ ID NOS: 26-30. In some embodiments, the nucleic acid sequence further comprises a promoter sequence, for example selected from SEQ ID NOs 1-11. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81), and a Cas7 polypeptide (optionally SEQ ID NO: 82) (type I-C CRISPR-Cas system). In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID NO. 24. In some embodiments, the bacteriophage comprises a CRISPR system having at least 90% identity to SEQ ID No. 25.

CRISPR array

In some embodiments, a CRISPR array (crArray) 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 pseudomonas species. In some embodiments, endogenous or exogenous Cas6 processes the CRISPR array into mature crrnas. In some embodiments, exogenous Cas6 is introduced into the phage. In some embodiments, the phage comprises exogenous Cas6. In some embodiments, exogenous Cas6 is introduced into a pseudomonas species.

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 a spacer sequence at its 5 'end or its 3' end. In some embodiments, the CRISPR array is of any length and comprises any number of spacer nucleotide sequences alternating with the repetitive nucleotide sequences necessary to achieve the desired level of killing of pseudomonas species by targeting one or more essential genes. In some embodiments, the CRISPR array comprises, consists essentially of, or consists of 1 to about 100 spacer nucleotide sequences, each spacer nucleotide sequence linked at its 5 'end and its 3' end to a repeating nucleotide sequence. In some embodiments, a CRISPR array 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.

In some embodiments, a CRISPR array comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 83. In some embodiments, a CRISPR array comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 84. In some embodiments, a CRISPR array comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 85. In some embodiments, the CRISPR array comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 86. In some embodiments, a CRISPR array comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 87. In some embodiments, the CRISPR array is engineered into a Φkz virus. In some embodiments, the Φkz virus is p1194 or p4430. In some embodiments, the CRISPR array is engineered into Φkmv virus. In some embodiments, the Φkmv virus is p2167. In some embodiments, the CRISPR array is engineered into a brunesoid virus. In some embodiments, the brunesoid virus is p1695 or p3278. In some embodiments, the CRISPR array is engineered into a salsa Mu Na viral genus. In some embodiments, the salsa Mu Na virus is p1772, p2131, p2132 or p2973. In some embodiments, the CRISPR array is engineered into a pluravirus. In some embodiments, the pluravirus is p1106, p1587, p1835, p2037, p2363, p2421 or pb1. In some embodiments, the CRISPR array is engineered into a southern national virus. In some embodiments, the CRISPR array is engineered into an arbek virus. In some embodiments, the CRISPR array is engineered into a begalvirus. In some embodiments, the CRISPR array is engineered into the Bei Telei virus. In some embodiments, the CRISPR array is engineered into a casadaban virus. In some embodiments, the CRISPR array is engineered into a sitex virus. In some embodiments, the CRISPR array is engineered into a vesicular virus. In some embodiments, the CRISPR array is engineered into a detrilus virus. In some embodiments, the CRISPR array is engineered into an elv virus. In some embodiments, the CRISPR array is engineered into holo Wei Bingdu. In some embodiments, the CRISPR array is engineered into a chimaphila virus. In some embodiments, the CRISPR array is engineered into a livina virus. In some embodiments, the CRISPR array is engineered into Lu Saipu theirs. In some embodiments, the CRISPR array is engineered into a niprenavirus. In some embodiments, the CRISPR array is engineered into a parkinsonism virus. In some embodiments, the CRISPR array is engineered into a pamekt virus. In some embodiments, the CRISPR array is engineered into a boidem virus. In some embodiments, the CRISPR array is engineered into a filteirus. In some embodiments, the CRISPR array is engineered into the pri Mo Liji virus. In some embodiments, the CRISPR array is engineered into a septicemia martensii virus. In some embodiments, the CRISPR array is engineered into a starbuch virus. In some embodiments, the CRISPR array is engineered into a telthebai virus. In some embodiments, the CRISPR array is engineered into a johnsonian virus. In some embodiments, the CRISPR array is engineered into a atactic trie virus.

Spacer sequences

In some embodiments, the spacer sequence is complementary to a target nucleotide sequence in a pseudomonas species. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence in pseudomonas aeruginosa. 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 non-essential gene. In some embodiments, the target nucleotide sequence is a non-coding sequence. In some embodiments, the non-coding 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 pseudomonas species. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence of a sequence present in a pseudomonas species. In some embodiments, the spacer sequence is complementary to a target nucleotide sequence that comprises all or part of a promoter sequence of an essential gene. In some embodiments, the spacer sequence comprises one, two, three, four, or five mismatches compared to the target nucleotide sequence. In some embodiments, the mismatches are consecutive. In some embodiments, the mismatches are discontinuous. In some embodiments, the spacer sequence has 70% complementarity to the target nucleotide sequence. In some embodiments, the spacer sequence has 80% complementarity to the target nucleotide sequence. In some embodiments, the spacer sequence has 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementarity to the 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 the target nucleotide sequence that is at least about 8 nucleotides to about 150 nucleotides in length. In some embodiments, the spacer sequence has complete complementarity or substantial complementarity over a region of the 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 the target nucleotide sequence, while the 3' region of the spacer is substantially complementary to the target nucleotide sequence, and thus the total 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 (seed region) of the 20 nucleotide spacer sequence are 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 at the 3 'end of the spacer sequence are 100% complementary to the target nucleotide sequence, while in some embodiments the first 7 to 10 nucleotides in the 3' end of the spacer sequence are substantially complementary to the target nucleotide sequence, while the remaining nucleotides in the 5 'region of the spacer sequence are at least about 50% complementary to the target nucleotide sequence (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 greater), while in some embodiments the first 7 to 10 nucleotides in the 3' end 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 are 100% complementary to the target nucleotide sequence, while in some embodiments the first 7 to 10 nucleotides in the 5' end of the spacer sequence are at least about 10% complementary to the 5 'end of the target nucleotide sequence, while in some embodiments the 5' end of the spacer sequence are at least about 10% complementary to the 5 'end of the target nucleotide sequence, e.g., at least about 10% complementary to the 5' end of the spacer sequence, while in some embodiments the 5 'end of the spacer sequence are at least about 50% to about 99% complementary to the 5' nucleotide sequence, while in some embodiments the first 7 to the 5 'end of the spacer sequence are at least about 10% complementary to the 5' end, the first 20 nucleotides at the 5' end) has about 75% or greater complementarity (75% to about 100% complementarity) to the target nucleotide sequence, while the remainder of the spacer sequence has about 50% or greater complementarity to the target nucleotide sequence. In some embodiments, the first 8 nucleotides at the 5' end of the spacer sequence have 100% complementarity or have one or two mutations to the target nucleotide sequence, and thus have about 88% complementarity or about 75% complementarity, respectively, with the remainder of the spacer nucleotide sequence having at least about 50% or greater complementarity to the target nucleotide sequence.

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 of the present invention, in some embodiments, the spacer nucleotide sequence is 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 about 35, 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, at least about 130, at least about 140, at least about 150 nucleotides, or more, and any value or range therein. In some embodiments, the pseudomonas aeruginosa type I-C Cas system has a spacer length of about 30 to 39 nucleotides, about 31 to about 38 nucleotides, about 32 to about 37 nucleotides, about 33 to about 36 nucleotides, about 34 to about 35 nucleotides, or about 35 nucleotides. In some embodiments, the pseudomonas aeruginosa type I-C Cas system has a spacer length of about 34 nucleotides. In some embodiments, the pseudomonas aeruginosa type I-C Cas system has at least about 10, at least about 15, at least about 20, at least 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 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least 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.

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 any one of SEQ ID NOs 12-23, 31-74 or 88-120. In some cases, the spacer sequence comprises at least or about 95% homology to any of SEQ ID NOs 12-23, 31-74 or 88-120. In some cases, the spacer sequence comprises at least or about 97% homology to any of SEQ ID NOS 12-23, 31-74 or 88-120. In some cases, the spacer sequence comprises at least or about 99% homology to any of SEQ ID NOS 12-23, 31-74 or 88-120. In some cases, the spacer sequence comprises 100% homology with any of SEQ ID NOS 12-23, 31-74 or 88-120. In some cases, the spacer sequence comprises 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, or at least a portion of more than 34 nucleotides having any of SEQ ID NOs 12-23, 31-74, or 88-120.

The term "sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) within a comparison window. The term "percent sequence identity" is calculated by: comparing the two optimally aligned sequences within a comparison window, determining the number of positions in the two sequences at which the same nucleobase (e.g., A, T, C, G, U or I) occurs to yield the number of matched positions, dividing the number of matched positions by the total number of positions within the comparison window (i.e., window size), and multiplying the result by 100 to yield the percent sequence identity.

The term "homology" or "similarity" between two proteins is determined by comparing the amino acid sequences of one protein sequence with the other and conservative amino acid substitutions thereof. Similarity can be determined by programs well known in the art, such as the BLAST program (basic local alignment search tool of the national center for bioinformation).

In some embodiments, the identity of two or more spacer sequences of a CRISPR array is the same. In some embodiments, the identity of two or more spacer sequences of a CRISPR array is different. In some embodiments, the identity of two or more spacer sequences of a CRISPR array is different, but complementary to one or more target nucleotide sequences. In some embodiments, the identity of two or more spacer sequences of a CRISPR array is different and complementary to one or more target nucleotide sequences that are overlapping sequences. In some embodiments, the identity of two or more spacer sequences of a CRISPR array is different and 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 contiguous nucleotides in length, immediately adjacent to the PAM sequence in the genome of the organism (PAM sequence immediately 3' of the target region). In some embodiments, the target nucleotide sequence is located near or flanking PAM (protospacer adjacent motif). In some embodiments, two or more sequences of a CRISPR array comprise at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of SEQ ID NOS: 12-23, 31-74 or 88-120. In some cases, two or more sequences of the CRISPR array comprise at least or about 95% homology to any of SEQ ID NOS: 12-23, 31-74 or 88-120. In some cases, two or more sequences of the CRISPR array comprise at least or about 97% homology to any of SEQ ID NOS: 12-23, 31-74 or 88-120. In some cases, two or more sequences of the CRISPR array comprise at least or about 99% homology to any of SEQ ID NOS: 12-23, 31-74 or 88-120. In some cases, two or more sequences of the CRISPR array comprise 100% homology to any of SEQ ID NOS: 12-23, 31-74 or 88-120. In some cases, two or more sequences of a CRISPR array comprise 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, or at least a portion of more than 34 nucleotides having any of SEQ ID NOs 12-23, 31-74, or 88-120. In some embodiments, the CRISPR array is engineered into a Φkz virus. In some embodiments, the Φkz virus is p1194 or p4430. In some embodiments, the CRISPR array is engineered into Φkmv virus. In some embodiments, the Φkmv virus is p2167. In some embodiments, the CRISPR array is engineered into a brunesoid virus. In some embodiments, the brunesoid virus is p1695 or p3278. In some embodiments, the CRISPR array is engineered into a salsa Mu Na viral genus. In some embodiments, the salsa Mu Na virus is p1772, p2131, p2132 or p2973. In some embodiments, the CRISPR array is engineered into a pluravirus. In some embodiments, the pluravirus is p1106, p1587, p1835, p2037, p2363, p2421 or pb1. In some embodiments, the CRISPR array is engineered into a southern national virus. In some embodiments, the CRISPR array is engineered into an arbek virus. In some embodiments, the CRISPR array is engineered into a begalvirus. In some embodiments, the CRISPR array is engineered into the Bei Telei virus. In some embodiments, the CRISPR array is engineered into a casadaban virus. In some embodiments, the CRISPR array is engineered into a sitex virus. In some embodiments, the CRISPR array is engineered into a vesicular virus. In some embodiments, the CRISPR array is engineered into a detrilus virus. In some embodiments, the CRISPR array is engineered into an elv virus. In some embodiments, the CRISPR array is engineered into holo Wei Bingdu. In some embodiments, the CRISPR array is engineered into a chimaphila virus. In some embodiments, the CRISPR array is engineered into a livina virus. In some embodiments, the CRISPR array is engineered into Lu Saipu theirs. In some embodiments, the CRISPR array is engineered into a niprenavirus. In some embodiments, the CRISPR array is engineered into a parkinsonism virus. In some embodiments, the CRISPR array is engineered into a pamekt virus. In some embodiments, the CRISPR array is engineered into a boidem virus. In some embodiments, the CRISPR array is engineered into a filteirus. In some embodiments, the CRISPR array is engineered into the pri Mo Liji virus. In some embodiments, the CRISPR array is engineered into a septicemia martensii virus. In some embodiments, the CRISPR array is engineered into a starbuch virus. In some embodiments, the CRISPR array is engineered into a telthebai virus. In some embodiments, the CRISPR array is engineered into a johnsonian virus. In some embodiments, the CRISPR array is engineered into a atactic trie virus.

In some embodiments, the CRISPR array comprises a first spacer sequence comprising 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: 12-15; a second spacer sequence comprising 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 16-19; a third spacer sequence comprising 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.20-23, wherein the first, second and third spacer sequences comprise 0-8 nucleotide modifications. In some cases, the first spacer sequence comprises at least or about 97% homology to any of SEQ ID NOs 12-15; the second spacer sequence comprises at least or about 97% homology to any of SEQ ID NOS.16-19; and the third spacer sequence comprises at least or about 97% homology to any of SEQ ID NOS.20-23. In some cases, the first spacer sequence comprises at least or about 99% homology to any of SEQ ID NOs 12-15; the second spacer sequence comprises at least or about 99% homology to any of SEQ ID NOS.16-19; and the third spacer sequence comprises at least or about 99% homology with any of SEQ ID NOS.20-23. In some cases, the first spacer sequence comprises at least or about 100% homology to any of SEQ ID NOs 12-15; the second spacer sequence comprises at least or about 100% homology to any of SEQ ID NOS.16-19; and the third spacer sequence comprises at least or about 100% homology with any of SEQ ID NOS.20-23. In some embodiments, the CRISPR array is engineered into a Φkz virus. In some embodiments, the Φkz virus is p1194 or p4430. In some embodiments, the CRISPR array is engineered into Φkmv virus. In some embodiments, the Φkmv virus is p2167. In some embodiments, the CRISPR array is engineered into a brunesoid virus. In some embodiments, the brunesoid virus is p1695 or p3278. In some embodiments, the CRISPR array is engineered into a salsa Mu Na viral genus. In some embodiments, the salsa Mu Na virus is p1772, p2131, p2132 or p2973. In some embodiments, the CRISPR array is engineered into a pluravirus. In some embodiments, the pluravirus is p1106, p1587, p1835, p2037, p2363, p2421 or pb1. In some embodiments, the CRISPR array is engineered into a southern national virus. In some embodiments, the CRISPR array is engineered into an arbek virus. In some embodiments, the CRISPR array is engineered into a begalvirus. In some embodiments, the CRISPR array is engineered into the Bei Telei virus. In some embodiments, the CRISPR array is engineered into a casadaban virus. In some embodiments, the CRISPR array is engineered into a sitex virus. In some embodiments, the CRISPR array is engineered into a vesicular virus. In some embodiments, the CRISPR array is engineered into a detrilus virus. In some embodiments, the CRISPR array is engineered into an elv virus. In some embodiments, the CRISPR array is engineered into holo Wei Bingdu. In some embodiments, the CRISPR array is engineered into a chimaphila virus. In some embodiments, the CRISPR array is engineered into a livina virus. In some embodiments, the CRISPR array is engineered into Lu Saipu theirs. In some embodiments, the CRISPR array is engineered into a niprenavirus. In some embodiments, the CRISPR array is engineered into a parkinsonism virus. In some embodiments, the CRISPR array is engineered into a pamekt virus. In some embodiments, the CRISPR array is engineered into a boidem virus. In some embodiments, the CRISPR array is engineered into a filteirus. In some embodiments, the CRISPR array is engineered into the pri Mo Liji virus. In some embodiments, the CRISPR array is engineered into a septicemia martensii virus. In some embodiments, the CRISPR array is engineered into a starbuch virus. In some embodiments, the CRISPR array is engineered into a telthebai virus. In some embodiments, the CRISPR array is engineered into a johnsonian virus. In some embodiments, the CRISPR array is engineered into a atactic trie virus.

In some embodiments, the CRISPR array comprises a first spacer 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. 12; a second spacer 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. 16; a third spacer 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. 20, wherein the first, second and third spacer sequences comprise 0-8 nucleotide modifications. In some cases, the first spacer sequence comprises at least or about 97% homology to SEQ ID NO. 12; the second spacer sequence comprises at least or about 97% homology to SEQ ID NO. 16; and the third spacer sequence comprises at least or about 97% homology to SEQ ID NO. 20. In some cases, the first spacer sequence comprises at least or about 99% homology to SEQ ID NO. 12; the second spacer sequence comprises at least or about 99% homology to SEQ ID NO. 16; and the third spacer sequence comprises at least or about 99% homology with SEQ ID NO. 20. In some cases, the first spacer sequence comprises at least or about 100% homology to SEQ ID NO. 12; the second spacer sequence comprises at least or about 100% homology to SEQ ID NO. 16; and the third spacer sequence comprises at least or about 100% homology with SEQ ID NO. 20. In some embodiments, the CRISPR array is engineered into a Φkz virus. In some embodiments, the Φkz virus is p1194 or p4430. In some embodiments, the CRISPR array is engineered into Φkmv virus. In some embodiments, the Φkmv virus is p2167. In some embodiments, the CRISPR array is engineered into a brunesoid virus. In some embodiments, the brunesoid virus is p1695 or p3278. In some embodiments, the CRISPR array is engineered into a salsa Mu Na viral genus. In some embodiments, the salsa Mu Na virus is p1772, p2131, p2132 or p2973. In some embodiments, the CRISPR array is engineered into a pluravirus. In some embodiments, the pluravirus is p1106, p1587, p1835, p2037, p2363, p2421 or pb1. In some embodiments, the CRISPR array is engineered into a southern national virus. In some embodiments, the CRISPR array is engineered into an arbek virus. In some embodiments, the CRISPR array is engineered into a begalvirus. In some embodiments, the CRISPR array is engineered into the Bei Telei virus. In some embodiments, the CRISPR array is engineered into a casadaban virus. In some embodiments, the CRISPR array is engineered into a sitex virus. In some embodiments, the CRISPR array is engineered into a vesicular virus. In some embodiments, the CRISPR array is engineered into a detrilus virus. In some embodiments, the CRISPR array is engineered into an elv virus. In some embodiments, the CRISPR array is engineered into holo Wei Bingdu. In some embodiments, the CRISPR array is engineered into a chimaphila virus. In some embodiments, the CRISPR array is engineered into a livina virus. In some embodiments, the CRISPR array is engineered into Lu Saipu theirs. In some embodiments, the CRISPR array is engineered into a niprenavirus. In some embodiments, the CRISPR array is engineered into a parkinsonism virus. In some embodiments, the CRISPR array is engineered into a pamekt virus. In some embodiments, the CRISPR array is engineered into a boidem virus. In some embodiments, the CRISPR array is engineered into a filteirus. In some embodiments, the CRISPR array is engineered into the pri Mo Liji virus. In some embodiments, the CRISPR array is engineered into a septicemia martensii virus. In some embodiments, the CRISPR array is engineered into a starbuch virus. In some embodiments, the CRISPR array is engineered into a telthebai virus. In some embodiments, the CRISPR array is engineered into a johnsonian virus. In some embodiments, the CRISPR array is engineered into a atactic trie virus.

In some embodiments, the CRISPR array comprises a first spacer 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. 13; a second spacer 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. 17; a third spacer 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. 21, wherein the first, second and third spacer sequences comprise 0-8 nucleotide modifications. In some cases, the first spacer sequence comprises at least or about 97% homology to SEQ ID NO. 13; the second spacer sequence comprises at least or about 97% homology to SEQ ID NO. 17; and the third spacer sequence comprises at least or about 97% homology with SEQ ID NO. 21. In some cases, the first spacer sequence comprises at least or about 99% homology to SEQ ID NO. 13; the second spacer sequence comprises at least or about 99% homology to SEQ ID NO. 17; and the third spacer sequence comprises at least or about 99% homology with SEQ ID NO. 21. In some cases, the first spacer sequence comprises at least or about 100% homology to SEQ ID NO. 13; the second spacer sequence comprises at least or about 100% homology with SEQ ID NO. 17; and the third spacer sequence comprises at least or about 100% homology with SEQ ID NO. 21. In some embodiments, the CRISPR array is engineered into a Φkz virus. In some embodiments, the Φkz virus is p1194 or p4430. In some embodiments, the CRISPR array is engineered into Φkmv virus. In some embodiments, the Φkmv virus is p2167. In some embodiments, the CRISPR array is engineered into a brunesoid virus. In some embodiments, the brunesoid virus is p1695 or p3278. In some embodiments, the CRISPR array is engineered into a salsa Mu Na viral genus. In some embodiments, the salsa Mu Na virus is p1772, p2131, p2132 or p2973. In some embodiments, the CRISPR array is engineered into a pluravirus. In some embodiments, the pluravirus is p1106, p1587, p1835, p2037, p2363, p2421 or pb1. In some embodiments, the CRISPR array is engineered into a southern national virus. In some embodiments, the CRISPR array is engineered into an arbek virus. In some embodiments, the CRISPR array is engineered into a begalvirus. In some embodiments, the CRISPR array is engineered into the Bei Telei virus. In some embodiments, the CRISPR array is engineered into a casadaban virus. In some embodiments, the CRISPR array is engineered into a sitex virus. In some embodiments, the CRISPR array is engineered into a vesicular virus. In some embodiments, the CRISPR array is engineered into a detrilus virus. In some embodiments, the CRISPR array is engineered into an elv virus. In some embodiments, the CRISPR array is engineered into holo Wei Bingdu. In some embodiments, the CRISPR array is engineered into a chimaphila virus. In some embodiments, the CRISPR array is engineered into a livina virus. In some embodiments, the CRISPR array is engineered into Lu Saipu theirs. In some embodiments, the CRISPR array is engineered into a niprenavirus. In some embodiments, the CRISPR array is engineered into a parkinsonism virus. In some embodiments, the CRISPR array is engineered into a pamekt virus. In some embodiments, the CRISPR array is engineered into a boidem virus. In some embodiments, the CRISPR array is engineered into a filteirus. In some embodiments, the CRISPR array is engineered into the pri Mo Liji virus. In some embodiments, the CRISPR array is engineered into a septicemia martensii virus. In some embodiments, the CRISPR array is engineered into a starbuch virus. In some embodiments, the CRISPR array is engineered into a telthebai virus. In some embodiments, the CRISPR array is engineered into a johnsonian virus. In some embodiments, the CRISPR array is engineered into a atactic trie virus.

In some embodiments, the CRISPR array comprises a first spacer 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. 14; a second spacer 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. 18; a third spacer 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, wherein the first, second and third spacer sequences comprise 0-8 nucleotide modifications. In some cases, the first spacer sequence comprises at least or about 97% homology to SEQ ID NO. 14; the second spacer sequence comprises at least or about 97% homology to SEQ ID NO. 18; and the third spacer sequence comprises at least or about 97% homology with SEQ ID NO. 22. In some cases, the first spacer sequence comprises at least or about 99% homology to SEQ ID NO. 14; the second spacer sequence comprises at least or about 99% homology with SEQ ID NO. 18; and the third spacer sequence comprises at least or about 99% homology with SEQ ID NO. 22. In some cases, the first spacer sequence comprises at least or about 100% homology to SEQ ID NO. 14; the second spacer sequence comprises at least or about 100% homology with SEQ ID NO. 18; and the third spacer sequence comprises at least or about 100% homology with SEQ ID NO. 22. In some embodiments, the CRISPR array is engineered into a Φkz virus. In some embodiments, the Φkz virus is p1194 or p4430. In some embodiments, the CRISPR array is engineered into Φkmv virus. In some embodiments, the Φkmv virus is p2167. In some embodiments, the CRISPR array is engineered into a brunesoid virus. In some embodiments, the brunesoid virus is p1695 or p3278. In some embodiments, the CRISPR array is engineered into a salsa Mu Na viral genus. In some embodiments, the salsa Mu Na virus is p1772, p2131, p2132 or p2973. In some embodiments, the CRISPR array is engineered into a pluravirus. In some embodiments, the pluravirus is p1106, p1587, p1835, p2037, p2363, p2421 or pb1. In some embodiments, the CRISPR array is engineered into a southern national virus. In some embodiments, the CRISPR array is engineered into an arbek virus. In some embodiments, the CRISPR array is engineered into a begalvirus. In some embodiments, the CRISPR array is engineered into the Bei Telei virus. In some embodiments, the CRISPR array is engineered into a casadaban virus. In some embodiments, the CRISPR array is engineered into a sitex virus. In some embodiments, the CRISPR array is engineered into a vesicular virus. In some embodiments, the CRISPR array is engineered into a detrilus virus. In some embodiments, the CRISPR array is engineered into an elv virus. In some embodiments, the CRISPR array is engineered into holo Wei Bingdu. In some embodiments, the CRISPR array is engineered into a chimaphila virus. In some embodiments, the CRISPR array is engineered into a livina virus. In some embodiments, the CRISPR array is engineered into Lu Saipu theirs. In some embodiments, the CRISPR array is engineered into a niprenavirus. In some embodiments, the CRISPR array is engineered into a parkinsonism virus. In some embodiments, the CRISPR array is engineered into a pamekt virus. In some embodiments, the CRISPR array is engineered into a boidem virus. In some embodiments, the CRISPR array is engineered into a filteirus. In some embodiments, the CRISPR array is engineered into the pri Mo Liji virus. In some embodiments, the CRISPR array is engineered into a septicemia martensii virus. In some embodiments, the CRISPR array is engineered into a starbuch virus. In some embodiments, the CRISPR array is engineered into a telthebai virus. In some embodiments, the CRISPR array is engineered into a johnsonian virus. In some embodiments, the CRISPR array is engineered into a atactic trie virus.

In some embodiments, the CRISPR array comprises a first spacer 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. 15; a second spacer 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. 19; a third spacer 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, wherein the first, second and third spacer sequences comprise 0-8 nucleotide modifications. In some cases, the first spacer sequence comprises at least or about 97% homology to SEQ ID NO. 15; the second spacer sequence comprises at least or about 97% homology to SEQ ID NO. 19; and the third spacer sequence comprises at least or about 97% homology with SEQ ID NO. 23. In some cases, the first spacer sequence comprises at least or about 99% homology to SEQ ID NO. 15; the second spacer sequence comprises at least or about 99% homology with SEQ ID NO. 19; and the third spacer sequence comprises at least or about 99% homology with SEQ ID NO. 23. In some cases, the first spacer sequence comprises at least or about 100% homology to SEQ ID NO. 15; the second spacer sequence comprises at least or about 100% homology with SEQ ID NO. 19; and the third spacer sequence comprises at least or about 100% homology with SEQ ID NO. 23. In some embodiments, the CRISPR array is engineered into a Φkz virus. In some embodiments, the Φkz virus is p1194 or p4430. In some embodiments, the CRISPR array is engineered into Φkmv virus. In some embodiments, the Φkmv virus is p2167. In some embodiments, the CRISPR array is engineered into a brunesoid virus. In some embodiments, the brunesoid virus is p1695 or p3278. In some embodiments, the CRISPR array is engineered into a salsa Mu Na viral genus. In some embodiments, the salsa Mu Na virus is p1772, p2131, p2132 or p2973. In some embodiments, the CRISPR array is engineered into a pluravirus. In some embodiments, the pluravirus is p1106, p1587, p1835, p2037, p2363, p2421 or pb1. In some embodiments, the CRISPR array is engineered into a southern national virus. In some embodiments, the CRISPR array is engineered into an arbek virus. In some embodiments, the CRISPR array is engineered into a begalvirus. In some embodiments, the CRISPR array is engineered into the Bei Telei virus. In some embodiments, the CRISPR array is engineered into a casadaban virus. In some embodiments, the CRISPR array is engineered into a sitex virus. In some embodiments, the CRISPR array is engineered into a vesicular virus. In some embodiments, the CRISPR array is engineered into a detrilus virus. In some embodiments, the CRISPR array is engineered into an elv virus. In some embodiments, the CRISPR array is engineered into holo Wei Bingdu. In some embodiments, the CRISPR array is engineered into a chimaphila virus. In some embodiments, the CRISPR array is engineered into a livina virus. In some embodiments, the CRISPR array is engineered into Lu Saipu theirs. In some embodiments, the CRISPR array is engineered into a niprenavirus. In some embodiments, the CRISPR array is engineered into a parkinsonism virus. In some embodiments, the CRISPR array is engineered into a pamekt virus. In some embodiments, the CRISPR array is engineered into a boidem virus. In some embodiments, the CRISPR array is engineered into a filteirus. In some embodiments, the CRISPR array is engineered into the pri Mo Liji virus. In some embodiments, the CRISPR array is engineered into a septicemia martensii virus. In some embodiments, the CRISPR array is engineered into a starbuch virus. In some embodiments, the CRISPR array is engineered into a telthebai virus. In some embodiments, the CRISPR array is engineered into a johnsonian virus. In some embodiments, the CRISPR array is engineered into a atactic trie virus.

In some embodiments, the spacers are combined in a plurality, clusters, within one or more bacteriophage, e.g., engineered bacteriophage. The bacteriophage or bacteriophage cocktail may comprise one or more arrays having a first spacer sequence comprising 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 12-15; a second spacer sequence comprising 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 16-19; a third spacer sequence comprising 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.20-23. In some embodiments, a bacteriophage cocktail may comprise one or more arrays having a first spacer 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. 12; a second or third spacer 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 embodiments, a bacteriophage cocktail may comprise one or more arrays having a first spacer 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. 13; a second or third spacer 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 embodiments, a bacteriophage cocktail may comprise one or more arrays having a first spacer 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. 14; a second or third spacer 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. 21. In some embodiments, a bacteriophage cocktail may comprise one or more arrays having a first spacer 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. 15; a second or third spacer 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. 20. In some embodiments, a bacteriophage cocktail may comprise one or more arrays having a first spacer 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. 16; a second or third spacer 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. 19. In some embodiments, a bacteriophage cocktail may comprise one or more arrays having a first spacer 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. 17; a second or third spacer 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. 18. In some embodiments, multiple bacteriophage are used together. In some embodiments, the plurality of bacteriophage used together target the same or different bacteria within a sample or subject. In some embodiments, cocktails comprising multiple bacteriophages are used together. In some embodiments, the cocktail comprises at least 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, or 27 phages selected from table 1A. In some embodiments, the cocktail comprises 2 phages selected from table 1A. In some embodiments, the cocktail comprises wild-type or engineered Φkz virus. In some embodiments, the Φkz virus is p1194 or p4430. In some embodiments, the cocktail comprises wild-type or engineered Φkmv virus. In some embodiments, the Φkmv virus is p2167. In some embodiments, the cocktail comprises wild-type or engineered brunesoid virus. In some embodiments, the brunesoid virus is p1695 or p3278. In some embodiments, the cocktail comprises wild-type or engineered salsa Mu Na virus. In some embodiments, the salsa Mu Na virus is p1772, p2131, p2132 or p2973. In some embodiments, the cocktail comprises wild-type or engineered pluraviruses. In some embodiments, the pluravirus is p1106, p1587, p1835, p2037, p2363, p2421 or pb1. In some embodiments, the cocktail comprises wild-type or engineered southern national virus. In some embodiments, the cocktail comprises wild-type or engineered arbitral virus. In some embodiments, the cocktail comprises wild-type or engineered begalviruses. In some embodiments, the cocktail comprises wild-type or engineered Bei Telei virus. In some embodiments, the cocktail comprises wild-type or engineered kazaban virus. In some embodiments, the cocktail comprises wild-type or engineered cetuximab virus. In some embodiments, the cocktail comprises wild-type or engineered vesicular virus. In some embodiments, the cocktail comprises wild-type or engineered de tei virus. In some embodiments, the cocktail comprises wild-type or engineered elviruses. In some embodiments, the cocktail comprises wild-type or engineered holo Wei Bingdu. In some embodiments, the cocktail comprises wild-type or engineered chimaphila virus. In some embodiments, the cocktail comprises wild-type or engineered livinoviruses. In some embodiments, the cocktail comprises wild-type or engineered Lu Saipu pedicles Ma Bingdu. In some embodiments, the cocktail comprises wild-type or engineered niprenavirus. In some embodiments, the cocktail comprises wild-type or engineered parkinsonism. In some embodiments, the cocktail comprises wild-type or engineered pamekt virus. In some embodiments, the cocktail comprises wild-type or engineered boenkem virus. In some embodiments, the cocktail comprises wild-type or engineered filteiruses. In some embodiments, the cocktail comprises wild-type or engineered pri Mo Liji virus. In some embodiments, the cocktail comprises wild-type or engineered septemamate virus. In some embodiments, the cocktail comprises wild-type or engineered starbuch virus. In some embodiments, the cocktail comprises wild-type or engineered telthebai virus. In some embodiments, the cocktail comprises wild-type or engineered about subviruses. In some embodiments, the cocktail comprises wild-type or engineered ataxia virus.

In some embodiments, the cocktail comprises a cocktail selected from table 6A. In some embodiments, at least one bacteriophage in the cocktail comprises a CRISPR array. In some embodiments, at least 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, or 27 bacteriophage present in the cocktail comprises a CRISPR array. In some embodiments, at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding a cascades 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, 21, 22, 23, 24, 25, 26, or 27 bacteriophage present in the cocktail comprises a nucleic acid sequence encoding a cascades polypeptide. In some embodiments, at least one bacteriophage in the cocktail comprises a nucleic acid sequence encoding a Cas3 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, 21, 22, 23, 24, 25, 26, or 27 bacteriophage present in the cocktail comprises a nucleic acid sequence encoding a Cas3 polypeptide. In some embodiments, the cocktail comprises p1106e003, p1835e002, p1772e005, and p2131e002. In some embodiments, the cocktail further comprises p1194. In some embodiments, the cocktail further comprises p1695. In some embodiments, the cocktail further comprises p4430. In some embodiments, the cocktail comprises p1106e003, p1835e002, p1772e005, p2131e002, p1194, and p1695. In some embodiments, the cocktail comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430, and p1695.

PAM sequences are present in the target gene in the vicinity of the region to which the spacer sequence binds, because the spacer sequence is complementary to the region; and identifies the initiation point for base pairing with the spacer nucleotide sequence. The exact PAM sequence required varies between each different CRISPR-Cas system and is identified by established bioinformatics and experimental procedures. Non-limiting examples of PAM include CCA, CCT, CCG, TTC, AAG, AGG, ATG, GAG and/or CC. For type I systems, PAM is located directly 5 'of the sequence that matches the spacer, and thus 3' of the sequence that base pairs with the spacer nucleotide sequence, and is directly recognized by cascades. Once the protospacer sequence is identified, cascades typically recruit endonuclease Cas3, which cleaves and degrades the target DNA. For type II systems, cas 9/sgrnas require PAM to form an R loop to interrogate specific DNA sequences through their watson-crick pairing of the guide RNA with the genome. PAM specificity is a function of the DNA binding specificity of the Cas9 protein (e.g., the protospacer adjacent motif recognition domain at the C-terminus of Cas 9)

In some embodiments, the target nucleotide sequence in the bacteria 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, the target nucleotide sequence comprises, consists essentially of, or consists of all or part of a nucleotide sequence encoding a promoter of an essential gene or its complement. In some embodiments, the spacer nucleotide sequence is complementary to a promoter of an essential gene or a portion thereof. In some embodiments, the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding or non-coding strand of an essential gene. In some embodiments, the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding for the transcribed region of the essential gene.

In some embodiments, the essential gene is any gene of an organism that is critical to its survival. However, "necessary" is highly dependent on the environment in which the organism is living. For example, the genes required to digest starch are only necessary if starch is the sole source of energy. In some embodiments, the target nucleotide sequence comprises all or part of a promoter sequence of the target gene. In some embodiments, the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding strand of the transcribed region of the target gene. In some embodiments, the target nucleotide sequence comprises at least a portion of an essential gene required for the survival of a pseudomonas species. In some embodiments, the target nucleotide sequence comprises a highly conserved non-coding sequence or intergenic sequence. In some embodiments, the target sequence is an intergenic sequence located between the essential gene rpmF and the conserved hypothetical protein. In some embodiments, the essential gene is Tsf, acpP, gapA, 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, tRNAasn or metK. In some embodiments, the essential gene is dnaA, ftsA, gyrB, dnaN, glnS or rpoB. In some embodiments, the target sequence is the boundary between PA4325 (hypothetical protein), PA1310 (phnW, pyruvate aminotransferase), or PA2970 (rpmF, 50S ribosomal protein L32) and PA2971 (conserved hypothetical protein). In some embodiments, a non-essential gene is any gene of an organism that is not critical to survival. However, "nonessential" is highly dependent on the environment in which the organism is living.

In some embodiments, non-limiting examples of target nucleotide sequences of interest include target nucleotide sequences encoding transcriptional regulators, translational regulators, polymerase genes, metabolic enzymes, transporters, rnases, proteases, DNA replicases, DNA modification or degradation enzymes, regulatory RNAs, transfer RNAs, or ribosomal RNAs. In some embodiments, the target nucleotide sequence is from a gene involved in cell division, cellular structure, metabolism, motility, pathogenicity, toxicity, or antibiotic resistance. In some embodiments, the target nucleotide sequence is from a hypothetical gene whose function has not been characterized. Thus, for example, these genes are any genes from any bacteria.

In some embodiments, suitable spacer sequences for whole construct phages are identified by locating a representative genome search set, searching the genome with relevant parameters, and determining the quality of the spacer for CRISPR engineered phages.

First, a suitable representative genomic search set is located and obtained for the organism/species/target of interest. In some embodiments, the representative gene set may be found in various databases, including, but not limited to, the NCBI gene library or the PATRIC database. The NCBI gene library is one of the largest databases available, which contains a mixture of reference and submitted genomes of nearly all sequenced organisms to date. In particular, the PATRIC (pathological systems resource integration center) database provides additional comprehensive genomic resources for pathogenic organisms and provides attention to clinically relevant strains and genomes associated with pharmaceutical products. Both databases allow for batch downloading of genomes via FTP (file transfer protocol) servers, enabling fast and programmed data set acquisition

Next, the genome is searched using the relevant parameters to locate the appropriate spacer sequence. In some embodiments, the genome is read end-to-end in forward and reverse complement orientations to locate a contiguous DNA fragment containing PAM (protospacer adjacent motif) sites. The spacer sequence will be an N-length DNA sequence (depending on the CRISPR system type) adjacent to 3 'or 5' of the PAM site, where N is specific for the Cas system of interest and is typically known in advance. In some embodiments, characterizing the PAM sequence and the spacer sequence is performed during discovery and initial study of the Cas system. In some embodiments, each observed PAM adjacent spacer is saved to a file and/or database for downstream use. The exact PAM sequence required varies between each different CRISPR-Cas system and is identified by established bioinformatics and experimental procedures.

Next, the mass of the spacer used in CRISPR engineered phage was determined. In some embodiments, each observed spacer is evaluated to determine how many of them are present in the evaluated genome. In some embodiments, the observed spacers are evaluated to see how many times they may occur in each given genome. In some embodiments, the presence of a spacer at more than one location per genome is advantageous because if a mutation occurs, the Cas system may not recognize the target site and each additional "backup" site increases the likelihood that a suitable non-mutated target site will exist. In some embodiments, the observed spacers are evaluated to determine whether they are present in the functionally annotated region of the genome. If such information is available, the functional annotation may be further evaluated to determine if such regions of the genome are "necessary" for the survival and function of the organism. By focusing on the spacer present in all or nearly all of the estimated genomes of interest (> = 99%), spacer selection can be widely applied to many targeted genomes. If there is a large pool of conserved spacer selections, the spacers that occur in genomic regions with known functions may be preferentially selected, given higher priority if these genomic regions are "essential" for survival and occur more than 1 time in each genome.

In some embodiments, the spacer sequence of the full construct phage is verified. In some embodiments, the first step comprises identifying plasmids that replicate in the organism, species, or target of interest. In some embodiments, the plasmid has a selectable marker. In some embodiments, the selectable marker is an antibiotic resistance gene. In some embodiments, the expression cassette comprises a nucleotide sequence of a selectable marker. In some embodiments, the selectable marker is adenine deaminase (ada), blasticidin S deaminase (Bsr, BSD), bleomycin binding protein (Ble), neomycin phosphotransferase (neo), histidinol dehydrogenase (hisD), glutamine Synthase (GS), dihydrofolate reductase (dhfr), cytosine deaminase (codA), puromycin N-acetyltransferase (Pac) or hygromycin B phosphotransferase (Hph), ampicillin, chloramphenicol, kanamycin, tetracycline, polymyxin B, erythromycin, carbenicillin, streptomycin, spectinomycin, puromycin N-acetyltransferase (Pac), or gecomycin (Sh bla). In some embodiments, the selectable marker is a gene that is involved in thymidylate synthase, thymidine kinase, dihydrofolate reductase, or glutamine synthase. In some embodiments, the selectable marker is a gene encoding a fluorescent protein.

In some embodiments, the second step comprises inserting the genes encoding the Cas system into a plasmid such that they will be expressed in the organism, species, or target of interest. In some embodiments, a promoter is provided upstream of the Cas system. In some embodiments, the promoter is recognized by an organism, species, or target of interest to drive expression of the Cas system. Exemplary promoters include, but are not limited to, L-arabinose inducible (araBAD, P _BAD ) Promoters, 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), a anhydrotetracycline inducible (tetA) promoter, trp, ipp, phoA, recA, proU, cst-1, cadA, nar, ipp-lac, cspA, 11-lac operon, T3-lac operon, T4 gene 32, T5-lac operon, nprM-lac operon, vhb, protein a, corynebacterium-escherichia coli-like promoter, thr, horn, diphtheria toxin promoter, sig a, sig B, nusG, soxS, katb, alpha-amylase (Pamy), ptms, P43 (comprising two overlapping RNA polymerase sigma factor recognition sites, σΑ, σ beta), ptms, P43, rplK-rplA, ferredoxin promoter, and/or xylose promoter. In some embodiments, the promoter is bba_j23102, bba_j23104, or Bba_j23109. In some embodiments, the promoter is derived from an organism, species, or target bacteria, such as an endogenous CRISPR promoter, an endogenous Cas operator promoter, p16, plpp, or ptat. In some embodiments, the promoter is a phage promoter, such as a promoter of gp105 or gp 245. In some embodiments, a Ribosome Binding Site (RBS) is provided between the promoter and the Cas system. In some embodiments, the RBS is recognized by an organism, species, or target of interest.

In some embodiments, the third step comprises providing a genome targeting spacer into the plasmid. In some embodiments, the genome targeting spacer uses bioinformatic identification. In some embodiments, the genome targeting spacer is provided upstream of the repeat-spacer-repeat. In some embodiments, a promoter is provided. In some embodiments, the promoter is recognized by an organism, species, or target of interest to drive expression of the crRNA. In some embodiments, the cloning of the third step comprises using an organism or species that is not targeted by the cloned spacer.

In some embodiments, the fourth step comprises providing the non-target spacer into a plasmid expressing the Cas system. In some embodiments, the non-target spacer comprises a random sequence. In some embodiments, the non-target spacer comprises a sequence that does not comprise a targeting site in the genome of the organism, species or target of interest. In some embodiments, the non-target spacer sequence is determined using bioinformatics to not include a target site in the genome of the organism, species, or target of interest. In some embodiments, the non-target spacer sequence is provided upstream of the repeat-spacer-repeat. In some embodiments, a promoter is provided. In some embodiments, the promoter is recognized by an organism, species, or target of interest to drive expression of the crRNA.

In some embodiments, the fifth step comprises determining the efficacy of each spacer generated. In some embodiments, the killing efficacy is determined. In some embodiments, the efficacy of each spacer to target the bacterial genome is determined. In some embodiments, the transfer rate of a plasmid comprising a non-targeting spacer is reduced to about 2/3, about 1/2, 1/5, 1/10, 1/20, 1/40, 1/60, 1/80, or up to about 1/100 as compared to a plasmid comprising the spacer.

Repeated nucleotide sequences

In some embodiments, the repeated nucleotide sequence of the CRISPR array comprises the nucleotide sequence of any known repeated nucleotide sequence of a CRISPR-Cas system. In some embodiments, the repetitive nucleotide sequence is a synthetic sequence comprising a naturally repeated secondary structure (e.g., an internal hairpin) from a CRISPR-Cas system. In some embodiments, the repeated nucleotide sequences differ from one another based on the known repeated nucleotide sequences of the CRISPR-Cas system. In some embodiments, the repeated nucleotide sequences are each composed of a different secondary structure (e.g., an internal hairpin) from the natural repeat of the CRISPR-Cas system. In some embodiments, the repeated nucleotide sequence is a combination of different repeated nucleotide sequences operable with a CRISPR-Cas system.

In some embodiments, the spacer sequence is linked at its 5 'end to the 3' end of the 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 the repeat sequence. In some embodiments, about 1 to about 8, about 1 to about 10, about 1 to about 15 nucleotides of the repeat sequence are part of the 3' end of the repeat sequence. In some embodiments, the spacer nucleotide sequence is linked at its 3 'end to the 5' end of the 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 the repeat sequence. In some embodiments, about 1 to about 8, about 1 to about 10, about 1 to about 15 nucleotides of the repeat sequence are part of the 5' end of the repeat sequence.

In some embodiments, the spacer nucleotide sequence is linked at its 5 'end to a first repeat sequence and at its 3' end to a second repeat sequence to form a repeat-spacer-repeat sequence. In some embodiments, the spacer sequence is linked at its 5 'end to the 3' end of the first repeat sequence and at its 3 'end to the 5' end of the second repeat sequence, wherein the spacer sequence and the second repeat sequence repeat to form a repeat- (spacer-repeat) n sequence such that n is any integer from 1 to 100. In some embodiments, the 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.

In some embodiments, the repeat sequence has identity or substantial identity to a repeat sequence from a wild-type CRISPR locus. In some embodiments, the repeat sequence is a repeat sequence present 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 consecutive nucleotides of the 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, 28, 29, 30, 31, 32, 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 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, 20 to 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, 24 to 35, 25 to 35, 26 to 35, 27 to 35, 28 to 35, 29 to 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 pseudomonas aeruginosa type I-C Cas system. In some embodiments, the pseudomonas aeruginosa type I-C Cas system has a repeat length of about 25 to 38 nucleotides.

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 any one of SEQ ID NOS.26-30. In some cases, the repeat sequence comprises at least or about 95% homology to any of SEQ ID NOS.26-30. In some cases, the repeat sequence comprises at least or about 97% homology to any of SEQ ID NOS.26-30. In some cases, the repeat sequence comprises at least or about 99% homology to any of SEQ ID NOS.26-30. In some cases, the repeat sequence comprises 100% homology with any of SEQ ID NOS.26-30. In some cases, the repeat sequence comprises 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, or at least a portion of more than 32 nucleotides having any of SEQ ID nos. 26-30. In some embodiments, the repeat is engineered into the Φkz virus. In some embodiments, the Φkz virus is p1194 or p4430. In some embodiments, the repeat is engineered into Φkmv virus. In some embodiments, the Φkmv virus is p2167. In some embodiments, the repeat is engineered into a brunesoid virus. In some embodiments, the brunesoid virus is p1695 or p3278. In some embodiments, the repeats are engineered into a salsa Mu Na viral genus. In some embodiments, the salsa Mu Na virus is p1772, p2131, p2132 or p2973. In some embodiments, the repeat is engineered into the pluravirus. In some embodiments, the pluravirus is p1106, p1587, p1835, p2037, p2363, p2421 or pb1. In some embodiments, the replicates are engineered into a southern national virus. In some embodiments, the replicates are engineered into an arbitral virus. In some embodiments, the CRISPR array is engineered into a begalvirus. In some embodiments, the repeat is engineered into the Bei Telei virus. In some embodiments, the replicates are engineered into the casadaban virus. In some embodiments, the replicates are engineered into a sitex virus. In some embodiments, the CRISPR array is engineered into a vesicular virus. In some embodiments, the replicates are engineered into a detril virus. In some embodiments, the repeat is engineered into an elv virus. In some embodiments, the CRISPR array is engineered into holo Wei Bingdu. In some embodiments, the replicates are engineered into the chimaphila virus. In some embodiments, the repeat is engineered into the livina virus. In some embodiments, the replicates are engineered into Lu Saipu thema virus. In some embodiments, the repeat is engineered into the nipagin virus. In some embodiments, the repeat is engineered into the parkinsonism virus. In some embodiments, the repeat is engineered into pamek's virus. In some embodiments, the repeat is engineered into the Bondum virus. In some embodiments, the replicates are engineered into the filteirus. In some embodiments, the repeats are engineered into the pler Mo Liji virus. In some embodiments, the replicates are engineered into the septemamate thunder virus. In some embodiments, the repeat is engineered into a starbuch virus. In some embodiments, the repeat is engineered into the telthebai virus. In some embodiments, the repeat is engineered into about a subvirus. In some embodiments, the repeat is engineered into the ataxia virus. In some embodiments, the repeat is part of a CRISPR array engineered into a bacteriophage.

I-type CRISPR-Cas system

In some embodiments, the type I CRISPR-Cas system is a type I-A system, a type I-B system, a type I-C system, a type I-D system, a type I-E system, or a type I-F 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 comprises a cascades polypeptide. Type I cascades process CRISPR arrays to produce processed RNAs, which are then used to bind the complex to target sequences complementary to spacers in the processed RNAs. In some embodiments, the type I Cascade complex is a type I-A Cascade polypeptide, type I-B Cascade polypeptide, type I-C Cascade polypeptide, type I-D Cascade polypeptide, type I-E Cascade polypeptide, type I-F Cascade polypeptide, or type I-U Cascade polypeptide.

In some embodiments, a type I cascades complex comprises: (a) A nucleotide sequence encoding a Cas7 (Csa 2) polypeptide, a nucleotide sequence encoding a Cas8a1 (Csx 13) polypeptide or a Cas8a2 (Csx 9) 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); (b) A nucleotide sequence encoding a Cas6B polypeptide, a nucleotide sequence encoding a Cas8B (Csh 1) polypeptide, a nucleotide sequence encoding a Cas7 (Csh 2) polypeptide, and a nucleotide sequence encoding a Cas5 polypeptide (type I-B); (c) A nucleotide sequence encoding a Cas5d polypeptide, a nucleotide sequence encoding a Cas8C (Csd 1) polypeptide, and a nucleotide sequence encoding a Cas7 (Csd 2) polypeptide (type I-C); (d) A nucleotide sequence encoding a Casl Od (Csc 3) polypeptide, a nucleotide sequence encoding a Csc2 polypeptide, a nucleotide sequence encoding a Csc1 polypeptide, and a nucleotide sequence encoding a Cas6D polypeptide (type I-D); (e) A nucleotide sequence encoding a Cse1 (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); and/or (F) a nucleotide sequence encoding a Cys1 polypeptide, a nucleotide sequence encoding a Cys2 polypeptide, a nucleotide sequence encoding a Cas7 (Cys 3) polypeptide, and a nucleotide sequence encoding a Cas6F polypeptide (type I-F).

In some embodiments, the type I CRISPR-Cas system is externally derived from a target bacterium. In some embodiments, the exogenous type I CRISPR-Cas system comprises (a) a nucleotide sequence encoding a Cas7 (Csa 2) polypeptide, a nucleotide sequence encoding a Cas8a1 (Csx 13) polypeptide or a Cas8a2 (Csx 9) 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 that does not have nuclease activity (type I-a); (b) A nucleotide sequence encoding a Cas6B polypeptide, a nucleotide sequence encoding a Cas8B (Csh 1) polypeptide, a nucleotide sequence encoding a Cas7 (Csh 2) polypeptide, and a nucleotide sequence encoding a Cas5 polypeptide (type I-B); (c) A nucleotide sequence encoding a Cas5d polypeptide, a nucleotide sequence encoding a Cas8C (Csd 1) polypeptide, and a nucleotide sequence encoding a Cas7 (Csd 2) polypeptide (type I-C); (d) A nucleotide sequence encoding a Casl Od (Csc 3) polypeptide, a nucleotide sequence encoding a Csc2 polypeptide, a nucleotide sequence encoding a Csc1 polypeptide, and a nucleotide sequence encoding a Cas6D polypeptide (type I-D); (e) A nucleotide sequence encoding a Cse1 (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); and/or (F) a nucleotide sequence encoding a Cys1 polypeptide, a nucleotide sequence encoding a Cys2 polypeptide, a nucleotide sequence encoding a Cas7 (Cys 3) polypeptide, and a nucleotide sequence encoding a Cas6F polypeptide (type I-F).

In some embodiments, a type I CRISPR-Cas system exogenous to a target bacterium comprises a nucleotide sequence encoding a Cas5d polypeptide, a nucleotide sequence encoding a Cas8C (Csd 1) polypeptide, and a nucleotide sequence encoding a Cas7 (Csd 2) polypeptide (type I-C).

Bacteriophage of bacteria

In some embodiments, the bacteriophage is an obligate lytic bacteriophage. In some embodiments, the bacteriophage is a temperate bacteriophage that retains a lysogen. In some embodiments, the bacteriophage is a temperate bacteriophage in which some lysogenic genes have been removed, replaced, or inactivated. In some embodiments, the bacteriophage is a temperate bacteriophage whose lysogenic genes have been removed, replaced, or inactivated, thereby rendering the bacteriophage lytic.

In some embodiments, the bacteriophage targets certain species of pseudomonas. In some embodiments, the bacteriophage targets pseudomonas aeruginosa. In some embodiments, the bacteriophage specifically targets certain species of pseudomonas as compared to other bacterial species. In some embodiments, the bacteriophage targets certain species of pseudomonas in the absence of a CRISPR-Cas system. In some embodiments, the bacteriophage binds to lipopolysaccharide. In some embodiments, the bacteriophage binds to type IV pili. In some embodiments, the bacteriophage binds to the outer membrane porin OprM. In some embodiments, the target is a bacteriophage that infects bacteria. In some embodiments, the target refers to a bacteriophage that kills bacteria. In some embodiments, a specific target refers to a bacteriophage that infects a first species of bacteria but not a second species of bacteria. In some embodiments, a specific target refers to a bacteriophage that kills a first species of bacteria but not a second species of bacteria.

In some embodiments, the bacteriophage herein is or is engineered from a bacteriophage that infects pseudomonas. In some embodiments, the bacterial phage infected with pseudomonas is Φkz virus, Φkmv virus, brunesoid virus, salsa Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cetrimexo virus, vesicular virus, detrile virus, ehrlift virus, holo Wei Bingdu, chimaphila virus, livina virus, lu Saipu pedicle Ma Bingdu, niprina virus, parkona virus, pamex virus, boenjim virus, filteirus, pri Mo Liji virus, septembotrio virus, starbuchner virus, takitty virus, jojojoba virus, atameria virus, or prana virus. In some embodiments, the bacteriophage that infects pseudomonas comprises a wild-type pna phage subtype listed in table 5A, wherein the phage infects pseudomonas target, such as with a positive (+) tag (e.g., phage p1106 infects b 002548). In some embodiments, the bacterial phage that infects pseudomonas comprises an engineered pna phage subtype listed in table 5A, wherein the phage infects a target pseudomonas, such as with a positive (+) tag (e.g., p1106e003 infects b 002548). In some embodiments, the bacterial phage infected with pseudomonas comprises a wild-type salsa Mu Na virus phage subtype, an engineered salsa Mu Na virus phage subtype, a wild-type Φkz virus, a wild-type Φkmv virus, or a wild-type brunesabout virus, e.g., as set forth in table 5B, wherein the phage infects a target pseudomonas, e.g., marked with a plus (+) sign. As listed in table 5A, the wild-type pna phage subtypes may be p1106, p1587, p1835, p2037, p2363, p2421 and/or pb1, while the engineered pna phage subtypes may be p1106e003, p1587e002, p1835e002, p2037e002, p2363e003 and/or p2421e002. As listed in table 5B, the wild-type salsa Mu Na viral phage subtypes may be p1772, p2131, p2132, and/or p2973, the engineered salsa Mu Na viral phage subtypes may be pb1e002, p1772e005, p2131e002, p2132e002, and/or p2973e002, the wild-type Φkz viral phage subtypes may be p1194 and/or p4430, the wild-type Φkmv viral phage subtypes may be p2167, and the wild-type brunesoid viral phage subtypes may be p1695 and p3278. In some embodiments, the bacteriophage that infects pseudomonas is a southern national virus. In some embodiments, the bacteriophage that infects pseudomonas is an arbek virus. In some embodiments, the bacteriophage that infects pseudomonas is a begalvirus. In some embodiments, the bacteriophage that infects pseudomonas is a Bei Telei virus. In some embodiments, the bacterial phage that infects pseudomonas is a casadaban virus. In some embodiments, the bacteriophage that infects pseudomonas is a cetuximab virus. In some embodiments, the bacteriophage that infects pseudomonas is a vesicular virus. In some embodiments, the bacteriophage that infects pseudomonas is a detrilus virus. In some embodiments, the bacteriophage that infects pseudomonas is an elv virus. In some embodiments, the bacteriophage that infects pseudomonas is holo Wei Bingdu. In some embodiments, the bacteriophage that infects pseudomonas is a chimaphila virus. In some embodiments, the bacteriophage that infects pseudomonas is a livina virus. In some embodiments, the bacteriophage that infects pseudomonas is Lu Saipu pedicle Ma Bingdu. In some embodiments, the bacteriophage that infects pseudomonas is niprena virus. In some embodiments, the bacteriophage that infects pseudomonas is a pecona virus. In some embodiments, the bacteriophage that infects pseudomonas is pamek's virus. In some embodiments, the bacteriophage that infects pseudomonas is a boenkem virus. In some embodiments, the bacteriophage that infects pseudomonas is a filteirus. In some embodiments, the bacteriophage that infects pseudomonas is the pri Mo Liji virus. In some embodiments, the bacteriophage that infects pseudomonas is a septoria martevirus. In some embodiments, the bacteriophage that infects pseudomonas is a starbuch virus. In some embodiments, the bacteriophage that infects pseudomonas is a telthebai virus. In some embodiments, the bacteriophage that infects pseudomonas is about subvirus. In some embodiments, the bacteriophage that infects pseudomonas is a atactic trie virus. In some embodiments, the bacterial phage that infects pseudomonas kills pseudomonas. In some embodiments, the bacterial phage that infects pseudomonas does not infect staphylococcus aureus. In some embodiments, the bacterial phage that infects pseudomonas does not kill staphylococcus aureus. In some embodiments, the Pseudomonas killing bacteriophage does not infect Staphylococcus aureus. In some embodiments, the pseudomonas killing bacteriophage does not kill staphylococcus aureus. In some embodiments, the bacterial phage infected with pseudomonas does not infect klebsiella pneumoniae. In some embodiments, the bacterial phage infected with pseudomonas does not kill klebsiella pneumoniae. In some embodiments, the pseudomonas killing bacteriophage does not infect klebsiella pneumoniae. In some embodiments, the pseudomonas killing bacteriophage does not kill klebsiella pneumoniae. In some embodiments, the bacterial phage that infects pseudomonas does not infect enterococcus faecium. In some embodiments, the bacterial phage that infects pseudomonas does not kill enterococcus faecium. In some embodiments, the bacterial phage that kills pseudomonas does not infect enterococcus faecium. In some embodiments, the bacterial phage that kills pseudomonas does not kill enterococcus faecium. In some embodiments, the bacterial phage infected with pseudomonas does not infect enterobacter cloacae. In some embodiments, the bacterial phage infected with pseudomonas does not kill enterobacter cloacae. In some embodiments, the bacterial phage that kills pseudomonas does not infect enterobacter cloacae. In some embodiments, the bacterial phage that kills pseudomonas does not kill enterobacter cloacae. In some embodiments, the bacterial phage infected with pseudomonas does not infect acinetobacter baumannii. In some embodiments, the bacterial phage infected with pseudomonas does not kill acinetobacter baumannii. In some embodiments, the pseudomonas killing bacteriophage does not infect acinetobacter baumannii. In some embodiments, the pseudomonas killing bacteriophage does not kill acinetobacter baumannii. In some embodiments, the bacterial phage that infects pseudomonas does not infect staphylococcus epidermidis. In some embodiments, the bacterial phage infected with pseudomonas does not kill staphylococcus epidermidis. In some embodiments, the Pseudomonas killing bacteriophage does not infect Staphylococcus epidermidis. In some embodiments, the pseudomonas killing bacteriophage does not kill staphylococcus epidermidis. In some embodiments, the combination of bacteriophages infects pseudomonas. As one non-limiting example, the combination infects at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of pseudomonas in table 5A. As one non-limiting example, the combination infects at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of pseudomonas in table 5B. As one non-limiting example, the combination infects at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of pseudomonas in table 6B. In some embodiments, the combination of bacteriophages kills pseudomonas. As one non-limiting example, the combination kills at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the pseudomonas in table 5A. As one non-limiting example, the combination kills at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the pseudomonas in table 5B. As one non-limiting example, the combination kills at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the pseudomonas in table 6B.

In some embodiments, the bacteriophage is present in a cocktail comprising other bacteriophage, wherein each of the bacteriophage does not disrupt the function of the other bacteriophage in the cocktail.

In some embodiments, the bacteriophage is Φkz virus, Φkmv virus, british virus, sal Mu Na virus, south China virus, arbutus virus, begol virus, bei Telei virus, casadaban virus, cetuximab virus, vesicular virus, detriline virus, halloysite virus, holo Wei Bingdu, chikungunya virus, livina virus, lu Saipu ti Ma Bingdu, nipenv, parkenna virus, pamex virus, boenmer virus, filteirus, pri Mo Liji virus, sapetrex virus, starbuchner virus, telteirus, johne virus, atairia virus, or prina virus. In some embodiments, the bacteriophage is a Φkz virus. In some embodiments, the bacteriophage is Φkmv virus. In some embodiments, the bacteriophage is a brunesoid virus. In some embodiments, the bacteriophage is a sal Mu Na virus. In some embodiments, the bacteriophage is a pluravirus. In some embodiments, the bacteriophage comprises a CRISPR-Cas3 system. In some embodiments, the bacteriophage includes, but is not limited to, p1106 (ATCC accession number PTA-127024), p1194 (ATCC accession number PTA-127025), p1587 (ATCC accession number PTA-127027), p1695 (ATCC accession number PTA-127028), p1772 (ATCC accession number PTA-127030), p1835 (ATCC accession number PTA-127032), p2037 (ATCC accession number PTA-127034), p2131 (ATCC accession number PTA-127036), p2132 (ATCC accession number PTA-127038), p2167 (ATCC accession number PTA-127039), p2363 (ATCC accession number PTA-127041), p2421 (ATCC accession number PTA-127043), p2973 (ATCC accession number PTA-127045), p3278 (ATCC accession number PTA-127046), p4430 (ATCC accession number PTA-127047) or PB1 (ATCC accession number PTA-127049), which targets Pseudomonas species.

In some embodiments, the bacteriophage is p1106, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1106. In some embodiments, the bacteriophage is a p1106 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is p1106e003 (ATCC accession number PTA-127023). In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p1106e 003.

In some embodiments, the bacteriophage is p1194, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1194. In some embodiments, the bacteriophage is a p1194 bacteriophage comprising a CRISPR-Cas system.

In some embodiments, the bacteriophage is p1587, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1587. In some embodiments, the bacteriophage is a p1587 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is p1587e002 (ATCC accession number PTA-127026). In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to 01587e 002.

In some embodiments, the bacteriophage is p1695, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1695. In some embodiments, the bacteriophage is a p1695 bacteriophage comprising a CRISPR-Cas system.

In some embodiments, the bacteriophage is p1772, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1772. In some embodiments, the bacteriophage is a p1772 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is p1772e005 (ATCC accession number PTA-127029). In some embodiments, the bacteriophage comprises at least 70%, 775%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p1772e 005.

In some embodiments, the bacteriophage is p1835, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1835. In some embodiments, the bacteriophage is a p1835 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is p1835e002 (ATCC accession number PTA-127026). In some embodiments, the bacteriophage comprises at least 70%, 775%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p1835e 002.

In some embodiments, the bacteriophage is p2037, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2037. In some embodiments, the bacteriophage is a p2037 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is p2037e002 (ATCC accession number PTA-127033). In some embodiments, the bacteriophage comprises at least 70%, 775%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p2037e 002.

In some embodiments, the bacteriophage is p2131, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2131. In some embodiments, the bacteriophage is a p2131 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is p2131 (ATCC accession number PTA-127035). In some embodiments, the bacteriophage comprises at least 70%, 775%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2131.

In some embodiments, the bacteriophage is p2132, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2132. In some embodiments, the bacteriophage is a p2132 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is p2132e002 (ATCC accession number PTA-127037). In some embodiments, the bacteriophage comprises at least 70%, 775%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p2132e 002.

In some embodiments, the bacteriophage is p2167, or a mutant thereof that retains the ability to target a certain species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2167. In some embodiments, the bacteriophage is a p2167 bacteriophage comprising a CRISPR-Cas system.

In some embodiments, the bacteriophage is p2163, or a mutant thereof that retains the ability to target a certain species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2163. In some embodiments, the bacteriophage is a p2163 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is p2163e003 (ATCC accession No. PTA-127040). In some embodiments, the bacteriophage comprises at least 70%, 775%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p2163e 003.

In some embodiments, the bacteriophage is p2421, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2421. In some embodiments, the bacteriophage is a p2421 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is p2141e002 (ATCC accession No. PTA-127042). In some embodiments, the bacteriophage comprises at least 70%, 775%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p2141e 0002.

In some embodiments, the bacteriophage is p2973, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2973. In some embodiments, the bacteriophage is a p2973 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is p2973e002 (ATCC accession number PTA-127044). In some embodiments, the bacteriophage comprises at least 70%, 775%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p2973e 002.

In some embodiments, the bacteriophage is p3278, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 3278. In some embodiments, the bacteriophage is a p3278 bacteriophage comprising a CRISPR-Cas system.

In some embodiments, the bacteriophage is p4430, or a mutant thereof that retains the ability to target a species of pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 4430. In some embodiments, the bacteriophage is a p1106 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is PB1, or a mutant thereof that retains the ability to target a species of Pseudomonas. In some embodiments, the bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to PB 1. In some embodiments, the bacteriophage is a PB1 bacteriophage comprising a CRISPR-Cas system. In some embodiments, the bacteriophage is PB1e002 (ATCC accession No. PTA-127049). In some embodiments, the bacteriophage comprises at least 70%, 775%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to PB1e 002.

In some embodiments, the bacteriophage comprises a bacteriophage listed in table 1A, or a mutant thereof that retains the ability to target a species of pseudomonas.

Also disclosed herein is a cocktail comprising two or more bacteriophage. In some embodiments, the two or more bacteriophage are selected from the following lineages: Φkz virus, Φkmv virus, brunejo virus, sal Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cet-ter-chi virus, vesicular virus, detrilavirus, hallo Wei Bingdu, eriosema virus, lirana virus, lu Saipu pedicle Ma Bingdu, nipena virus, parkenna virus, pamex virus, boende-mer virus, filtei virus, pri Mo Liji virus, sapendamate virus, starbuch virus, teltei virus, jojo-subunit virus, ataegre-trie virus, or prana virus. In some embodiments, the cocktail comprises at least six bacteriophage, wherein the bacteriophage comprises Φkz virus, Φkmv virus, brunesoid virus, sal Mu Na virus, and plura virus. In some embodiments, the cocktail comprises at least one pluravirus, at least one salsa Mu Na virus, at least one Φkz virus, and at least one brunesabout virus. In some embodiments, the at least one bacteriophage of the cocktail comprises a CRISPR-Cas system. In some embodiments, at least two bacteriophage of the cocktail comprise a CRISPR-Cas system. In some embodiments, the 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, the at least one bacteriophage of the cocktail does not comprise a CRISPR-Cas system. In some embodiments, the at least two bacteriophage of the cocktail do not comprise a CRISPR-Cas system.

In some embodiments, the cocktail comprises at least two bacteriophage, wherein the bacteriophage comprises p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB1, or two or more bacteriophage thereof. In some embodiments, the cocktail comprises a first bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to p1106e 003. In some embodiments, the cocktail comprises a second bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to p1835e 002. In some embodiments, the cocktail comprises a third bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to p1772e 005. In some embodiments, the cocktail comprises a fourth bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to p2131e 002. In some embodiments, the cocktail comprises a fifth bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1194. In some embodiments, the cocktail comprises a fifth bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 4430. In some embodiments, the cocktail comprises a fifth bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1695. In some embodiments, the cocktail comprises a sixth bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 4430. In some embodiments, the cocktail comprises a sixth bacteriophage comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1695.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1106. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1106. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1106, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1106, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1106, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1106, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1106, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1587. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1106, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1106, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1106, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1106, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1106, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1695. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1106, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1106, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1106, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1106, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1106, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1772. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1106, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1106, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1106, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1106, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1106, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 1835. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1106, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1106, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of a peptide associated with p1194, p1587, p1695, p1772, p1106, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1106, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1106, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2037. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p1106, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p1106, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p1106, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p1106, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p1106, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2131. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p1106, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p1106, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p1106, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p1106, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p1106, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2132. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p1106, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p1106, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p1106, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p1106, p2167, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p1106, p2167, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2167. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p1106, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p1106, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p1106, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p1106, p2363, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p1106, p2363, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2363. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p1106, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p1106, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p1106, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p1106, p2421, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p1106, p2421, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2421. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p1106, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p1106, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p1106, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p1106, p2973, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p1106, p2973, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 2973. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p1106, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p1106, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p1106, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p1106, p3278, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p1106, p3278, p4430 or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 3278. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p1106, p4430, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p1106, p4430, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p1106, p4430, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p1106, p4430, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p1106, p4430, or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to p 4430. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p1106, or PB 1. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p1106, or PB 1. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p1106, or PB 1. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p1106, or PB 1. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p1106, or PB 1. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

In some embodiments, the first bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% sequence identity to PB 1. In some embodiments, the cocktail comprises a second bacteriophage, wherein the second bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or p 1106. In some embodiments, the cocktail comprises a third bacteriophage, wherein the third bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or p 1106. In some embodiments, the cocktail comprises a fourth bacteriophage, wherein the fourth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or p 1106. In some embodiments, the cocktail comprises a fifth bacteriophage, wherein the fifth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or p 1106. In some embodiments, the cocktail comprises a sixth bacteriophage, wherein the sixth bacteriophage comprises at least 70%, 75%, 80%, 85%, 90%95%, 96%, 97%, 98%, 99% or 100% of p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or p 1106. In some embodiments, at least one, two, three, or four bacteriophage comprise a CRISPR-Cas system. In some embodiments, at least one or both bacteriophage does not comprise a CRISPR Cas system.

Pseudomonas species

In some embodiments, the bacterium is pseudomonas. In some embodiments, the bacterium is pseudomonas aeruginosa.

In some embodiments, the pseudomonas species causes, contributes to, and/or causes complications of an infection, disease or condition, and the compositions and methods described herein are used to treat the infection, disease or condition. In some embodiments, the infection, disease, or condition is acute or chronic. In some embodiments, the infection, disease, or condition is localized or systemic. In some embodiments, the infection, disease, or condition is idiopathic. In some embodiments, the infection, disease or condition is obtained by a variety of means including, but not limited to, respiratory inhalation, ingestion, skin and wound infection, blood flow infection, middle ear infection, gastrointestinal tract infection, peritoneal infection, urinary tract infection, genitourinary tract infection, oral soft tissue infection, intra-abdominal infection, epidermal or mucosal absorption, ocular infection (including contact lens contamination), endocarditis, infection in cystic fibrosis, infection of indwelling medical devices such as joint prostheses, dental implants, catheters and cardiac implants, sexual contact and/or hospital acquired and ventilator-associated bacterial pneumonia. In some embodiments, the pseudomonas species causes urinary tract infection. In some embodiments, the pseudomonas species causes and/or exacerbates an inflammatory disease. In some embodiments, the pseudomonas species causes and/or exacerbates an autoimmune disease. In some embodiments, the pseudomonas species causes and/or exacerbates Inflammatory Bowel Disease (IBD). In some embodiments, the pseudomonas species causes and/or exacerbates psoriasis. In some embodiments, the pseudomonas species cause and/or exacerbate Psoriatic Arthritis (PA). In some embodiments, the pseudomonas species cause and/or exacerbate Rheumatoid Arthritis (RA). In some embodiments, the pseudomonas species causes and/or exacerbates Systemic Lupus Erythematosus (SLE). In some embodiments, the pseudomonas species causes and/or exacerbates Multiple Sclerosis (MS). In some embodiments, the pseudomonas species causes and/or exacerbates graves' disease. In some embodiments, the pseudomonas species causes and/or aggravates hashimoto thyroiditis. In some embodiments, the pseudomonas species cause and/or exacerbate myasthenia gravis. In some embodiments, the pseudomonas species causes and/or exacerbates vasculitis. In some embodiments, the pseudomonas species causes and/or aggravates cancer. In some embodiments, the pseudomonas species causes and/or aggravates cancer progression. In some embodiments, the pseudomonas species causes and/or aggravates cancer metastasis. In some embodiments, the pseudomonas species causes and/or exacerbates resistance to cancer therapy. In some embodiments, therapies for treating cancer include, but are not limited to, chemotherapy, immunotherapy, hormonal therapy, targeted drug therapy, and/or radiation therapy. In some embodiments, the cancer develops in an organ including, but not limited to, anus, bladder, blood and blood components, bone marrow, brain, breast, cervix, colon and rectum, esophagus, kidney, larynx, lymphatic system, muscle (i.e., soft tissue), mouth and pharynx, ovary, pancreas, prostate, skin, small intestine, stomach, testes, thyroid, uterus, and/or vulva. In some embodiments, the pseudomonas species cause and/or exacerbate a Central Nervous System (CNS) disorder. In some embodiments, the pseudomonas species cause and/or exacerbate attention deficit/hyperactivity disorder (ADHD). In some embodiments, the pseudomonas species causes and/or exacerbates autism. In some embodiments, the pseudomonas species causes and/or exacerbates bipolar disorder. In some embodiments, the pseudomonas species causes and/or exacerbates major depression. In some embodiments, the pseudomonas species causes and/or aggravates epilepsy. In some embodiments, the pseudomonas species causes and/or exacerbates neurodegenerative disorders, including, but not limited to, alzheimer's disease, huntington's disease, and/or parkinson's disease. In some embodiments, the compositions disclosed herein, such as one or more bacteriophages, engineered bacteriophages, or bacteriophage cocktails described herein, are used to treat a disease or condition or any of the symptoms associated with the disease or condition. In some embodiments, the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are used in combination with one or more other drugs for treating or alleviating one or more conditions associated with a disease, treating the disease or condition described above or a symptom associated with the disease described above.

Cystic fibrosis and cystic fibrosis related bronchiectasis are associated with pseudomonas aeruginosa infection. See, e.g., p.farrell et al, radiology, volume 252, phase 2, pages 534-543 (2009). In some embodiments, one or more bacteriophage is administered to a patient suffering from cystic fibrosis or cystic fibrosis-related bronchiectasis. In some embodiments, a combination of two or more bacteriophage is administered to a patient suffering from cystic fibrosis or cystic fibrosis-related bronchiectasis. In some embodiments, administration of the bacteriophage to a patient suffering from cystic fibrosis or cystic fibrosis-related bronchiectasis results in a reduction of bacterial load in the patient. In some embodiments, the reduction in bacterial load results in a clinical improvement in patients suffering from cystic fibrosis or cystic fibrosis related bronchiectasis.

Non-cystic fibrosis bronchiectasis is associated with pseudomonas aeruginosa infection. See, e.g., R.Wilson et al, respiratory Medicine, vol.117, pages 179-189 (2016). In some embodiments, one or more bacteriophage is administered to a patient suffering from non-cystic fibrosis bronchiectasis. In some embodiments, a combination of two or more bacteriophage is administered to a patient suffering from non-cystic fibrosis bronchiectasis. In some embodiments, administering a bacteriophage to a patient having non-cystic fibrosis bronchiectasis results in a reduction of bacterial load in the patient. In some embodiments, the reduction in bacterial load results in clinical improvement in patients with non-cystic fibrosis bronchiectasis.

In some embodiments, the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are administered to a subject suffering from a pseudomonas aeruginosa infection or a disease caused directly or indirectly by pseudomonas aeruginosa. In some embodiments, the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are used to treat a blood stream infection caused by pseudomonas aeruginosa. In some embodiments, the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are suitable for use in treating respiratory inhalation, ingestion, skin, and wound infections caused by pseudomonas aeruginosa. In some embodiments, the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are used to treat middle ear infections, gastrointestinal infections, peritoneal infections, urinary tract infections, genitourinary tract infections, oral soft tissue infections, intra-abdominal infections, epidermal or mucosal absorption, ocular infections (including contact lens contamination), urinary tract infections 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 pneumonia. In some embodiments, the pseudomonas species causes and/or exacerbates an inflammatory disease, and the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are used to treat an inflammatory disease. In some embodiments, the pseudomonas species causes and/or exacerbates an autoimmune disease, and the compositions disclosed herein, one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails as described herein, are used to treat an autoimmune disease. In some embodiments, the pseudomonas species causes and/or aggravates Inflammatory Bowel Disease (IBD), and the compositions disclosed herein, one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails as described herein, are used to treat IBD. In some embodiments, the bacteriophage is selected from table 1A, table 5A, and/or table 5B. In some embodiments, the compositions disclosed herein, such as one or more bacteriophages, engineered bacteriophages or bacteriophage cocktails described herein, are used to treat a disease, reducing bacterial load. In some embodiments, the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are used to treat a disease, reducing inflammation. In some embodiments, the compositions disclosed herein, such as one or more bacteriophages, engineered bacteriophages or bacteriophage cocktails described herein, are used to treat a disease, alleviate one or more symptoms associated with a bacterial infection or one or more sequelae of a bacterial infection.

In some embodiments, the treatment is administered as an intravenous or intramuscular drug. In some embodiments, the treatment is administered via the oral route. In some embodiments, the treatment is administered via a nebulizer. In some embodiments, the treatment is administered via a patient-operable nebulizer. In some embodiments, the treatment is administered via a metered dose nebulizer. In some embodiments, the treatment is administered in combination with one or more other drugs or therapeutic agents.

In some embodiments, the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are used to treat cancer. In some embodiments, the bacteriophage is selected from table 1A, table 5A, and/or table 5B.

In some embodiments, the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are used to treat pneumonia. In some embodiments, the bacteriophage is selected from table 1A, table 5A, and/or table 5B.

In some embodiments, the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are used to treat cystic fibrosis bronchiectasis. In some embodiments, administration of the bacteriophage to a patient suffering from cystic fibrosis or cystic fibrosis-related bronchiectasis results in a reduction of bacterial load in the patient. In some embodiments, the reduction in bacterial load results in clinical improvement in patients with cystic fibrosis bronchiectasis. In some embodiments, the bacteriophage is selected from table 1A, table 5A, and/or table 5B. In some embodiments, the treatment is administered as an intravenous or intramuscular drug. In some embodiments, the treatment is administered via the oral route. In some embodiments, the treatment is administered via a nebulizer. In some embodiments, the treatment is administered via a patient-operable nebulizer. In some embodiments, the treatment is administered via a metered dose nebulizer. In some embodiments, the treatment is administered in combination with one or more other drugs or therapeutic agents.

In some embodiments, the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are used to treat non-cystic fibrosis bronchiectasis. In some embodiments, administration of the bacteriophage to a patient suffering from non-cystic fibrosis bronchiectasis or fibrosis-related bronchiectasis results in a reduction of bacterial load in the patient. In some embodiments, the reduction in bacterial load results in clinical improvement in patients with non-cystic fibrosis bronchiectasis, fibrosis-related bronchiectasis. In some embodiments, the bacteriophage is selected from table 1A, table 5A, and/or table 5B. In some embodiments, the treatment is administered as an intravenous or intramuscular drug. In some embodiments, the treatment is administered via the oral route. In some embodiments, the treatment is administered via a nebulizer. In some embodiments, the treatment is administered via a patient-operable nebulizer. In some embodiments, the treatment is administered via a metered dose nebulizer. In some embodiments, the treatment is administered in combination with one or more other drugs or therapeutic agents.

In some embodiments, the treatment comprises a composition, such as a pharmaceutical composition comprising one or more bacteriophages (e.g., a bacteriophage cocktail), wherein the bacteriophage (e.g., bacteriophage cocktail) in the composition is from the following lineages: Φkz virus, Φkmv virus, brunejo virus, sal Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cet-ter-chi virus, vesicular virus, detrilavirus, hallo Wei Bingdu, eriosema virus, lirana virus, lu Saipu pedicle Ma Bingdu, nipena virus, parkenna virus, pamex virus, boende-mer virus, filtei virus, pri Mo Liji virus, sapendamate virus, starbuch virus, teltei virus, jojo-subunit virus, ataegre-trie virus, or prana virus. In some embodiments, the composition comprises a bacteriophage cocktail 511. In some embodiments, the composition comprises a bacteriophage cocktail PACK512. In some embodiments, provided herein is a pharmaceutical composition, wherein the pharmaceutical composition comprises at least six bacteriophage, wherein the bacteriophage is from p1106e003, p1835e002, p1772e005, p2131e002, p4430, and p1695.

Insertion site

In some embodiments, insertion of the nucleic acid sequence into a bacteriophage retains 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 the 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 a non-essential and/or lysogenic gene with a nucleic acid sequence does not affect the lytic activity of the bacteriophage. In some embodiments, replacement of a non-essential and/or lysogenic gene with a nucleic acid sequence retains the lytic activity of the bacteriophage. In some embodiments, replacement of a non-essential and/or lysogenic gene with a nucleic acid sequence enhances the lytic activity of the bacteriophage. In some embodiments, replacement of a non-essential and/or lysogenic gene with a nucleic acid sequence confers lytic properties to a lysogenic bacteriophage.

In some embodiments, the nucleic acid sequence is introduced into the bacteriophage genome at a first location, and one or more non-essential and/or lysogenic genes are removed and/or inactivated separately from the bacteriophage genome at different locations. In some embodiments, the nucleic acid sequence is introduced into the bacteriophage genome at a first location, and one or more non-essential and/or lysogenic genes are removed and/or inactivated from the bacteriophage genome at a plurality of different locations, respectively. 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, removal and/or inactivation of one or more non-essential and/or lysogenic genes retains the lytic activity of the bacteriophage. In some embodiments, removal of one or more non-essential and/or lysogenic genes renders the lysogenic bacteriophage a lytic bacteriophage.

In some embodiments, the bacteriophage is a temperate bacteriophage that has been rendered lytic by any of the foregoing means. In some embodiments, the temperate bacteriophage is rendered lytic by removal, replacement or inactivation of one or more lysogenic genes. In some embodiments, the lytic activity of the bacteriophage is due to removal, replacement, or inactivation of at least one lysogenic gene. In some embodiments, the lysogenic genes play a role in maintaining the lysogenic cycle in the bacteriophage. In some embodiments, the lysogenic genes play a role in establishing the lysogenic cycle in the bacteriophage. In some embodiments, lysogenic genes play a role in establishing and maintaining lysogenic cycles in the bacteriophage. In some embodiments, the lysogenic gene is a repressor gene. In some embodiments, the lysogenic gene is a cI repressor gene. In some embodiments, the lysogenic gene is an activator gene. In some embodiments, the lysogenic gene is a cII gene. In some embodiments, the lysogenic gene is the lexA gene. In some embodiments, the lysogenic gene is the int (integrase) gene. In some embodiments, two or more lysogenic genes are removed, replaced, or inactivated to cause arrest of the bacteriophage lysogenic cycle and/or induction of the lytic cycle. In some embodiments, the temperate bacteriophage is rendered lytic by insertion of one or more lytic genes. In some embodiments, the temperate bacteriophage is rendered lytic by insertion of one or more genes that help induce a lytic cycle. In some embodiments, the temperate bacteriophage is rendered lytic by altering the expression of one or more genes that help induce a lytic cycle. In some embodiments, the temperate bacteriophage phenotypically changes from lysogenic bacteriophage to lytic bacteriophage. In some embodiments, the temperate bacteriophage is rendered lytic by an environmental change. In some embodiments, environmental changes include, but are not limited to, changes in temperature, pH, or nutrients; exposure to antibiotics, hydrogen peroxide, foreign DNA or DNA damaging agents; the presence of organic carbon; and the presence of heavy metals, for example in the form of chromium (VI). In some embodiments, the temperate bacteriophage that is rendered lytic is prevented from reverting to a lysogenic state. In some embodiments, the return of the temperate bacteriophage to the lysogenic state that is conferred with lytic properties is prevented by the self-targeting activity of the CRISPR array that is first introduced. In some embodiments, the temperate bacteriophage that is conferred with lytic properties is prevented from reverting to lysogenic state by the introduction of additional CRISPR arrays. In some embodiments, the bacteriophage does not confer any new properties to the pseudomonas species other than cell death caused by the lytic activity of the bacteriophage and/or the activity of the first or second CRISPR array.

Nonessential genes

Transcriptional activator

In some embodiments, the nucleic acid sequence further comprises a transcriptional activator. In some embodiments, the encoded transcriptional activator modulates expression of a gene of interest within a pseudomonas species. In some embodiments, the transcriptional activator activates expression of a gene of interest within a pseudomonas species, whether exogenous or endogenous. In some embodiments, the transcriptional activator activates an expressed gene of interest within a pseudomonas species by disrupting the activity of one or more inhibitory elements within the pseudomonas species. In some embodiments, the inhibitory element comprises a transcriptional repressor. In some embodiments, the suppression element comprises a global transcriptional repressor. In some embodiments, the inhibitory element is a histone-like nuclear structure (H-NS) protein or a homolog or functional fragment thereof. In some embodiments, the inhibitory element is leucine-reactive regulatory protein (LRP). In some embodiments, the inhibitory element is a CodY protein.

In some bacteria, CRISPR-Cas systems are poorly expressed and are considered silent under most environmental conditions. In these bacteria, modulation of the CRISPR-Cas system is the result of the activity of transcriptional regulators, such as histone-like nuclear structure (H-NS) proteins, which are widely involved in the transcriptional regulation of the host genome. H-NS controls host transcriptional regulation by multimerizing along AT-rich sites, resulting in DNA bending. In some bacteria, modulation of the CRISPR-Cas3 operon is modulated by H-NS.

Similarly, in some bacteria, repression of the CRISPR-Cas system is controlled by an inhibitory element, such as leucine-reactive regulatory protein (LRP). LRP is associated with binding of regions upstream and downstream of the transcription initiation site. Notably, the activity of LRP to modulate expression of CRISPR-Cas system varies from bacterium to bacterium. Unlike H-NS, which have broad inter-species repression activity, LRP has been demonstrated to regulate expression of the host CRISPR-Cas system in different ways. Thus, in some cases, LRP reflects a host-specific way of modulating CRISPR-Cas system expression in different bacteria.

In some cases, repression of the CRISPR-Cas system is also controlled by the inhibitory element CodY. CodY is a GTP-sensing transcriptional repressor that functions by DNA binding. The intracellular concentration of GTP serves as an indicator of the nutritional status of the environment. Under normal culture conditions, GTP is abundant and binds to CodY to repress transcriptional activity. However, as the GTP concentration decreases, codY becomes less active in binding DNA, allowing transcription of previously repressed genes. Thus, codY can act as a stringent global transcriptional repressor.

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 reacts to the environmental trophic status of the bacteria. Under normal conditions, expression of LeuO is poor. However, leuO is up-regulated when amino acids starve and/or reach the stationary phase of the bacterial life cycle. Increased expression of LeuO results in antagonism of H-NS at overlapping promoter regions, thereby affecting gene expression. Overexpression of LeuO upregulates expression of the CRISPR-Cas system.

In some embodiments, expression of LeuO results in disruption of the inhibitory element. In some embodiments, disruption of the inhibitory element due to expression of LeuO derepresses transcription of the CRISPR-Cas system. In some embodiments, expression of LeuO derepresses transcription of the CRISPR-Cas system due to H-NS activity. In some embodiments, disruption of the inhibitory element due to expression of LeuO results in increased expression of the CRISPR-Cas system. In some embodiments, an increase in expression of the CRISPR-Cas system due to disruption of the inhibitory element by expression of LeuO results in an increase in CRISPR-Cas processing of a nucleic acid sequence comprising the CRISPR array. In some embodiments, an increase in expression of the CRISPR-Cas system due to disruption of the inhibitory element by expression of LeuO results in an increase in CRISPR-Cas processing of a nucleic acid sequence comprising the CRISPR array, thereby increasing the level of mortality of the CRISPR array to bacteria. In some embodiments, the transcriptional activator causes an increase in the activity of the bacteriophage and/or CRISPR-Cas system.

Adjusting element

In some embodiments, the nucleic acid sequences are operably associated with a plurality of promoters, terminators and other regulatory elements for expression in a variety of 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 to the CRISPR array. Any promoter useful for the present disclosure is used, and includes, for example, promoters that function with the organism of interest, as well as constitutive, inducible, developmentally regulated, tissue specific/preferred promoters, and the like, as disclosed herein. The regulatory elements used herein are endogenous or heterologous. In some embodiments, endogenous regulatory elements derived from an organism's subject are inserted into a genetic background in which they are not naturally occurring (e.g., at a location in the genome that is different from that in which they naturally occur), thereby producing recombinant or unnatural nucleic acids.

In some embodiments, expression of the nucleic acid sequence is constitutive, inducible, time-regulated, developmentally regulated, or chemically regulated. In some embodiments, expression of the nucleic acid sequence is altered to constitutive, inducible, time-regulated, developmentally-regulated, or chemically-regulated by operably linking the nucleic acid sequence to a promoter that functions in the organism of interest. In some embodiments, repression is made reversible by operably linking the nucleic acid sequence to an inducible promoter that functions in the organism of interest. The choice of promoters disclosed herein will vary, depending on the quantitative, temporal and spatial requirements of expression, and also on the host cell to be transformed.

Exemplary promoters for use in the methods, bacteriophages and compositions disclosed herein include promoters that function in bacteria. For example, L-arabinose inducible (araBAD, P _BAD ) Promoters, 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), a anhydrotetracycline inducible (tetA) promoter, trp, ipp, phoA, recA, proU, cst-1, cadA, nar, ipp-lac, cspA, 11-lac operon, T3-lac operon, T4 gene 32, T5-lac operon, nprM-lac operon, vhb, protein a, corynebacterium-escherichia coli-like promoter, thr, horn, diphtheria toxin promoter, sig a, sig B, nusG, soxS, katb, alpha-amylase (Pamy), ptms, P43 (comprising two overlapping RNA polymerase sigma factor recognition sites, σΑ, σ beta), ptms, P43, rplK-rplA, ferredoxin promoter, and/or xylose promoter. In some embodiments, the promoter is the bba_j23102 promoter. In some embodiments, the promoter functions in a broad range of bacteria such as bba_j23104, bba_j23109, and the like. In some embodiments, the promoter is derived from a target bacterium, such as an endogenous CRISPR promoter, an endogenous Cas operator promoter, p16, plpp, or ptat. In some embodiments, the promoter is a phage promoter, such as a promoter of gp105 or gp 245.

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 1-11. In some cases, the promoter comprises at least or about 95% homology to any of SEQ ID NOs 1-11. In some cases, the promoter comprises at least or about 97% homology to any of SEQ ID NOs 1-11. In some cases, the promoter comprises at least or about 99% homology to any of SEQ ID NOS.1-11. In some cases, the promoter comprises 100% homology with any of SEQ ID NOS.1-11. In some cases, the promoter comprises at least a portion of 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 nos. 1-11. In some cases, the promoter comprises at least a portion of 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 nos. 1-11.

In some embodiments, an inducible promoter is used. In some embodiments, chemically regulated promoters are used to regulate expression of genes in an organism by the application of exogenous chemical regulators. The use of chemically regulated promoters allows RNA and/or polypeptides encoded by nucleic acid sequences to be synthesized only when the organism is treated, for example, with an inducing chemical. In some embodiments using chemically inducible promoters, the application of the chemical induces gene expression. In some embodiments using chemically-repressed promoters, the application of a chemical represses gene expression. In some embodiments, the promoter is a light-inducible promoter, wherein application of light of a particular wavelength induces gene expression. In some embodiments, the promoter is a light-repressed promoter, wherein the application of light of a particular wavelength represses gene expression.

Expression cassette

In some embodiments, the nucleic acid sequence is or is present in an expression cassette. In some embodiments, the expression cassette is designed to express a nucleic acid sequence disclosed herein. In some embodiments, the nucleic acid sequence is an expression cassette encoding a component of the CRISPR-Cas system. In some embodiments, the nucleic acid sequence is an expression cassette encoding a component 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 an operable component of a type I CRISPR-Cas system (including cascades and Cas 3). In some embodiments, the nucleic acid sequence is an expression cassette encoding an operable component of a type I CRISPR-Cas system (including crRNA, cascade and Cas 3).

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, the expression cassette is naturally occurring, but has been obtained in a recombinant form that can be used for heterologous expression.

In some embodiments, the expression cassette comprises transcriptional and/or translational termination regions (i.e., termination regions) that function in the selected host cell. In some embodiments, the termination region is responsible for terminating transcription beyond the heterologous nucleic acid sequence of interest and for proper mRNA polyadenylation. In some embodiments, the termination region is derived from the transcription initiation region, from the operably linked nucleic acid sequence of interest, from the host cell, or from another source (i.e., exogenous or heterologous promoter, nucleic acid sequence of interest, host, or any combination thereof). In some embodiments, the terminator is operably linked to the nucleic acid sequences disclosed herein.

In some embodiments, the expression cassette comprises a nucleotide sequence of a selectable marker. In some embodiments, the nucleotide sequence encodes a selectable or screenable marker, depending on whether the marker confers a trait that is selected by chemical means, such as by using a selection agent (e.g., an antibiotic), or whether the marker is merely a trait that is identified by observation or testing, such as by screening (e.g., fluorescence).

Carrier body

In addition to expression cassettes, the nucleic acid sequences disclosed herein (e.g., nucleic acid sequences comprising a CRISPR array) are used in conjunction with a vector. The vector comprises a nucleic acid molecule comprising a nucleotide sequence to be transferred, delivered or introduced. Non-limiting examples of general classes of vectors include, but are not limited to, viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, gossip vectors, bacteriophage, artificial chromosomes, or agrobacterium binary vectors in double-stranded or single-stranded linear or circular form that may or may not be self-transferring or mobile. The vector converts a prokaryotic or eukaryotic host by integration into the cell genome or by presence extrachromosomal (e.g., an autonomously replicating plasmid with an origin of replication). In addition, shuttle vectors are included, which refers to DNA vectors that are capable of replication, either naturally or by design, in two different host organisms. In some embodiments, the shuttle vector replicates in actinomycetes and bacteria and/or eukaryotes. In some embodiments, the nucleic acid in the vector is under the control of and operably linked to a suitable promoter or other regulatory element for transcription in a host cell. In some embodiments, the vector is a bifunctional expression vector that functions in a variety of hosts.

Codon optimization

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 using a species-specific codon usage table for codon usage bias. The codon usage table was generated based on sequence analysis of the highest expressed gene of the species of interest. When the nucleotide sequence is to be expressed in the nucleus, the codon usage table is generated based on sequence analysis of highly expressed nuclear genes of the species of interest. The modification of the nucleotide sequence is determined by comparing the species-specific codon usage table with codons present in the native polynucleotide sequence. Codon optimization of a nucleotide sequence results in a nucleotide sequence that has 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%, etc.) to the native nucleotide sequence, but which still encodes a polypeptide having the same function as the polypeptide encoded by the original nucleotide sequence. In some embodiments, the nucleic acid sequences of the present disclosure are codon optimized for expression in an organism/species of interest.

Transformation

In some embodiments, the nucleic acid sequences and/or expression cassettes disclosed herein are transiently expressed and/or stably incorporated into the genome of a host organism. In some embodiments, the nucleic acid sequences and/or expression cassettes disclosed herein are introduced into cells by any method known to those of skill in the art. Exemplary transformation methods include transformation via electroporation of competent cells, passive uptake of competent cells, chemical transformation of competent cells, and any other electrical, chemical, physical (mechanical), and/or biological mechanism that results in the introduction of nucleic acids into cells, including any combination thereof. In some embodiments, transformation of the cell comprises nuclear transformation. In some embodiments, transformation of the cells includes plasmid transformation and conjugation.

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 located on the same or different nucleic acid constructs. In some embodiments, the nucleotide sequence is introduced into the cell of interest in a single transformation event or in multiple independent transformation events.

Antimicrobial agents and peptides

In some embodiments, the bacteriophage disclosed herein is further genetically modified to express an antibacterial peptide, a functional fragment of an antibacterial peptide, or a lytic gene. In some embodiments, the bacteriophage disclosed herein expresses at least one antimicrobial agent or peptide disclosed herein. In some embodiments, the bacteriophage disclosed herein comprises a nucleic acid sequence encoding an antibiotic enzyme, wherein a protein product of the nucleic acid sequence targets a bacteriophage resistant bacterium. In some embodiments, the bacteriophage comprises a nucleic acid encoding an enzyme that facilitates decomposition or degradation of the biofilm matrix. In some embodiments, the bacteriophage disclosed herein comprises a nucleic acid encoding a disperse protein 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, pectinase, peptide glutaminase, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, or lyase. In some embodiments, the enzyme is selected from the group consisting of cellulases, such as the glycosyl hydroxylase family of cellulases, such as the glycosyl hydroxylase 5 family of enzymes, also known as cellulase a; polyglucosamine (PGA) depolymerase; and colonic acid depolymerases such as 1, 4-L-fucose hydrolase, cola acid, depolymerase, DNase I or combinations thereof. In some embodiments, the bacteriophage disclosed herein secretes an enzyme disclosed herein.

In some embodiments, the antimicrobial agent or peptide is expressed and/or secreted by a bacteriophage disclosed herein. In some embodiments, the bacteriophage disclosed herein secretes and expresses an antibiotic, such as ampicillin, penicillin, a penicillin derivative, cephalosporin, monobactam, carbapenem, ofloxacin, ciprofloxacin, levofloxacin, gatifloxacin, norfloxacin, lomefloxacin, trovafloxacin, moxifloxacin, sparfloxacin, gemifloxacin, pazufloxacin, or any antibiotic disclosed herein. In some embodiments, the 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 pseudomonas species. In some embodiments, the bacteriophage disclosed herein comprises a nucleic acid sequence encoding a peptide, a nucleic acid sequence encoding an antibacterial peptide, a peptide expressing an antibacterial peptide or secretion assisting or enhancing the activity of the first and/or second type I CRISPR-Cas system.

Application method

In certain embodiments, disclosed herein are methods of killing a pseudomonas species comprising introducing any one of the bacteriophage disclosed herein into the pseudomonas species.

In certain embodiments, further disclosed herein are methods of modifying a mixed population of bacterial cells having a first bacterial species comprising a target nucleotide sequence in an essential gene and a second bacterial species not comprising a target nucleotide sequence in an essential gene, comprising introducing any of the bacterial phages disclosed herein into the mixed population of bacterial cells.

In certain embodiments, also disclosed herein are methods of treating a disease or condition in an individual in need thereof, the method comprising administering to the individual any of the bacteriophage disclosed herein.

In some embodiments, the pseudomonas species are killed solely by the lytic activity of the bacteriophage. In some embodiments, the pseudomonas species are killed solely by the activity of the CRISPR-Cas system. In some embodiments, the pseudomonas species are killed by: the CRISPR-Cas system processes the CRISPR array to produce processed crrnas that are capable of directing CRISPR-Cas based endonuclease activity and/or cleavage at a target nucleotide sequence in a target gene of a bacterium.

In some embodiments, the pseudomonas species is killed by a combination of lytic activity of the bacteriophage and activity of the type I CRISPR-Cas system. In some embodiments, the killing of pseudomonas species by the activity of the type I CRISPR-Cas system is independent of the lytic activity of the bacteriophage. In some embodiments, the activity of the type I CRISPR-Cas system complements 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.

In some embodiments, the lytic activity of the bacteriophage and the activity of the type I CRISPR-Cas system are synergistic. In some embodiments, synergistic activity is defined as an activity that results in a higher level of phage killing than the additive combination of the lytic activity of a bacteriophage and a type I CRISPR-Cas system. In some embodiments, the lytic activity of the bacteriophage is modulated by the concentration of the bacteriophage. In some embodiments, the activity of the type I CRISPR-Cas system is modulated by the concentration of bacteriophage.

In some embodiments, by increasing the concentration of bacteriophage applied to the bacteria, the synergistic killing of the bacteria is adjusted to more favor killing by the lytic activity of the bacteriophage over the activity of the first CRISPR-Cas system. In some embodiments, by reducing the concentration of bacteriophage applied to the bacteria, the synergistic killing of the bacteria is adjusted such that the lytic activity by the bacteriophage is less favorable to killing than the activity of the CRISPR-Cas system. In some embodiments, lytic replication allows for amplification and killing of target bacteria at low concentrations. In some embodiments, at high concentrations, phage amplification is not required. In some embodiments, the synergistic killing of bacteria is modulated to favor killing over the lytic activity of the bacteriophage by the activity of the CRISPR-Cas system by altering the number, length, composition, identity, or any combination thereof of the spacers to increase the lethality of the CRISPR array. In some embodiments, the synergistic killing of bacteria is modulated to be less conducive to killing by the activity of the CRISPR-Cas system than the lytic activity of the bacteriophage by altering the number, length, composition, identity, or any combination thereof of the spacers to reduce the lethality of the CRISPR array.

In one aspect, provided herein is a method of treating a pseudomonas infection in a subject, the method comprising administering to the subject a composition comprising a bacteriophage, wherein the bacteriophage comprises a nucleic acid sequence encoding a type I CRISPR-Cas system that causes cell death by targeting and degrading a pseudomonas bacterial genome. In some embodiments, a CRISPR-Cas system targeting pseudomonas bacteria comprises a CRISPR array comprising one or more spacer sequences complementary to a target nucleotide sequence in a pseudomonas species; a cascades polypeptide; and Cas3 polypeptides. In some embodiments, the one or more spacer sequences comprise at least one of SEQ ID NOS 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS 12-23, 31-74, or 88-120. 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 one or more spacer sequences at its 5 'end or its 3' end. In some embodiments, the repeat sequence comprises at least about 90% sequence identity to any one of SEQ ID NOS.26-30. In some embodiments, the CRISPR array comprises at least about 90% sequence identity to the sequences set forth in FIGS. 1A-1E or SEQ ID NOS: 83-87. In some embodiments, the target nucleotide sequence comprises a coding sequence. In some embodiments, the target nucleotide sequence comprises a non-coding sequence or an intergenic sequence. In some embodiments, the target nucleotide sequence comprises all or part of a promoter sequence. In some embodiments, the promoter sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 1-11. In some embodiments, the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding strand of the transcribed region of the essential gene.

In one embodiment, the essential gene is Tsf, acpP, gapA, 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 or metK. In some embodiments, the cascades polypeptide forms a cascades of an I-A type CRISPR-Cas system, an I-B type CRISPR-Cas system, an I-C type CRISPR-Cas system, an I-D type CRISPR-Cas system, an I-E type CRISPR-Cas system, or an I-F type CRISPR-Cas system. In some embodiments, the cascades complex comprises: (i) A Cas7 polypeptide, a Cas8a1 polypeptide or a Cas8a2 polypeptide, a Cas5 polypeptide, a Csa5 polypeptide, and a Cas6a polypeptide, wherein the Cas3 polypeptide comprises a Cas3' polypeptide and a Cas3 "polypeptide having no nuclease activity (type I-a CRISPR-Cas system); (ii) Cas6B polypeptides, cas8B polypeptides, cas7 polypeptides, and Cas5 polypeptides (type I-B CRISPR-Cas system); (iii) Cas5d polypeptides, cas8C polypeptides, and Cas7 polypeptides (type I-C CRISPR-Cas system); (iv) Cas10D polypeptide, csc2 polypeptide, csc1 polypeptide, cas6D polypeptide (I-D CRISPR-Cas system); (v) Cse1, cse2, cas7, cas5, and Cas6E polypeptides (I-E CRISPR-Cas system); (vi) Csy1 polypeptides, csy2 polypeptides, csy3 polypeptides, and Csy4 polypeptides (type I-F CRISPR-Cas system). In some embodiments, the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81), and a Cas7 polypeptide (optionally SEQ ID NO: 82) (type I-C CRISPR-Cas system). In some embodiments, the nucleic acid sequence further comprises a promoter sequence.

In one embodiment, provided herein is a method of treating a selected group of subjects having a pseudomonas infection. In one embodiment, the subject is refractory to one or more commonly used therapies, such as therapies comprising one or more antibiotic compounds.

In one embodiment, the selected group of subjects is identified as being infected with a strain of MDR of a species of the genus Pseudomonas. In one embodiment, the selected group of subjects is identified as immunocompromised subjects. In some embodiments, the infection is a nosocomial infection. In some embodiments, the infection is a persistent or recurrent infection. In some embodiments, the subject is symptomatic. In some embodiments, the subject suffers from chronic pseudomonas-induced infections and diseases. In one embodiment, the composition is administered to the subject as a single dose.

In some embodiments, provided herein is a method of treating a subject having a pseudomonas infection by administering a composition, e.g., a pharmaceutical composition comprising one or more bacteriophage (e.g., a bacteriophage cocktail), wherein the bacteriophage (e.g., bacteriophage cocktail) in the composition is from the following lineages: Φkz virus, Φkmv virus, brunejo virus, sal Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cet-ter-chi virus, vesicular virus, detrilavirus, hallo Wei Bingdu, eriosema virus, lirana virus, lu Saipu pedicle Ma Bingdu, nipena virus, parkenna virus, pamex virus, boende-mer virus, filtei virus, pri Mo Liji virus, sapendamate virus, starbuch virus, teltei virus, jojo-subunit virus, ataegre-trie virus, or prana virus. In some embodiments, the composition comprises a bacteriophage cocktail 511. In some embodiments, the composition comprises a bacteriophage cocktail PACK512. In some embodiments, provided herein is a pharmaceutical composition, wherein the pharmaceutical composition comprises at least six bacteriophage, wherein the bacteriophage is from p1106e003, p1835e002, p1772e005, p2131e002, p4430, and p1695. In some embodiments, the compositions disclosed herein, such as one or more bacteriophage, engineered bacteriophage, or bacteriophage cocktails described herein, are used to treat cancer. In some embodiments, the bacteriophage is selected from table 1A, table 5A, and/or table 5B.

In some embodiments, the treatment is administered as an intravenous or intramuscular drug. In some embodiments, the treatment is administered via the oral route. In some embodiments, the treatment is administered via a nebulizer. In some embodiments, the treatment is administered via a patient-operable nebulizer. In some embodiments, the treatment is administered via a metered dose nebulizer.

In some embodiments, the treatment is administered in combination with one or more other drugs or therapeutic agents, for example, antibiotics such as tobramycin. In some embodiments, exemplary therapeutic agents co-administered with a composition comprising one or more bacteriophage may also be an antibiotic, such as ampicillin, penicillin, a penicillin derivative, cephalosporin, monobactam, carbapenem, ofloxacin, ciprofloxacin, levofloxacin, gatifloxacin, norfloxacin, lomefloxacin, trovafloxacin, moxifloxacin, sparfloxacin, gemifloxacin, pazufloxacin, or any antibiotic disclosed herein. In some embodiments, the additional therapeutic agent comprises a drug for improving airway function. In some embodiments, the additional therapeutic agent comprises a drug for reducing airway responsiveness. In some embodiments, the additional therapeutic agent comprises a drug for reducing airway inflammation. In some embodiments, the additional therapeutic agent comprises a bronchodilator. In some embodiments, the additional therapeutic agent comprises a drug for increasing oxygen utilization. In some embodiments, the additional therapeutic agent comprises a drug for reducing airway mucus production. In some embodiments, the additional therapeutic agent comprises dnase. In some embodiments, the additional therapeutic agent is saline. In some embodiments, the additional therapeutic agent is a therapeutic method comprising cough exercises, e.g., for treating cystic fibrosis.

In one embodiment, the composition is administered to the subject more than once, e.g., in multiple amounts. In one embodiment, the composition is administered to the subject in 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses. In one embodiment, the composition is administered to the subject once daily, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once weekly. In one embodiment, the composition is administered to the subject once a day 10. In one embodiment, the composition is administered to the subject once a 12 day period. In one embodiment, the composition is administered to the subject once every 2 weeks. In one embodiment, the composition is administered to the subject once every 3 weeks. In one embodiment, the composition is administered to the subject once a month.

In one embodiment, the first composition is administered to the subject in multiple doses over a period of one month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or longer.

In one aspect, provided herein is a method of treating a pseudomonas infection in a subject, the method comprising administering to the subject a first composition comprising a bacteriophage, wherein the bacteriophage comprises a nucleic acid sequence encoding a type I CRISPR-Cas system targeting a pseudomonas bacterium; and administering a second therapeutic agent to the subject. In some embodiments, the second therapeutic agent is an antibiotic or an antibacterial composition. In one embodiment, the first composition and the second therapeutic agent are administered on the same day. In one embodiment, the first composition and the second therapeutic agent are administered on a non-same day.

Route of administration and dosage

The dosage and duration of administration of the compositions disclosed herein will depend on a variety of factors, including the age of the subject, the weight of the subject, and the tolerance of the phage. In some embodiments, the bacteriophage disclosed herein is administered to a subject intra-arterially, intravenously, intra-urethrally, intramuscularly, orally, subcutaneously, by inhalation, or any combination thereof. In some embodiments, the bacteriophage disclosed herein is administered to a subject by oral administration. In some embodiments, the bacteriophage disclosed herein is administered to a patient by topical, skin, transdermal, transmucosal, implant, sublingual, buccal, rectal, vaginal, ocular, otic, or nasal administration. In some embodiments, the bacteriophage disclosed herein is administered to a subject by any combination of the aforementioned routes of administration. In some embodiments, the bacteriophage disclosed herein is administered to a subject by inhalation. In some embodiments, the bacteriophage disclosed herein is administered to a subject by inhalation using a nebulizer.

In some embodiments, the compositions and methods described herein are used to treat pulmonary infection or disease. In some embodiments, the pulmonary infection or disease is cystic fibrosis. In some embodiments, administering the composition comprising the bacteriophage to a patient suffering from cystic fibrosis or cystic fibrosis-related bronchiectasis is via a nebulizer. In some embodiments, the treatment is administered via a patient-operable nebulizer. In some embodiments, the treatment is administered via a metered dose nebulizer. In some embodiments, the treatment is administered in combination with one or more other drugs or therapeutic agents, such as antibiotics or bronchodilators. In some embodiments, the treatment is a bacteriophage, a bacteriophage composition, and/or a bacteriophage cocktail as described herein. For example, a composition comprising p1106e003, p1835e002, p1772e005, p2131e002, p4430 and p 1695. The composition may be in an nebulizable formulation for pulmonary delivery.

In some embodiments, between 10 is administered to a subject ³ And 10 ²⁰ Phage dose between PFUs. In some embodimentsIn a regimen, a subject is administered between 10 ³ And 10 ¹⁰ Phage dose between PFUs. In some embodiments, between 10 is administered to a subject ⁶ And 10 ²⁰ Phage dose between PFUs. In some embodiments, between 10 is administered to a subject ⁶ And 10 ¹⁰ Phage dose between PFUs. For example, in some embodiments, a composition comprising a bacteriophage is used to provide a composition of between 10 ³ And 10 ¹¹ The amount between PFUs is administered to a subject. In some embodiments, the composition comprising bacteriophage is at 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 is administered to a subject. In some embodiments, the composition comprising bacteriophage is less than 10 ¹ The amount of PFU is administered to the subject. In some embodiments, the composition comprising bacteriophage is present at between 10 ¹ And 10 ⁸ 、10 ⁴ And 10 ⁹ 、10 ⁵ And 10 ¹⁰ Or 10 ⁷ And 10 ¹¹ The amount between PFUs is administered to a subject. In some embodiments, a composition comprising two or more bacteriophages is administered to a subject, wherein each bacteriophage is present at 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 Or more. In some embodiments, a composition comprising two or more bacteriophages is administered to a subject, wherein each bacteriophage is present at less than 10 ¹ The PFU is administered in an amount. In some embodiments, the administration to a subject comprises two of Or a combination of more bacteriophages, wherein each bacteriophage is present in an amount of between 10 ¹ And 10 ⁸ 、10 ⁴ And 10 ⁹ 、10 ⁵ And 10 ¹⁰ Or 10 ⁷ And 10 ¹¹ The amount between PFUs is administered.

In some embodiments, the bacteriophage or 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, or 24 times per day. In some embodiments, the bacteriophage or mixture is administered to a subject in need thereof at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per week. In some embodiments, the bacteriophage or mixture is administered to a subject in need thereof at least 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 per month. In some embodiments, the bacteriophage or mixture is administered to a subject in need thereof every 2, 4, 6, 8, 10, 12, 14, 18, 20, 22, or 24 hours.

In some embodiments, the compositions disclosed herein (bacteriophage) are administered before, during, or after the occurrence of a disease or condition. In some embodiments, the time for administering the bacteriophage containing composition is different. In some embodiments, the pharmaceutical composition is used as a prophylactic agent and is continuously administered to a subject having a condition or predisposition to a disease, in order to prevent the occurrence of the disease or condition. In some embodiments, the pharmaceutical composition is administered to the subject as soon as possible during or after the onset of symptoms. In some embodiments, administration of the composition begins within 48 hours prior to onset of symptoms, within 24 hours prior to onset of symptoms, within 6 hours prior to onset of symptoms, or within 3 hours of onset of symptoms. In some embodiments, the initial administration of the composition uses any of the formulations described herein via any feasible route, e.g., by any of the routes described herein. In some embodiments, the composition is administered as soon as practicable after the onset of the disease or condition is detected or suspected, such as, for example, about 1 month to about 3 months, for the length of time necessary to treat the disease. In some embodiments, the length of treatment will vary from subject to subject.

Bacterial infection

In certain embodiments, disclosed herein are methods of treating a bacterial infection. In some embodiments, the bacteriophage disclosed herein treats or prevents a disease or condition in a human or animal subject that is mediated or caused by a bacterium as disclosed herein. In some embodiments, the bacteriophage disclosed herein treats or prevents a disease or condition in a human or animal subject caused or exacerbated by a bacterium as disclosed herein. Such bacteria are typically in contact with tissue of a subject, including: intestinal, oral, pulmonary, axillary, ocular, vaginal, anal, otic, nasal or laryngeal tissues. In some embodiments, the bacterial infection is treated by modulating the activity of the bacteria and/or by directly killing the bacteria.

In some embodiments, the target bacteria are pseudomonas. In some embodiments, the bacterium is pseudomonas aeruginosa.

In some embodiments, the one or more target bacteria present in the population of bacteria are pathogenic. In some embodiments, the pathogen is urinary tract pathogenic. In some embodiments, the pathogen is a pulmonary pathogen. In some embodiments, the pathogen is a blood stream pathogen.

In some embodiments, the bacteriophage disclosed herein is used to treat an infection, disease, or condition in the pulmonary system of a subject. In some embodiments, the bacteriophage is used to modulate and/or kill target bacteria within the pulmonary microbiome of a subject. In some embodiments, the bacteriophage is used to selectively modulate and/or kill one or more target bacteria from a plurality of bacteria within a pulmonary microbiome of a subject. In some embodiments, the bacteriophage is used to selectively modulate and/or kill one or more target pathogenic bacteria from a plurality of bacteria within a pulmonary microbiome of a subject.

In some embodiments, the bacteriophage disclosed herein is used to treat an infection, disease, or condition in the urinary tract of a subject. In some embodiments, the bacteriophage is used to modulate and/or kill a target bacterium within the urinary tract flora of a subject. In some embodiments, the bacteriophage is used to selectively modulate and/or kill one or more target urinary tract pathogenic bacteria from a plurality of bacteria within the urinary tract flora of a subject.

In some embodiments, the bacteriophage disclosed herein is used to treat an infection, disease, or condition on the skin of a subject. In some embodiments, the bacteriophage is used to modulate and/or kill target bacteria on the skin of a subject.

In some embodiments, the bacteriophage disclosed herein is used to treat an infection, disease, or condition on a mucosa of a subject. In some embodiments, the bacteriophage is used to modulate and/or kill a target bacterium on a subject's mucosa.

In some embodiments, the pathogen is antibiotic-resistant.

In some embodiments, the one or more target bacteria present in the population of bacteria form a biofilm. In some embodiments, the biofilm comprises a pathogenic bacteria. In some embodiments, the bacteriophage disclosed herein is used to treat a biofilm.

In some embodiments, the bacteriophage treats acne and other related skin infections.

In some embodiments, the pseudomonas species is a multi-drug resistant (MDR) bacterial strain. The MDR strain is a bacterial strain that is resistant to at least one antibiotic. In some embodiments, the bacterial strain is resistant to antibiotics such as cephalosporins, fluoroquinolones, carbapenems, colistins, aminoglycosides, vancomycin, streptomycin, and methicillin. In some embodiments, the bacterial strain is resistant to an antibiotic, such as cefpirane, ceftaroline, clindamycin, daphnetin, daptomycin, linezolid, mupirocin, olanzapine, tedizolid, telavancin, tigecycline, vancomycin, aminoglycoside, carbapenem, ceftazidime, cefepime, cefpirane, fluoroquinolone, piperacillin, ticarcillin, linezolid, streptoamycin, tigecycline, daptomycin, or any combination thereof. In some embodiments, the MDR strain is pseudomonas aeruginosa.

In some embodiments, the bacterium is a pseudomonas species. In some embodiments, the bacterium is pseudomonas aeruginosa. In some embodiments, the methods and compositions disclosed herein are used for veterinary and medical applications and research applications.

In some embodiments, the bacterial infection is present in a subject suffering from cystic fibrosis. In some embodiments, the bacterial infection is present in a subject suffering from non-cystic fibrosis bronchiectasis. In some embodiments, the bacterial infection is present in a subject suffering from pneumonia. In some embodiments, the bacterial infection contributes to pneumonia. As non-limiting examples, pneumonia is hospital-acquired pneumonia, ventilator-acquired pneumonia, community-acquired pneumonia, or healthcare-acquired pneumonia. In some embodiments, the bacterial infection is a hematological infection (BSI).

In some embodiments, the methods described herein comprise administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an antibiotic. In some embodiments, the antibiotic comprises tobramycin.

Microbiome

"microbiome", "microbiota" and "microbiota" are used interchangeably hereinafter and refer to the ecological community of microorganisms living on or in the body surface, body cavity and body fluids of a subject. Non-limiting examples of microbiome habitats include: intestinal tract, colon, skin surface, skin pores, vaginal cavity, umbilical region, conjunctival region, intestinal region, stomach, nasal cavity, and passage, gastrointestinal tract, genitourinary tract, saliva, mucous and feces. In some embodiments, the microbiome includes microbiome materials including, but not limited to, bacteria, archaea, protist, fungi, and viruses. In some embodiments, the microbial material comprises gram negative bacteria. In some embodiments, the microbial material comprises gram positive bacteria. In some embodiments, the microbial material comprises Proteus, actinomyces, bacteroides, or Thick-walled bacteria.

In some embodiments, the bacteriophage as disclosed herein is used to modulate or kill a target bacterium within a microbiome of a subject. In some embodiments, the bacteriophage is used to modulate and/or kill target bacteria within the microbiome by a CRISPR-Cas system, lytic activity, or a combination thereof. In some embodiments, the bacteriophage is used to modulate and/or kill a target bacterium within a microbiome of a subject. In some embodiments, the bacteriophage is used to selectively modulate and/or kill one or more target bacteria from a plurality of bacteria within a microbiome of a subject.

In some embodiments, the bacteriophage is used to modulate or kill a single or multiple target bacteria within a subject's pulmonary microbiome. Alteration of the pulmonary microbiome (e.g., dysbiosis) increases the risk of health conditions such as diabetes, psychotic disorders, ulcerative colitis, colorectal cancer, autoimmune disorders, obesity, diabetes, central nervous system diseases, and inflammatory bowel disease.

In some embodiments, the bacteriophage disclosed herein is administered to a subject to promote a healthy microbiome. In some embodiments, the bacteriophage disclosed herein is administered to a subject to restore the subject's microbiome to a health promoting microbiome composition. In some embodiments, a composition comprising a bacteriophage disclosed herein comprises a prebiotic or third agent. In some embodiments, the microbiome-related disease or disorder is treated by a bacteriophage disclosed herein.

Environmental therapy

In some embodiments, the bacteriophage disclosed herein is further used in food and agricultural hygiene (including meat, fruit, and vegetable hygiene), hospital hygiene, home hygiene, vehicle and equipment hygiene, industrial hygiene, and the like. In some embodiments, the bacteriophage disclosed herein is used to remove antibiotic resistance or other undesirable pathogens from medicine, veterinary medicine, animal husbandry, or any other environment where bacteria are transferred to humans or animals.

Environmental applications of phage in healthcare facilities are used in devices such as endoscopes and environments such as ICU, which are potential sources of nosocomial infections due to pathogens that are difficult or impossible to disinfect. In some embodiments, the phage disclosed herein are used to treat equipment or environments inhabited by bacteria that are resistant to common disinfectants. In some embodiments, the phage compositions disclosed herein are used to disinfect inanimate objects. In some embodiments, the phage titer is sprayed, smeared or poured onto the environment disclosed herein with an aqueous solution. In some embodiments, the solutions described herein comprise 10 ¹ -10 ²⁰ Plaque Forming Units (PFU)/ml. In some embodiments, the bacteriophage disclosed herein is applied by an aerosolization reagent comprising a dry dispersing agent to aid in the distribution of the bacteriophage into the environment. In some embodiments, the object is immersed in a solution containing the bacteriophage disclosed herein.

(Hygiene)

In some embodiments, the bacteriophage disclosed herein are used as a hygienic agent in various fields. Although the term "phage" or "bacteriophage" may be used, it should be noted that this term should be construed broadly to include single bacteriophage, multiple bacteriophage, e.g., a mixture of bacteriophages, and mixtures of bacteriophages with agents such as disinfectants, detergents, surfactants, water, etc., where appropriate.

In some embodiments, the bacteriophage is used to sterilize hospital facilities, including operating rooms, wards, waiting rooms, laboratories, or other various hospital equipment. In some embodiments, the apparatus includes an electrocardiograph, a ventilator, a cardiovascular assist device, an intra-aortic balloon pump, an infusion device, other patient care devices, a television, a monitor, a remote control, a telephone, a bed, and the like. In some cases, the bacteriophage is applied through an aerosol canister. In some embodiments, the bacteriophage is applied by rubbing the bacteriophage onto an object with a transfer vehicle.

In some embodiments, the bacteriophage described herein is used in conjunction with a patient care device. In some embodiments, the bacteriophage is used in conjunction with a conventional ventilator or respiratory therapy device to clean the inner and outer surfaces from patient to patient. Examples of ventilators include devices to support ventilation during surgery, devices to support ventilation of disabled patients, and the like. In some embodiments, conventional therapies include automatic or motorized devices, or manual bag-type devices, such as those commonly found in emergency rooms and ambulances. In some embodiments, respiratory therapy includes an inhaler to introduce a drug, such as a bronchodilator commonly used for chronic obstructive pulmonary disease or asthma; or a device for maintaining airway patency, such as a cpap device.

In some embodiments, the bacteriophage described herein is used to clean surfaces and treat colonized populations in areas where highly infectious bacterial diseases such as meningitis or intestinal infections are present.

In some embodiments, the water supply is treated with a composition disclosed herein. In some embodiments, the bacteriophage disclosed herein is used to treat contaminated water, water present in tanks, wells, reservoirs, tanks, water conduits, pipes and similar water distribution devices. In some embodiments, the bacteriophage is applied to industrial storage tanks, where water, oil, cooling fluids, and other fluids are collected in a collection tank. In some embodiments, the bacteriophage disclosed herein is periodically introduced into an industrial storage tank to reduce bacterial growth.

In some embodiments, the bacteriophage disclosed herein is used to sterilize living areas, such as houses, independent apartments, private apartments, dormitories, or any living area. In some embodiments, the bacteriophage is used to sterilize public areas such as theatres, concert halls, museums, train stations, airports, pet areas such as pet beds or litter boxes, and the like. In this case, the bacteriophage is dispensed from conventional devices, including pump sprayers, aerosol containers, spray bottles, pre-moistened wipes, etc., which are applied directly to (e.g., sprayed onto) the area to be sterilized or transferred to the area via a transfer vehicle such as a towel, sponge, etc. In some embodiments, the phage disclosed herein are applied to various rooms of a house, including kitchens, bedrooms, bathrooms, garages, basements, and the like. In some embodiments, the phage disclosed herein are in the same manner as conventional cleaners. In some embodiments, the phage is applied in combination with (before, after, or simultaneously with) a conventional cleaner, provided that the conventional cleaner is formulated to maintain sufficient bacteriophage biological activity.

In some embodiments, the bacteriophage disclosed herein is added to a component of a paper product during processing of the paper product or after processing is complete. Paper products to which the bacteriophage disclosed herein have been added include, but are not limited to, paper towels, toilet paper, wet tissues.

Food safety

In some embodiments, the bacteriophage described herein is used in any food product or nutritional supplement to prevent contamination. Examples of food or pharmaceutical products are milk, yoghurt, curd, cheese, fermented milks, milk based fermented products, ice-creams, fermented cereal based products, milk based powders, infant formulas or tablets, liquid suspensions, dry oral supplements, wet oral supplements or dry tube feeds.

The broad concept of bacteriophage hygiene is applicable to other agricultural applications and organisms. Agricultural products, including fruits and vegetables, dairy products, and other agricultural products. For example, freshly cut agricultural products are often contaminated with pathogenic bacteria when they reach the processing plant. This results in an outbreak of food borne diseases that can be traced back to agricultural products. In some embodiments, the application of bacteriophage preparations (preparations) to agricultural products significantly reduces or eliminates the likelihood of food borne diseases by the application of a single bacteriophage or mixture of bacteriophages specific to the bacterial species associated with the food borne disease. In some embodiments, the bacteriophage is applied at different stages of production and processing to reduce bacterial contamination at the point or prevent contamination at a subsequent point.

In some embodiments, the specific bacteriophage is applied to produce in restaurants, groceries, produce distribution centers. In some embodiments, the bacteriophage disclosed herein is applied periodically or continuously to the fruit and vegetable contents of the salad dressing. In some embodiments, the application of the bacteriophage to a salad dressing or to sterilize the exterior of the food item is a spraying or sprinkling process or a cleaning process.

In some embodiments, the bacteriophage described herein is used in a substrate or support medium containing packaging containing meat, agricultural products, cut fruits and vegetables, and other food products. In some embodiments, a polymer suitable for packaging is impregnated with a bacteriophage formulation.

In some embodiments, the bacteriophage described herein is used in farms and livestock feed. In some embodiments, in a farm that raises livestock, the bacteriophage is provided to the livestock in its drinking water, food, or both. In some embodiments, the bacteriophage described herein is sprayed onto carcasses and used to disinfect slaughter houses.

The use of specific bacteriophages as biocontrol agents on agricultural products has many advantages. For example, bacteriophages are natural, non-toxic products that do not disrupt the ecological balance of the natural microbial community as common chemical bactericides, but specifically solubilize targeted food-borne pathogens. Because bacteriophages, unlike chemical bactericides, are natural products that evolve with their host bacteria, new phages that are active against the most recently emerged resistant bacteria can be rapidly identified when needed, and identification of new effective bactericides is a much longer process that takes years.

Pharmaceutical composition

In certain embodiments, disclosed herein are pharmaceutical compositions comprising (a) a nucleic acid sequence as disclosed herein; and (b) a pharmaceutically acceptable excipient. In certain embodiments, also disclosed herein are pharmaceutical compositions comprising (a) a bacteriophage as disclosed herein; and (b) a pharmaceutically acceptable excipient. In certain embodiments, further disclosed herein are pharmaceutical compositions comprising (a) a composition as disclosed herein; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient comprises a surfactant. In some embodiments, the pharmaceutically acceptable excipient is a buffer.

In some embodiments, the present 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 agents discussed above in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions or methods disclosed herein treat pseudomonas bacterial infections. In some embodiments, the bacterial infection is a pseudomonas aeruginosa blood stream infection. In some embodiments, the bacterial infection is a pseudomonas aeruginosa respiratory tract infection. In some embodiments, the pharmaceutical compositions of the methods disclosed herein treat cystic fibrosis related bronchiectasis. In some embodiments, the pharmaceutical compositions or methods disclosed herein treat non-cystic fibrosis related bronchiectasis. In some embodiments, the pharmaceutical compositions of the methods disclosed herein treat malignant otitis externa, endophthalmitis, endocarditis, meningitis, pneumonia, or sepsis.

In some embodiments, the compositions disclosed herein include pharmaceutical agents, carriers, adjuvants, dispersants, diluents, and the like.

In some embodiments, the bacteriophage disclosed herein is formulated for administration in a pharmaceutical carrier according to a suitable method. In some embodiments, according to the manufacture of the pharmaceutical compositions of the present disclosure, the bacteriophage is in particular admixed with an acceptable carrier. In some embodiments, the carrier is solid (including powder) or liquid, or both, and is preferably formulated as a unit dose composition. In some embodiments, one or more bacteriophage is incorporated into a composition disclosed herein, which is prepared by any suitable pharmaceutical method.

In some embodiments, a method of treating a disease in a subject comprises administering to the 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 bacteriophage is administered to a human subject or animal in need thereof by any means known in the art.

In some embodiments, methods and compositions suitable for applying the bacteriophage disclosed herein to a surface of an object or subject include aqueous solutions. In some embodiments, such aqueous solutions are sprayed onto the surface of an object or subject. In some embodiments, the aqueous solution is used to irrigate and clean a physical wound of a subject from foreign debris including bacteria.

In some embodiments, methods and compositions suitable for nasal administration or other administration to the lungs of a subject include any suitable means, such as administration by an aerosol suspension of respirable particles comprising a bacteriophage composition inhaled by the subject. In some embodiments, the respirable particles are liquid or solid. As used herein, "aerosol" includes any airborne suspension phase that is capable of being inhaled into the bronchioles or nasal passages. In some embodiments, the aerosol of liquid particles is produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer or a mesh nebulizer. In some embodiments, the solid particle aerosol comprising the composition is produced by any solid particle drug aerosol generator by techniques known in the pharmaceutical arts.

A nebulizer is a liquid aerosol generator that converts a bulk liquid, typically a water-based composition, into a mist or cloud of small droplets having a Mass Median Aerodynamic Diameter (MMAD) of less than 5 microns in diameter, which may be inhaled into the lower respiratory tract. The bulk liquid contains the therapeutic agent particles or therapeutic agent solution and any necessary excipients. When the aerosol cloud is inhaled, the droplets carry the therapeutic agent into the nose, upper airway, or deep lung.

Pneumatic (jet) atomizers use a pressurized gas supply as the driving force for the atomization of a liquid. The compressed gas is delivered through a nozzle or orifice to create a low pressure field that entrains surrounding bulk liquid and shears it into a film or filament. The film or filament is unstable and breaks up into small droplets that are carried by the compressed gas stream into the inhalation. Baffles inserted into the plume of droplets screen out larger droplets and return them to the bulk liquid reservoir. Examples include PARIOr->Atomizer, devilbsiss->Atomizer and Boehringer Ingelheim->An inhaler.

Electromechanical atomizers use electrically generated mechanical forces to atomize liquids. The electromechanical driving force is applied by vibrating the bulk liquid at ultrasonic frequencies or by forcing the bulk liquid through small holes in the film. The force produces a thin liquid film or stream of filaments that breaks up into small droplets to form a slow moving aerosol stream that may be entrained in the respiratory stream.

One form of electromechanical atomizer is an ultrasonic atomizer in which a bulk liquid is coupled to a vibrator that oscillates at a frequency in the ultrasonic range. The coupling being achieved by direct contact of the liquid with a vibrator, e.g. a plate or ring in a receiving cup, or by bringing a bulk liquid The drop is placed on a solid vibration projector (projector). The vibration produces a circular upstanding film which breaks up into droplets at its edges to atomize the liquid. Examples includeBeetle +.>Atomizer, octive Tech +.>Atomizer and John BunnAn atomizer. Another form of electromechanical atomizer is a mesh atomizer in which a bulk liquid is driven through a mesh or membrane having small pores with diameters of 2 to 8 microns to produce filaments that immediately break up into small droplets. In some designs, by using a solenoid plunger driver +.>Applying pressure, or forcing the liquid through the mesh by sandwiching the liquid between a piezoelectric vibrating plate and the mesh, which results in an oscillating pumping action (>AerovectRx、TouchSpray ^TM ). In a second design type, the mesh is vibrated back and forth through an upstanding column of liquid to pump it through the holes. Examples include->AeroNeb/>PARI/>Omron/>And Aradigm

In some embodiments, the bacteriophage disclosed herein is for oral administration. In some embodiments, the bacteriophage is 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 (sublingual) administration include lozenges comprising bacteriophage in a flavored base, typically sucrose and acacia or tragacanth; and lozenges comprising bacteriophage in an inert matrix such as gelatin and glycerol or sucrose and acacia.

In some embodiments, the methods and compositions of the present disclosure are suitable for parenteral administration, including sterile aqueous and non-aqueous injection solutions of bacteriophage. In some embodiments, these formulations are isotonic with the blood of the intended recipient. In some embodiments, these formulations comprise antioxidants, buffers, bacteriostats, and solutes that render the composition isotonic with the blood of the intended recipient. In some embodiments, the aqueous and non-aqueous sterile suspensions comprise a suspending agent and a thickening agent. In some embodiments, the compositions disclosed herein are present in unit-dose or multi-dose containers (e.g., sealed ampoules and vials) and are stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, e.g., saline or water for injection, immediately prior to use.

In some embodiments, methods and compositions suitable for rectal administration are presented as unit dose suppositories. In some embodiments, these are prepared by mixing the bacteriophage with one or more conventional solid carriers (e.g., 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 ointments, creams, lotions, pastes, gels, sprays, aerosols or oils. In some embodiments, the carrier used includes petrolatum, lanolin, polyethylene glycols, alcohols, transdermal enhancers, and combinations of two or more thereof.

In some embodiments, methods and compositions suitable for transdermal administration are presented in discrete patches that are adapted to remain in intimate contact with the epidermis of the recipient for a prolonged period of time.

In some embodiments, the bacteriophage disclosed herein is administered to a subject in a therapeutically effective amount. In some embodiments, at least one bacteriophage composition disclosed herein is formulated into a pharmaceutical formulation. In some embodiments, the 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 cases, the pharmaceutical formulation comprises a bacteriophage as described herein and at least one of an excipient, diluent, or carrier.

In some embodiments, the pharmaceutical formulation comprises an excipient. Excipients are described in Handbook of Pharmaceutical Excipients, american Pharmaceutical Association (1986) and include, but are not limited to, solvents, dispersion media, diluents or other liquid vehicles, dispersing or suspending aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders and lubricants.

Non-limiting examples of suitable excipients include, but are not limited to, buffers, preservatives, stabilizers, binders, compactors, lubricants, chelating agents, dispersion enhancing agents, disintegrants, flavoring agents, sweeteners, colorants.

In some embodiments, the excipient is a buffer. Non-limiting examples of suitable buffers include, but are not limited to, sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. In some embodiments, the pharmaceutical formulation comprises any one or more of the buffers listed: sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, 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.

In some embodiments, the excipient is a preservative. Non-limiting examples of suitable preservatives include, but are not limited to, antioxidants such as alpha-tocopherol and ascorbate; and antimicrobial agents such as parabens, chlorobutanol, and phenol. In some embodiments, antioxidants include, but are not limited to, ethylenediamine tetraacetic acid (EDTA), citric acid, ascorbic acid, butylated Hydroxytoluene (BHT), butylated Hydroxyanisole (BHA), sodium sulfite, para-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, and N-acetylcysteine. In some embodiments, the preservative comprises validamycin A, TL-3, sodium orthovanadate, sodium fluoride, N-a-tosyl-Phe-chloromethylketone, N-a-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, protease inhibitors, reducing agents, alkylating agents, antimicrobial agents, oxidase inhibitors, or other inhibitors.

In some embodiments, the pharmaceutical formulation comprises a binder as an excipient. Non-limiting examples of suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxazolidone, polyvinyl alcohol, C ₁₂ -C ₁₈ Fatty acid alcohols, polyethylene glycols, polyols, sugars, oligosaccharides, and combinations thereof.

In some embodiments, the binder for the pharmaceutical formulation is selected from starches such as potato starch, corn starch, wheat starch; sugars such as sucrose, glucose, dextrose, lactose, maltodextrin; natural and synthetic gums; gelatin; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose; polyvinylpyrrolidone (povidone); polyethylene glycol (PEG); a wax; calcium carbonate; a calcium phosphate; alcohols such as sorbitol, xylitol, mannitol, and water or combinations thereof.

In some embodiments, the pharmaceutical formulation comprises a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, refined hydrogenated vegetable oil (sterotex), polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil. In some embodiments, the lubricant in the pharmaceutical formulation is selected from metal stearates (e.g., magnesium stearate, calcium stearate, aluminum stearate), fatty acid esters (e.g., sodium stearyl fumarate), fatty acids (e.g., stearic acid), fatty alcohols, glyceryl behenate, mineral oil, paraffin, hydrogenated vegetable oil, leucine, polyethylene glycol (PEG), metal lauryl sulfate (e.g., sodium lauryl sulfate, magnesium lauryl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

In some embodiments, the excipient comprises a flavoring agent. In some embodiments, the flavoring agent comprises a natural oil; extracts from plants, leaves, flowers and fruits; and combinations thereof.

In some embodiments, the excipient comprises a sweetener. Non-limiting examples of suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts, such as sodium salts; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; stevia rebaudiana (stevioside); chloro derivatives of sucrose, such as sucralose; and sugar alcohols such as sorbitol, mannitol, xylitol (sylitol), and the like.

In some cases, the pharmaceutical formulation comprises a colorant. Non-limiting examples of suitable colorants include food, drug and cosmetic colorants (FD & C), drug and cosmetic colorants (D & C), and topical drug and cosmetic colorants (ext.d & C).

In some embodiments, the pharmaceutical formulations disclosed herein comprise a chelator. In some embodiments, the chelating agent comprises ethylenediamine-N, N' -tetraacetic acid (EDTA); disodium, trisodium, tetrasodium, dipotassium, tripotassium, dilithium and diammonium salts of EDTA; barium, calcium, cobalt, copper, dysprosium, europium, iron, indium, lanthanum, magnesium, manganese, nickel, samarium, strontium, or zinc chelates of EDTA.

In some cases, the pharmaceutical formulation comprises a diluent. Non-limiting examples of diluents include water, glycerol, methanol, ethanol, and other similar biocompatible diluents. In some embodiments, the diluent is an aqueous acid, such as acetic acid, citric acid, maleic acid, hydrochloric acid, phosphoric acid, nitric acid, sulfuric acid, or the like.

In some embodiments, the pharmaceutical formulation comprises a surfactant. In some embodiments, the surfactant is selected from, but is not limited to, polyoxyethylene sorbitan fatty acid esters (polysorbates), sodium lauryl sulfate, 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, fatty acid sugar esters, fatty acid glycerides, or combinations thereof.

In some cases, the pharmaceutical formulation comprises an additional pharmaceutical agent. In some embodiments, the additional pharmaceutical agent is an antibiotic agent. In some embodiments, the antibiotic agent is an aminoglycoside, ansamycin, carbacephem, carbapenem, cephalosporin (including first, second, third, fourth and fifth generation cephalosporins), lincosamine, macrolide, monomycins, nitrofurans, quinolones, penicillins, sulfonamides, polypeptides, or tetracyclines.

In some embodiments, the antibiotic agent described herein is an aminoglycoside, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, or paromomycin. In some embodiments, the antibiotic agent described herein is an ansamycin, such as geldanamycin or herbimycin.

In some embodiments, the antibiotic agent described herein is a carbacephem, such as chlorocarba-cephem. In some embodiments, the antibiotic agent described herein is a carbapenem, such as ertapenem, doripenem, imipenem/cilastat Ding Huomei ropine.

In some embodiments, the antibiotic agent described herein is a cephalosporin (first generation), such as cefadroxil, cefazolin, cefalexin, or Cefalotin, or alternatively is a cephalosporin (second generation), such as cefaclor, cefamandole, cefoxitin, cefprozil, or cefuroxime. In some embodiments, the antibiotic agent is a cephalosporin (third generation) such as cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftibuten, ceftizoxime, and ceftriaxone; or a cephalosporin (fourth generation), such as cefepime or cefpirane.

In some embodiments, the antibiotic agents described herein are lincomamines, such as clindamycin and azithromycin; or macrolides such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, vinegared marcomycin, telithromycin and spectinomycin.

In some embodiments, the antibiotic agent described herein is a monomycin, such as aztreonam; or nitrofurans, such as furazolidone or nitrofurantoin.

In some embodiments, the antibiotic agent described herein is a penicillin, such as amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, penicillin G or V, piperacillin, temoxicillin, and ticarcillin.

In some embodiments, the antibiotic agent described herein is a sulfonamide, such as sulfamuron, sulfonamide Ke Yiding (sulfonamide), sulfoacetamide, sulfadiazine, silver sulfadiazine, sulfamethoxazole, sulfadimethisoxazole (sulfafanilimide), sulfasalazine, sulfamethoxazole, trimethoprim, or trimethoprim-sulfamethoxazole (TMP-SMX).

In some embodiments, the antibiotic agent described herein is a quinolone such as ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, nalidixic acid, norfloxacin, ofloxacin, trovafloxacin, glafloxacin, sparfloxacin, and temafloxacin.

In some embodiments, the antibiotic agent described herein is a polypeptide, such as bacitracin, colistin, or polymyxin B.

In some embodiments, the antibiotic agent described herein is a tetracycline, such as demeclocycline, doxycycline, minocycline, or oxytetracycline.

The embodiments listed:

1. a bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system, the type I CRISPR-Cas system comprising:

(a) A CRISPR array comprising one or more spacer sequences complementary to a target nucleotide sequence in a pseudomonas species;

(b) A cascades polypeptide; and

(c) Cas3 polypeptide.

2. The bacteriophage of embodiment 1, wherein said one or more spacer sequences comprise at least one of SEQ ID NOs 12 to 23, 31 to 74 or 88 to 120 or comprise at least 90% sequence identity to any one of SEQ ID NOs 12 to 23, 31 to 74 or 88 to 120.

3. The bacteriophage of any one of embodiments 1-2, wherein said CRISPR array further comprises at least one repeat sequence.

4. The bacteriophage of embodiment 3, wherein said at least one repeat sequence is operably linked to said one or more spacer sequences at its 5 'end or its 3' end.

5. The bacteriophage of any one of embodiments 3 to 4, wherein said repeat sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 26 to 30.

6. The bacteriophage of any one of embodiments 1 to 5, wherein said CRISPR array comprises at least about 90% sequence identity to the sequence set forth in fig. 1A-1E or SEQ ID No. 83-87.

7. The bacteriophage of any one of embodiments 1 to 6, wherein said target nucleotide sequence comprises a coding sequence.

8. The bacteriophage of any one of embodiments 1 to 6, wherein said target nucleotide sequence comprises a non-coding sequence or an intergenic sequence.

9. The bacteriophage of any one of embodiments 1 to 6, wherein said target nucleotide sequence comprises all or part of a promoter sequence.

10. The bacteriophage of embodiment 9, wherein said promoter sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 1 to 11.

11. The bacteriophage of embodiment 1, wherein said target nucleotide sequence comprises all or a portion of a nucleotide sequence located on the coding strand of the transcribed region of the essential gene.

12. The bacteriophage of embodiment 11, wherein said essential gene is Tsf, acpP, gapA, 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.

13. The bacteriophage of any one of embodiments 1 to 12, wherein said cascades polypeptide forms a cascades 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.

14. The bacteriophage of embodiment 13, wherein said cascades complex comprises:

(i) Cas5d polypeptides, cas8C polypeptides, and Cas7 polypeptides (type I-C CRISPR-Cas system); (ii) Cas6B polypeptides, cas8B polypeptides, cas7 polypeptides, and Cas5 polypeptides (type I-B CRISPR-Cas system);

(iii) A Cas7 polypeptide, a Cas8a1 polypeptide or a Cas8a2 polypeptide, a Cas5 polypeptide, a Csa5 polypeptide, and a Cas6a polypeptide, wherein the Cas3 polypeptide comprises a Cas3' polypeptide and a Cas3 "polypeptide having no nuclease activity (type I-a CRISPR-Cas system);

(iv) Cas10D polypeptide, csc2 polypeptide, csc1 polypeptide, cas6D polypeptide (I-D CRISPR-Cas system);

(v) Cse1, cse2, cas7, cas5, and Cas6E polypeptides (I-E CRISPR-Cas system); or (vi) Csy1 polypeptide, csy2 polypeptide, csy3 polypeptide and Csy4 polypeptide (type I-F CRISPR-Cas system).

15. The bacteriophage of embodiment 13, wherein the cascades comprise a Cas5d polypeptide (optionally SEQ ID NO: 80), a Cas8C polypeptide (optionally SEQ ID NO: 81) and a Cas7 (optionally SEQ ID NO: 82) polypeptide (type I-C CRISPR-Cas system).

16. The bacteriophage of any one of embodiments 1 to 15, wherein said nucleic acid sequence further comprises a promoter sequence.

17. The bacteriophage of any one of embodiments 1 to 16, wherein said bacteriophage is an obligate lytic bacteriophage.

18. The bacteriophage of any one of embodiments 1 to 16, wherein said bacteriophage is a temperate bacteriophage that is conferred lytic.

19. The bacteriophage of embodiment 18, wherein said temperate bacteriophage is rendered lytic by removal, replacement or inactivation of a lysogenic gene.

20. The bacteriophage of any one of embodiments 17 to 19, wherein said pseudomonas species is killed only by the lytic activity of said bacteriophage.

21. The bacteriophage of any one of embodiments 1 to 19, wherein said pseudomonas species is killed by the activity of the CRISPR-Cas system only.

22. The bacteriophage of any one of embodiments 17 to 19, wherein said pseudomonas species is killed by a combination of lytic activity of said bacteriophage and activity of said CRISPR-Cas system.

23. The bacteriophage of embodiment 22, wherein the killing by activity of the CRISPR-Cas system of the pseudomonas species is independent of the lytic activity of the bacteriophage.

24. The bacteriophage of embodiment 22, wherein the activity of the CRISPR-Cas system supplements or enhances the lytic activity of the bacteriophage.

25. The bacteriophage of embodiment 22, wherein the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic.

26. The bacteriophage of any one of embodiments 17 to 25, wherein the lytic activity of the bacteriophage, the activity of the CRISPR-Cas system, or both is modulated by the concentration of the bacteriophage.

27. The bacteriophage of any one of embodiments 1 to 26, wherein said bacteriophage infects a plurality of bacterial strains of said pseudomonas species.

28. The bacteriophage of any one of embodiments 1 to 27, wherein the bacteriophage comprises Φkz virus, Φkmv virus, brunijo virus, sal Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cetex virus, vesicular virus, detrilavirus, aether virus, holo Wei Bingdu, eriosema virus, liimage virus, lu Saipu pedicle Ma Bingdu, niponer virus, parkona virus, pamex virus, boennom virus, filteirus, pri Mo Liji virus, septumamateur virus, starbucona virus, taerty virus, joinferior virus, ataegrina virus, or prana virus.

29. The bacteriophage of embodiment 28, wherein said bacteriophage comprises at least 80% sequence identity to a bacteriophage selected from p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB1, or two or more thereof.

30. The bacteriophage of embodiment 29, wherein the bacteriophage comprises p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132e002, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more thereof.

31. The bacteriophage of any one of embodiments 1 to 30, wherein said nucleic acid sequence is inserted into a non-essential bacteriophage gene.

32. A pharmaceutical composition comprising:

(a) The bacteriophage of any one of embodiments 1 to 31; and

(b) Pharmaceutically acceptable excipients.

33. The pharmaceutical composition of embodiment 32, wherein the pharmaceutical composition comprises at least two bacteriophage.

34. The pharmaceutical composition according to embodiment 33, wherein the bacteriophage is from the following lineages: Φkz virus, Φkmv virus, brunejo virus, sal Mu Na virus, south China virus, arblet virus, begol virus, bei Telei virus, casadaban virus, cet-ter-chi virus, vesicular virus, detrilavirus, hallo Wei Bingdu, eriosema virus, lirana virus, lu Saipu pedicle Ma Bingdu, nipena virus, parkenna virus, pamex virus, boende-mer virus, filtei virus, pri Mo Liji virus, sapendamate virus, starbuch virus, teltei virus, joja virus, ataconvalien virus, and prana virus.

35. The pharmaceutical composition of embodiment 33, wherein the pharmaceutical composition comprises at least six bacteriophage, wherein the bacteriophage comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430 and p1695.

36. The pharmaceutical composition of any of embodiments 32-35, wherein the pharmaceutical composition is in the form of a tablet, capsule, liquid, syrup, oral formulation, intravenous formulation, intranasal formulation, ophthalmic formulation, otic formulation, subcutaneous formulation, topical formulation, transdermal formulation, transmucosal formulation, inhalable respiratory formulation, suppository, lyophilized formulation, nebulizable formulation, and any combination thereof.

37. A method of killing a pseudomonas species comprising introducing into the target bacterium a nucleic acid sequence encoding a type I CRISPR-Cas system from a bacteriophage comprising:

(a) A CRISPR array comprising one or more spacer sequences complementary to a target nucleotide sequence in said pseudomonas species;

(b) A cascades polypeptide; and

(c) Cas3 polypeptide.

38. The method of embodiment 37, wherein the one or more spacer sequences comprise at least one of SEQ ID NOS: 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS: 12-23, 31-74, or 88-120.

39. The method of any one of embodiments 37-38, wherein the CRISPR array further comprises at least one repeat sequence.

40. The method of embodiment 39, wherein the at least one repeat sequence is operably linked to the one or more spacer sequences at its 5 'end or its 3' end.

41. The method of any of embodiments 39-40, wherein the repeat sequence comprises at least about 90% sequence identity to any of SEQ ID NOS: 26-30.

42. The method of any of embodiments 37-41, wherein the CRISPR array comprises at least about 90% sequence identity to the sequence set forth in figures 1A-1E or SEQ ID No. 83-87.

43. The method of any one of embodiments 37-42, wherein the target nucleotide sequence comprises a coding sequence.

44. The method of any one of embodiments 37-42, wherein the target nucleotide sequence comprises a non-coding sequence or an intergenic sequence.

45. The method of any one of embodiments 37-42, wherein the target nucleotide sequence comprises all or part of a promoter sequence.

46. The method of embodiment 45, wherein the promoter sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 1-11.

47. The method according to any one of embodiments 37-43, wherein the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding strand of the transcribed region of the essential gene.

48. The method according to embodiment 47, wherein the essential gene is Tsf, acpP, gapA, 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, tRNAasn or metK.

49. The method of any of embodiments 37-48, wherein the cascades polypeptide forms a cascades complex of an I-a type CRISPR-Cas system, an I-B type CRISPR-Cas system, an I-C type CRISPR-Cas system, an I-D type CRISPR-Cas system, an I-E type CRISPR-Cas system, or an I-F type CRISPR-Cas system.

50. The method of embodiment 49, wherein the cascades complex comprises:

(i) A Cas7 polypeptide, a Cas8a1 polypeptide or a Cas8a2 polypeptide, a Cas5 polypeptide, a Csa5 polypeptide, and a Cas6a polypeptide, wherein the Cas3 polypeptide comprises a Cas3' polypeptide and a Cas3 "polypeptide having no nuclease activity (type I-a CRISPR-Cas system);

(ii) Cas6B polypeptides, cas8B polypeptides, cas7 polypeptides, and Cas5 polypeptides (type I-B CRISPR-Cas system);

(iii) Cas5d polypeptides, cas8C polypeptides, and Cas7 polypeptides (type I-C CRISPR-Cas system); (iv) Cas10D polypeptide, csc2 polypeptide, csc1 polypeptide, cas6D polypeptide (I-D CRISPR-Cas system);

(v) Cse1, cse2, cas7, cas5, and Cas6E polypeptides (I-E CRISPR-Cas system); or (b)

(vi) Csy1 polypeptides, csy2 polypeptides, csy3 polypeptides, and Csy4 polypeptides (type I-F CRISPR-Cas system).

51. The method of embodiment 49, wherein the cascades comprise a Cas5d polypeptide (optionally SEQ ID No. 80), a Cas8C polypeptide (optionally SEQ ID No. 81) and a Cas7 polypeptide (optionally SEQ ID No. 82) (type I-C CRISPR-Cas system).

52. The method of any one of embodiments 37-51, wherein the nucleic acid sequence further comprises a promoter sequence.

53. The method according to any one of embodiments 37-52, wherein the bacteriophage is an obligate lytic bacteriophage.

54. The method of any one of embodiments 37-52, wherein the bacteriophage is a temperate bacteriophage that is conferred lytic.

55. The method of embodiment 54, wherein the temperate bacteriophage is rendered lytic by removal, replacement or inactivation of a lysogenic gene.

56. The method of any one of embodiments 37-55, wherein the pseudomonas species is killed by only the activity of the CRISPR-Cas system.

57. The method of any of embodiments 53-55, wherein the pseudomonas species is killed by the lytic activity of the bacteriophage in combination with the activity of the CRISPR-Cas system.

58. The method of embodiment 57, wherein the killing of the pseudomonas species by the activity of the CRISPR-Cas system is independent of the lytic activity of the bacteriophage.

59. The method of embodiment 57, wherein the activity of the CRISPR-Cas system complements or enhances the lytic activity of the bacteriophage.

60. The method of embodiment 57, wherein the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic.

61. The method of any one of embodiments 53-60, wherein the lytic activity of the bacteriophage, the activity of the CRISPR-Cas system, or both, is modulated by the concentration of the bacteriophage.

62. The method of any one of embodiments 37-61, wherein the bacteriophage infects a plurality of bacterial strains of the pseudomonas species.

63. The method of any one of embodiments 37-62, wherein the bacteriophage comprises at least 80% identity to p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB1, or two or more phages thereof.

64. The method of embodiment 63, wherein the bacteriophage comprises at least 80% identity to p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132e002, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more thereof.

65. The method according to any one of embodiments 37-64, wherein the nucleic acid sequence is inserted at or near the location of a non-essential bacteriophage gene.

66. The method of any one of embodiments 37-65, wherein the mixed population of bacterial cells comprises the pseudomonas species.

67. The method of any one of embodiments 37-66, further comprising administering at least one additional bacteriophage.

68. The method of embodiment 67, comprising administering at least six bacteriophage, wherein the bacteriophage comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430 and p1695.

69. A method of treating a disease or condition in an individual in need thereof, the method comprising administering to the individual a bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising:

(a) A CRISPR array;

(b) A cascades polypeptide comprising one or more spacer sequences complementary to a target nucleotide sequence in a pseudomonas species; and

(c) Cas3 polypeptide.

70. The method of embodiment 69, wherein the one or more spacer sequences comprise at least one of SEQ ID NOS: 12-23, 31-74, or 88-120, or comprise at least 90% sequence identity with any one of SEQ ID NOS: 12-23, 31-74, or 88-120.

71. The method of any one of embodiments 69-70 wherein the CRISPR array further comprises at least one repeat.

72. The method of embodiment 71, wherein the at least one repeat sequence is operably linked to the one or more spacer sequences at its 5 'end or its 3' end.

73. The method of any of embodiments 71-72, wherein the repeat sequence comprises at least about 90% sequence identity to any of SEQ ID NOs 26-30.

74. The method of any of embodiments 69-73 wherein the CRISPR array comprises at least about 90% sequence identity to the sequence shown in figures 1A-1E or SEQ ID No. 83-87.

75. The method of any one of embodiments 69-74 wherein the target nucleotide sequence comprises a coding sequence.

76. The method of any one of embodiments 69-74 wherein the target nucleotide sequence comprises a non-coding sequence or an intergenic sequence.

77. The method of any one of embodiments 69-74 wherein the target nucleic acid sequence comprises all or part of a promoter sequence.

78. The method of embodiment 77, wherein said promoter sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 1-11.

79. The method according to any of embodiments 69-74, wherein the target nucleotide sequence comprises all or part of a nucleotide sequence located on the coding strand of the transcribed region of the essential gene.

80. The method according to embodiment 79, wherein the essential gene is Tsf, acpP, gapA, 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, tRNAasn or metK.

81. The method of any of embodiments 69-80 wherein the cascades polypeptide forms a cascades complex of an I-a type CRISPR-Cas system, an I-B type CRISPR-Cas system, an I-C type CRISPR-Cas system, an I-D type CRISPR-Cas system, an I-E type CRISPR-Cas system, or an I-F type CRISPR-Cas system.

82. The method of embodiment 81, wherein the cascades complex comprises:

(i) Cas5d polypeptides, cas8C polypeptides, and Cas7 polypeptides (type I-C CRISPR-Cas system);

83. The method of embodiment 82, wherein the cascades comprise a Cas5d polypeptide (optionally SEQ ID No. 80), a Cas8C polypeptide (optionally SEQ ID No. 81) and a Cas7 polypeptide (optionally SEQ ID No. 82) (type I-C CRISPR-Cas system).

84. The method of any one of embodiments 69-83 wherein the nucleic acid sequence further comprises a promoter sequence.

85. The method according to any of embodiments 69-84, wherein the bacteriophage is an obligate lytic bacteriophage.

86. The method according to any of embodiments 69-84, wherein the bacteriophage is a temperate bacteriophage that is conferred lytic.

87. The method of embodiment 86, wherein the temperate bacteriophage is rendered lytic by removal, replacement or inactivation of a lysogenic gene.

88. The method of any of embodiments 69-87 wherein the pseudomonas species is killed by only the activity of the CRISPR-Cas system.

89. The method of any one of embodiments 85-87, wherein the pseudomonas species is killed by the lytic activity of the bacteriophage in combination with the activity of the CRISPR-Cas system.

90. The method of embodiment 89, wherein the killing of the pseudomonas species by the activity of the CRISPR-Cas system is independent of the lytic activity of the bacteriophage.

91. The method of embodiment 89, wherein the activity of the CRISPR-Cas system complements or enhances the lytic activity of the bacteriophage.

92. The method of embodiment 89, wherein the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic.

93. The method of any one of embodiments 85-92, wherein the lytic activity of the bacteriophage, the activity of the CRISPR-Cas system, or both, is modulated by the concentration of the bacteriophage

94. The method of any one of embodiments 69-93 wherein the bacteriophage infects a plurality of bacterial strains.

95. The method of any one of embodiments 69-87, wherein the bacteriophage comprises at least 80% identity to p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430, or PB1, or two or more phages thereof.

96. The method of embodiment 95, wherein the bacteriophage comprises at least 80% identity to p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132e002, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more thereof.

97. The method according to any one of embodiments 69-96, wherein the nucleic acid sequence is inserted at or near the location of a non-essential bacteriophage gene.

98. The method of any one of embodiments 69-97 further comprising administering at least one additional bacteriophage.

99. The method of embodiment 98, comprising administering at least six bacteriophage, wherein the bacteriophage comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430 and p1695.

100. The method according to any one of embodiments 69-99, wherein the disease or condition is a bacterial infection, cystic fibrosis, non-cystic fibrosis bronchiectasis, or pneumonia.

101. The method of embodiment 100, wherein the bacterial infection is associated with cystic fibrosis or non-cystic fibrosis bronchiectasis, or wherein the bacterial infection is a blood flow infection.

102. The method according to any one of embodiments 69-101, wherein the pseudomonas species responsible for the disease or condition is a drug resistant pseudomonas species.

103. The method of embodiment 102, wherein the drug-resistant pseudomonas species is resistant to at least one antibiotic.

104. The method according to any one of embodiments 69-103, wherein the pseudomonas species responsible for the disease or condition is a multidrug resistant pseudomonas species.

105. The method of embodiment 104, wherein the multi-drug resistant pseudomonas species is resistant to at least one antibiotic.

106. The method of any one of embodiments 103 or 105, wherein the antibiotic comprises a cephalosporin, fluoroquinolone, carbapenem, colistin, aminoglycoside, vancomycin, streptomycin, or methicillin.

107. The method according to any one of embodiments 69-106, wherein the pseudomonas species is pseudomonas aeruginosa.

108. The method of any of embodiments 69-107 wherein the administration is intra-arterial, intravenous, intra-urethral, intramuscular, oral, subcutaneous, inhalation, topical, skin, transdermal, transmucosal, implant, sublingual, buccal, rectal, vaginal, ocular, aural, or nasal administration, or any combination thereof.

109. The method of any one of embodiments 69-108 further comprising administering an additional therapeutic agent.

110. The method of embodiment 109, wherein the additional therapeutic agent comprises tobramycin.

111. The method of any one of embodiments 69-110 wherein the individual is a mammal.

112. A bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system, the type I CRISPR-Cas system comprising:

(b) A cascades polypeptide comprising Cas5, cas8c, and Cas 7; and

(c) Cas3 polypeptide.

113. The bacteriophage of embodiment 112, wherein said one or more spacer sequences comprise at least one of SEQ ID NOs 12 to 23, 31 to 74 or 88 to 120 or comprise at least 90% sequence identity to any one of SEQ ID NOs 12 to 23, 31 to 74 or 88 to 120.

114. The bacteriophage of any one of embodiments 112 to 113, wherein said CRISPR array further comprises at least one repeat sequence.

115. The bacteriophage of embodiment 114, wherein said at least one repeat sequence is operably linked to said one or more spacer sequences at its 5 'end or its 3' end.

116. The bacteriophage of any one of embodiments 112 to 115, wherein said repeat sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 26 to 30.

117. The bacteriophage of any one of embodiments 112 to 116, wherein said CRISPR array comprises at least about 90% sequence identity to the sequence set forth in figures 1A-1E or SEQ ID No. 83-87.

118. The bacteriophage of any one of embodiments 112 to 117, wherein said target nucleotide sequence comprises a coding sequence.

119. The bacteriophage of any one of embodiments 112 to 117, wherein said target nucleotide sequence comprises a non-coding sequence or an intergenic sequence.

120. The bacteriophage of any one of embodiments 112 to 117, wherein said target nucleotide sequence comprises all or part of a promoter sequence.

121. The bacteriophage of embodiment 120, wherein said promoter sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 1 to 11.

122. The bacteriophage of any one of embodiments 112 to 121, wherein said target nucleotide sequence comprises all or a portion of a nucleotide sequence located on the coding strand of the transcribed region of the essential gene.

123. The bacteriophage of embodiment 122, wherein said essential gene is Tsf, acpP, gapA, 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.

124. The bacteriophage of any one of embodiments 112 to 123, wherein said nucleic acid sequence further comprises a promoter sequence.

125. The bacteriophage of any one of embodiments 112 to 124, wherein said bacteriophage is an obligate lytic bacteriophage.

126. The bacteriophage of any one of embodiments 112 to 124, wherein said bacteriophage is a temperate bacteriophage that is conferred lytic.

127. The bacteriophage of embodiment 126, wherein said temperate bacteriophage is rendered lytic by removal, replacement or inactivation of a lysogenic gene.

128. The bacteriophage of any one of embodiments 125 to 127, wherein said pseudomonas species is killed only by the lytic activity of said bacteriophage.

129. The bacteriophage of any one of embodiments 125-127, wherein said pseudomonas species is killed by only the activity of said CRISPR-Cas system.

130. The bacteriophage of any one of embodiments 125 to 127, wherein said pseudomonas species is killed by a combination of lytic activity of said bacteriophage and activity of said CRISPR-Cas system.

131. The bacteriophage of embodiment 130, wherein the active killing by said CRISPR-Cas system of said pseudomonas species is independent of the lytic activity of said bacteriophage.

132. The bacteriophage of embodiment 130, wherein the activity of the CRISPR-Cas system supplements or enhances the lytic activity of the bacteriophage.

133. The bacteriophage of embodiment 130, wherein the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic.

134. The bacteriophage of any one of embodiments 125-133, wherein the lytic activity of said bacteriophage, the activity of said CRISPR-Cas system, or both is modulated by the concentration of said bacteriophage

135. The bacteriophage of any one of embodiments 112 to 134, wherein said bacteriophage infects a plurality of bacterial strains.

136. The bacteriophage of any one of embodiments 112 to 135, wherein said bacteriophage comprises at least 80% identity to p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB1, or two or more thereof.

137. The bacteriophage of embodiment 136, wherein said bacteriophage comprises at least 80% identity to p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132e002, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more thereof. 138. The bacteriophage of any one of embodiments 112 to 137, wherein said nucleic acid sequence is inserted into a non-essential bacteriophage gene.

139. A pharmaceutical composition comprising:

(a) The bacteriophage of any one of embodiments 112 to 138; and

(b) Pharmaceutically acceptable excipients.

140. The pharmaceutical composition of embodiment 139, wherein the pharmaceutical composition comprises at least two bacteriophage.

141. The pharmaceutical composition of embodiment 140, wherein the pharmaceutical composition comprises at least six bacteriophage, wherein the bacteriophage comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430 and p1695.

142. The pharmaceutical composition of any of embodiments 139-141, wherein the pharmaceutical composition is in the form of a tablet, capsule, liquid, syrup, oral formulation, intravenous formulation, intranasal formulation, ophthalmic formulation, otic formulation, subcutaneous formulation, topical formulation, transdermal formulation, transmucosal formulation, inhalable respiratory formulation, suppository, lyophilized formulation, nebulizable formulation, and any combination thereof.

143. A method of sterilizing a surface in need thereof, the method comprising applying to the surface a bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising:

(a) A CRISPR array;

(c) Cas3 polypeptide.

144. The method of embodiment 143, wherein the surface is a hospital surface, a vehicle surface, a device surface, or an industrial surface.

145. A method of preventing contamination in a food product or nutritional supplement, the method comprising administering to the food product or nutritional supplement a bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system comprising:

(a) A CRISPR array;

(c) Cas3 polypeptide.

146. The method of embodiment 145, wherein the food product or nutritional supplement comprises milk, yogurt, curd, cheese, fermented milks, milk based fermented products, ice cream, fermented cereal based products, milk based powders, infant formulas or tablets, liquid suspensions, dry oral supplements, wet oral supplements, or dry tube feeds.

147. A bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system, the type I CRISPR-Cas system comprising:

(a) A CRISPR array comprising a spacer sequence complementary to a target nucleotide sequence in a pseudomonas species, wherein said spacer sequence comprises SEQ ID NOs 12, 16 and 20;

(b) A cascades polypeptide; and

(c) Cas3 polypeptide.

148. A bacteriophage comprising at least 80% sequence identity to a bacteriophage selected from p1106, p1194, p1587, p1695, p1772, p1835, p2037, p2131, p2132, p2167, p2363, p2421, p2973, p3278, p4430 or PB1, or two or more thereof.

149. The bacteriophage of embodiment 148, wherein said bacteriophage comprises at least 80% identity to p1106e003, p1106wt, p1194wt, p1587e002, p1587wt, p1695wt, p1772e005, p1772wt, p1835e002, p1835wt, p2037e002, p2037wt, p2131e002, p2131wt, p2132e002, p2132wt, p2167wt, p2363e003, p2363wt, p2421e002, p2421wt, p2973e002, p2973wt, p3278wt, p4430wt, PB1e002 or PB1wt, or two or more thereof. 150. The bacteriophage of embodiment 148, further comprising

(a) A CRISPR array;

(c) Cas3 polypeptide.

151. The bacteriophage of embodiment 150, wherein said one or more spacer sequences comprise at least one of SEQ ID NOs 12 to 23, 31 to 74 or 88 to 120 or comprise at least 90% sequence identity to any one of SEQ ID NOs 12 to 23, 31 to 74 or 88 to 120.

152. The bacteriophage of any one of embodiments 150 to 151, wherein said CRISPR array further comprises at least one repeat sequence.

153. The bacteriophage of embodiment 151, wherein said at least one repeat sequence is operably linked to said one or more spacer sequences at its 5 'end or its 3' end.

154. The bacteriophage of any one of embodiments 151 to 153, wherein said repeat sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 26 to 30.

155. The bacteriophage of any one of embodiments 151 to 154, wherein said CRISPR array comprises at least about 90% sequence identity to the sequence set forth in fig. 1A-1E or SEQ ID No. 83-87.

156. The bacteriophage of any one of embodiments 151 to 155, wherein said target nucleotide sequence comprises a coding sequence.

157. The bacteriophage of any one of embodiments 151 to 156, wherein said target nucleotide sequence comprises a non-coding sequence or an intergenic sequence.

158. The bacteriophage of any one of embodiments 151 to 157, wherein said target nucleotide sequence comprises all or a portion of a promoter sequence.

159. The bacteriophage of embodiment 158, wherein said promoter sequence comprises at least about 90% sequence identity to any one of SEQ ID NOs 1 to 11.

160. A composition comprising at least four bacteriophage comprising:

(a) A first bacteriophage comprising at least 80% sequence identity to p1106e 003;

(b) A second bacteriophage comprising at least 80% sequence identity to p1835e 002;

(c) A third bacteriophage comprising at least 80% sequence identity to p1772e 005; and

(d) A fourth bacteriophage comprising at least 80% sequence identity to p2131e 002.

161. The composition of embodiment 160, further comprising a fifth bacteriophage comprising at least 80% sequence identity to p 1194.

162. The composition of embodiment 160, further comprising a fifth bacteriophage comprising at least 80% sequence identity to p 1695.

163. The composition of embodiment 160, further comprising a fifth bacteriophage comprising at least 80% sequence identity to p 4430.

164. The composition of embodiment 161 or 163, further comprising a sixth bacteriophage comprising at least 80% sequence identity to p 1695.

Examples

Example 1: engineered phage for use in the present application

The bacteriophage is engineered to contain the crArray and Cas constructs. Table 1A depicts the components of phage used in the following applications. Table 1B depicts the sequences of promoters used to drive expression of the crArray and Cas promoters. Table 1C depicts the sequences of spacer sequences in crArray for targeting specific sites. In addition, FIGS. 1A-1E depict the sequences and alignment of crArray1-crArray5 used in the examples below. The full sequence of the combined crArray1 and pseudomonas type I C CRISPR insertions is shown in fig. 1F-1K and table 1D. The full sequence of the combined crArray3 and pseudomonas type I C CRISPR insertions is shown in fig. 1L-1Q and table 1D.

Table 1A: assemblies of phages

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Table 1B: promoter sequence

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Table 1C: spacer sequences for targeting certain species of pseudomonas

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Table 1D: paIC insertion sequence

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Example 2: the exogenous Cas operon and crRNA spacer kill bacteria.

Pseudomonas aeruginosa strains with functional type I-C Cas operons were transformed with either Cas alone or crRNA containing plasmids. In the presence of an endogenous I-C type Cas system, expression of crRNA results in the bacteria self-targeting and degrading their own DNA. The number of transformants was determined by counting the number of colonies grown on agar plates with plasmid-specific antibiotic selection. The only bacteria that can form colonies are those that both acquire plasmids and survive the targeting. These data indicate that exogenous Cas expression improves self-targeting relative to endogenous systems and functions in the absence of endogenous systems, as seen in fig. 2A. The upper two figures show the results of transforming Cas-only plasmids, plasmids containing a single targeting spacer, or plasmids containing a 3-spacer targeting array into strains containing an endogenous I-C type Cas system. In this experimental set-up, the spacer or array alone works with the endogenous Cas system and is sufficient to kill most transformants. The lower panel shows that adding an exogenous type I-C Cas operon to the crRNA plasmid further enhances killing upon transformation, where the bacterial level is present below the detection level.

The plasmid was transformed into a pseudomonas aeruginosa strain that did not contain a functional type I-C Cas operon. The transformed plasmid expresses only the spacer array, or expresses the spacer array and the type I-C Cas operon. Figure 2B shows the number of bacterial transformants obtained per mL of Cas operon null mutant transformed into pseudomonas aeruginosa strain B1121. Array 1 targets bacteria, while array 2 is a non-targeted control. The different plasmids were normalized to the empty vector control plasmid with respect to molar concentration. When cells are transformed with Cas and targeting array 1, the number of transformants detected is reduced. Cells transfected with either targeting array 1 alone or Cas and non-targeting array 2 did not show a reduction in the number of transformants compared to the number of transformants received with empty vector.

Plasmids containing individual spacers or unique arrays were transformed into pseudomonas aeruginosa strain b1121 with endogenous type I-C Cas system or Cas operon null mutants of the same strain. Cell death was observed in b1121 transfected cells, but not in Cas operon null mutants. As depicted in fig. 2C, these data indicate that the individual spacers targeting rpoB and ftsA, as well as arrays 3 and 4, are able to function with endogenous type I-C Cas systems to kill cells.

Example 3: stability of phage engineered with CRISPR-Cas full constructs

FIG. 3A depicts a schematic representation of the genome of wild-type phage p1772 and its engineered variants. Bars below the genome axis indicate the region of the genome that was removed and replaced. The schematic below the phage genome shows the DNA used to replace the WT phage gene in the deleted region. CRISPR arrays crArray 1, crArray 3, and crArray 4 target bacteria and are expected to kill bacteria in the presence of active type I-C Cas systems. crArray2 is made from a non-targeting spacer, but is structurally identical to the three targeting arrays, and serves as a control to demonstrate type I Cas-specific self-targeting activity.

Phage carrying CRISPR-Cas3 constructs were serially passaged to assess the stability of the repeats contained in the phage genome. P1772e005 was continuously amplified on Pseudomonas aeruginosa strain b1126 (type I-F strain). Amplification was performed as a one-step amplification in which 50uL of bacterial overnight culture was added to 5mL of LB in a 15mL falcon tube followed immediately by 1 uL of prior lysate. The mixture was grown in a shaking incubator at 37℃for 10-16 hours. After incubation, phage-bacteria mixtures were centrifuged at 5,000rcf for 10min, and the supernatant was filtered through a 0.45 μm syringe filter and stored at 4 ℃. For amplification nine, eight serial ten-fold dilutions were spotted onto soft agar overlays of strains b1121 or b 1126. Individual plaques were picked from each plate with a pipette and transferred to 200 μl PBS to obtain amplified nine. Ten microliters of amplified nine was added to 50 μ L b1121 or b1126 overnight and 5mL LB, then grown for 16 hours, followed by centrifugation and filtration. For sanger sequencing, phage DNA was amplified from lysates by PCR using primers flanking the engineered site. Mulberry and NGS sequencing confirmed the stability and integrity of the CRISPR-Cas3 construct when loaded onto phage genomes (shown in FIG. 3B).

Example 4 bacteriophage morphology with CRISPR construct

1.5mL of the crude lysate was centrifuged at 24,000Xg at 4℃for 1hr. A portion of the supernatant (about 1.4 mL) was gently discarded, and 1mL of ammonium acetate (0.1M, pH 7.5) was added to the remaining lysate, followed by centrifugation. This step was performed twice. The washed phage samples were visualized by negative staining transmission electron microscopy. A glow discharge polyvinyl formal/carbon coated 400 mesh copper grid (Ted Pella, inc., redding, CA) was floated on 25 μl sample suspension droplets for five minutes, quickly transferred to two drops of deionized water, then transferred to one drop of 2% aqueous uranyl acetate, and stained for 30 seconds. The grid was blotted dry with filter paper and air dried. The samples were observed using a JEOL JEM-1230 transmission electron microscope operating at 80kV and images were taken using a Gatan Orius SC1000 CCD camera with Gatan Microscopy Suite 3.0.0 software. The results are illustrated in fig. 4. After modification, there was no significant observable change in phage morphology.

Example 5 amplification of phage engineered with CRISPR-Cas full constructs

P1772wt (wild type) and engineered variants p1772e004 (Cas system only) and p1772e005 (targeting crarray1+cas system) were mixed with exponentially growing b1126 cultures with multiplicity of infection (MOI) of 1. Samples were collected for Plaque Forming Unit (PFU) counting (fig. 5A-5B) and RNA isolation and quantification at 0min, 15min, 30min, 1h, 2h, 4h, 7h, 10min and 24h post infection. For PFU counts, samples collected at each time point were filtered through a 0.45 μm filter to isolate phage from host bacteria. A soft agar overlay was prepared as described for slide 3. A 10-fold serial dilution of phage samples was spotted onto the cover layer and incubated at 37 ℃. The following day, plaques were counted and used to calculate PFU/mL in the initial sample. Based on these data, no significant differences in phage growth pattern were observed and each phage reached a similar maximum titer.

P1772wt, p1772e004 and p1772e005 were diluted to a particle count of 1e6 and each individual phage was used to infect a set of 34 different bacteria at a MOI of 0.01. Optical Density (OD) readings at 600nm wavelength were taken per hour over a period of 20 hours. The resulting OD readings are used to generate a bacterial growth curve in the presence of one of the three phages. The area under the curve (AUC) for each growth curve was calculated using integration, with a smaller AUC when phage was added indicating a reduction in bacterial load. The host range was determined by monitoring the OD600 (turbidity) of the culture over time to obtain a bacterial growth curve, wherein the bottom of the graph indicates the initial amount of phage introduced (input phage titer in plaque forming units/ml). AUC of a given strain in the presence and absence of phage was compared. Fig. 5C illustrates the AUC ratio, where the AUC calculation for strain growth in the presence of phage is divided by the AUC for strain growth in the absence of phage. Each row represents a unique bacterial strain. Darker values in the heat map indicate a more reduced bacterial load. If (AUC in the presence of phage)/(AUC in the absence of phage) is less than 0.65, phage is considered to infect a given strain. The heat map of AUC ratios shows that in this assay, the engineered phage variants have a host range comparable to the wild-type parent. The host ranges of p1772wt, p1772e004 and p1772e005 are similar to each other and within the measurement error. Host range confirmation of AUC hits by plaque suggests no difference between WT and CRISPR-Cas 3.

Table 2 shows data from representative growth experiments of two unique engineered phages containing the full construct, parry 3 (targeting crarray3+cas system) and parry 4 (targeting crarray4+cas system). In this assay, amplification was performed by inoculating LB growth medium with single colony bacteria and adding phage as indicated in the "input PFU/mL" column. The amplifications were incubated overnight. After incubation, bacteria were removed by filtration and phage titer in lysates was quantified by soft agar overlay method. Titers of lysates are indicated in the "output PFU/mL" column. These data indicate that the engineered phage replicates efficiently. These data also demonstrate the relative accuracy of titration measurements.

Table 2: growth of pArray3 and pArray4

Example 6 expression of CRISPR-Cas System in engineered phages

This example shows that Cas system and crArray are successfully expressed from phage genome. Fig. 6A depicts the arrangement of spacer arrays (crArray) and Cas operons engineered into p1772 and other phages described herein. The arrow represents the binding position of the primer pair for quantitative reverse transcription PCR (qRT-PCR) analysis of gene expression.

P1772wt (wild type) without Cas operon was used as control. For RNA isolation, samples collected at each time point were added directly to rnadetect. The samples were incubated for 5 minutes at room temperature, centrifuged at 5000 Xg for 10 minutes, and the supernatant was discarded. The precipitate was stored at-80 ℃. RNA was then isolated using the Qiagen Rneasy mini kit. cDNA was synthesized using the BioRad iScript cDNA synthesis kit. qRT-PCR was performed using BioRad SsoAdvanced Universal SYBR Green Supermix. All data are averages of two biological replicates. The pseudomonas aeruginosa gene rpsH was used as housekeeping gene and each data point was compared to the cell-only control at the same time point, fold change of 2 ^-ΔΔCt 。

FIGS. 6B-6D show the relative expression levels of indicated RNA after infection of the Pseudomonas aeruginosa strain with different variants of bacteriophage p 1772. The data in these figures are expressed as fold change in expression compared to vehicle control in the absence of phage. Each time point was normalized to the uninfected control of that time point. The change in bacterial concentration was explained by normalizing the samples using the bacterial housekeeping gene rpsH. These data indicate that phage produce crArray, cas3, and Cas8c transcripts upon infection with pseudomonas aeruginosa. Bacterial hosts used in panels B-D contain an endogenous Cas operon, so the difference between p1772e005 (targeting the crarray1+cas system) and p1772wt represents an increase in phage-mediated expression relative to endogenous expression.

FIG. 6E shows the relative expression of cas3 mRNA from different engineered phage genomes. These data indicate that at 1h post-infection Cas3 expression from p1772e005 is more than Cas3 expression from the other two engineered phages p2131e002 (targeting crarray1+cas system) and p2132e002 (targeting crarray1+cas system). However, 24h after infection, phage expression was close to the same amount of cas 3. These data were calculated by comparing cas3 RNA expression to the amount of phage gDNA and normalizing to p1772e005 at 1h post infection. The bacterial strains used in these assays are Cas-free and therefore do not contribute from endogenous Cas3 expression.

Example 7 phage lytic Activity engineered with CRISPR-Cas full constructs

By combining 100. Mu.L of saturated overnight culture of p1772 indicator strain b1121 with 6mL of 0.375% agar containing 10mM MgCl ₂ And 10mM CaCl ₂ Is mixed with LB to prepare a top agar overlay. After the top agar solidified, 2 μl drops of serial 10-fold dilutions of p1772wt (wild-type) and p1772e004 (Cas system only) and p1772e005 (crarray1+cas system targeted) were spotted onto the surface of the top agar. Plates were incubated at 37℃for 18h and then imaged using a Keyence BZ-X800 microscope at 4-fold and 10-fold magnification. FIG. 7 shows the improved plaque morphology of the p1772 phage. The morphology of wild-type phage was observed to produce a blurred plaque, while the engineered variant p1772e005 produced a similarly sized plaque with a blurred halo but clear at the center. This data shows that p1772e005 kills bacteria more completely than p1772 wt.

Example 8: phages containing crArray and Cas operons kill bacteria more effectively than phages containing only crArray

P1772 wild-type and engineered phages were mixed with log-growing bacteria and immediately plated at 2ul spot on LB agar. The ratio of phage to bacteria was varied by serial dilution so that the amount of bacteria remained constant at each dilution, but the amount of phage was 1 to 4 dilutions. At the highest dilution, the multiplicity of infection (MOI) was 100, meaning that there were approximately 100 phages per bacteria. In fig. 8A, p1772e005 (targeting crarray1+cas system) and p1772e006 (targeting crarray1 only) consistently killed most of the bacteria present in the type I-C strain after overnight incubation, as indicated by the lack or near lack of growing bacterial colonies in those spots, while wild type phage did not control bacterial colony formation. Thus, wild type and p1772e004 (Cas system only) are unable to control bacterial replication even though the MOI is 100. Fig. 8B shows that, due to the endogenous Cas system in the bacteria, p1772e006 kills bacteria more effectively than the wild type in this bacterial strain, however it appears to be less effective than phage (p 1772e 005) that also contains an exogenous Cas system. This is because with prolonged incubation, more bacteria form colonies in the spots exposed to p1772e006 than to p1772e 005.

Fig. 8C is a quantification of individual MOI from the same type of assay performed in fig. 8A-8B. In contrast to fig. 8A-8B, the bacterial strain in fig. C has no endogenous Cas system, but has a genomically integrated copy of the mCherry gene. Plates were imaged and fluorescence from each spot was quantified. Results with MOI of 1.5 are shown, but MOI above 0.4 all have results consistent with MOI of 1.5. Due to the lack of an endogenous Cas system, the crArray phage (p 1772e 006) alone behaves similarly to the wild-type phage. The fully engineered phage containing non-targeted crArray (p 1772e 008) was also not improved over the wild type. However, the fully engineered phage containing the targeting crArray (p 1772e 005) inhibited cell growth to a significantly greater extent than any other phage variant. This data suggests that the fully engineered variants do not require an endogenous Cas system to function.

Pseudomonas aeruginosa strain with active endogenous I-C type Cas system (b 1121) was grown to mid-log and infected with phage in liquid culture at indicated multiplicity of infection (MOI). In all cases, the phage successfully killed the bacteria as depicted in fig. 9A-9C, as indicated by a decrease in Colony Forming Units (CFU)/mL recovered compared to the uninfected control. p1772e005 (targeting crarray1+cas system) and p1772e006 (targeting crArray1 only) were more effective at killing bacteria than wild-type phage. There was no activity improvement of p1772e004 (Cas system only) relative to p1772wt (wild-type) or self-targeted variants, indicating that self-targeting crRNA and type I CRISPR-Cas modules are both necessary to improve phage efficacy. Notably, p1772e006 and p1772e005 have the same level of killing, suggesting that the engineered phage variant is able to act as a kill by targeting the Cas system from phage-expressing bacteria in the presence of a compatible and active Cas system. The dashed lines in these figures represent the limit of detection (LOD) of the assay. Samples from which colonies were not obtained are shown at LOD.

Example 9: multiple different pseudomonas aeruginosa targeting spacers improve phage efficacy

P1772 wild-type and engineered phage variants were mixed with logarithmically growing mCherry expressing bacteria and immediately plated onto LB agar. The ratio of phage to bacteria was varied by serial dilution of phage so that the amount of bacteria in each spot remained constant, but the amount of phage varied. The highest multiplicity of infection (MOI) was 100, meaning that there were approximately 100 phages per bacterium. After overnight incubation, bacterial growth was recorded by bright field and mCherry fluorescence imaging of the plates. The sample is quantized based on the images.

Five different phage variants were used to determine the effect of lytic phage delivery of non-targeted crrnas with exogenous I-C type Cas system (p 1772e 008), self-targeted crrnas without exogenous CRISPR-Cas system only (p 1772e 006), two different self-targeted crarrays delivered with exogenous I-C type Cas system (pArray 3 and pArray 4), and parental wild-type phage (p 1772 wt). In these assays, a pseudomonas aeruginosa strain lacking any endogenous Cas system and the indicated phage were mixed in indicated ratios and immediately plated on LB plates. The host bacterial strain used in these assays is Cas-free and has a chromosomally integrated mCherry gene to facilitate the observation and quantification of bacteria by measuring relative fluorescence. The results are depicted in fig. 10A-10B. Darker spots represent higher bacterial growth. The numbers to the right of each picture represent the multiplicity of infection (MOI). At the highest MOI, there are approximately 100 phages per 1 bacterium. These plate images show that at higher MOI, phages pArray3 and pArray4 (both p1772 phages encoding active I-C Cas systems, each with a unique array of three different self-targeting spacers) were more effective in killing pseudomonas aeruginosa than p1772wt, phages with Cas operon and non-targeting spacer (p 1772e 008) or p1772 (p 1772e 006) containing crary but no exogenous Cas system. As expected, crRNA phage (p 1772e 006) alone did not improve phage efficacy because the bacteria did not have an endogenous Cas system.

Fig. 10C is a higher resolution view in the box in fig. 10A and highlights the difference between the fully engineered phage (pArray 3) and phage with only crArray without Cas operon (p 1772e 006). In the bottom row (MOI 0.00610), pArray3 formed a clearer plaque than p1772e006 (i.e., the light spot in the pArray3 sample was brighter). In the top row (MOI 0.0244), pArray3 inhibited bacterial growth better than p1772e006 (dark spots).

FIGS. 10D-10E show quantification of the relative amounts of fluorescent bacteria present under the fourth row (MOI 1.5) of corresponding fluorescent images of the same plate shown in FIGS. 10A and 10B. Consistent with bright field images, samples treated with pArray3 and pArray4 had significantly less fluorescent signal (indicative of loss of viable bacterial cells) than samples treated with wild-type phage or non-targeted (p 1772e 008) and CrArray-only (p 1772e 006) engineered phage at an MOI of about 1.5.

Example 10: efficacy of crArray/Cas inserts with different promoters driving expression of Cas operon.

P1772 wild-type and engineered phage variants were mixed with logarithmically growing mCherry expressing bacteria and immediately plated onto LB agar. The ratio of phage to bacteria was varied by serial dilution of phage so that the amount of bacteria in each spot remained constant, but the amount of phage varied. The highest multiplicity of infection (MOI) was 100, meaning that there were approximately 100 phages per bacterium. After overnight incubation, bacterial growth was recorded by bright field and mCherry fluorescence imaging of the plates. The sample is quantized based on the images. Figure 11A shows bacterial killing of p1772 wild type and multiple engineered variants of phage containing Cas system and crArray expressed by different promoters. All engineered phage variants have the same structure as p1772e005 (see fig. 3A), except for the identity of the promoter driving expression of the Cas operon. p1772e016 uses a promoter that drives the endogenous type I-C Cas system in pseudomonas aeruginosa. p1772e005, p1772e017 and p1772e021 all use E.coli promoters or derivatives of E.coli bacterial promoters. p1772e018, p1772e022 and p1772e023 all used the pseudomonas aeruginosa bacterial promoter. p1772e019 and p1772e020 used the pseudomonas aeruginosa phage promoter. Both plates were from the same assay, while the controls (p 1772wt and p1772e 005) were from the same phage-bacteria mixture before plating at each point. The bacterial host strain used was a Cas-free pseudomonas aeruginosa strain expressed from a chromosome. Individual images were obtained using a 4-fold objective and bright field illumination, and then stitched together to obtain the images shown here. Fig. 11B shows quantification under the fourth row (MOI-1.5) of the corresponding fluorescence image of the same plate shown in fig. 11A. As indicated by significantly fewer fluorescent signals (indicative of loss of living bacterial cells) compared to p1772wt, a difference in overall efficacy was observed between the different promoters used.

Example 11: a variety of different phages have improved log reduction efficacy

Wild-type and engineered phage variants were mixed with logarithmically growing mCherry expressing bacteria and immediately plated onto LB agar. The results shown are from a multiplicity of infection (MOI) of 1.5, meaning that there are approximately 1.5 phages per single bacterium. After overnight incubation, bacterial growth was recorded by mCherry fluorescence imaging of the plates. The samples were quantified as depicted in fig. 12A-12B based on these images. Two unique wild-type phages (p 2131 and p 2973) and their engineered counterparts containing Cas system and crArray1 (p 2132e002 and p2973e 002) were tested. At an MOI of about 1.5, the engineered phage had much fewer viable bacteria than the wild-type phage. These results indicate that phage-delivered Cas systems function in a variety of unique phages.

Example 12: efficacy of spacer array/Cas insertion in surrogate phage and pseudomonas strains

A set of pseudomonas strains was assayed with p4209wt (wild type) and p4209e002 (targeting crarray1+cas system). Briefly, log-early bacterial cultures were mixed with phage to obtain Final titers listed in the figures. Samples were plated immediately (t=0 h) and after incubation for 3h and 24h at 37 ℃. Plates were imaged and differences between wild type and full construct variants were tabulated. P4209 wild-type and engineered phage variants were mixed with log-growing bacteria and plated onto LB agar immediately or incubated in liquid for the indicated amount of time prior to plating. The ratio of phage to bacteria was varied by serial dilution of phage so that the amount of bacteria in each spot remained constant, but the amount of phage varied. As bacteria replicate and yield to phage, the relative ratio of phage to bacteria changes during the course of the experiment. After overnight incubation, bacterial growth was recorded by imaging the plates. The label at the top of each set of images represents the Cas type of the bacterial strain shown in the image. The results of this assay are shown in fig. 13A. In strain b2550 at all time points, p4209e002 was found at 1×10 ⁹ The PFU/mL titer completely inhibited bacterial growth, which was comparable to p4209wt at the same titer. In strain b2631, t=0h, for 1×10 ⁵ No growth was observed for p4209e002 at the titer of PFU/mL, whereas significantly more growth was observed for p4209wt at the same titer. In the same strain at t=3h, no growth was observed for p4209e002 at any titer, whereas growth was seen for p4209wt at all titers. In strain b2816, t=0h, compared to p4209wt at the same titer, for 1×10 ⁹ Slightly less growth was observed at the titer of PFU/mL, p4209e 002. In the same strain at t=3h, for 1×10 ⁹ Very little growth was observed for p4209e002 at the titer of PFU/mL, whereas there was significant growth for p4209wt at the same titer. In strain b2825 with t=0h, for 1×10 ⁷ No growth was observed for p4209e002 at the titer of PFU/mL, whereas significant growth was observed for p4209wt at the same titer. In the same strain at t=3h, for 1×10 ⁹ PFU/mL and 1X 10 ⁷ Some growth was observed for p4209e002 at the titer of PFU/mL, while significantly more growth was observed for p4209wt at the same titer. Taken together, these data indicate that the unique phage p4209e002 is pseudo-against several unrelated copper-greenThe monad strains have improved Cas and crRNA spacer activity.

P4209wt, p4209e001 (Cas system only) and p4209e002 (crarray1+cas system targeted) were plaque performed on multiple bacterial strains to examine plaque efficiency. Pseudomonas aeruginosa strain b1121 equally supports all variants and is provided as a titer reference. On pseudomonas aeruginosa strain b2631, the plaque level of the wild-type variant was significantly reduced, with only the Cas variant completely free of plaque, while the plaque efficiency of the fully engineered variant was not lost, comparable to b 1121. On pseudomonas aeruginosa strain b2816, neither wild type nor Cas variant alone, showed any evidence of activity, whereas the fully engineered variant produced a transparent region. On pseudomonas aeruginosa strain b2825, the wild-type and Cas-only variants significantly reduced plaque efficiency, while the fully engineered variants maintained efficiency comparable to b 1121. both b2631 and b2825 show examples of engineered events (insertion of Cas system) with detrimental effects, i.e. a decrease in plaque efficiency (b 2631) or a decrease in plaque transparency (b 2825). In both cases, the addition of a targeting crArray (which renders the Cas system active) not only saves the decrease in activity, but also improves the activity beyond that seen in the wild-type parent. The label at the bottom of the plate image indicates the bacterial strain shown in the image and the type of endogenous Cas system it contains. These results further support that Cas systems and targeting crarrays improve the ability of phage to replicate and kill individual bacterial strains.

EXAMPLE 13 in vivo efficacy Studies

Figure 15A outlines materials and methods for in vivo efficacy modeling with p1772wt (wild-type) and p1772e005 (targeting crarray1+cas system). Female ICR mice from Envigo were neutropenia via two intraperitoneal injections of cyclophosphamide (150 mg/kg and 100mg/kg, respectively) on day-4 and day-1. After induction of neutropenia, mice were infected with pseudomonas aeruginosa b1121 by a single intramuscular injection. Previous model development determined that-5 e6 CFU was the ideal inoculum for this particular strain. Mice were treated with vehicle (1xtbs+10 mm salt), p1772wt or p1772e005 3h after infection (p.i) by a single intramuscular injection in the infected thigh. The lower left table details the total PFU delivered to each infected thigh in each experiment. Mice were euthanized and thigh muscle harvested at indicated time points post inoculation. The thigh was homogenized using a bead mill system. Homogenates were serially diluted and plated for CFU quantification. The homogenate was also filtered through a 0.45um filter. The filtrate was serially diluted and plated on b1121 overlay plates for PFU quantification. All CFU and PFU measurements were normalized to g tissue.

For each experiment, bacterial Colony Forming Units (CFU) and phage Plaque Forming Units (PFU) are shown. Figures 15B-15C show phage efficacy in mice that were intramuscularly administered with phage. Thigh muscle tissue was harvested at the indicated time points. Both replicates showed that the fully engineered phage reduced colonization to a greater extent than the wild-type phage. Fig. 15D shows phage efficacy in mice administered phage intravenously. Thigh muscle tissue was harvested at the indicated time points. The fully engineered phage destroyed the bacteria to a greater extent than the wild-type phage. Taken together, these data from fig. 15B-15D indicate that phage delivered by different routes enter the thigh and kill bacteria. At each time point, the CFU/g thigh tissue of mice treated with fully engineered phage was lower than mice treated with wild-type phage. Fig. 15E is a schematic diagram showing experimental design modeling of the dose-response of phage therapy in a mouse infection model. This experiment was performed similarly to the experiment shown in fig. 15A, but additionally included an antibiotic treatment group to represent the current standard of care. Phage doses were also titrated between the different groups.

Fig. 15F shows the results of treatment with different doses of phage or antibiotics in mice. Taken together, these data indicate that engineered p1772e005 is more effective than p1772wt in the mouse infection model. Furthermore, the engineered phage perform better than the antibiotic therapy administered. In graphs B-D and F, the data are shown as mean ± SEM. * p <0.05, < p <0.01, < p <0.001, < p <0.0001. Statistical significance was determined using one-way ANOVA and multiple comparisons or two-way ANOVA and base tests.

EXAMPLE 14 in vivo efficacy Studies

Cultures of b1121 were grown overnight and back diluted to LB+10mM MgCl ₂ +10mM CaCl ₂ And grown to an OD600 of 0.45. Cultures were separated and treated with LB/salt (cell control only), p1772e005 (moi=0.1), PB1e002 (moi=0.1) or cocktails of p1772e005+pb1e002 (moi=0.1 per phage). All samples were incubated in microtiter plates with shaking at 37℃for 24h and the OD at 630nm was measured every 10 minutes. Data are presented as an average of 12 replicates. Error bars represent standard deviation. The data indicate that the cocktail of the two full-construct phages inhibited culture rebound to a greater extent than either phage itself. Fig. 16 shows the synergy between p1772e005 and PB1e 002.

Example 15: activity of different repeat sequences

Pseudomonas aeruginosa cultures were transformed with vectors comprising different repeat sequences. The vector is an empty vector pUCP19 (empty vector), or a pUCP19 vector containing a spacer comprising a pseudomonas type I C Cas system and targeting the gyrB gene, said spacer flanking the repeat sequence listed in table 3. An aliquot was removed from each test condition, diluted and spotted to count bacterial CFU.

Table 3: repeated sequence

The results of this assay are depicted in fig. 17. Specific sequences result in different numbers of transformants. Both repeat 1 and repeat 3 resulted in lower numbers of transformants compared to empty vector or bacteria transformed with either repeat 2, repeat 4 or repeat 5. This indicates that the sequence of the repeat sequence affects the efficacy of phage targeting in pseudomonas cultures.

Example 16: design and validation of spacer sequences for targeting target bacteria

Spacer design

The spacer sequence was designed using the following scheme. First, a suitable search set of representative genomes of organisms/species/targets of interest is obtained. Examples of suitable databases include the NCBI gene library and the PATRIC (pathology system resource integration center) database. The genome is downloaded in batches via an FTP (file transfer protocol) server, thus enabling fast and programmed data set acquisition.

The genome is searched with relevant parameters to locate the appropriate spacer sequence. The genome can be read end-to-end in both forward and reverse complement orientations to locate a continuous DNA fragment containing PAM (protospacer adjacent motif) sites. The spacer sequence will be a DNA sequence of N length 3' adjacent to the PAM site, where N is specific for the Cas system of interest and is typically known in advance. Characterization of PAM sequences and spacer sequences is typically performed during discovery and preliminary studies of Cas systems. Each observed PAM adjacent spacer may be saved to a file and/or database for downstream use.

Next, the mass of the spacer used in CRISPR engineered phage was determined using the following procedure. First, each observed spacer can be evaluated to determine how many of them are present in the evaluated genome. The observed spacers may additionally be evaluated to see how many times they may occur in each given genome. The presence of a spacer at more than one location per genome may be advantageous because if a mutation occurs, the Cas system may not recognize the target site and each additional "backup" site increases the likelihood that a suitable non-mutated target location will exist. The observed spacers can be evaluated to determine if they are present in the functionally annotated region of the genome. If such information is available, the functional annotation may be further evaluated to determine if such regions of the genome are "necessary" for the survival and function of the organism. Focusing on the spacers present in all or almost all of the estimated genomes of interest (> =99) ensured broad applicability to demonstrate spacer selection. If there is a large pool of conserved spacer selections, the spacers that occur in genomic regions with known functions may be preferentially selected, given higher priority if these genomic regions are "essential" for survival and occur more than 1 time in each genome.

Spacer verification

The identified spacer subsequence may then be verified by completing the following procedure. First, a plasmid that replicates in an organism of interest and has a selectable marker (e.g., an antibiotic resistance gene) is identified. The genes encoding the Cas system are inserted into plasmids such that they will be expressed in the organism of interest. Upstream of the Cas system, a promoter is included that is recognized by the organism of interest to drive expression of the Cas system. A Ribosome Binding Site (RBS) is included between the promoter and Cas system that is recognized by the organism of interest.

Next, the genome targeting spacer that has been bioinformatically identified is inserted into a plasmid expressing the Cas system. Upstream of the repeat-spacer-repeat, a promoter is included that is recognized by the organism of interest to drive expression of crRNA. Examples of such promoters are listed in table 1B. Such cloning must be performed in organisms or strains that are not targeted by the cloned spacer.

Next, the non-targeting spacer is inserted into the plasmid expressing the Cas system. The sequence of this spacer may be randomly generated and then bioinformatically demonstrated that there are no targeting sites in the genome of the organism of interest. Upstream of the repeat-spacer-repeat, a promoter is included that is recognized by the organism of interest to drive expression of crRNA.

Next, the killing efficacy of each test spacer was determined. Plasmids listed in table 4 were normalized to the same molar concentration. Each plasmid is transferred to the organism of interest by transformation, conjugation, or any other method of introducing the plasmid into the cell. The transformed cells are plated onto a suitable selective medium (e.g., antibiotic-containing agar). After the cells grew into colonies, colonies generated by each different plasmid transfer were counted. Plasmids containing targeting spacers transfer at significantly lower rates than control plasmids containing non-targeting spacers are considered successful in targeting bacterial genomes.

Table 4: plasmids used and controls

Plasmid(s)	Function of
		Empty skeleton carrier	Control of plasmid transfer efficiency
Vectors containing Cas systems	Control of Cas system toxicity
		Vectors containing Cas systems and non-targeting spacers	Contrast to off-target Effect
Vectors containing Cas systems and targeting spacers	Test sample

Example 17: purified pseudomonas aeruginosa phage host range assay

Data were obtained for purified Pseudomonas aeruginosa phage reporting optimal results from combined liquid and plaque host range assays. The end result is the median of binary hits across liquid and plaque host ranges for a given phage plus strain combination. Liquid host range involves adding 5uL of frozen, OD controlled culture material, 5uL of phage material of known titer and 40uL of growth medium to one well of a 364 well plate, as well as appropriate cultures, phages and medium-only controls. Plates were incubated at 37 ℃ for 20 hours while shaking, and OD600 readings were taken per hour by a liquid processor. The results were calculated by determining the ratio between the area under the curve (AUC) of the phage added samples and their corresponding controls. Samples with AUC ratios below 0.65 were considered positive (+) hits, while AUC ratios greater than or equal to 0.65 were negative (-) hits. Bacterial strains of interest are cultured and screened against prophages for plaque host range assays. The phage of interest was serially diluted 50-fold in 1 XPBS, from undiluted to 50-3, by microtiter plates. The agar overlay of the strain used as the titer host was poured and allowed to stand overnight. The next day lysates of the bacterial strains of interest are spotted. After 15-20min, plates were imaged using Hamilton-STAR-C062, either manually counted, or converted, background subtracted, and counted through an internally developed image analysis pipeline. Samples with a positive (+) number of plaque forming units were considered hits. The results of this assay involving pseudomonas aeruginosa, wild-type and engineered pna phage subtypes are listed in table 5A. The results of this assay involving pseudomonas aeruginosa, wild-type salsa Mu Na virus phage subtype, engineered salsa Mu Na virus phage subtype, wild-type Φkz virus, wild-type Φkmv virus and wild-type brunesabout virus are listed in table 5B. As listed in table 5A, wild-type pna phage subtypes are p1106, p1587, p1835, p2037, p2363, p2421 and pb1, and engineered pna phage subtypes are p1106e003, p1587e002, p1835e002, p2037e002, p2363e003 and p2421e002. As listed in table 5B, wild-type salsa Mu Na viral phage subtypes are p1772, p2131, p2132 and p2973, engineered salsa Mu Na viral phage subtypes are pb1e002, p1772e005, p2131e002, p2132e002 and p2973e002, wild-type Φkz viral phage subtypes are p1194 and p4430, wild-type Φkmv viral phage subtype is p2167, and wild-type bruneshop viral phage subtypes are p1695 and p3278.

Table 5A: pseudomonas aeruginosa phage host range

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Table 5B: pseudomonas aeruginosa phage host range

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Example 18: pseudomonas aeruginosa cocktail phage host range assay

Data were obtained for pseudomonas aeruginosa cocktail phage reporting optimal results from combined fluid and plaque host range assays. The end result is the median of binary hits across liquid and plaque host ranges for a given phage plus strain combination. Liquid host range involves adding 5uL of frozen, OD controlled culture material, 5uL of phage material of known titer and 40uL of growth medium to one well of a 364 well plate, as well as appropriate cultures, phages and medium-only controls. Plates were incubated at 37 ℃ for 20 hours while shaking, and OD600 readings were taken per hour by a liquid processor. The results were calculated by determining the ratio between the Area Under Coverage (AUC) of the phage added samples and their corresponding controls. Samples with AUC ratios below 0.65 were considered positive (+) hits, while AUC ratios greater than or equal to 0.65 were negative (-) hits. Bacterial strains of interest are cultured and screened against prophages for plaque host range assays. The phage of interest was serially diluted 50-fold in 1 XPBS, from undiluted to 50-3, by microtiter plates. The agar overlay of the strain used as the titer host was poured and allowed to stand overnight. The next day lysates of the bacterial strains of interest are spotted. After 15-20min, plates were imaged using Hamilton-STAR-C062, either manually counted, or converted, background subtracted, and counted through an internally developed image analysis pipeline. Samples with a positive (+) number of plaque forming units were considered hits. The detailed composition of phage cocktails CK000125, CK000239, CK000240, CK000241, CK000511, and CK000512 (also referred to as PACK512, cocktail 512, CK00512, CK 512) are listed in table 6A. As listed in table 6B, phage cocktail ck000125 comprises p1106e003, p1835e002, p1772e005 and p2131e002. As listed in table 6B, phage cocktail ck000239 comprises p1106e003, p1835e002, p1772e005, p2131e002 and p1194. As listed in table 6B, phage cocktail ck000240 comprises p1106e003, p1835e002, p1772e005, p2131e002 and p4430. As listed in table 6B, phage cocktail ck000241 comprises p1106e003, p1835e002, p1772e005, p2131e002 and p1695. As listed in table 6B, phage cocktail ck000511 comprises p1106e003, p1835e002, p1772e005, p2131e002, p1194 and p1695. As listed in table 6B, phage cocktail ck000512 comprises p1106e003, p1835e002, p1772e005, p2131e002, p4430 and p1695.

Table 6A: host range phage cocktail composition

The results of this assay involving pseudomonas aeruginosa and phage cocktails ck000125, ck000239, ck000240, ck000241, ck000511 and ck000512 are listed in table 6A. Overall, the increase in positive (+) hits as listed on table 6B shows an increase in host range in the phage cocktail assay as compared to the phage assays alone listed in table 5A and table 5B. As listed in table 6B, cocktail ck000512 pseudomonas aeruginosa host range data continued to increase.

As shown in table 6C, the host ranges of cocktails CK000125 and CK00512 were tested in 284 different pseudomonas bacterial isolates. 111 isolates were from Cystic Fibrosis (CF) and 85 isolates were from non-cystic fibrosis bronchiectasis (NCFB). Of 284 pseudomonas isolates, 95 were multi-resistant, with 49 of the isolates from CF and 9 from NCFB. The host range of both cocktails was greater than 85%, with cocktail CK00512 being 100% for all MDF isolates.

Table 6B: pseudomonas aeruginosa cocktail phage host range

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Table 6C: cocktail host range

Example 19: off-target plaque pseudomonas aeruginosa cocktail phage assay

As shown on table 7, the pseudomonas aeruginosa cocktail phage did not exhibit off-target plaques. Except b1233/PAO1, pseudomonas aeruginosa cocktail phage plaques on b2631 or b1121, as not all phages infect this bacterial strain. Pseudomonas aeruginosa cocktails CK000511 and CK000512 (PACK 512); constitutive phages p1835e002, p1106e003, p1772e005, p2131e002, p1194, p1695 and p4430; positive control phages plaque on off-target ESKAPE species. As indicated by (-), no off-target plaques were observed from any of the phages tested. As detailed in Table 7, the data obtained indicate that phage only hit (+) P.aeruginosa.

Table 7: off-target plaque phage assay

Example 20: pseudomonas aeruginosa cocktail effective against Pseudomonas aeruginosa respiratory tract/cystic fibrosis isolates

Comparison of CRISPR phage and wild type phage

Challenge (challenge) inoculantIs prepared from

Bacterial isolates, tobramycin MIC and challenge inoculums are described in table 8. The study used Pseudomonas aeruginosa isolate b1121. A round of bacterial stock was scraped from the surface of the frozen vials of culture and streaked out on several TSA plates. After 16h growth at 37℃the inoculum was removed from the plate and suspended in PBS, pH 7.2 (Gibco 20012027) and adjusted to OD650 _nm 1.0 + -0.3. In the same study, two cohorts of animals were challenged with an inoculum prepared separately.

Table 8: bacterial strains

CF, cystic fibrosis; CFU, colony forming units; MIC, minimum inhibitory concentration; TOB, tobramycin.

Treatment of mice in acute lower respiratory infection model (LRTI)

Tobramycin (75 mg/kg/day, HED 6 mg/kg/day) was tested as a comparator. Tobramycin sulfate (Xgen, batch AZ 1240B) in dH ₂ O was prepared and administered subcutaneously in 0.2 mL. Treatment was started 1h after infection and continued at 12 hour intervals twice daily (BID) for a total of 4 doses. Tobramycin was freshly prepared daily and stored at 4 ℃ between doses. The individual bacteriophages were diluted to 4.25E+09PFU/mL in PBS, pH 7.4, such that the final concentration of each individual phage was 10.7Log10 PFU/dose. Phage were delivered intranasally under anesthesia 2h after challenge. Additional doses were administered 8, 24 and 32h after challenge. Animals were randomized at treatment and divided into two treatment groups. In both groups, animals were treated with either p1772WT or p1772 FC. Tobramycin and PBS were included as controls in both queues.

Murine pneumonia model

Female C57BL/6J mice (Jackson Laboratory, bar Harbor Maine) of 7-8 weeks old without the specific pathogen were anesthetized with 3% isoflurane and maintained at 3 liters/min of oxygen prior to inoculation into both nostrils with 0.050mL of Pseudomonas aeruginosa suspension b 1121. Mice were placed in the supine position in cages and allowed to recover from anesthesia. 24 and 48h after attack by CO ₂ Animals were euthanized by asphyxiation, and the lungs were aseptically removed and placed in a homogenization tube. Animals dying of moribund were humanly euthanized and counted as dead. The high dose group was determined by MFD (maximum feasible dose; targeting 10 for the highest strain) ^10-11 PFU/mL). Necropsy for general observation and creation of a standard tissue panel for histopathological study; the second lung lobe may be used for PD (PCR).

CFU assay in tissue

The lungs were harvested into soft tissue homogenates containing 1.4mm ceramic beads (VWR 10158-610) and 1mL PBS, pH7.4 (Gibco 10010023). Homogenizing the tissue for 20s, thenStanding for 10s, and homogenizing for 20s. Homogenates were serially diluted 1:100 into 0.9% sterile saline (BBL, 221819) and plated onto Tryptic Soy Agar (TSA) plates for CFU counting using the spiral plate method. After incubation for 20-22h at 37℃CFU was determined. Bacterial count is expressed as Log ₁₀ CFU/gram was organized and data from both studies were pooled for analysis.

Figure 18E shows the efficacy of CRISPR phage p1772FC (p 1772E 005) compared to wild type phage.

Comparison of phage cocktails and individual phages

These studies used Pseudomonas aeruginosa isolates b1121, b2631 or b3144. Pseudomonas aeruginosa suspension isolates were prepared for challenge. Pseudomonas aeruginosa b3144 was diluted 1:3 in PBS, pH 7.2, and Pseudomonas aeruginosa b2631 was diluted 1:10 in PBS. Isolate b1121 was not diluted after normalization to OD650 nm=1.0.

Tobramycin treatment was prepared and administered as depicted in fig. 18A. Animals were dosed BID at 12 hour intervals for a total of 3 doses. Treatment controls included tobramycin, tobramycin plus PACK512 and PBS. Based on phage titer and EU/mL, cocktail 512 (also referred to as PACK 512) (described in table 6A) was diluted to the maximum concentration available and delivered intranasally under anesthesia 2h after challenge. Additional doses were administered 8, 24h after challenge. Animals were randomized at treatment and divided into two treatment groups. In cohort one, animals were treated with cocktail and phage alone. In cohort two, animals were treated with PACK 512. Tobramycin, tobramycin plus PACK512 and PBS were included in both queues and the data from both studies were pooled for analysis.

Female C57BL/6J mice (Jackson Laboratory, bar Harbor Maine) of 7-8 weeks old without the specific pathogen were anesthetized with 3% isoflurane and maintained at 3 liters/min of oxygen prior to inoculation with 0.050mL of Pseudomonas aeruginosa suspension into both nostrils. Mice were placed in the supine position in cages and allowed to recover from anesthesia. During recovery, the cages were placed on a heating pad at 105°f until the animals were fully awake and able to walk.

Copper in useLungs were harvested 24 and 48h after pseudomonas suspension challenge as described above, except after serial dilutions, homogenates were plated on Pseudomonas Inhibition Agar (PIA) plates for CFU counting using spiral plate method. After incubation at 37℃for 16-22h, the CFU of each animal was determined. Bacterial count is expressed as Log ₁₀ CFU/gram and data were analyzed using t-test, mann-whitney test, analysis of variance (ANOVA) or log rank test. All statistical analyses were performed using GraphPad Prism version 8.0. p value<0.05 was considered statistically significant.

As depicted in fig. 18B-18D, treatment with cocktail and combination of cocktail and tobramycin resulted in levels of pseudomonas aeruginosa for all 3 test strains below the detection level. Furthermore, treatment with cocktails resulted in a significant decrease in the CFU detected compared to treatment with tobramycin alone.

In a similar assay as described above, phage cocktail ck00125 was also tested. Fig. 19A shows an assay design for testing in vivo efficacy of CK00125 (CK 125) in an acute LRTI model. Figures 19B-19D demonstrate a statistically significant reduction in bacterial load in all three tested pseudomonas aeruginosa strains 24 hours as early as after challenge with CK125 alone. When combined with tobramycin, the bacterial level was below the detection level. Thus, it can be seen that the cocktail is almost completely depleted of bacteria, similar to that observed with ck00512 (PACK 512) cocktail.

Example 21: comparison of efficacy of cocktails compared to phage alone

Materials and methods:

challenge inoculumIs prepared from

The study used Pseudomonas aeruginosa isolates b1121, b2631 or b3144. Isolates for challenge were prepared as described above, with the following modifications. Pseudomonas aeruginosa b3144 was diluted 1:3 in PBS, pH 7.2, and Pseudomonas aeruginosa b2631 was diluted 1:10 in PBS. Isolate b1121 was not diluted after normalization to OD650 nm=1.0.

Treatment of

Tobramycin was prepared and administered as described above, except that animals were given BID at 12 hour intervals for a total of 3 doses. Based on phage titer and EU/mL (Table 2), individual phage and cocktail 512 (PACK 512) were diluted to the maximum concentration available. Phage concentrations were identical in the cocktail alone. Phage were delivered intranasally under anesthesia 2h after challenge. Additional doses were administered 8, 24h after challenge. Animals were randomized at treatment and divided into two treatment groups. In cohort one, animals were treated with cocktail and phage alone. In cohort two, animals were treated with PACK 512. Tobramycin, tobramycin plus PACK512 and PBS were included in both queues and the data from both studies were pooled for analysis.

Murine pneumonia model

Female C57BL/6J mice (Jackson Laboratory, bar Harbor Maine) of 7-8 weeks old without the specific pathogen were anesthetized with 3% isoflurane and maintained at 3 liters/min of oxygen prior to inoculation with 0.050mL of Pseudomonas aeruginosa suspension into both nostrils. Mice were placed in the supine position in cages and allowed to recover from anesthesia. During recovery, the cage was placed on a 105F heating pad until the animal was fully awake and able to walk

To understand the benefits of cocktail PACK512 (also referred to as ck 00512) over engineered phage alone, comparisons were made between phages P1106FC, P1772FC, P1835FC, P2131FC, P1685WT, P4430WT and cocktail 512 (PACK 512) with or without tobramycin. The experimental setup is shown in fig. 18F. The results of pseudomonas aeruginosa b2631, b3144 and p1121 shown in fig. 18G, 18H and 18I, respectively, indicate that PACK512 showed better efficacy than single phage in the acute LRTI model, with or without tobramycin treatment.

CFU assay in tissue

Lungs were harvested as described above, except that after serial dilutions, homogenates were plated on Pseudomonas Inhibition Agar (PIA) plates for CFU counting using spiral plate method. After incubation at 37℃for 16-22h, the CFU of each animal was determined. Bacterial count is expressed as Log ₁₀ CFU/gram tissue.

Statistical analysis

Data were analyzed using t-test, mann-whitney test, analysis of variance (ANOVA) or log rank test. All statistical analyses were performed using GraphPad Prism version 8.0. p-values <0.05 were considered statistically significant.

Example 21: influence of cocktail on Pseudomonas aeruginosa biofilm

In this example, the effect of phage cocktails on Pseudomonas biofilms was tested. The measurement device is shown in fig. 20A. As shown in fig. 20B, cocktail CK125 exhibited anti-biofilm activity against preformed biofilms, as indicated by high levels of bacterial inhibition at low MOI for 24 and 48 hours of biofilm treatment with cocktail; and are effective in a plurality of pseudomonas strains. PACK512 cocktails also showed inhibitory activity against preformed (fig. 22) and newly formed biofilm (MBIC) and planktonic bacteria (MIC) from all three key strains (b 1121, b2631, b 3144).

Example 22: bactericidal activity of cocktails in the presence of mucins

Mucin is a glycoprotein which is abundant in mucus in healthy and sick people. Cystic fibrosis is characterized by the overproduction of mucus by airway and lung epithelial cells, which primarily form a barrier for the therapeutic agent to enter the cells. In this experiment, airway epithelial tissue derived from healthy people was cultured for 1 month; and mucin was measured, followed by bacterial infection of the cultures for 30 minutes and then followed by phage cocktail. After 19.5 hours incubation, samples were collected and bacterial load and phage load were determined. As shown in fig. 22 and 23, phage cocktail ck125 and PACK512 (respectively) successfully reduced bacterial load in cell cultures infected with pseudomonas aeruginosa b1121 (left) from respiratory tract isolates and b2631 (right) from CF patient isolates. Mucin levels at the time of bacterial addition were 1.3±0.09mg/mL (n=3 't=0 harvest' migration cell (transwell) and n=2 geometric mean of sample dilution/migration cell) as detected by the alcian blue assay.

EXAMPLE 23 persistence of phages in vivo

To understand the time course of phage persisting in vivo, the change in phage levels over time was determined in the LRTI mouse model. It was observed that the level of each phage was high despite the bacterial clearance. Exemplary results are shown in fig. 24, where the persistence of phage is demonstrated in quantity by plaque assay and qPCR determination of individual phage copy number at 32h post-treatment. This indicates that phage are not easily cleared from the system.

Phage Genome Copy (GC) levels per dose were determined by qPCR, multiplied by the number of doses administered and divided by the geometric mean of lung weights within each treatment group.

PFU/dose was estimated based on PFU/mL titer of NME phage stocks and expected PFU/dose in formulated cocktail, and then divided by the geometric mean of lung weights within each treatment group.

Sequence listing

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While preferred embodiments of the present disclosure 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. Many changes, modifications and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. The following claims are intended to define the scope of the present disclosure and thus cover methods and structures within the scope of these claims and their equivalents.

Sequence listing

<110> rocos bioscience Co

<120> phage composition for Pseudomonas comprising CRISPR-CAS system and method of using the same

<130> 53240-743.601

<140>

<141>

<150> 63/184,728

<151> 2021-05-05

<150> 63/110,288

<151> 2020-11-05

<160> 120

<170> patent in version 3.5

<210> 1

<211> 97

<212> DNA

<213> unknown

<220>

<223> unknown description:

phage genome sequence

<400> 1

acaagcggca cattgtgcct attgcgaatt aggcacaatg tgcctaatct aacgtcatgc 60

cagccacaac ggcgaggcgc caagaaggat agaagcc 97

<210> 2

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 2

tttacagcta gctcagtcct agggactgtg ctagc 35

<210> 3

<211> 155

<212> DNA

<213> Pseudomonas aeruginosa

<400> 3

gatttttttc gggtgaggtt gcgggctgtt cggtaggttt ataaacactg ctatccaaag 60

ctatggacac gctcggctac gagaacagtt ggcgtgatgg cctctagcaa ttagattgtt 120

atgcgacatc cgcagacttg gcagggagcg cacct 155

<210> 4

<211> 35

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 4

tttacggcta gctcagtcct aggtatagtg ctagc 35

<210> 5

<211> 199

<212> DNA

<213> Pseudomonas aeruginosa

<400> 5

atccgaggga tacgggcctt gtcagcacgg tgttgctaat gagagccttt gcccgggcaa 60

tagtacgggc agtgtgtagc ggattgaaag acgctgaatc actgacaggc atgaagacta 120

tcgatagagt ctgatagtgt cgccgccgca cagcggatag agtccacagt cattgaagtg 180

ttaatccgcg atcaagctc 199

<210> 6

<211> 216

<212> DNA

<213> unknown

<220>

<223> unknown description:

phage genome sequence

<400> 6

gacctagctt ttatagcggg tttcgtggtt tatagcccat tgaaaaaaat ctcacatcta 60

tatcacaggt gtgcactcgt tcccgaaagg ttctgagtct acttgatcaa gtattgaaat 120

accatcgtaa aggaaaaaga catgtctatt cgtgatagcg aaaacaacaa cggccaacag 180

cagcagaccg cgcaaactgc cgcccccgcc ccgcaa 216

<210> 7

<211> 182

<212> DNA

<213> unknown

<220>

<223> unknown description:

phage genome sequence

<400> 7

ttcaatttaa gtagtaacga ggtcagcccg gaatctttgg gtattcttaa ggtatttctg 60

actcagtgtg gttgggacag cttcactgta cattgcactg gatttgttaa tttcttatac 120

cggggcacca tgggcagcaa atcgtgttac gaattccgtc taaccaataa gcgagctaaa 180

ta 182

<210> 8

<211> 105

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Polynucleotide

<400> 8

cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 60

caataaccct gataaatgct tcaataatat tgaaaaagga agagt 105

<210> 9

<211> 161

<212> DNA

<213> Pseudomonas aeruginosa

<400> 9

cttcaagaat tcgtattgac cccatagaca gcttcgtcga cgcccgtccc ggcccccttg 60

ggcttgccgg acggcttatg tcatgatggc gccaccctcg caggttcaag gccggctttc 120

ttcctctatg aacaaatccc ttgcgctgac tacgtaatca c 161

<210> 10

<211> 207

<212> DNA

<213> Pseudomonas aeruginosa

<400> 10

cttcaagaat tcggggtatt cctgatcctg cgccgctagc gccgcgcacg gccactaggc 60

ccgcgccgat agccagtcgc gctcccggct ggcacactac tcccatttcc gccggaaacg 120

cgcgcaacgt accggcaacg aacgtggaaa gaccatgaaa gactggctgg atgagattca 180

ctggaacgcc gtgacctacg tatgcac 207

<210> 11

<211> 59

<212> DNA

<213> Escherichia coli

<400> 11

gaaaattatt ttaaatttcc tctagtcagg ccggaataac tccctataat gcgacacca 59

<210> 12

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 12

agaagggtca gggccatgcg gtttttcctc tgtg 34

<210> 13

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 13

gctcgactgg tcggtaacca cttgtgtgtg gtga 34

<210> 14

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 14

ggtgctgacc gaggacgaga aggaactggg cgtg 34

<210> 15

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 15

gatgacacca acccggccaa ggaagaccag gagt 34

<210> 16

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 16

gagaccgaag agaacgtgcc gaccaccgcc gctg 34

<210> 17

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 17

cagtgcatgg cagcgaacgc cgagagccga cacc 34

<210> 18

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 18

tccgcgatga gctgccgtcc caacaattca acac 34

<210> 19

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 19

aacgcgaagc cctgttgaaa ccgctgcaac tggt 34

<210> 20

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 20

tgctgaacag ccatgattga ttaactccta aacg 34

<210> 21

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 21

cgtaaaccta atgggcctga tctacagtaa tcta 34

<210> 22

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 22

accaccgaga cgcccacacc gtgcaagccg ccgg 34

<210> 23

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 23

ctatcgcgaa ttcctgcagg ctggcgcaac caag 34

<210> 24

<211> 6024

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Polynucleotide

<400> 24

acaagcggca cattgtgcct attgcgaatt aggcacaatg tgcctaatct aacgtcatgc 60

cagccacaac ggcgaggcgc caagaaggat agaagccgtc gcgccccgca cgggcgcgtg 120

gattgaaacg gtgctgaccg aggacgagaa ggaactgggc gtggtcgcgc cccgcacggg 180

cgcgtggatt gaaactccgc gatgagctgc cgtcccaaca attcaacacg tcgcgccccg 240

cacgggcgcg tggattgaaa caccaccgag acgcccacac cgtgcaagcc gccgggtcgc 300

gccccgcacg ggcgcgtgga ttgaaaccat gcaagcttgg cgtaggccgc ttcgtcccta 360

tcaaagcttg gagtttacag ctagctcagt cctagggact gtgctagcat taaagaggag 420

aaaatggacg cggaggctag cgatactcac ttttttgctc actccacctt aaaggcagat 480

cgcagcgatt ggcagcctct ggtcgagcat ctacaggctg ttgcccgttt ggcaggagag 540

aaggctgcct tcttcggcgg cggtgaatta gctgctcttg ctggtctgtt gcatgacttg 600

ggtaaataca ctgacgagtt tcagcggcgt attgcgggtg atgccatccg tgtcgatcac 660

tctactcgcg gggccatact ggcggtagaa cgctatggcg cgctaggtca attgctagcc 720

tacggcatcg ctggccacca tgccgggttg gccaatggcc gcgaggctgg tgagcgaact 780

gccttggtcg accgcctgaa aggggttggg ctgccacggt tattggaggg gtggtgcgtg 840

gaaatcgtgc tacccgagcg ccttcaacca ccgccactaa aagcgcgcct ggaaagaggt 900

ttctttcagt tggcctttct tggccggatg ctcttttcct gcttggttga tgcggattat 960

ctagataccg aagccttcta ccaccgcgtc gaaggacggc gctcccttcg cgagcaagcg 1020

cggccgacct tggccgagtt acgcgcagcc cttgatcggc atctgactga gttcaaggga 1080

gatacgccgg tcaaccgcgt tcgcggggag atattggccg gcgtgcgcgg caaggcgagc 1140

gaacttcccg ggctgttttc tctcacagtg cccacaggag gcggcaagac cctggcctct 1200

ctggctttcg ccctggatca cgctctagct catgggctgc gccgggtgat ctacgtgatt 1260

cccttcacta gcatcgtcga gcagaacgct gcggtattcc gtcgtgcact cggggcctta 1320

ggcgaagagg cggtgctgga gcatcacagc gccttcgttg atgaccgccg gcagagcctg 1380

gaggccaaga agaaactgaa cctagcgatg gagaactggg acgcgcctat cgtggtgacc 1440

actgcagtgc agttcttcga aagcctgttt gccgaccgtc cagcccagtg ccgcaagcta 1500

cacaacatcg ccggcagcgt ggtgattctt gacgaggcac agaccctacc gctcaagctg 1560

ttgcggccct gcgttgccgc ccttgatgaa ctggcgctca actaccgttg tagcccagtt 1620

ctctgtactg ccacgcagcc agcgcttcaa tcgccggatt tcatcggtgg gctgcaggac 1680

gtacgtgagc tggcgcccga gccgcagcgg ctgttccggg agttggtgcg ggtacgaata 1740

cggacattgg gcccgctcga agatgcggcc ttgactgagc agatcgccag gcgtgaacaa 1800

gtgctgtgca tcgtcaacaa tcgacgccag gcccgtgcgc tctatgagtc gcttgccgag 1860

ttgcccggtg cccgccatct caccaccctg atgtgcgcca agcaccgtag cagcgtgctg 1920

gccgaggtgc gccagatgct caaaaagggg gagccctgtc gcctggtggc cacctcgctg 1980

atcgaggccg gtgtggatgt ggattttccc gtggtactgc gtgccgaggc tggattggat 2040

tccatcgccc aggccgcggg acgctgcaat cgcgaaggca agcggccgct ggccgaaagc 2100

gaggtgctgg tgttcgccgc ggccaattct gactgggcgc cacccgagga actcaagcag 2160

ttcgcccagg ccgcccgcga agtgatgcgc ctgcacccgg atgattgcct gtccatggcg 2220

gccatcgagc ggtattttcg catactgtac tggcagaagg gcgcggagga gttggatgcg 2280

ggtaacctgc tcggcctgat tgagagaggc cggctcgatg gcctgcccta cgagactttg 2340

gccaccaagt tccgcatgat cgacagcctt caactgccgg tgatcatccc atttgatgac 2400

gaggccagag cagccctgcg cgagctggag ttcgccgacg gctgcgccgc catcgcccgt 2460

cgcctgcagc catatctggt gcagatgcca cgcaagggtt atcaggcatt gcgggaagcc 2520

ggtgcgatcc aggcggcggc aggtacgcgt tatggtgagc agtttatggc gttggtcaac 2580

cctgatctgt atcaccacca attcgggttg cactgggata atccggcctt tgtcagcagc 2640

gagcggctat gttggtagtc gggacgcgca acagcggcct ggcctggatg atgtgaaagg 2700

gagggccgat ggcctacgga attcgcttaa tggtctgggg cgagcgtgcc tgcttcaccc 2760

gcccggaaat gaaggtggaa cgcgtctctt acgatgcgat cacgccgtcc gccgcgcgcg 2820

gcattctcga ggctatccac tggaagccgg cgattcgctg ggtggtggat cgcattcaag 2880

tgcttaagcc gatccgcttc gaatccatcc ggcgcaacga ggtcggcggc aagctgtccg 2940

ctgtcagcgt cggtaaggca atgaaggccg ggcgtactaa tggtctggtg aatctggtcg 3000

aggaggatcg ccagcagcgc gcgactactc tgctgcgcga tgtctcctat gtcatcgagg 3060

cgcatttcga gatgactgac agggctggcg ccgacgatac ggtgggcaag catctggata 3120

tcttcaaccg tcgcgcacgg aaggggcagt gcttccatac accctgccta ggcgtgcgcg 3180

agtttccggc cagttttcgg ttgctggaag agggcagtgc cgagcctgaa gtcgatgcct 3240

ttctgcgcgg cgagcgtgat ctgggctgga tgctgcatga cattgacttc gccgatggca 3300

tgaccccgca cttcttccgt gccctgatgc gcgatgggct gatcgaggtg ccggccttca 3360

gggcggcaga ggacaaggca tgatcctttc ggccctcaat gactattatc agcgactgct 3420

ggagcggggt gaagcgaata tctcaccctt cggctacagc caagaaaaga tcagttacgc 3480

cctgctgctg tccgcacaag gagagttgct ggacgtgcag gacattcgct tgctctctgg 3540

caagaagcct caacccaggc ttatgagtgt gccgcagccg gagaagcgca cctcgggcat 3600

caagtccaac gtactgtggg acaagaccag ctatgtgctg ggtgttagtg ccaagggcgg 3660

agagcgtact cagcaggagc acgagtcctt caagacgctg caccggcaga tcttggttgg 3720

ggaaggcgac cccggtctgc aggccttgct ccagttcctc gactgttggc agccggagca 3780

gttcaagccc ccgctgttca gcgaagcaat gctcgacagc aacttagtgt tccgcctaga 3840

cggccaacaa cgctatctgc acgagactcc ggcggccctg gcgttgcgta cccggctgtt 3900

ggccgacggc gacagccgcg aggggctgtg cctagtctgc ggccaacgtc agccgttggc 3960

gcgcctgcat ccagcggtca agggcgtcaa tggtgcccag agttcggggg cttccatcgt 4020

ctccttcaac ctcgacgctt tttcctccta cggcaagagc cagggggaaa atgctccggt 4080

ctccgaacag gccgcctttg cctacaccac ggtgctcaac catttgttgc gtcgcgacga 4140

gcacaaccgc cagcgcctgc agattggcga cgcgagtgtg gtgttctggg cgcaggcgga 4200

tactcctgct caggtggccg ccgccgagtc gaccttctgg aacctgctgg agccacccgc 4260

agatgatggt caggaagcgg aaaagctgcg cggcgtgctg gatgctgtgg ccacggggcg 4320

gcccttgcat gagctcgact cgctaatgga ggaaggtacc cgcatttttg tgttagggct 4380

ggcgcccaat acctcgcgac tgtccattcg gttctgggca gtcgatagcc ttgcggtatt 4440

cacccagcat ctggccgagc atttccggga tatgcacctt gagcctctgc cctggaagac 4500

ggagccggcc atctggcgct tgctctatgc taccgcgccc agtcgtgacg gcagagccaa 4560

gaccgaagac gtactcccac aactggccgg tgaaatgacc cgcgccatcc tgaccggcag 4620

ccgctatccg cgcagtttgc tagccaacct gatcatgcgc atgcgtgccg acggcgacgt 4680

ctctggcata cgcgtcgcgc tgtgcaaggc cgtgctcgct cgcgaggcac gcctgagcgg 4740

caaaattcac caagaggagc tacctatgag tctcgacaag gacgccagca accccggcta 4800

tcgcttgggg aggctgttcg ccgtgttgga aggcgcccag cgcgcagccc tgggcgacag 4860

ggtcaatgcc actatccgtg accgctacta cggtgccgcg tccagcacgc cagccacggt 4920

tttcccgata ctgctgcgca acacacaaaa ccacttggcc aagctgcgca aggagaagcc 4980

cggactagca gtgaacctag agcgcgatat aggcgaaatc attgacggta tgcagagcca 5040

attcccgcgt tgcctgcgcc tggaggacca gggacgcttt gctattggtt actaccaaca 5100

ggcccaggcc cgtttcaacc gtggccccga ttccgtcgag taaggagcag aagaatgacc 5160

gccatctcca accgctacga gttcgtttac ctctttgatg tcagcaatgg caatcccaat 5220

ggcgacccgg atgctggcaa catgccgcgt ctcgatccgg aaaccaacca ggggttggtc 5280

actgacgttt gcctcaagcg caagatccgc aactacgtca gcctggagca ggaaagtgcc 5340

cccggctatg ccatctatat gcaggaaaaa tccgtgctga ataaccagca caaacaggcc 5400

tacgaggcgc tcggtatcga gtcagaggca aagaaactgc ccaaggacga agccaaggcg 5460

cgcgaactga cctcttggat gtgcaagaac ttcttcgatg tgcgtgcttt cggggcggtg 5520

atgaccaccg agattaatgc cggccaggtg cgtggaccga tccaactggc attcgccacg 5580

tctatcgacc cggtattgcc tatggaggta tccatcaccc gcatggcggt gactaacgaa 5640

aaggatttgg agaaggaacg caccatggga cgcaagcaca tcgtgcctta cggcttgtac 5700

cgcgcccatg gtttcatctc tgccaagttg gccgagcgaa ccggcttttc cgacgacgac 5760

ttggaactgc tatggcgcgc tttggccaat atgttcgaac acgaccgctc ggcggcacgt 5820

ggcgagatgg cagcgcgcaa gttgatcgtc ttcaagcatg agcatgccat gggcaatgca 5880

cccgcccatg tgctgttcgg cagcgttaag gtcgagcgag tcgaggggga cgcagttaca 5940

ccagcacgcg gtttccagga ttaccgtgtc agcatcgatg cggaagctct gcctcagggc 6000

gtgagcgtgc gcgagtacct ctag 6024

<210> 25

<211> 6024

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Polynucleotide

<400> 25

acaagcggca cattgtgcct attgcgaatt aggcacaatg tgcctaatct aacgtcatgc 60

cagccacaac ggcgaggcgc caagaaggat agaagccgtc gcgccccgca cgggcgcgtg 120

gattgaaaca gaagggtcag ggccatgcgg tttttcctct gtggtcgcgc cccgcacggg 180

cgcgtggatt gaaacgagac cgaagagaac gtgccgacca ccgccgctgg tcgcgccccg 240

cacgggcgcg tggattgaaa ctgctgaaca gccatgattg attaactcct aaacggtcgc 300

gccccgcacg ggcgcgtgga ttgaaaccat gcaagcttgg cgtaggccgc ttcgtcccta 360

tcaaagcttg gagtttacag ctagctcagt cctagggact gtgctagcat taaagaggag 420

aaaatggacg cggaggctag cgatactcac ttttttgctc actccacctt aaaggcagat 480

cgcagcgatt ggcagcctct ggtcgagcat ctacaggctg ttgcccgttt ggcaggagag 540

aaggctgcct tcttcggcgg cggtgaatta gctgctcttg ctggtctgtt gcatgacttg 600

ggtaaataca ctgacgagtt tcagcggcgt attgcgggtg atgccatccg tgtcgatcac 660

tctactcgcg gggccatact ggcggtagaa cgctatggcg cgctaggtca attgctagcc 720

tacggcatcg ctggccacca tgccgggttg gccaatggcc gcgaggctgg tgagcgaact 780

gccttggtcg accgcctgaa aggggttggg ctgccacggt tattggaggg gtggtgcgtg 840

gaaatcgtgc tacccgagcg ccttcaacca ccgccactaa aagcgcgcct ggaaagaggt 900

ttctttcagt tggcctttct tggccggatg ctcttttcct gcttggttga tgcggattat 960

ctagataccg aagccttcta ccaccgcgtc gaaggacggc gctcccttcg cgagcaagcg 1020

cggccgacct tggccgagtt acgcgcagcc cttgatcggc atctgactga gttcaaggga 1080

gatacgccgg tcaaccgcgt tcgcggggag atattggccg gcgtgcgcgg caaggcgagc 1140

gaacttcccg ggctgttttc tctcacagtg cccacaggag gcggcaagac cctggcctct 1200

ctggctttcg ccctggatca cgctctagct catgggctgc gccgggtgat ctacgtgatt 1260

cccttcacta gcatcgtcga gcagaacgct gcggtattcc gtcgtgcact cggggcctta 1320

ggcgaagagg cggtgctgga gcatcacagc gccttcgttg atgaccgccg gcagagcctg 1380

gaggccaaga agaaactgaa cctagcgatg gagaactggg acgcgcctat cgtggtgacc 1440

actgcagtgc agttcttcga aagcctgttt gccgaccgtc cagcccagtg ccgcaagcta 1500

cacaacatcg ccggcagcgt ggtgattctt gacgaggcac agaccctacc gctcaagctg 1560

ttgcggccct gcgttgccgc ccttgatgaa ctggcgctca actaccgttg tagcccagtt 1620

ctctgtactg ccacgcagcc agcgcttcaa tcgccggatt tcatcggtgg gctgcaggac 1680

gtacgtgagc tggcgcccga gccgcagcgg ctgttccggg agttggtgcg ggtacgaata 1740

cggacattgg gcccgctcga agatgcggcc ttgactgagc agatcgccag gcgtgaacaa 1800

gtgctgtgca tcgtcaacaa tcgacgccag gcccgtgcgc tctatgagtc gcttgccgag 1860

ttgcccggtg cccgccatct caccaccctg atgtgcgcca agcaccgtag cagcgtgctg 1920

gccgaggtgc gccagatgct caaaaagggg gagccctgtc gcctggtggc cacctcgctg 1980

atcgaggccg gtgtggatgt ggattttccc gtggtactgc gtgccgaggc tggattggat 2040

tccatcgccc aggccgcggg acgctgcaat cgcgaaggca agcggccgct ggccgaaagc 2100

gaggtgctgg tgttcgccgc ggccaattct gactgggcgc cacccgagga actcaagcag 2160

ttcgcccagg ccgcccgcga agtgatgcgc ctgcacccgg atgattgcct gtccatggcg 2220

gccatcgagc ggtattttcg catactgtac tggcagaagg gcgcggagga gttggatgcg 2280

ggtaacctgc tcggcctgat tgagagaggc cggctcgatg gcctgcccta cgagactttg 2340

gccaccaagt tccgcatgat cgacagcctt caactgccgg tgatcatccc atttgatgac 2400

gaggccagag cagccctgcg cgagctggag ttcgccgacg gctgcgccgc catcgcccgt 2460

cgcctgcagc catatctggt gcagatgcca cgcaagggtt atcaggcatt gcgggaagcc 2520

ggtgcgatcc aggcggcggc aggtacgcgt tatggtgagc agtttatggc gttggtcaac 2580

cctgatctgt atcaccacca attcgggttg cactgggata atccggcctt tgtcagcagc 2640

gagcggctat gttggtagtc gggacgcgca acagcggcct ggcctggatg atgtgaaagg 2700

gagggccgat ggcctacgga attcgcttaa tggtctgggg cgagcgtgcc tgcttcaccc 2760

gcccggaaat gaaggtggaa cgcgtctctt acgatgcgat cacgccgtcc gccgcgcgcg 2820

gcattctcga ggctatccac tggaagccgg cgattcgctg ggtggtggat cgcattcaag 2880

tgcttaagcc gatccgcttc gaatccatcc ggcgcaacga ggtcggcggc aagctgtccg 2940

ctgtcagcgt cggtaaggca atgaaggccg ggcgtactaa tggtctggtg aatctggtcg 3000

aggaggatcg ccagcagcgc gcgactactc tgctgcgcga tgtctcctat gtcatcgagg 3060

cgcatttcga gatgactgac agggctggcg ccgacgatac ggtgggcaag catctggata 3120

tcttcaaccg tcgcgcacgg aaggggcagt gcttccatac accctgccta ggcgtgcgcg 3180

agtttccggc cagttttcgg ttgctggaag agggcagtgc cgagcctgaa gtcgatgcct 3240

ttctgcgcgg cgagcgtgat ctgggctgga tgctgcatga cattgacttc gccgatggca 3300

tgaccccgca cttcttccgt gccctgatgc gcgatgggct gatcgaggtg ccggccttca 3360

gggcggcaga ggacaaggca tgatcctttc ggccctcaat gactattatc agcgactgct 3420

ggagcggggt gaagcgaata tctcaccctt cggctacagc caagaaaaga tcagttacgc 3480

cctgctgctg tccgcacaag gagagttgct ggacgtgcag gacattcgct tgctctctgg 3540

caagaagcct caacccaggc ttatgagtgt gccgcagccg gagaagcgca cctcgggcat 3600

caagtccaac gtactgtggg acaagaccag ctatgtgctg ggtgttagtg ccaagggcgg 3660

agagcgtact cagcaggagc acgagtcctt caagacgctg caccggcaga tcttggttgg 3720

ggaaggcgac cccggtctgc aggccttgct ccagttcctc gactgttggc agccggagca 3780

gttcaagccc ccgctgttca gcgaagcaat gctcgacagc aacttagtgt tccgcctaga 3840

cggccaacaa cgctatctgc acgagactcc ggcggccctg gcgttgcgta cccggctgtt 3900

ggccgacggc gacagccgcg aggggctgtg cctagtctgc ggccaacgtc agccgttggc 3960

gcgcctgcat ccagcggtca agggcgtcaa tggtgcccag agttcggggg cttccatcgt 4020

ctccttcaac ctcgacgctt tttcctccta cggcaagagc cagggggaaa atgctccggt 4080

ctccgaacag gccgcctttg cctacaccac ggtgctcaac catttgttgc gtcgcgacga 4140

gcacaaccgc cagcgcctgc agattggcga cgcgagtgtg gtgttctggg cgcaggcgga 4200

tactcctgct caggtggccg ccgccgagtc gaccttctgg aacctgctgg agccacccgc 4260

agatgatggt caggaagcgg aaaagctgcg cggcgtgctg gatgctgtgg ccacggggcg 4320

gcccttgcat gagctcgact cgctaatgga ggaaggtacc cgcatttttg tgttagggct 4380

ggcgcccaat acctcgcgac tgtccattcg gttctgggca gtcgatagcc ttgcggtatt 4440

cacccagcat ctggccgagc atttccggga tatgcacctt gagcctctgc cctggaagac 4500

ggagccggcc atctggcgct tgctctatgc taccgcgccc agtcgtgacg gcagagccaa 4560

gaccgaagac gtactcccac aactggccgg tgaaatgacc cgcgccatcc tgaccggcag 4620

ccgctatccg cgcagtttgc tagccaacct gatcatgcgc atgcgtgccg acggcgacgt 4680

ctctggcata cgcgtcgcgc tgtgcaaggc cgtgctcgct cgcgaggcac gcctgagcgg 4740

caaaattcac caagaggagc tacctatgag tctcgacaag gacgccagca accccggcta 4800

tcgcttgggg aggctgttcg ccgtgttgga aggcgcccag cgcgcagccc tgggcgacag 4860

ggtcaatgcc actatccgtg accgctacta cggtgccgcg tccagcacgc cagccacggt 4920

tttcccgata ctgctgcgca acacacaaaa ccacttggcc aagctgcgca aggagaagcc 4980

cggactagca gtgaacctag agcgcgatat aggcgaaatc attgacggta tgcagagcca 5040

attcccgcgt tgcctgcgcc tggaggacca gggacgcttt gctattggtt actaccaaca 5100

ggcccaggcc cgtttcaacc gtggccccga ttccgtcgag taaggagcag aagaatgacc 5160

gccatctcca accgctacga gttcgtttac ctctttgatg tcagcaatgg caatcccaat 5220

ggcgacccgg atgctggcaa catgccgcgt ctcgatccgg aaaccaacca ggggttggtc 5280

actgacgttt gcctcaagcg caagatccgc aactacgtca gcctggagca ggaaagtgcc 5340

cccggctatg ccatctatat gcaggaaaaa tccgtgctga ataaccagca caaacaggcc 5400

tacgaggcgc tcggtatcga gtcagaggca aagaaactgc ccaaggacga agccaaggcg 5460

cgcgaactga cctcttggat gtgcaagaac ttcttcgatg tgcgtgcttt cggggcggtg 5520

atgaccaccg agattaatgc cggccaggtg cgtggaccga tccaactggc attcgccacg 5580

tctatcgacc cggtattgcc tatggaggta tccatcaccc gcatggcggt gactaacgaa 5640

aaggatttgg agaaggaacg caccatggga cgcaagcaca tcgtgcctta cggcttgtac 5700

cgcgcccatg gtttcatctc tgccaagttg gccgagcgaa ccggcttttc cgacgacgac 5760

ttggaactgc tatggcgcgc tttggccaat atgttcgaac acgaccgctc ggcggcacgt 5820

ggcgagatgg cagcgcgcaa gttgatcgtc ttcaagcatg agcatgccat gggcaatgca 5880

cccgcccatg tgctgttcgg cagcgttaag gtcgagcgag tcgaggggga cgcagttaca 5940

ccagcacgcg gtttccagga ttaccgtgtc agcatcgatg cggaagctct gcctcagggc 6000

gtgagcgtgc gcgagtacct ctag 6024

<210> 26

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 26

gtcgcgcccc gcacgggcgc gtggattgaa ac 32

<210> 27

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 27

gtcgcgcccc gcacgggcgc gtggagtgaa ag 32

<210> 28

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 28

gtcgcgcccc gcacgggtgc gtggattgaa ac 32

<210> 29

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 29

gtcgcgcccc gcatgggcgc gtggattgaa ca 32

<210> 30

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 30

gtcgcgccct acgcgggcgc gtggagtgaa ag 32

<210> 31

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 31

cgccagatgc ccgagctgat cgagcgtggc taca 34

<210> 32

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 32

gaaccaacgc atggcaggat caaaacctgc tgcc 34

<210> 33

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 33

gccgccggag gtctcgttgc ggtaacgggt cgca 34

<210> 34

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 34

cgggttgccg cctttcaggt tgaccacgac accg 34

<210> 35

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 35

cagagccatc accagctcga cggtgtcaag ggag 34

<210> 36

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 36

accagccgcg acaaagccgc tgcccacctg cagg 34

<210> 37

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 37

cgtgtggagt tggaaaatgg gcacgtcgtc accg 34

<210> 38

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 38

agcggctgcc ccggagcgat cgcttgcgcg acgt 34

<210> 39

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 39

cctttgacac ccagtaccgt cacggtgacg tcgt 34

<210> 40

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 40

ttcatcgacg tgctcttcga tgaagcgctc gtcg 34

<210> 41

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 41

ggtgctgacc gaggacgaga aggaactggg cgtg 34

<210> 42

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 42

aacatcatcg acacccccgg ccacgtcgac ttca 34

<210> 43

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 43

tggtggaaca cgtcgccata ggtgaccttg ccga 34

<210> 44

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 44

cagccggccc agtcggactc gtccatgccg tcct 34

<210> 45

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 45

ggcttgggct tgtcgccgct atcggccacg cgac 34

<210> 46

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 46

tccgcgatga gctgccgtcc caacaattca acac 34

<210> 47

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 47

cacctggtgg aacatcggcg agtgggtcag gtcg 34

<210> 48

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 48

agctcgatac catgaacagt gctacccacc ggga 34

<210> 49

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 49

tcgcgctcgt ccggcatcga ccagcccatc cgcc 34

<210> 50

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 50

cacgcccatg gcgaaaccga cacccggggt cggc 34

<210> 51

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 51

cagcgcttga tggtgcccgc gaagccctta ccct 34

<210> 52

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 52

gccgctacct ggacgacaac ggcttcctcg acgt 34

<210> 53

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 53

cgccagatgc ccgagctgat cgagcgtggc taca 34

<210> 54

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 54

accaccgaga cgcccacacc gtgcaagccg ccgg 34

<210> 55

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 55

gatgacacca acccggccaa ggaagaccag gagt 34

<210> 56

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 56

ctggtacagg atgatgccgt aggtgggctt gagc 34

<210> 57

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 57

gggatccaga gtgccgttgg tttccaggtc cagg 34

<210> 58

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 58

cccgtcggct accaccgtta gttccagggc ttgc 34

<210> 59

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 59

cttgatcggc ttgccgttct cgtcgagcat ggcg 34

<210> 60

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 60

gtaggtggca tagtcgatat cggcgcgcag ggtg 34

<210> 61

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 61

gtaatcaacc ggatgggaga agccgaggga cagg 34

<210> 62

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 62

tgggcgatgg gcgataccgg accctgcggt ccct 34

<210> 63

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 63

gacgccatcg gcgccgacct cgaggccaag ggcc 34

<210> 64

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 64

cagcgcttcg taggaggtgg ccggatcgac gatc 34

<210> 65

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 65

agacccaggg tgtccagctt ggcaaccagg ccct 34

<210> 66

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 66

ctggtattcg gagagcagct tctcgtgctc cagg 34

<210> 67

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 67

cgggccggcc tgggtcagca gggtcaggtc ggat 34

<210> 68

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 68

ggcgggttgt gctcggtgtg gtacacgcgg ccgg 34

<210> 69

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 69

agagcgcgaa cggcggactc gcggcccggg cccg 34

<210> 70

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 70

ttggctgcat cgatgttgcc ggtggcacct tcgc 34

<210> 71

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 71

gaagctggcc cccggcggcg gcgtcagccg gccg 34

<210> 72

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 72

gaacccccga cacccttttg aggtgtactc cctt 34

<210> 73

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 73

cgcgatagct cagtcggtag agcaaatgac tgtt 34

<210> 74

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 74

cgggttgtgc tgggtggaca gcaccaccgc atcg 34

<210> 75

<211> 2235

<212> DNA

<213> Pseudomonas aeruginosa

<400> 75

atggacgcgg aggctagcga tactcacttt tttgctcact ccaccttaaa ggcagatcgc 60

agcgattggc agcctctggt cgagcatcta caggctgttg cccgtttggc aggagagaag 120

gctgccttct tcggcggcgg tgaattagct gctcttgctg gtctgttgca tgacttgggt 180

aaatacactg acgagtttca gcggcgtatt gcgggtgatg ccatccgtgt cgatcactct 240

actcgcgggg ccatactggc ggtagaacgc tatggcgcgc taggtcaatt gctagcctac 300

ggcatcgctg gccaccatgc cgggttggcc aatggccgcg aggctggtga gcgaactgcc 360

ttggtcgacc gcctgaaagg ggttgggctg ccacggttat tggaggggtg gtgcgtggaa 420

atcgtgctac ccgagcgcct tcaaccaccg ccactaaaag cgcgcctgga aagaggtttc 480

tttcagttgg cctttcttgg ccggatgctc ttttcctgct tggttgatgc ggattatcta 540

gataccgaag ccttctacca ccgcgtcgaa ggacggcgct cccttcgcga gcaagcgcgg 600

ccgaccttgg ccgagttacg cgcagccctt gatcggcatc tgactgagtt caagggagat 660

acgccggtca accgcgttcg cggggagata ttggccggcg tgcgcggcaa ggcgagcgaa 720

cttcccgggc tgttttctct cacagtgccc acaggaggcg gcaagaccct ggcctctctg 780

gctttcgccc tggatcacgc tctagctcat gggctgcgcc gggtgatcta cgtgattccc 840

ttcactagca tcgtcgagca gaacgctgcg gtattccgtc gtgcactcgg ggccttaggc 900

gaagaggcgg tgctggagca tcacagcgcc ttcgttgatg accgccggca gagcctggag 960

gccaagaaga aactgaacct agcgatggag aactgggacg cgcctatcgt ggtgaccact 1020

gcagtgcagt tcttcgaaag cctgtttgcc gaccgtccag cccagtgccg caagctacac 1080

aacatcgccg gcagcgtggt gattcttgac gaggcacaga ccctaccgct caagctgttg 1140

cggccctgcg ttgccgccct tgatgaactg gcgctcaact accgttgtag cccagttctc 1200

tgtactgcca cgcagccagc gcttcaatcg ccggatttca tcggtgggct gcaggacgta 1260

cgtgagctgg cgcccgagcc gcagcggctg ttccgggagt tggtgcgggt acgaatacgg 1320

acattgggcc cgctcgaaga tgcggccttg actgagcaga tcgccaggcg tgaacaagtg 1380

ctgtgcatcg tcaacaatcg acgccaggcc cgtgcgctct atgagtcgct tgccgagttg 1440

cccggtgccc gccatctcac caccctgatg tgcgccaagc accgtagcag cgtgctggcc 1500

gaggtgcgcc agatgctcaa aaagggggag ccctgtcgcc tggtggccac ctcgctgatc 1560

gaggccggtg tggatgtgga ttttcccgtg gtactgcgtg ccgaggctgg attggattcc 1620

atcgcccagg ccgcgggacg ctgcaatcgc gaaggcaagc ggccgctggc cgaaagcgag 1680

gtgctggtgt tcgccgcggc caattctgac tgggcgccac ccgaggaact caagcagttc 1740

gcccaggccg cccgcgaagt gatgcgcctg cacccggatg attgcctgtc catggcggcc 1800

atcgagcggt attttcgcat actgtactgg cagaagggcg cggaggagtt ggatgcgggt 1860

aacctgctcg gcctgattga gagaggccgg ctcgatggcc tgccctacga gactttggcc 1920

accaagttcc gcatgatcga cagccttcaa ctgccggtga tcatcccatt tgatgacgag 1980

gccagagcag ccctgcgcga gctggagttc gccgacggct gcgccgccat cgcccgtcgc 2040

ctgcagccat atctggtgca gatgccacgc aagggttatc aggcattgcg ggaagccggt 2100

gcgatccagg cggcggcagg tacgcgttat ggtgagcagt ttatggcgtt ggtcaaccct 2160

gatctgtatc accaccaatt cgggttgcac tgggataatc cggcctttgt cagcagcgag 2220

cggctatgtt ggtag 2235

<210> 76

<211> 675

<212> DNA

<213> Pseudomonas aeruginosa

<400> 76

atggcctacg gaattcgctt aatggtctgg ggcgagcgtg cctgcttcac ccgcccggaa 60

atgaaggtgg aacgcgtctc ttacgatgcg atcacgccgt ccgccgcgcg cggcattctc 120

gaggctatcc actggaagcc ggcgattcgc tgggtggtgg atcgcattca agtgcttaag 180

ccgatccgct tcgaatccat ccggcgcaac gaggtcggcg gcaagctgtc cgctgtcagc 240

gtcggtaagg caatgaaggc cgggcgtact aatggtctgg tgaatctggt cgaggaggat 300

cgccagcagc gcgcgactac tctgctgcgc gatgtctcct atgtcatcga ggcgcatttc 360

gagatgactg acagggctgg cgccgacgat acggtgggca agcatctgga tatcttcaac 420

cgtcgcgcac ggaaggggca gtgcttccat acaccctgcc taggcgtgcg cgagtttccg 480

gccagttttc ggttgctgga agagggcagt gccgagcctg aagtcgatgc ctttctgcgc 540

ggcgagcgtg atctgggctg gatgctgcat gacattgact tcgccgatgg catgaccccg 600

cacttcttcc gtgccctgat gcgcgatggg ctgatcgagg tgccggcctt cagggcggca 660

gaggacaagg catga 675

<210> 77

<211> 1764

<212> DNA

<213> Pseudomonas aeruginosa

<400> 77

atgatccttt cggccctcaa tgactattat cagcgactgc tggagcgggg tgaagcgaat 60

atctcaccct tcggctacag ccaagaaaag atcagttacg ccctgctgct gtccgcacaa 120

ggagagttgc tggacgtgca ggacattcgc ttgctctctg gcaagaagcc tcaacccagg 180

cttatgagtg tgccgcagcc ggagaagcgc acctcgggca tcaagtccaa cgtactgtgg 240

gacaagacca gctatgtgct gggtgttagt gccaagggcg gagagcgtac tcagcaggag 300

cacgagtcct tcaagacgct gcaccggcag atcttggttg gggaaggcga ccccggtctg 360

caggccttgc tccagttcct cgactgttgg cagccggagc agttcaagcc cccgctgttc 420

agcgaagcaa tgctcgacag caacttagtg ttccgcctag acggccaaca acgctatctg 480

cacgagactc cggcggccct ggcgttgcgt acccggctgt tggccgacgg cgacagccgc 540

gaggggctgt gcctagtctg cggccaacgt cagccgttgg cgcgcctgca tccagcggtc 600

aagggcgtca atggtgccca gagttcgggg gcttccatcg tctccttcaa cctcgacgct 660

ttttcctcct acggcaagag ccagggggaa aatgctccgg tctccgaaca ggccgccttt 720

gcctacacca cggtgctcaa ccatttgttg cgtcgcgacg agcacaaccg ccagcgcctg 780

cagattggcg acgcgagtgt ggtgttctgg gcgcaggcgg atactcctgc tcaggtggcc 840

gccgccgagt cgaccttctg gaacctgctg gagccacccg cagatgatgg tcaggaagcg 900

gaaaagctgc gcggcgtgct ggatgctgtg gccacggggc ggcccttgca tgagctcgac 960

tcgctaatgg aggaaggtac ccgcattttt gtgttagggc tggcgcccaa tacctcgcga 1020

ctgtccattc ggttctgggc agtcgatagc cttgcggtat tcacccagca tctggccgag 1080

catttccggg atatgcacct tgagcctctg ccctggaaga cggagccggc catctggcgc 1140

ttgctctatg ctaccgcgcc cagtcgtgac ggcagagcca agaccgaaga cgtactccca 1200

caactggccg gtgaaatgac ccgcgccatc ctgaccggca gccgctatcc gcgcagtttg 1260

ctagccaacc tgatcatgcg catgcgtgcc gacggcgacg tctctggcat acgcgtcgcg 1320

ctgtgcaagg ccgtgctcgc tcgcgaggca cgcctgagcg gcaaaattca ccaagaggag 1380

ctacctatga gtctcgacaa ggacgccagc aaccccggct atcgcttggg gaggctgttc 1440

gccgtgttgg aaggcgccca gcgcgcagcc ctgggcgaca gggtcaatgc cactatccgt 1500

gaccgctact acggtgccgc gtccagcacg ccagccacgg ttttcccgat actgctgcgc 1560

aacacacaaa accacttggc caagctgcgc aaggagaagc ccggactagc agtgaaccta 1620

gagcgcgata taggcgaaat cattgacggt atgcagagcc aattcccgcg ttgcctgcgc 1680

ctggaggacc agggacgctt tgctattggt tactaccaac aggcccaggc ccgtttcaac 1740

cgtggccccg attccgtcga gtaa 1764

<210> 78

<211> 870

<212> DNA

<213> Pseudomonas aeruginosa

<400> 78

atgaccgcca tctccaaccg ctacgagttc gtttacctct ttgatgtcag caatggcaat 60

cccaatggcg acccggatgc tggcaacatg ccgcgtctcg atccggaaac caaccagggg 120

ttggtcactg acgtttgcct caagcgcaag atccgcaact acgtcagcct ggagcaggaa 180

agtgcccccg gctatgccat ctatatgcag gaaaaatccg tgctgaataa ccagcacaaa 240

caggcctacg aggcgctcgg tatcgagtca gaggcaaaga aactgcccaa ggacgaagcc 300

aaggcgcgcg aactgacctc ttggatgtgc aagaacttct tcgatgtgcg tgctttcggg 360

gcggtgatga ccaccgagat taatgccggc caggtgcgtg gaccgatcca actggcattc 420

gccacgtcta tcgacccggt attgcctatg gaggtatcca tcacccgcat ggcggtgact 480

aacgaaaagg atttggagaa ggaacgcacc atgggacgca agcacatcgt gccttacggc 540

ttgtaccgcg cccatggttt catctctgcc aagttggccg agcgaaccgg cttttccgac 600

gacgacttgg aactgctatg gcgcgctttg gccaatatgt tcgaacacga ccgctcggcg 660

gcacgtggcg agatggcagc gcgcaagttg atcgtcttca agcatgagca tgccatgggc 720

aatgcacccg cccatgtgct gttcggcagc gttaaggtcg agcgagtcga gggggacgca 780

gttacaccag cacgcggttt ccaggattac cgtgtcagca tcgatgcgga agctctgcct 840

cagggcgtga gcgtgcgcga gtacctctag 870

<210> 79

<211> 744

<212> PRT

<213> Pseudomonas aeruginosa

<400> 79

Met Asp Ala Glu Ala Ser Asp Thr His Phe Phe Ala His Ser Thr Leu

1 5 10 15

Lys Ala Asp Arg Ser Asp Trp Gln Pro Leu Val Glu His Leu Gln Ala

20 25 30

Val Ala Arg Leu Ala Gly Glu Lys Ala Ala Phe Phe Gly Gly Gly Glu

35 40 45

Leu Ala Ala Leu Ala Gly Leu Leu His Asp Leu Gly Lys Tyr Thr Asp

50 55 60

Glu Phe Gln Arg Arg Ile Ala Gly Asp Ala Ile Arg Val Asp His Ser

65 70 75 80

Thr Arg Gly Ala Ile Leu Ala Val Glu Arg Tyr Gly Ala Leu Gly Gln

85 90 95

Leu Leu Ala Tyr Gly Ile Ala Gly His His Ala Gly Leu Ala Asn Gly

100 105 110

Arg Glu Ala Gly Glu Arg Thr Ala Leu Val Asp Arg Leu Lys Gly Val

115 120 125

Gly Leu Pro Arg Leu Leu Glu Gly Trp Cys Val Glu Ile Val Leu Pro

130 135 140

Glu Arg Leu Gln Pro Pro Pro Leu Lys Ala Arg Leu Glu Arg Gly Phe

145 150 155 160

Phe Gln Leu Ala Phe Leu Gly Arg Met Leu Phe Ser Cys Leu Val Asp

165 170 175

Ala Asp Tyr Leu Asp Thr Glu Ala Phe Tyr His Arg Val Glu Gly Arg

180 185 190

Arg Ser Leu Arg Glu Gln Ala Arg Pro Thr Leu Ala Glu Leu Arg Ala

195 200 205

Ala Leu Asp Arg His Leu Thr Glu Phe Lys Gly Asp Thr Pro Val Asn

210 215 220

Arg Val Arg Gly Glu Ile Leu Ala Gly Val Arg Gly Lys Ala Ser Glu

225 230 235 240

Leu Pro Gly Leu Phe Ser Leu Thr Val Pro Thr Gly Gly Gly Lys Thr

245 250 255

Leu Ala Ser Leu Ala Phe Ala Leu Asp His Ala Leu Ala His Gly Leu

260 265 270

Arg Arg Val Ile Tyr Val Ile Pro Phe Thr Ser Ile Val Glu Gln Asn

275 280 285

Ala Ala Val Phe Arg Arg Ala Leu Gly Ala Leu Gly Glu Glu Ala Val

290 295 300

Leu Glu His His Ser Ala Phe Val Asp Asp Arg Arg Gln Ser Leu Glu

305 310 315 320

Ala Lys Lys Lys Leu Asn Leu Ala Met Glu Asn Trp Asp Ala Pro Ile

325 330 335

Val Val Thr Thr Ala Val Gln Phe Phe Glu Ser Leu Phe Ala Asp Arg

340 345 350

Pro Ala Gln Cys Arg Lys Leu His Asn Ile Ala Gly Ser Val Val Ile

355 360 365

Leu Asp Glu Ala Gln Thr Leu Pro Leu Lys Leu Leu Arg Pro Cys Val

370 375 380

Ala Ala Leu Asp Glu Leu Ala Leu Asn Tyr Arg Cys Ser Pro Val Leu

385 390 395 400

Cys Thr Ala Thr Gln Pro Ala Leu Gln Ser Pro Asp Phe Ile Gly Gly

405 410 415

Leu Gln Asp Val Arg Glu Leu Ala Pro Glu Pro Gln Arg Leu Phe Arg

420 425 430

Glu Leu Val Arg Val Arg Ile Arg Thr Leu Gly Pro Leu Glu Asp Ala

435 440 445

Ala Leu Thr Glu Gln Ile Ala Arg Arg Glu Gln Val Leu Cys Ile Val

450 455 460

Asn Asn Arg Arg Gln Ala Arg Ala Leu Tyr Glu Ser Leu Ala Glu Leu

465 470 475 480

Pro Gly Ala Arg His Leu Thr Thr Leu Met Cys Ala Lys His Arg Ser

485 490 495

Ser Val Leu Ala Glu Val Arg Gln Met Leu Lys Lys Gly Glu Pro Cys

500 505 510

Arg Leu Val Ala Thr Ser Leu Ile Glu Ala Gly Val Asp Val Asp Phe

515 520 525

Pro Val Val Leu Arg Ala Glu Ala Gly Leu Asp Ser Ile Ala Gln Ala

530 535 540

Ala Gly Arg Cys Asn Arg Glu Gly Lys Arg Pro Leu Ala Glu Ser Glu

545 550 555 560

Val Leu Val Phe Ala Ala Ala Asn Ser Asp Trp Ala Pro Pro Glu Glu

565 570 575

Leu Lys Gln Phe Ala Gln Ala Ala Arg Glu Val Met Arg Leu His Pro

580 585 590

Asp Asp Cys Leu Ser Met Ala Ala Ile Glu Arg Tyr Phe Arg Ile Leu

595 600 605

Tyr Trp Gln Lys Gly Ala Glu Glu Leu Asp Ala Gly Asn Leu Leu Gly

610 615 620

Leu Ile Glu Arg Gly Arg Leu Asp Gly Leu Pro Tyr Glu Thr Leu Ala

625 630 635 640

Thr Lys Phe Arg Met Ile Asp Ser Leu Gln Leu Pro Val Ile Ile Pro

645 650 655

Phe Asp Asp Glu Ala Arg Ala Ala Leu Arg Glu Leu Glu Phe Ala Asp

660 665 670

Gly Cys Ala Ala Ile Ala Arg Arg Leu Gln Pro Tyr Leu Val Gln Met

675 680 685

Pro Arg Lys Gly Tyr Gln Ala Leu Arg Glu Ala Gly Ala Ile Gln Ala

690 695 700

Ala Ala Gly Thr Arg Tyr Gly Glu Gln Phe Met Ala Leu Val Asn Pro

705 710 715 720

Asp Leu Tyr His His Gln Phe Gly Leu His Trp Asp Asn Pro Ala Phe

725 730 735

Val Ser Ser Glu Arg Leu Cys Trp

740

<210> 80

<211> 224

<212> PRT

<213> Pseudomonas aeruginosa

<400> 80

Met Ala Tyr Gly Ile Arg Leu Met Val Trp Gly Glu Arg Ala Cys Phe

1 5 10 15

Thr Arg Pro Glu Met Lys Val Glu Arg Val Ser Tyr Asp Ala Ile Thr

20 25 30

Pro Ser Ala Ala Arg Gly Ile Leu Glu Ala Ile His Trp Lys Pro Ala

35 40 45

Ile Arg Trp Val Val Asp Arg Ile Gln Val Leu Lys Pro Ile Arg Phe

50 55 60

Glu Ser Ile Arg Arg Asn Glu Val Gly Gly Lys Leu Ser Ala Val Ser

65 70 75 80

Val Gly Lys Ala Met Lys Ala Gly Arg Thr Asn Gly Leu Val Asn Leu

85 90 95

Val Glu Glu Asp Arg Gln Gln Arg Ala Thr Thr Leu Leu Arg Asp Val

100 105 110

Ser Tyr Val Ile Glu Ala His Phe Glu Met Thr Asp Arg Ala Gly Ala

115 120 125

Asp Asp Thr Val Gly Lys His Leu Asp Ile Phe Asn Arg Arg Ala Arg

130 135 140

Lys Gly Gln Cys Phe His Thr Pro Cys Leu Gly Val Arg Glu Phe Pro

145 150 155 160

Ala Ser Phe Arg Leu Leu Glu Glu Gly Ser Ala Glu Pro Glu Val Asp

165 170 175

Ala Phe Leu Arg Gly Glu Arg Asp Leu Gly Trp Met Leu His Asp Ile

180 185 190

Asp Phe Ala Asp Gly Met Thr Pro His Phe Phe Arg Ala Leu Met Arg

195 200 205

Asp Gly Leu Ile Glu Val Pro Ala Phe Arg Ala Ala Glu Asp Lys Ala

210 215 220

<210> 81

<211> 587

<212> PRT

<213> Pseudomonas aeruginosa

<400> 81

Met Ile Leu Ser Ala Leu Asn Asp Tyr Tyr Gln Arg Leu Leu Glu Arg

1 5 10 15

Gly Glu Ala Asn Ile Ser Pro Phe Gly Tyr Ser Gln Glu Lys Ile Ser

20 25 30

Tyr Ala Leu Leu Leu Ser Ala Gln Gly Glu Leu Leu Asp Val Gln Asp

35 40 45

Ile Arg Leu Leu Ser Gly Lys Lys Pro Gln Pro Arg Leu Met Ser Val

50 55 60

Pro Gln Pro Glu Lys Arg Thr Ser Gly Ile Lys Ser Asn Val Leu Trp

65 70 75 80

Asp Lys Thr Ser Tyr Val Leu Gly Val Ser Ala Lys Gly Gly Glu Arg

85 90 95

Thr Gln Gln Glu His Glu Ser Phe Lys Thr Leu His Arg Gln Ile Leu

100 105 110

Val Gly Glu Gly Asp Pro Gly Leu Gln Ala Leu Leu Gln Phe Leu Asp

115 120 125

Cys Trp Gln Pro Glu Gln Phe Lys Pro Pro Leu Phe Ser Glu Ala Met

130 135 140

Leu Asp Ser Asn Leu Val Phe Arg Leu Asp Gly Gln Gln Arg Tyr Leu

145 150 155 160

His Glu Thr Pro Ala Ala Leu Ala Leu Arg Thr Arg Leu Leu Ala Asp

165 170 175

Gly Asp Ser Arg Glu Gly Leu Cys Leu Val Cys Gly Gln Arg Gln Pro

180 185 190

Leu Ala Arg Leu His Pro Ala Val Lys Gly Val Asn Gly Ala Gln Ser

195 200 205

Ser Gly Ala Ser Ile Val Ser Phe Asn Leu Asp Ala Phe Ser Ser Tyr

210 215 220

Gly Lys Ser Gln Gly Glu Asn Ala Pro Val Ser Glu Gln Ala Ala Phe

225 230 235 240

Ala Tyr Thr Thr Val Leu Asn His Leu Leu Arg Arg Asp Glu His Asn

245 250 255

Arg Gln Arg Leu Gln Ile Gly Asp Ala Ser Val Val Phe Trp Ala Gln

260 265 270

Ala Asp Thr Pro Ala Gln Val Ala Ala Ala Glu Ser Thr Phe Trp Asn

275 280 285

Leu Leu Glu Pro Pro Ala Asp Asp Gly Gln Glu Ala Glu Lys Leu Arg

290 295 300

Gly Val Leu Asp Ala Val Ala Thr Gly Arg Pro Leu His Glu Leu Asp

305 310 315 320

Ser Leu Met Glu Glu Gly Thr Arg Ile Phe Val Leu Gly Leu Ala Pro

325 330 335

Asn Thr Ser Arg Leu Ser Ile Arg Phe Trp Ala Val Asp Ser Leu Ala

340 345 350

Val Phe Thr Gln His Leu Ala Glu His Phe Arg Asp Met His Leu Glu

355 360 365

Pro Leu Pro Trp Lys Thr Glu Pro Ala Ile Trp Arg Leu Leu Tyr Ala

370 375 380

Thr Ala Pro Ser Arg Asp Gly Arg Ala Lys Thr Glu Asp Val Leu Pro

385 390 395 400

Gln Leu Ala Gly Glu Met Thr Arg Ala Ile Leu Thr Gly Ser Arg Tyr

405 410 415

Pro Arg Ser Leu Leu Ala Asn Leu Ile Met Arg Met Arg Ala Asp Gly

420 425 430

Asp Val Ser Gly Ile Arg Val Ala Leu Cys Lys Ala Val Leu Ala Arg

435 440 445

Glu Ala Arg Leu Ser Gly Lys Ile His Gln Glu Glu Leu Pro Met Ser

450 455 460

Leu Asp Lys Asp Ala Ser Asn Pro Gly Tyr Arg Leu Gly Arg Leu Phe

465 470 475 480

Ala Val Leu Glu Gly Ala Gln Arg Ala Ala Leu Gly Asp Arg Val Asn

485 490 495

Ala Thr Ile Arg Asp Arg Tyr Tyr Gly Ala Ala Ser Ser Thr Pro Ala

500 505 510

Thr Val Phe Pro Ile Leu Leu Arg Asn Thr Gln Asn His Leu Ala Lys

515 520 525

Leu Arg Lys Glu Lys Pro Gly Leu Ala Val Asn Leu Glu Arg Asp Ile

530 535 540

Gly Glu Ile Ile Asp Gly Met Gln Ser Gln Phe Pro Arg Cys Leu Arg

545 550 555 560

Leu Glu Asp Gln Gly Arg Phe Ala Ile Gly Tyr Tyr Gln Gln Ala Gln

565 570 575

Ala Arg Phe Asn Arg Gly Pro Asp Ser Val Glu

580 585

<210> 82

<211> 289

<212> PRT

<213> Pseudomonas aeruginosa

<400> 82

Met Thr Ala Ile Ser Asn Arg Tyr Glu Phe Val Tyr Leu Phe Asp Val

1 5 10 15

Ser Asn Gly Asn Pro Asn Gly Asp Pro Asp Ala Gly Asn Met Pro Arg

20 25 30

Leu Asp Pro Glu Thr Asn Gln Gly Leu Val Thr Asp Val Cys Leu Lys

35 40 45

Arg Lys Ile Arg Asn Tyr Val Ser Leu Glu Gln Glu Ser Ala Pro Gly

50 55 60

Tyr Ala Ile Tyr Met Gln Glu Lys Ser Val Leu Asn Asn Gln His Lys

65 70 75 80

Gln Ala Tyr Glu Ala Leu Gly Ile Glu Ser Glu Ala Lys Lys Leu Pro

85 90 95

Lys Asp Glu Ala Lys Ala Arg Glu Leu Thr Ser Trp Met Cys Lys Asn

100 105 110

Phe Phe Asp Val Arg Ala Phe Gly Ala Val Met Thr Thr Glu Ile Asn

115 120 125

Ala Gly Gln Val Arg Gly Pro Ile Gln Leu Ala Phe Ala Thr Ser Ile

130 135 140

Asp Pro Val Leu Pro Met Glu Val Ser Ile Thr Arg Met Ala Val Thr

145 150 155 160

Asn Glu Lys Asp Leu Glu Lys Glu Arg Thr Met Gly Arg Lys His Ile

165 170 175

Val Pro Tyr Gly Leu Tyr Arg Ala His Gly Phe Ile Ser Ala Lys Leu

180 185 190

Ala Glu Arg Thr Gly Phe Ser Asp Asp Asp Leu Glu Leu Leu Trp Arg

195 200 205

Ala Leu Ala Asn Met Phe Glu His Asp Arg Ser Ala Ala Arg Gly Glu

210 215 220

Met Ala Ala Arg Lys Leu Ile Val Phe Lys His Glu His Ala Met Gly

225 230 235 240

Asn Ala Pro Ala His Val Leu Phe Gly Ser Val Lys Val Glu Arg Val

245 250 255

Glu Gly Asp Ala Val Thr Pro Ala Arg Gly Phe Gln Asp Tyr Arg Val

260 265 270

Ser Ile Asp Ala Glu Ala Leu Pro Gln Gly Val Ser Val Arg Glu Tyr

275 280 285

Leu

<210> 83

<211> 369

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Polynucleotide

<400> 83

acaagcggca cattgtgcct attgcgaatt aggcacaatg tgcctaatct aacgtcatgc 60

cagccacaac ggcgaggcgc caagaaggat agaagccgtc gcgccccgca cgggcgcgtg 120

gattgaaaca gaagggtcag ggccatgcgg tttttcctct gtggtcgcgc cccgcacggg 180

cgcgtggatt gaaacgagac cgaagagaac gtgccgacca ccgccgctgg tcgcgccccg 240

cacgggcgcg tggattgaaa ctgctgaaca gccatgattg attaactcct aaacggtcgc 300

gccccgcacg ggcgcgtgga ttgaaaccat gcaagcttgg cgtagcttcg tccctatcaa 360

agcttggag 369

<210> 84

<211> 373

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Polynucleotide

<400> 84

acaagcggca cattgtgcct attgcgaatt aggcacaatg tgcctaatct aacgtcatgc 60

cagccacaac ggcgaggcgc caagaaggat agaagccgtc gcgccccgca cgggcgcgtg 120

gattgaaacg ctcgactggt cggtaaccac ttgtgtgtgg tgagtcgcgc cccgcacggg 180

cgcgtggatt gaaaccagtg catggcagcg aacgccgaga gccgacaccg tcgcgccccg 240

cacgggcgcg tggattgaaa ccgtaaacct aatgggcctg atctacagta atctagtcgc 300

gccccgcacg ggcgcgtgga ttgaaaccat gcaagcttgg cgtaggccgc ttcgtcccta 360

tcaaagcttg gag 373

<210> 85

<211> 369

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Polynucleotide

<400> 85

acaagcggca cattgtgcct attgcgaatt aggcacaatg tgcctaatct aacgtcatgc 60

cagccacaac ggcgaggcgc caagaaggat agaagccgtc gcgccccgca cgggcgcgtg 120

gattgaaacg gtgctgaccg aggacgagaa ggaactgggc gtggtcgcgc cccgcacggg 180

cgcgtggatt gaaactccgc gatgagctgc cgtcccaaca attcaacacg tcgcgccccg 240

cacgggcgcg tggattgaaa caccaccgag acgcccacac cgtgcaagcc gccgggtcgc 300

gccccgcacg ggcgcgtgga ttgaaaccat gcaagcttgg cgtagcttcg tccctatcaa 360

agcttggag 369

<210> 86

<211> 369

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Polynucleotide

<400> 86

acaagcggca cattgtgcct attgcgaatt aggcacaatg tgcctaatct aacgtcatgc 60

cagccacaac ggcgaggcgc caagaaggat agaagccgtc gcgccccgca cgggcgcgtg 120

gattgaaacg atgacaccaa cccggccaag gaagaccagg agtgtcgcgc cccgcacggg 180

cgcgtggatt gaaacaacgc gaagccctgt tgaaaccgct gcaactggtg tcgcgccccg 240

cacgggcgcg tggattgaaa cctatcgcga attcctgcag gctggcgcaa ccaaggtcgc 300

gccccgcacg ggcgcgtgga ttgaaaccat gcaagcttgg cgtagcttcg tccctatcaa 360

agcttggag 369

<210> 87

<211> 177

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Polynucleotide

<400> 87

gaaaattatt ttaaatttcc tctagtcagg ccggaataac tccctataat gcgacaccag 60

tcgcgccccg cacgggcgcg tggattgaaa catttatcac aaaaggattg ttcgatgtcc 120

aacaagtcgc gccccgcacg ggcgcgtgga ttgaaacgca ctcccgttct ggataat 177

<210> 88

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (11)..(11)

<223> a、c、t

g

<220>

<221> modified base

<222> (14)..(14)

<223> a, c, t or g

<220>

<221> modified base

<222> (32)..(32)

<223> a, c, t or g

<400> 88

agaagggtca nggncatgcg gtttttcctc tntg 34

<210> 89

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (7)..(7)

<223> a, c, t or g

<220>

<221> modified base

<222> (14)..(14)

<223> a, c, t or g

<220>

<221> modified base

<222> (32)..(32)

<223> a, c, t or g

<400> 89

agaaggntca gggncatgcg gtttttcctc tntg 34

<210> 90

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (7)..(7)

<223> a, c, t or g

<220>

<221> modified base

<222> (11)..(11)

<223> a, c, t or g

<220>

<221> modified base

<222> (32)..(32)

<223> a, c, t or g

<400> 90

agaaggntca nggccatgcg gtttttcctc tntg 34

<210> 91

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (7)..(7)

<223> a, c, t or g

<220>

<221> modified base

<222> (11)..(11)

<223> a, c, t or g

<220>

<221> modified base

<222> (14)..(14)

<223> a, c, t or g

<400> 91

agaaggntca nggncatgcg gtttttcctc tgtg 34

<210> 92

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (7)..(7)

<223> a, c, t or g

<220>

<221> modified base

<222> (11)..(11)

<223> a, c, t or g

<220>

<221> modified base

<222> (14)..(14)

<223> a, c, t or g

<220>

<221> modified base

<222> (32)..(32)

<223> a, c, t or g

<400> 92

agaaggntca nggncatgcg gtttttcctc tntg 34

<210> 93

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (14)..(14)

<223> a, c, t or g

<400> 93

agaagggtca gggncatgcg gtttttcctc tgtg 34

<210> 94

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 94

agaagggtca gggtcatgcg gtttttcctc tgtg 34

<210> 95

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (32)..(32)

<223> a, c, t or g

<400> 95

agaagggtca gggccatgcg gtttttcctc tntg 34

<210> 96

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 96

agaagggtca gggccatgcg gtttttcctc tatg 34

<210> 97

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (7)..(7)

<223> a, c, t or g

<400> 97

agaaggntca gggccatgcg gtttttcctc tgtg 34

<210> 98

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 98

agaaggatca gggccatgcg gtttttcctc tgtg 34

<210> 99

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (7)..(7)

<223> a, c, t or g

<220>

<221> modified base

<222> (11)..(11)

<223> a, c, t or g

<400> 99

agaaggntca nggccatgcg gtttttcctc tgtg 34

<210> 100

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 100

agaaggatca aggccatgcg gtttttcctc tgtg 34

<210> 101

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (32)..(32)

<223> a, c, t or g

<400> 101

agaagggtca gggccatgcg gtttttcctc tntg 34

<210> 102

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 102

agaagggtca gggccatgcg gtttttcctc tatg 34

<210> 103

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (13)..(13)

<223> a, c, t or g

<220>

<221> modified base

<222> (15)..(15)

<223> a, c, t or g

<220>

<221> modified base

<222> (27)..(27)

<223> a, c, t or g

<400> 103

gagaccgaag agnangtgcc gaccacngcc gctg 34

<210> 104

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (15)..(15)

<223> a, c, t or g

<400> 104

gagaccgaag agaangtgcc gaccaccgcc gctg 34

<210> 105

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 105

gagaccgaag agaatgtgcc gaccaccgcc gctg 34

<210> 106

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (27)..(27)

<223> a, c, t or g

<400> 106

gagaccgaag agaacgtgcc gaccacngcc gctg 34

<210> 107

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 107

gagaccgaag agaacgtgcc gaccactgcc gctg 34

<210> 108

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (13)..(13)

<223> a, c, t or g

<400> 108

gagaccgaag agnacgtgcc gaccaccgcc gc 32

<210> 109

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 109

gagaccgaag aggacgtgcc gaccaccgcc gc 32

<210> 110

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (15)..(15)

<223> a, c, t or g

<220>

<221> modified base

<222> (17)..(19)

<223> a, c, t or g

<400> 110

tgctgaacag ccatnannna ttaactccta aacg 34

<210> 111

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (15)..(15)

<223> a, c, t or g

<400> 111

tgctgaacag ccatnattga ttaactccta aacg 34

<210> 112

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 112

tgctgaacag ccataattga ttaactccta aacg 34

<210> 113

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (15)..(15)

<223> a, c, t or g

<220>

<221> modified base

<222> (19)..(19)

<223> a, c, t or g

<400> 113

tgctgaacag ccatnattna ttaactccta aacg 34

<210> 114

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 114

tgctgaacag ccataattaa ttaactccta aacg 34

<210> 115

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (15)..(15)

<223> a, c, t or g

<220>

<221> modified base

<222> (17)..(17)

<223> a, c, t or g

<400> 115

tgctgaacag ccatnantga ttaactccta aacg 34

<210> 116

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 116

tgctgaacag ccataactga ttaactccta aacg 34

<210> 117

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (15)..(15)

<223> a, c, t or g

<220>

<221> modified base

<222> (18)..(19)

<223> a, c, t or g

<400> 117

tgctgaacag ccatnatnna ttaactccta aacg 34

<210> 118

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 118

tgctgaacag ccataatcaa ttaactccta aacg 34

<210> 119

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<220>

<221> modified base

<222> (15)..(15)

<223> a, c, t or g

<220>

<221> modified base

<222> (17)..(17)

<223> a, c, t or g

<220>

<221> modified base

<222> (19)..(19)

<223> a, c, t or g

<400> 119

tgctgaacag ccatnantna ttaactccta aacg 34

<210> 120

<211> 34

<212> DNA

<213> artificial sequence

<220>

<223> description of artificial sequence: synthesized

Oligonucleotides

<400> 120

tgctgaacag ccataactca ttaactccta aacg 34

Claims

(b) A cascades polypeptide; and

(c) Cas3 polypeptide.

2. The bacteriophage of claim 1, wherein said CRISPR array comprises a promoter sequence having at least about 90% sequence identity to any one of SEQ ID NOs 1-11.

3. The bacteriophage of claim 1 or claim 2, wherein said CRISPR array comprises a spacer sequence having at least 90% identity to any one of SEQ ID NOs 88-116 or 31-74.

4. The bacteriophage of claim 1 or claim 2, wherein said bacteriophage is a modified p1106, p1835, p1772 or p2131 bacteriophage.

5. A bacteriophage composition comprising a bacteriophage according to claim 1 or claim 2, further comprising p1695wt bacteriophage and/or p4430wt bacteriophage.

6. The bacteriophage of claim 1 or claim 2, wherein said cascades polypeptide forms a cascades complex of an I-C type CRISPR-Cas system, an I-B type CRISPR-Cas system, an I-a type CRISPR-Cas system, an I-D type CRISPR-Cas system, an I-E type CRISPR-Cas system, or an I-F type CRISPR-Cas system.

7. The bacteriophage of claim 1 or claim 2, wherein said cascades complex comprises:

8. The bacteriophage of claim 1 or claim 2, wherein said cascades comprise a Cas5d polypeptide (optionally SEQ ID No. 80), a Cas8C polypeptide (optionally SEQ ID No. 81) and a Cas7 polypeptide (optionally SEQ ID No. 82) (type I-C CRISPR-Cas system).

9. The bacteriophage of claim 1 or claim 2, wherein said Cas3 polypeptide comprises a sequence having at least about 90% identity to SEQ ID No. 79.

10. The bacteriophage of claim 1 or claim 2, wherein said bacteriophage infects a plurality of bacterial strains of a pseudomonas species.

11. A bacteriophage of claim 1 or claim 2, wherein the bacteriophage comprises Φkz virus (PhiKZvirus), Φkmv virus (PhiKMV virus), brunejosis virus (brunejosis virus), saquinavirus (Samunavirus), south China virus (Nankokuvirus), abilet virus (abijanvirs), begar virus (baikal virus), bei Telei virus (beitrevirus), kadaban virus (Casadabanvirus), cetex virus (citertrabanvirus), vesicular virus (cystolyvirus), de-trary virus (detrev), elvic virus (elvic), holrufiuja virus (hollayvirus), small hat virus (kochia virus), li Tu na virus (abibanawarus), lu Saipu pt Ma Bingdu (luzseim virus), niprevirus (bezebra virus), papyri virus (paepustus), papyri virus (papyristokura virus), or a combination of two or more viruses (pizebra virus), the virus (pizebra virus), the papyristokura virus (pizebra virus), the pizokura virus (pizebra virus), the pizebra virus (pizebra virus), the pizokukukur virus (pizei-kur virus (pikur).

12. A method of killing a pseudomonas species, the method comprising introducing into the target bacterium a nucleic acid sequence encoding a type I CRISPR-Cas system from a bacteriophage according to claim 1 or claim 2, wherein the target nucleotide sequence is present in the pseudomonas species.

13. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the individual the bacteriophage of claim 1 or claim 2, wherein the subject is infected with the pseudomonas species.

14. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject p1695wt bacteriophage and/or p4430wt bacteriophage.

15. The method of claim 14, wherein the subject is infected with the pseudomonas species.

16. The method of claim 13 or claim 14, wherein the disease or condition is a bacterial infection.

17. The method of claim 16, wherein the bacterial infection is associated with cystic fibrosis or non-cystic fibrosis bronchiectasis.

18. The method of claim 12, 13 or 15, wherein the pseudomonas species is a drug resistant pseudomonas species.

19. The method of claim 18, wherein the drug-resistant pseudomonas species is resistant to at least one antibiotic.

20. The method of claim 12, 13 or 15, wherein the pseudomonas species is a multidrug resistant pseudomonas species.

21. The method of claim 18, wherein the multi-drug resistant pseudomonas species is resistant to at least one antibiotic.

22. The method of claim 19 or claim 21, wherein the antibiotic comprises cephalosporin, fluoroquinolone, carbapenem, colistin, aminoglycoside, vancomycin, streptomycin, or methicillin.

23. The method of claim 12, 13 or 15, wherein the pseudomonas species is pseudomonas aeruginosa.

24. The method of claim 12, 13 or 15, wherein the bacteriophage is an obligate lytic bacteriophage or a temperate bacteriophage conferred lytic.

25. The method of claim 24, wherein the pseudomonas species is killed by the lytic activity of the bacteriophage and/or the activity of the CRISPR-Cas system.

26. The method of claim 24 or claim 25, wherein the lytic activity of the bacteriophage and the activity of the CRISPR-Cas system are synergistic.

27. A nucleic acid comprising SEQ ID No. 83 or a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 83.

28. A nucleic acid comprising SEQ ID No. 25 or a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 25.

29. A bacteriophage comprising a nucleic acid according to claim 27 or claim 28.

30. A bacteriophage comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to p1106e003, p1772e005, p1835e002, p2131e002, or two or more thereof, wherein the bacteriophage is recombinant.

31. The bacteriophage of claim 29 or claim 30, comprising

(a) A CRISPR array;

(b) A cascades polypeptide comprising one or more spacer sequences complementary to one or more target nucleotide sequences in a pseudomonas species; and

(c) Cas3 polypeptide.

32. The bacteriophage of claim 31, wherein said one or more spacer sequences comprise at least one of SEQ ID NOs 12, 16 and 20.

33. A bacteriophage comprising a nucleic acid sequence encoding a type I CRISPR-Cas system, the type I CRISPR-Cas system comprising:

(a) A CRISPR array comprising one or more spacer sequences complementary to one or more target nucleotide sequences in a pseudomonas species, wherein said one or more spacer sequences comprises at least one of SEQ ID NOs 12, 16 and 20;

(b) A cascades polypeptide; and

(c) Cas3 polypeptide.

34. The bacteriophage of claim 32 or claim 33, wherein said CRISPR array comprises SEQ ID No. 12.

35. The bacteriophage of claim 32 or claim 33, wherein said CRISPR array comprises SEQ ID No. 16.

36. The bacteriophage of claim 32 or claim 33, wherein said CRISPR array comprises SEQ ID No. 20.

37. A bacteriophage according to claim 32 or claim 33 comprising SEQ ID NOs 12, 16 and 20.

38. The bacteriophage of any one of claims 31 to 37, wherein said CRISPR array comprises a promoter sequence having at least about 90% sequence identity to any one of SEQ ID NOs 1 to 11.

39. The bacteriophage of any one of claims 31-38, wherein said cascades polypeptide forms a cascades complex of an I-C type CRISPR-Cas system, an I-B type CRISPR-Cas system, an I-a type CRISPR-Cas system, an I-D type CRISPR-Cas system, an I-E type CRISPR-Cas system, or an I-F type CRISPR-Cas system.

40. The bacteriophage of claim 39, wherein said cascades complex comprises:

41. The bacteriophage of claim 40, wherein said cascades comprise a Cas5d polypeptide (optionally SEQ ID No. 80), a Cas8C polypeptide (optionally SEQ ID No. 81) and a Cas7 polypeptide (optionally SEQ ID No. 82) (type I-C CRISPR-Cas system).

42. The bacteriophage of any one of claims 29 to 41, comprising SEQ ID No. 83 or a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 83.

43. The bacteriophage of any one of claims 29 to 42, comprising SEQ ID No. 25 or a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID No. 25.

44. The bacteriophage of any one of claims 29 to 43, wherein said bacteriophage comprises at least 85% or 90% identity to p1106e003, p1772e005, p1835e002, p2131e002, or two or more thereof.

45. A bacteriophage according to any one of claims 29 to 44, comprising:

46. The bacteriophage of claim 45, further comprising a fifth bacteriophage comprising at least 80% sequence identity to p 1695.

47. The bacteriophage of claim 45, further comprising a fifth bacteriophage comprising at least 80% sequence identity to p 4430.

48. The bacteriophage of claim 47, further comprising a sixth bacteriophage comprising at least 80% sequence identity to p 1695.

49. A method of killing a pseudomonas target bacterium, the method comprising administering to a subject in need thereof the nucleic acid of claim 27 or claim 28 or the bacteriophage of any one of claims 29-48.

50. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the nucleic acid of claim 27 or claim 28 or the bacteriophage of any one of claims 29-48.