CA3171200A1 - Multiplex crispr/cas system for modifying cell genomes - Google Patents

Abstract

The invention relates to methods of modifying cell genomes synergistically using multiple CRISPR/Cas systems. The invention also relates to compositions, crRNAs, Cas proteins and vectors for carrying out such methods.

Description

MULTIPLEX CRISPR/CAS SYSTEM FOR MODIFYING CELL GENOMES
TECHNICAL FIELD
[0001] The invention relates to methods of modifying cell genomes using multiple CRISPR/Cas systems. The invention also relates to compositions, crRNAs, Cas and vectors for carrying out such methods as disclosed herein.
BACKGROUND

[0002] The state of the art describes vectors and uses of these that employ CRISPR/Cas systems. For example, reference is made to W02020078893, W02019185551, W02019105821, W02017118598, US20180140698, US20170246221, US20180273940, US20160115488, US20180179547, US20170175142, US20160024510, US20150064138, US20170022499, US20160345578, US20180155729, US20180200342, W02017112620, W02018081502, PCT/EP2018/066954, PCT/EP2018/066980, PCT/EP2018/071454, EP3356533, EP3307872, W02020072253, W02020072254, W02020072250, W02020072248, W02019236566, W02019144061, W02017112620, W02015066119, EP16804164, W02019227080, EP3362571, EP3356533, W02016205623, EP3307872, US2016/0324938 and US2019/0160120 and equivalent publications by the US Patent and Trademark Office (USPTO) or WIPO, the disclosures of which are incorporated herein by reference.
SUMMARY OF THE INVENTION

[0003] The invention provides the following configurations.

[0004] In a First Configuration A method of modifying the genome of a cell, the method comprising (a) using a first CRISPR/Cas system to modify a first protospacer of the genome; and (b) using a second CRISPR/Cas system to modify a second protospacer of the genome, wherein the second protospacer is different to the first protospacer;
wherein the systems comprise different Cas and are provided simultaneously in the cell.
100051 In a Second Configuration A method of modifying the genome of one or more cells, the method comprising introducing into each cell components (a), (b), (c) or (d):-(a) first and second crRNAs;
(b) nucleic acid encoding first and second crRNAs, wherein the nucleic acid is expressed in the cell for producing the crRNAs;

(c) a first nucleic acid encoding a first crRNA, and a second nucleic acid encoding a second crRNA, wherein the nucleic acids are expressed in the cell for producing the crRNAs; or (d) a first crRNA and a nucleic acid encoding a second crRNA, wherein the nucleic acid is expressed in the cell for producing the second crRNA;
wherein for each cell (e) the first crRNA (crRNA1) is capable of guiding a first Cas (C1) to a protospacer sequence (PS1) comprised by the cell genome to modify PS1; and (f) the second crRNA (crRNA2) is capable of guiding a second Cas (C2) to a protospacer sequence (PS2) comprised by the cell genome to modify PS2;
(g) Cl and C2 are different;
(h) PS1 and PS2 are different; and (i) crRNA1, crRNA2. Cl and C2 are provided in the cell, whereby the genome of the cell is subjected to Cas modification.
In one aspect the invention uses the method to kill target cells. In another aspect the invention uses the method to edit the genomes of cells.
[0006] In a third Configuration A composition for use in a method treating or preventing a disease or condition in a human or animal subject that is mediated by target cells, the composition comprising components (a), (b) or (d):-(a) first and second crRNAs;
(b) nucleic acid encoding first and second crRNAs, wherein the nucleic acid is expressible in a target cell for producing the crRNAs;
(c) a first nucleic acid encoding a first crRNA, and a second nucleic acid encoding a second crRNA, wherein the nucleic acids are expressible in a target cell for producing the crRNAs; or (d) a first crRNA and a nucleic acid encoding a second crRNA, wherein the nucleic acid is expressible in a target cell for producing the second crRNA;
wherein (e) the first crRNA (crRNA1) is capable of guiding a first Cas (Cl) to a protospacer sequence (PS1) comprised by a target cell genome to modify PS1; and (f) the second crRNA (crRNA2) is capable of guiding a second Cas (C2) to a protospacer sequence (PS2) comprised by the target cell genome to modify PS2;
(g) Cl and C2 are different;
(h) PS1 and PS2 are different; and wherein the method comprises administering the composition to the subject whereby said components of the composition are introduced into target cells wherein crRNA1, crRNA2, Cl and C2 are provided in each cell and the gcnomc of each cell is subjected to Cas modification and the disease or condition is treated or prevented.

[0007] In a fourth Configuration A composition comprising components (a), (b) or (d):-(a) first and second crRNAs;
(b) nucleic acid encoding first and second crRNAs, wherein the nucleic acid is expressible in a target cell for producing the crRNAs;
(c) a first nucleic acid encoding a first crRNA, and a second nucleic acid encoding a second crRNA, wherein the nucleic acids are expressible in a target cell for producing the crRNAs; or (d) a first crRNA and a nucleic acid encoding a second crRNA, wherein the nucleic acid is expressible in a target cell for producing the second crRNA;
wherein (e) the first crRNA (crRNA1) is capable of guiding a first Cas (C1) to a protospacer sequence (PS1) comprised by a target cell genome to modify PS1; and (f) the second crRNA (crRNA2) is capable of guiding a second Cas (C2) to a protospacer sequence (PS2) comprised by the target cell genome to modify PS2;
(g) Cl and C2 are different;
(h) PS1 and PS2 are different; and wherein when said components of the composition are introduced into a target cell whereby crRNA1, crRNA2, Cl and C2 are provided in the cell, the genome of the cell is subjected to Cas modification.
[0008] Aspects provide:-The method of the invention for (a) producing synergistic Cas nuclease cutting of a cell genome;
(b) reducing a population of cells of a first species or strain by at least 100,000, 1,000,000 or 10,000,000-fold;
(c) killing at least at least 99%. 99.9%. 99.99%, 99.999%, 99.9999% or 99.99999% cells of a first species or strain comprised by a microbiome;
(d) producing synergistic Class 1 Cas modification of a cell genome; or (e) reducing bacterial cells of a first species or strain (eg, E coli cells) in a cell population by at least 105, 106 or 107 -fold, wherein the population comprises at least 100,000; 1,000,000; or 10,000,000 cells respectively.
100091 Aspects also provide pharmaceutical compositions, methods of making such compositions and medical methods using such compositions.
BRIEF DESCRIPTION OF THE DRAWINGS
[00010] Figure 1. Type T CRISPR-Cas system of E. coli and C. difficile targeting E. coli MG1655.
Layout of the CRISPR Guided VectorTm, CGVTm. (A) E. coli CRISPR-Cas CGV: ColE1 on, cas3 and cascade of E. coil, CRISPR array. (B) C. difficlle CRISPR Cas CGVs. Plasmid 1:
pSC101 on, cas3 and cascade of C. difficile. Plasmid 2: pC1oDF13 on, CRISPR array of C.
difficile.
[00011] Figure 2. Killing of E. coli MG1655 with type IE CRISPR-Cas system of E. coli and type I-B
CRISPR-Cas system of C. difficile. E. coil MG1655 harboring cas genes of C.
difficile was transformed with cognate CRISPR array and E. coli CRISPR-Cas CGV. Both CRISPR
systems together surprisingly synergistically killed 7 logio E. coli MG1655, compared to empty vectors.
Additionally, single transformations with the CGVs were tested. E. coli CRISPR-Cas system resulted in ¨4-log to reductions; C. diffiede CRISPR-Cas system resulted ¨3-log to (n=3).
DETAILED DESCRIPTION
[00012] The invention relates to methods of modifying cell genomes using multiple CRISPR/Cas systems. The invention also relates to compositions, crRNAs, Cas and vectors for carrying out such methods as disclosed herein.
1000131 The invention is useful to provide one or more of the following advantages:-(a) producing synergistic Cas nuclease cutting of a cell genome, (b) reducing a population of cells of a first species or strain by at least 100,000, 1,000,000 or 10,000,000-fold;
(c) killing at least at least 99%. 99.9%. 99.99%, 99.999%, 99.9999% or 99.99999% cells of a first species or strain comprised by a microbiome;
(d) producing synergistic Class 1 Cas modification of a cell genome; or (e) reducing bacterial cells of a first species or strain in a cell population by at least 105, 106 or 107-fold, wherein the population comprises at least 100,000; 1,000,000; or 10,000,000 cells respectively.
[00014] Advantageously, by reducing the number of target cells in a cell population, this may be beneficaial where the cells are undesirable (eg, detrimental to the health of a subject to which the method is applied, or detrimental to an ex vivo environment or in vitro cell sample to which the method is applied or the composition is administered). For example, the cells are cancer cells comprised by a patient and the multiple Cas cutting of the invention synergistically kills a very high number (cg, at least 99.999% or 105-fold) of the cells. By reducing the cells in this way, the number of seeder cells to re-grow the cancer is reduced. In another example, the cells may be bacterial or archaeal cells and by reducing the cells in this way, the number of seeder cells to re-grow an undesirable cell population will be reduced.
[00015] In a configuration, the invention provides in one aspect:-A method of modifying the genome of a cell, the method comprising (a) using a first CRISPR/Cas system to modify a first protospacer of the genome; and (b) using a second CRISPR/Cas system to modify a second protospacer of the genome, wherein the second protospacer is different to the first protospacer;
wherein the systems comprise different Cas and are used simultaneously.
[00016] Another aspect of the configuration provides:-A method of modifying the genome of one or more cells, the method comprising introducing into each cell components (a), (b), (c) or (d):-(a) first and second crRNAs;
(b) nucleic acid encoding first and second crRNAs, wherein the nucleic acid is expressed in the cell for producing the crRNAs;
(c) a first nucleic acid encoding a first crRNA, and a second nucleic acid encoding a second crRNA, wherein the nucleic acids are expressed in the cell for producing the crRNAs; or (d) a first crRNA and a nucleic acid encoding a second crRNA, wherein the nucleic acid is expressed in the cell for producing the second crRNA;
wherein for each cell (e) the first crRNA (crRNA1) is capable of guiding a first Cas (Cl) to a protospacer sequence (PS1) comprised by the cell genome to modify PS1; and (f) the second crRNA (crRNA2) is capable of guiding a second Cas (C2) to a protospacer sequence (PS2) comprised by the cell genome to modify PS2;
(g) Cl and C2 are different;
(h) PS1 and PS2 are different; and (i) crRNA1, crRNA2, Cl and C2 are provided in the cell, whereby the genome of the cell is subjected to Cas modification.
[00017] Optionally, Cl and/or C2 is a Cas nuclease, eg, Cl and C2 each is a Cas3. Optionally, Cl and/or C2 is a Cascade Cas, eg, CasA, CasB, CasC, CasD or CasE. Optionally, Cl is a Cas3 that operates in the cell with Cascade Cas, eg, one, more or all of CasA, B, C, D
and E.
[00018] Optionally, the crRNAs of component (a) are introduced simultaneously or sequentially.
Optionally, the nucleic acid and crRNA of component (c) are introduced simultaneously or sequentially. Optionally, the nucleic acids of component (d) arc introduced simultaneously or sequentially. The method, however, includes the presence of crRNA1, crRNA2, Cl and C2 at the same time in each cell, whereby multiple CRISPR/Cas systems are used to modify the genome.
crRNA1, crRNA2, CI and C2 are provided in the cell, whereby PSI and PS2 are subjected to Cas nuclease modification wherein the genome of the cell is modified.
[00019] Preferably, the first and second protospacers are different or comprised by different genes or intergenic sequences of the genome.

[00020] Modification of the genome may be cutting of nucleic acid of the genome, repression of transcription or translation of a gene comprised by the genome, upregulation of transcription or translation of a gene comprised by the genome, or editing of the genome (eg, to insert and/or delete one or more nucleic acid sequences). The invention may advantageously be useful for synergistically or efficiently cutting, modifying or editing the genome of each cell. In an example, DNA comprised by the genome is cut, modified or edited and/or RNA comprised by the genome is cut, modified or edited. In an example, DNA comprised by the genome is degraded (eg, in a process comprising Cos exo- or endonuclease activity) and/or RNA comprised by the genome is cut, modified or edited (eg, in a process comprising Cas exo- or endonuclease activity).
[00021] In an example, the component is comprised by a nucleic acid vector. In an example the component (a), (b), (c) or (d) is introduced into each cell by transfection, electroporation, transduction or conjugative transfer. For example, the vector is a virus or phage and the component is introduced by transduction. For example, the vector is a plasmid and the component is introduced by conjugation, transfection or electroporation. For example, the vector is a phagemid (optionally a phagemid comprised by a virus or phage) and the component is introduced by conjugation, transduction, transfection or elcctroporation. For example, the nucleic acid(s) of the component is(are) introduced by electroporation thereof. For example, a phage herein is a tailed phage. For example a phage herein is a lytic phage. For example, a phage herein is a non-lytic phage.
[00022] Optionally, the method is a recombineering method carried out in vitro, and for example the cell is an E coli cell.
[00023] Optionally, each said crRNA is encoded by a CRISPR array comprising first and second repeat sequences and a spacer sequence joining the repeat sequences.
Optionally, the nucleic acid of (b) comprises a CRISPR array encoding crRNA1 and crRNA2. Optionally, the first nucleic acid of (c) comprises a first CRISPR array encoding crRNA1 and the second nucleic acid comprises a second CRISPR array encoding crRNA2. Optionally, the nucleic acid of (b) comprises a CRISPR array encoding crRNA2.
[00024] In an example each repeat sequence is GAGTTCCCCGCGCCAGCGGGGATAAACCG or GTTTTATATTAACTAAGTGGTATGTAAAT. In an example, each protospacer or spacer sequence consists of from 15 to 70, 20 to 50, 17 to 45, 18 to 40, 18 to 35 or 20 to 40 contiguous nucleotides.
[00025] Optionally, Cosi and/or Cas2 are not introduced into each cell.
Optionally, each nucleic acid is devoid of nucleic acid sequence encoding Cosi and/or Cas2. Optionally, additionally Cas4 is not introduced into each cell, or each nucleic acid is devoid of a Cas4-encoding nucleic acid sequence.
Optionally, said introducing comprises (i) introducing into each cell an operon comprising nucleotide sequences encoding a type I Cas3 (wherein the Cas3 is Cl) and Cascade proteins under the control of a common constitutive promoter and/or introducing into each cell an operon comprising nucleotide sequences encoding a type I Cas3 (wherein the Cas3 is C2) and Cascade proteins under the control of a common constitutive promoter. In an example, Cl is a Type-IB Cas3 and/or C2 is a Type-IE Cas3.
Examples of suitable operons are disclosed in W02020078893 or US20200115716, the disclosures of which are expressly incorporated herein by reference for possible use in the present invention. The term -operon- is known to the skilled person such as relating to a functioning unit of DNA containing at least expressible 2 nucleotide sequences respectively encoding for an expression product (eg, a respective translatable mRNA), wherein the sequences are under common promoter control.
[00026] In an example, Cl is a Cas disclosed in W02019002218 and optionally the first crRNA is encoded by a CRISPR array comprising cognate repeat sequences, such as when Cl is a Cas (eg, Cas3) disclosed in W02019002218 the repeat sequences are the cognate repeat sequence disclosed in W02019002218. Additionally or alternatively, In an example, C2 is a Cas disclosed in W02019002218 and optionally the first crRNA is encoded by a CRISPR array comprising cognate repeat sequences, such as when C2 is a Cas (eg, Cas3) disclosed in W02019002218 the repeat sequences are the cognate repeat sequence disclosed in W02019002218. In an example, the first crRNA is encoded by an array comprising a repeat sequence disclosed in W02019002218 and/or the second crRNA is encoded by an array comprising a repeat sequence disclosed in W02019002218.
For example, one or more nucleotide sequences encoding one or more Cascade Cas (eg, which are cognate to C 1 or C2) are introduced into the cell, wherein the Cascade Cas are Cascade Cos disclosed in W02019002218. All of these disclosures in W02019002218 are expressly incorporated herein by reference for possible use in the present invention.
[00027] Optionally, (a) Cl is a Class 1 Cas and C2 is a Class 1 Cas;
(b) Cl is a Class 1 Cas and C2 is a Class 2 Cas;
(c) Cl is a Class 2 Cas and C2 is a Class 2 Cas;
(d) Cl is a Type I Cas (optionally Type I-A, B, C, D, E, F or U) and C2 is a Type I Cas (optionally Type I-A, B, C, D, E, F or U);
(e) Cl is a Type I (optionally Type I-A, B, C, D. E, F or U) or II Cas and C2 is a Type II Cas;
(f) Cl is a Type I (optionally Type I-A, B, C, D, E, F or U) or II Cas and C2 is a Type III Cas (optionally Type I-A or B);
(g) Cl is a Type I (optionally Type I-A, B, C, D, E, F or U) or II Cas and C2 is a Type IV Cas;
(h) Cl is a Type 1 (optionally Type I-A, B, C, D, E, F or U) or 11 Cas and C2 is a Type V Cas; or (i) Cl is a Type I or II Cas and C2 is a Type VI Cas.
[00028] Optionally. Cl and C2 are different Class 1 Cas selected from the Cas disclosed in Table 2.
Optionally, Cl is an E coli Cas (eg, Cas3) and C2 is a Cas selected from the Cas disclosed in Table 2.
Optionally, Cl is an C dificile Cas (eg, Cas3) and C2 is a Cas selected from the Cas disclosed in Table 2.
[00029] Optionally, Cl is a Type I-A, B, C, D, E, F or U Cas. Optionally, C2 is a Type I-A, B, C, D, E, F or U Cas.

[00030] Optionally, Cl is a Type I-A Cas and C2 is a Type I-B, C, E, F or U
Cas. Optionally, Cl is a Type I-B Cas and C2 is a Type I-B, C, E, F or U Cas. Optionally, CI is a Type I-C Cas and C2 is a Type I-B, C, E, F or U Cas. Optionally, Cl is a Type I-D Cas and C2 is a Type I-B, C, E, F or U Cas.
Optionally, Cl is a Type I-E Cas and C2 is a Type I-B, C, E, F or U Cos.
Optionally, Cl is a Type I-F
Cas and C2 is a Type I-B, C, E, F or U Cas. Optionally, CI is a Type I-U Cas and C2 is a Type I-B, C, E, F or U Cas.
[00031] Optionally, (a) Cl is a Type TB or C Cos and C2 is a Type I-E or F Cas (optionally Cl is a Type TB Cas3 and C2 is a Type IE Cas);
(b) Cl is a Type IC or C Cas and C2 is a Type I-E or F Cas (optionally Cl is a Type IC Cas3 and C2 is a Type IE Cas3); or (c) Cl is a Type II Cas9 and C2 is a Type I Cas3 (optionally C2 is an E coil Type IE or F Cas3;
or a C difficile Cas TB).
[00032] Optionally_ (a) Cl is a Cas3 (optionally a Type 1-A, B, C, D, E, F or U Cas3) and C2 is a Cas3 (optionally a Type I-A, B, C, D, E, F or U Cas3);
(b) Cl is a Cas9 and C2 is a Cas3 (optionally a Type I-A, B, C, D, E, F or U
Cas3);
(c) CI is a Cas3 (optionally a Type I-A, B, C, D, E, F or U Cas3) and C2 is a Cas10 (optionally Cas10 subtype A, B, C or D);
(d) Cl is a Cas9 and C2 is a Cas10 (optionally Cas10 subtype A, B, C or D);
(e) Cl is a Cas9 and C2 is a Cas12 (optionally Cas12a);
(f) Cl is a Cas3 (optionally a Type 1-A, B, C, D, E, F or U Cas3) and C2 is a Cas12 (optionally Cas12a);
(g) Cl is a Cas9 and C2 is a Cas13 (optionally Cas13a, Cas13b, Cas13c or Cas13d); or (h) Cl is a Cas3 (optionally a Type I-A, B, C, D, E, F or U Cas3) and C2 is a Cas13 (optionally Cas13a, Cas13b, Cas13c or Cas13d).
[00033] Optionally, PS1 and PS2 are protospacers comprised by (a) RNA and RNA respectively;
(b) DNA and RNA respectively;
(c) RNA and DNA respectively; or (d) DNA and DNA respectively.
[00034] Optionally, Cl is a Clostridiaceae Cas3 (optionally a C difficile Cas3, such as a Type T-B
Cas3) and C2 is an Enterobacteriaceae Cas3 (optionally an E coli Cas3, such as a Type I-E Cas3).

[00035] In an alternative, Cl and C2 are the same. In an alternative, Cl and C2 are the same type of Cas, eg, each is a Cas9, or each is a Cas3, or each is a Cas 1 2, or each is a Cas 1 3, or each is the same type of Cascade Cas.
[00036] Optionally, Cl is a Biostraticola, Buttiauxella, Cedecea, Citrobacter, Cronobacter, Enterobacillus, Enterobacter, Escherichia, Franconibacter, Gibbsiella, Izhakiella, Klebsiella, Kluyvera, Kosakonia, Leclercia, Lelliottia, Limnobaculum, Mangrovibacter, Metakosakonia, Pluralibacter, Pseudescherichia, Pseudocitrobacter, Raoultella or Rosenbergiella Cas (eg, Cas3 or Cascade Cas).
[00037] Optionally, Cl is a spCas9 (S pyogenes Cas9) or saCas9 (S aureus Cas9) and C2 is a Type I
Cas3 (optionally C2 is an E coli Type I-E or F Cas3).
[00038] Optionally, the modification is cutting of the genome, eg, cutting DNA
(eg, ssDNA or dsDNA) of the genome, RNA (eg, mRNA, crRNA, tracrRNA, tRNA, snRNA or rRNA, preferably mRNA), endonuclease cutting or exonuclease cutting, or cutting of one, but not both strands of dsDNA (double-stranded DNA) of the genome, or nicking of dsDNA of the genome.
[00039] Optionally_ PS 1 is a chromosomal sequence of the cell. Optionally, PS1 is an episomal (eg, plasmid) sequence of the cell.
[00040] Optionally, PS 1 is a chromosomal sequence of the cell and PS2 is a chromosomal sequence of the cell. Optionally, PS1 is a chromosomal sequence of the cell and PS2 is an episomal (eg, plasmid) sequence of the cell.
[00041] Optionally, each cell is a human, animal (ie, non-human animal), plant, yeast, fungus, amoeba, insect, mammalian, vertebrate, bird, fish, reptile, rodent, mouse, rat, livestock animal, cow, pig, sheep, goat, rabbit, frog, toad, protozoan, invertebrate, mollusc, fly, grass, tree, flowering plant, fruiting plant, crop plant, wheat, corn, maize, barley, potato, carrot or lichen cell. Optionally, each cell is a prokaryotic cell or eukaryotic cell. For example, each cell is a bacterial or archaeal cell, optionally an E coli cell or C difficile cell. In an embodiment, the cell or the cells are of a genus or species disclosed in Table 1. In an embodiment, the cell or the cells are gram positive cells. In an embodiment, the cell or the cells are gram negative cells.
[00042] Optionally, the step of introducing comprises infecting the cell with a virus (optionally a bacteriophage wherein the cell is a bacterial cell) or introducing a plasmid (optionally a conjugative plasmid) or introducing a phagcmid into thc cell, wherein the virus, plasmid or phagcmid encodes the crRNAs. Optionally, the virus, plasmid or phagemid encodes Cl and/or C2.
Optionally, the virus, plasmid or phagemid encodes one of said Cl and C2, and the other Cas is an endogenous Cas encoded by the genome of the cell. Optionally, the each of C 1 and C2 is an endogenous Cas encoded by the genome of the cell. In an example, the Cas is encoded by a chromosome of the cell.
[00043] Optionally Cl is a Cas3 and the virus or plasmid encodes a Cas5, Cas6, Cas7 and Cas8 (and optionally a Cas 11) that arc cognate to the Cas3. Additionally or alternatively, optionally C2 is a Cas3 and the virus or plasmid encodes a Cas5, Cas6, Cas7 and Cas8 (and optionally a Cash) that are cognate to the Cas3.
[00044] The Cas are simultaneously present in the cell and the Cas may cut the genome simultaneously or sequentially.
[00045] Optionally, the method comprises introducing into each cell or expressing in each cell at least 3, 4 or 5 different types of crRNAs wherein the types target different protospacer sequences comprised by the cell genome (e,g different chromosomal sequences). In an example, the cell is a bacterial or archaeal cell and the protospacers are comprised by the cell chromosome. For example, at least one or two of said crRNA types targets a respective chromosomal sequence and at least one or more of the crRNA types targets a sequence comprised by an episome (eg, a plasmid) of the cell, wherein the cell is a bacterial or archaeal cell. For example, the cell (eg, a human or mammalian cell) comprises a plurality of chromosomes and the crRNAs target protospacer sequences comprised by two or more of said chromosomes (eg, wherein the chromosomes are not members of the same diploid chromosomal pair).
[00046] For example, the method comprised introducing a nucleic acid into each cell, wherein the nucleic acid comprises, in 5' to 3' direction a nucleotide sequence encoding a Cas nuclease (eg, a cas3) and one or more sequences encoding one or more Cascade Cas (eg, cas8e, cas 11, cas7, cas5, and cas6; or cas6, cas8b, cas7, and cas5) that are operable with the Cas nuclease to modify a cognate protospacer sequence.
[00047] The nucleic acid(s) is(are) preferably devoid of an adaptation module.
Optionally, the module encodes a Cas 1 and a Cas2; or a Cas 1, a Cas2 and a Cas4 [00048] In an embodiment, a said nucleic acid comprises a CRISPR array encoding crRNAs, such as an array comprising at least 3, 4 or 5 spacer sequences targeting at least 3, 4 or 5 sequences of the cell respectively. For example, a plurality of chromosomal intergenic regions are targeted. Optionally, each spacer sequence consists of from 20 to 50, 20 to 40, 22 to 40, 25 to 40 or 30 to 35 consecutive nucleotides, eg, 32 or 37 nucleotides.
[00049] In an example, the array comprises the following spacer sequences (Spacers 1-3):
TGATTGACGGCTACGGTAAACCGGCAACGTTC;
GCTGTTAACGTACGTACCGCGCCGCATCCGGC; and CGGACTTAGTGCCAAAACATGGCATCGAAATT
separated by repeat sequence (ie, Spacer 1 ¨ repeat ¨ Spacer 2 ¨ repeat ¨
Spacer 3).
[00050] In another example, the array comprises 3, 4 or 5 of the following spacer sequences (Spacers 4-8):
GCCATAATCTGGATCAGGAAGTCTTCCTTATCCATAT;
GGCTTTACGCCAGCGACGTATTGCCACAGGAATAACT;
GGGGATAGCGCGCCTGGAGCGTGCGATAGAGACTTTG;

[00051] GGCATTTACCGACCAGCCCATCAGCAGTACAGCAAAC; and TCCTGAATCAAATCCGCCTGTGGCAGGCCATAGCCCG
separated by repeat sequence (ie, Spacer 4 - repeat - Spacer 5 - repeat -Spacer 6- repeat - Spacer 7 - repeat - Spacer 8).
[00052] Optionally, each repeat sequence consists of from 20 to 50, 20 to 40, 22 to 40, 25 to 40 or 30 to 35 consecutive nucleotides, eg, 29 nucleotides. For example, each repeat sequence consists of:
GAGTTCCCCGCGCCAGCGGGGATAAACCG (and optionally the Cas is/are E coli Cos). In another example, each repeat sequence consists of: (and optionally the Cas is/are C dificile Cas).
[00053] Optionally, each crRNA is expressed from the nucleic acid(s) under the control of a common or respective constitutive promoter.
[00054] Optionally, each Cas is expressed from the nucleic acid(s) under the control of a common or respective constitutive promoter. In an embodiment, the first crRNA and Cl are expressed under the control of a common constitutive promoter and/or the second crRNA and C2 are expressed under the control of a common constitutive promoter. For example, the promoters are the same promoter or they are different promoters. In an example, one, more of all of said promoters is a strong promoter.
A promoter may be any promoter disclosed in W02020078893 or U S20200115716, the disclosures of such promoters (and nucleic acids, operons and vectors comprising one or more such promoters) being expressly incorporated herein by reference for possible use in the present invention.
[00055] In an embodiment, a first plurality of different crRNAs are expressed in one or more of the in each cell, wherein each crRNA is operable with CS1 to guide modification of the genome and the plurality targets at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (preferably, at least 2, 3, 4 or 5; or exactly 2, 3, 4 or 5) different protospacers comprised by the genome of the cell;
and/or a second plurality of different crRNAs are expressed in each cell wherein each crRNA is operable with CS2 and the second plurality targets at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (preferably, at least 2, 3, 4 or 5; or exactly 2, 3, 4 or

5) different comprised by the genome of the cell. For example, the first plurality comprises from 2 to 10, eg, from 2 to 7, different crRNAs. For example, the second plurality comprises from 2 to 10, eg, from 2 to 7, different crRNAs.
[00056] Optionally, one or more or all of said cells are killed by the method.
Optionally, the growth or proliferation of one or more or all of said cells is reduced by the method.
Usefully, when the cell is a prokaryotic cell (eg, a bacterial or archaeal cell) the chromosome of the cell is cut by Cas. For example, a bacterial cell chromosome is cut by Cl and C2 and the cell is killed.
[00057] Optionally, the first crRNA (or each crRNA of said first plurality) is comprised by a guide RNA wherein the guide RNA further comprises a tracrRNA and/or the second crRNA
(or each crRNA of said second plurality) is comprised by a guide RNA wherein the guide RNA further comprises a tracrRNA. Optionally, the first crRNA (or each crRNA of said first plurality) is comprised by a chimaeric guide RNA and/or the second crRNA (or each crRNA of said second plurality) is comprised by a chimaeric guide RNA.
[00058] For example, the genome modification of a plurality of cells is cutting of genomic nucleic acid (eg, chromosomal DNA) and the cells are killed, wherein said killing of the plurality of cells is synergistic compared to killing using CI or C2 alone.
[00059] An aspect provides:-A method of killing or reducing the growth or proliferation of a plurality of cells (optionally prokaryotic cells, such as bacterial cells) of a first species or strain, the method comprising carrying out the method of the invention using the cells, wherein Cl and/or C2 is a Cas nuclease and the genomes of the cells are cut by Cas nuclease cutting and the cells are killed or the growth or proliferation of the cells is reduced.
1000601 Optionally, as exemplified herein, the method reduces the number of cells of said plurality at least 105, 106 or 107-fo1d, eg, between 105 and 107-fold, or between 105 and 10s-fold or between 105 and 109-fold. The skilled person will be familiar with determining fold-killing or reduction in cells, eg, using a cell sample that is representative of a microbiome or cell population. An illustrative example is given in the Examples below. For example, the extent of killing or reduction in growth or proliferation is determined using a cell sample, eg, a sample obtained from a subject to which the composition of the invention has been administered, or an environmental sample (eg, aqueous, water or soil sample) obtained from an environment (eg, a water source, waterway or field) that has been contacted with the composition of the invention.
1000611 For example, the method reduces the number of cells of said plurality at least 105, 106 or 107¨
fold and optionally the plurality comprises at least 100,000; 1,000,000; or 10,000,000 cells respectively.
[00062] Optionally, the plurality of cells is comprised by a cell population, wherein at least 5, 6 or 7 log10 of cells of the population are killed by the method, and optionally the plurality comprises at least 100,000; 1,000,000; or 10,000,000 cells respectively.
[00063] When a cell herein is a bacterial cell, it may be of a first species or genus selected from Table 1. Similarly, a plurality of cells herein may be cells which arc of a species or genus selected from Table 1.
[00064] Optionally, as exemplified herein, the method kills at least 99%.
99.9%. 99.99%, 99.999%, 99.9999% or 99.99999% cells of said plurality.
[00065] In an example, the method is carried out on a population (or said plurality) of said cells and the method kills, modifies or edits all (or essentially all) of the cells of said population (or said plurality). In an example, the method is carried out on a population (or said plurality) of said cells and the method kills, modifies or edits 100% (or about 100%) of the cells of said population (or plurality).

[00066] Optionally, the species is E coil or C difficile.
[00067] An aspect of the invention provides:-A method of editing the genome of one or more cells, the method comprising (a) modifying the genome of each cell by carrying out the method of the invention, wherein the genome is subjected to Cas cutting; and (b) inserting a nucleic acid at or adjacent to a Cos cut site in the genome and/or deleting a nucleic acid sequence from the genome at or adjacent to a Cas cut site in the genome, wherein a cell with an edited genome is produced; and (c) optionally isolating from the cell a nucleic acid comprising the insertion or the deletion; or sequencing a nucleic acid sequence of the cell wherein the nucleic acid sequence comprises the insertion or the deletion.
1000681 In an example, the method is carried out on a population of said cells, wherein the population comprises at least 100 of said cells and at least 90 or 99% of said cells are edited.
1000691 In an embodiment, the method is a method of recombineering, cg, in one or more E coil cells.
[00070] The insertion may be immediately adjacent to, or overlapping the cut site, or the insertion may be within lkb, 2kb or 200, 150, 100, 50, 25, 10 or 5 nucleotides of the cut site. For example, the nucleic acid is inserted by homologous recombination. In an embodiment, the nucleic acid is inserted by homologous recombination and replaces (the sequence is inserted in the place of genome sequence that is deleted) genome sequence of 1 to 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5kb, or 200, 150, 100, 50, 25, 10 or 5 nucleotides of the genome. For example, the deleted genome sequence flanks either side of the cut site, or is at the 5'- or 3'-side of the cut site. In an embodiment, the nucleic acid is inserted by homologous recombination and does not replace any genomic sequence.
[00071] The deletion may be immediately adjacent to, or overlapping the cut site, or the deletion may be within lkb, 2kb or 200, 150, 100, 50, 25, 10 or 5 nucleotides of the cut site. For example, deletion is a deletion of 1 to 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 2 or lkb, or 200, 150, 100, 50, 25, 10 or nucleotides of the genome. For example, the deleted genome sequence flanks either side of the cut site, or is at the 5'- or 3'-side of the cut site.
[00072] For example, the inserted nucleic acid is DNA. For example, the deleted nucleic acid is DNA, eg, chromosomal or episomal DNA).
[00073] For example, the inserted nucleic acid is at least (or no more than) 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 2 or Ikb; or 200, 150, 100, 50, 25, 10 or 5 consecutive nucleotides in length. For example, the deleted genomic nucleic acid is at least (or no more than) 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 2 or lkb; or 200, 150, 100, 50, 25, 10 or 5 consecutive nucleotides in length.

[00074] For example, the genomic sequence is DNA. For example, genomic DNA is deleted or replaced. For example, genomic DNA is deleted or replaced and the editing inserts DNA sequence into the genome (eg, at or flanking the cut site).
[00075] For example, the genomic sequence is RNA. For example, genomic RNA is deleted or replaced. For example, genomic RNA is deleted or replaced and the editing inserts RNA sequence into the genome (eg, at or flanking the cut site).
[00076] Optionally, the method further comprises (a) culturing the modified cell(s) to produce progeny thereof; and optionally isolating the progeny cells; or (b) inserting a sequence obtained from a cell in step (c) into a recipient cell and growing a cell line therefrom.
[00077] Optionally, the progeny cells or cell line expresses a protein, wherein the protein is encoded (all or in part) by a nucleotide sequence that comprises the inserted nucleic acid sequence, the method further comprising obtaining the expressed protein or isolating the expressed protein from the cells or cell line.
[00078] Optionally, the method further comprises combining the progeny cells, cell line or protein with a pharmaceutically acceptable carrier, diluent or excipient, thereby producing a pharmaceutical composition.
[00079] In an embodiment, the inserted nucleic acid comprises a transcription and/or translation regulatory element for controlling expression of one or more nucleic acid sequences of the edited genome that are adjacent to the insertion. For example, the inserted nucleic acid comprises a promoter, eg, a constitutive or strong promoter. In another example, the element is a transcription or translation terminator, eg, the inserted sequence comprises a stop codon. In this way, transcription of a gene (or a part of a gene) that is adjacent to the inserted sequence in the edited genome is terminated or prevented or reduced.
[00080] In an example, the deleted genomic sequence is a RNA (eg, mRNA) sequence. For example, the deletion of the RNA sequence reduces or prevents expression of an amino acid sequence in the cell, wherein the amino acid sequence is encoded by the deleted RNA sequence.
This may be useful for reducing or preventing expression in the cell of a protein comprising the amino acid sequence, such as where the protein is not desirable or required or detrimental to the cell or is a subject or environment that comprises the cell.
[00081] An aspect provides:-A method of treating or preventing a disease or condition in a human or animal subject, the method comprising (i) administering to the subject a pharmaceutical composition according to the invention wherein the composition comprises said protein, wherein the protein mediates treatment or prevention of the disease or condition; or (ii) administering to the subject a pharmaceutical composition according to the invention, wherein when the composition comprises said progeny cells or cell line, the cells or cell line expresses a protein or RNA in the subject, and wherein the protein or RNA
mediates treatment or prevention of the disease or condition.
[00082] Example diseases and conditions are disclosed below.
[00083] For example, the RNA encodes a therapeutic or prophylactic protein that is expressed in the subject. For example, the protein is a therapeutic or prophylactic protein.
The protein may exert a therapeutic or prophylactic cell by interacting with a further protein (eg, an endogenously-encoded protein) in the subject, or by interacting with a further cell of the subject.
[00084] Optionally, the protein is an antibiotic, antibacterial agent, enzyme, growth factor, antigen-binding protein (eg, an antibody or fragment thereof), hormone, blood component, cytokine, immune checkpoint modulator (eg, inhibitor or upregulator), analgesic, neurotransmitter, anti-inflammatory agent or anti-neoplastic agent.
1000851 Optionally, the plurality of cells is comprised by a microbiome sample, wherein the method is carried out in vitro and produces a modified cell sample in which cells of the first species or strain have been killed, the method further comprising combining the modified sample with a pharmaceutically acceptable carrier, diluent or excipient, thereby producing a pharmaceutical composition comprising a cell transplant. For example, the transplant may be administered to the gastrointestinal (GI) tract or gut of a human or animal subject, eg, by oral administration, or by rectal administration. For example the transsplant may be administered by vaginal administration.
[00086] Optionally, a microbiome herein is a gut, lung, kidney, urethral, bladder, blood, vaginal, eye, ear, nose, penile, bowel, liver, heart, tongue, hair or skin microbiome.
[00087] An aspect provides:-A method of treating or preventing a disease or condition in a human or animal subject, the method comprising administering to the subject a pharmaceutical composition of the invention.
[00088] An aspect provides:-An ex vivo or in vitro method of treating an environment or cell sample, the method comprising exposing the environment or sample to a composition of the invention, wherein cells comprised by the environment or sample arc modified, edited or killed, or the growth or proliferation of cells of the environment or sample is reduced.
For example, the cells are killed. For example, the cells are edited by the editing method of the invention. Optionally, the treated sample is administered to a human or animal subject or is contacted with an environment.

[00089] Optionally, the plurality of cells is comprised by an environmental sample (eg, an aqueous, water, oil, petroleum, soil or fluid (such as an air or liquid) sample). A
suitable environment may be contents of an industrial or laboratory apparatus or container, eg, a fermentation vessel.
[00090] Optionally, the method of the invention is carried out in vitro.
Optionally, the method of the invention is carried out ex vivo.
[00091] An aspect provides:-A composition for use in a method treating or preventing a disease or condition in a human or animal subject that is mediated by target cells, the composition comprising components (a), (b) or (d):-(a) first and second crRNAs;
(b) nucleic acid encoding first and second crRNAs, wherein the nucleic acid is expressible in a target cell for producing the crRNAs;
(c) a first nucleic acid encoding a first crRNA, and a second nucleic acid encoding a second crRNA, wherein the nucleic acids are expressible in a target cell for producing the crRNAs;
or (d) a first crRNA and a nucleic acid encoding a second crRNA, wherein the nucleic acid is expressible in a target cell for producing the second crRNA;
wherein (e) the first crRNA (crRNA1) is capable of guiding a first Cas (Cl) to a protospacer sequence (PS1) comprised by a target cell genome to modify PS1; and (f) the second crRNA (crRNA2) is capable of guiding a second Cas (C2) to a protospacer sequence (PS2) comprised by the target cell genome to modify PS2;
(g) Cl and C2 are different;
(h) PS1 and PS2 are different; and wherein the method comprises administering the composition to the subject whereby said components of the composition are introduced into target cells wherein crRNA1, crRNA2, Cl and C2 are provided in each cell and the genome of each cell is subjected to Cas modification and the disease or condition is treated or prevented.
[00092] Optionally, the treating or preventing compriscs carrying out the method of thc invention.
1000931 Optionally, the method is for reducing an infection of the subject by target cells (optionally wherein the target cells are pathogenic cells, such as pathogenic prokaryotic cells, such as pathogenic bacterial cells).
[00094] Optionally, the components are comprised by one (or one or more) nucleic acid vectors. In an example, each vector is a virus, phage, plasmid (eg, a conjugative plasmid), cosmid, phagemid or nanoparticic (cg, a liposomc).

[00095] In an example, any method herein is carried out on a population (or said plurality) of said cells, wherein the population comprises at least 100 of said cells and the genome of at least 90, 99, 99.9, 99.99, 99.999, 99.9999, 99.99999, 99.999999, 99.9999999, 99.99999999 or 99.999999999% of said cells are modified, eg, subjected to Cas nuclease cutting. In an embodiment, the population (or said plurality) comprises at least 1000 of said cells. In an embodiment, the population (or said plurality) comprises at least 10,000 of said cells. In an embodiment, the population (or said plurality) comprises at least 100,000 of said cells. In an embodiment, the population (or said plurality) comprises at least 1,000,000 of said cells. In an embodiment, the population (or said plurality) comprises at least 10,000,000 of said cells. In an embodiment, the population (or said plurality) comprises at least 100,000,000 of said cells. In an embodiment, the population (or said plurality) comprises at least 1000,000,000 of said cells. In an embodiment, the population (or said plurality) comprises at least 10,000,000,000 of said cells.
[00096] In an example, the population or said plurality is comprised by a microbiome of a human, animal (eg, a livestock animal or companion pet), plant or environment (eg, a waterway, soil, fluid microbiome).
[00097] An aspect provides:-A composition comprising components (a), (b) or (d):-(a) first and second crRNAs;
(b) nucleic acid encoding first and second crRNAs, wherein the nucleic acid is expressible in a target cell for producing the crRNAs;
(c) a first nucleic acid encoding a first crRNA, and a second nucleic acid encoding a second crRNA, wherein the nucleic acids are expressible in a target cell for producing the crRNAs; or (d) a first crRNA and a nucleic acid encoding a second crRNA, wherein the nucleic acid is expressible in a target cell for producing the second crRNA;
wherein (e) the first crRNA (crRNA1) is capable of guiding a first Cas (C1) to a protospacer sequence (PS1) comprised by a target cell genome to modify PS1; and (f) the second crRNA (crRNA2) is capable of guiding a second Cas (C2) to a protospacer sequence (PS2) comprised by the target cell genome to modify PS2;
(g) CI and C2 are different;
(h) PS1 and PS2 are different; and wherein when said components of the composition are introduced into a target cell whereby crRNA1, crRNA2, Cl and C2 are provided in the cell, the genome of the cell is subjected to Cas modification.
[00098] Optionally, the genome of each cell is edited or the cell is killed.
1000991 Optionally, each cell is a prokaryotic cell (optionally bacterial or archaeal cell).

[000100] Optionally, said nucleic acid(s) is(are) comprised by a virus (eg, an AAV, or cytomegalovirus, optionally wherein each cell is a mammalian cell, such as a human cell), phage (eg, wherein each cell is a bacterial cell), plasmid (optionally a conjugative plasmid, eg, wherein each cell is a bacterial cell), nanoparticle (eg, a liposome or gold particle) or phagemid (eg, wherein each cell is a bacterial cell).
[000101] When the nucleic acid is comprised by a virus, the cell may be a mammalian (eg, human or rodent, mouse or rat) cell, a bacterial cell, an archaeal cell or an amoeba cell. When the nucleic acid is comprised by a phage, the cell may be a bacterial cell.
[000102] Optionally, said nucleic acid(s) encode Cl and/or C2.
[000103] Optionally, Cl is a Type I Cas and said nucleic acid(s) encode one or more Cascade Cas that are operable with Cl and/or wherein C2 is a Type I Cas and said nucleic acid(s) encode one or more Cascade Cas that are operable with C2.
10001041 An aspect provides:-A pharmaceutical composition which is a composition according to the invention, wherein the composition comprises a pharmaceutically acceptable excipient, diluent or carrier.
[000105] The composition may be an aqueous composition. The composition may be a lyophilised or freeze-dried composition, eg, in a formulation that is suitable for inhaled delivery to the patient.
[000106] Optionally, the composition is comprised by a sterile medicament administration device, optionally a syringe, IV bag, intranasal delivery device, inhaler, nebuliser or rectal administration device). Optionally, the composition is comprised by a cosmetic product, dental hygiene product, personal hygiene product, laundry product, oil or petroleum additive, water additive, shampoo, hair conditioner, skin moisturizer, soap, hand detergent, clothes detergent, cleaning agent, environmental remediation agent, cooling agent (eg, an air cooling agent) or air treatment agent.
[000107] In an example the composition is comprised by a device for delivering the composition as a liquid or dry powder spray. This may be useful for administration topically to patients or for administration to large environmental areas, such as fields or waterways.
[000108] Optionally, the cells arc comprise by a gut, lung, kidney, urethral, bladder, blood, vaginal or skin microbiome of the subject.
[000109] Optionally, the method is carried out on a human or animal subject, wherein the cells are killed by the method and the killing upregulates or downregulates immune cells (optionally (i) upregulating CD8+, CD4+,TH1, TH2, TH17, NK cells, TILS, T regulatory or T
effector cells; or (ii) downregulating CDg+, CD4,'TH1, 'TH2, 'TH17, T regulatory or T effector cells) in the subject, thereby treating or preventing a disease or condition in the subject. In one preferred embodiment, CD8+, NK or TILS cells are upregulated, eg, wherein the disease or condition is a cancer. In one preferred embodiment, CD8+ or NK cells are upregulated, eg, wherein the disease or condition is a viral infection. In one preferred embodiment, TH1, TH2 or TH17 cells are downregulated, eg, wherein the disease or condition is an autoimmune or inflammatory disease or condition. For example, the disease or condition is a cancer or an autoimmune disease or condition. For example, the disease or condition is a cancer and CD8+ or T effector cells are upregulated in the subject and/or T regulatory cells are downregulated in the subject. For example, the disease or condition is an autoimmune disease or condition and CD 8+ or T effector cells are downregulated in the subject and/or T regulatory cells are upregulated in the subject.
[000110] Optionally, the method comprises introducing into each cell or expressing in each cell at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 (preferably, at least 2, 3, 4 or 5;
or exactly 2, 3, 4 or 5, or exactly 8, or at least 8) different types of crRNAs wherein the different types target different protospacer sequences comprised by the cell genome; and optionally wherein Cl and C2 are Class 1 Cas nucleases, eg, Cas 3 nucleases.
10001111 Optionally, the method comprises introducing into each cell a nucleic acid encoding a Cas3, Cas8e, Casll, Cas7, Cas5, and Cas6 (optionally, the Cas are E coli Cas) and/or a nucleic acid encoding a Cas3, Cas6, Cas8b, Cas7, and Cas5 (optionally, the Cas arc C
dijIcile Cas).
[000112] In another example, the method comprises introducing into each cell a nucleic acid encoding a Cas3, Cas8e, Casll, Cas7, Cas5, and a nucleic acid encoding a Cas9.
In another example, the method comprises introducing into each cell a nucleic acid encoding a Cas3, Cas6, Cas8b, Cas7, and Cas5 and a nucleic acid encoding a Cas9.
[000113] An aspect provides:
A method of modifying the genome of a cell, the method comprising (a) using a first CRISPR/Cas system to modify a first protospacer of the genome; and (b) using a second CRISPR/Cas system to modify a second protospacer of the genome, wherein the second protospacer is different to the first protospacer;
wherein the systems comprise different Cas and are provided simultaneously in the cell.
[000114] Optionally, the method comprises using a third CRISPR/Cas system to modify a third protospaccr of the genome, wherein the third protospacer is different to the first and second protospacers. For example, 3 different Cas3 are used; 3 different Cas9 are used; a Cas3 and two different Cas9 are used; or two different Cas3 and a Cas9 are used.
[000115] The method of claim 51, wherein the method is according to any one of claims 1 to 35.
[000116] In certain aspects:-The method of the invention is a method of (a) producing synergistic Cas nuclease cutting of a cell genome;
(b) reducing a population of cells of a first species or strain by at least 100,000, 1,000,000 or 10,000,000-fold;
(c) killing at least at least 99%. 99.9%. 99.99%, 99.999%, 99.9999% or 99.99999% cells of a first species or strain comprised by a microbiome;
(d) producing synergistic Class 1 Cas modification of a cell genome; or (e) reducing bacterial cells of a first species or strain (eg, E coli cells) in a cell population by at least 105, 106 or 107-fold, wherein the population comprises at least 100,000;
1,000,000; or 10,000,000 cells respectively.

Optionally, the disease or condition is selected from (a) A neurodegeneratiye disease or condition;
(b) A brain disease or condition;
(c) A CNS disease or condition-, (d) Memory loss or impairment;
(e) A heart or cardiovascular disease or condition, eg, heart attack, stroke or atrial fibrillation;
A liver disease or condition;
(g) A kidney disease or condition, eg, chronic kidney disease (CKD);
(h) A pancreas disease or condition;
(1) A lung disease or condition, eg, cystic fibrosis or COPD;
(1) A gastrointestinal disease or condition;
(k) A throat or oral cavity disease or condition;
(1) An ocular disease or condition;
(m) A genital disease or condition, eg, a vaginal, labial, penile or scrotal disease or condition;
(n) A sexually-transmissible disease or condition, eg, gonorrhea, HIV
infection, syphilis or Chlamydia infection;
(o) An ear disease or condition;
(13) A skin disease or condition;
(c1) A heart disease or condition;
(r) A nasal disease or condition (s) A haematological disease or condition, eg, anaemia, eg, anaemia of chronic disease or cancer;
(t) A viral infection;
(u) A pathogenic bacterial infection;
(v) A cancer;
(w) An autoimmunc disease or condition, cg, SLE;

(X) An inflammatory disease or condition, eg, rheumatoid arthritis, psoriasis, eczema, asthma, ulcerative colitis, colitis, Crohn's disease or IBD;
(y) Autism;
(z) ADHD;
(an) Bipolar disorder;
(bb) ALS [Amyotrophic Lateral Sclerosis];
(cc) Osteoarthritis;
(dd) A congenital or development defect or condition;
(ee) Miscarriage;
(if) A blood clotting condition;
(gg) Bronchitis;
(hh) Dry or wet AMD;
(ii) Neovascularisation (eg, of a tumour or in the eye);
(j1) Common cold;
(kk) Epilepsy;
(11) Fibrosis, eg, liver or lung fibrosis;
(mm) A fungal disease or condition, eg, thrush;
(nn) A metabolic disease or condition, eg, obesity, anorexia, diabetes, Type I or Type II diabetes.
(oo) Ulcer(s), eg, gastric ulceration or skin ulceration;
(1313) Dry skin;
(WI) Sjogren's syndrome;
(1-1) Cytokine storm;
(ss) Deafness, hearing loss or impairment;
(-11) Slow or fast metabolism (ie, slower or faster than average for the weight, sex and age of the subject);
(uu) Conception disorder, eg, infertility or low fertility;
(vv) Jaundice;
(ww) Skin rash;
(xx) Kawasaki Disease;
(yy) Lyme Disease;
(zz) An allergy, eg, a nut, grass, pollen, dust mite, cat or dog fur or dander allergy;
(aaa) Malaria, typhoid fever, tuberculosis or cholera;
(bbb) Depression;
(ccc) Mental retardation;
(ddd) Microcephaly;
(coo) Malnutrition;
(fff) Conjunctivitis;

(ggg) Pneumonia;
(hhh) Pulmonary embolism;
(iii) Pulmonary hypertension;
(jjj) A bone disorder;
(kkk) Sepsis or septic shock;
(111) Sinusitus;
(nimm) Stress (eg, occupational stress);
(nnn) Thalassaemia, anaemia, von Willebrand Disease, or haemophilia;
(000) Shingles or cold sore;
(ppp) Menstruation;
(qqq) Low sperm count.
NEURODEGENERATIVE OR CNS DISEASES OR CONDITIONS FOR TREATMENT OR
PREVENTION
[00113] In an example, a neurodegenerative or CNS disease or condition is selected from the group consisting of Alzheimer disease , geriopsychosis, Down syndrome, Parkinson's disease, Crcutzfeldt-jakob disease, diabetic neuropathy, Parkinson syndrome, Huntington's disease, Machado-Joseph disease, amyotrophic lateral sclerosis, diabetic neuropathy, and Creutzfeldt Creutzfeldt- Jakob disease. For example, the disease is Alzheimer disease. For example, the disease is Parkinson syndrome.
[00114] In an example, wherein the method of the invention is practised on a human or animal subject for treating a CNS or neurodegenerative disease or condition, the method causes downregulation of Treg cells in the subject, thereby promoting entry of systemic monocyte-derived macrophages and/or Treg cells across the choroid plexus into the brain of the subject, whereby the disease or condition (eg, Alzheimer's disease) is treated, prevented or progression thereof is reduced.
In an embodiment the method causes an increase of IFN-gamma in the CNS system (eg, in the brain and/or CSF) of the subject. In an example, the method restores nerve fibre and//or reduces the progression of nerve fibre damage. In an example, the method restores nerve myelin and//or reduces the progression of nerve myelin damage. In an example, the method of the invention treats or prevents a disease or condition disclosed in W02015136541 and/or the method can be used with any method disclosed in W02015136541 (the disclosure of this document is incorporated by reference herein in its entirety, eg, for providing disclosure of such methods, diseases, conditions and potential therapeutic agents that can be administered to the subject for effecting treatement and/or prevention of CNS and neurodegenerative diseases and conditions, eg, agents such as immune checkpoint inhibitors, eg, anti-PD-1, anti-PD-L1, anti-TIM3 or other antibodies disclosed therein).

CANCERS FOR TREATMENT OR PREVENTION
[00115] Cancers that may be treated include tumours that are not vascularized, or not substantially vascularized, as well as vascularized tumours. The cancers may comprise non-solid tumours (such as haematological tumours, for example, leukaemias and lymphomas) or may comprise solid tumours.
Types of cancers to be treated with the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukaemia or lymphoid malignancies, benign and malignant tumours, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumours/cancers and paediatric tumours/cancers are also included.
[00116] Haematologic cancers are cancers of the blood or bone marrow. Examples of haematological (or haematogenous) cancers include leukaemias, including acute leukaemias (such as acute lymphocytic leukaemia, acute myelocytic leukaemia, acute myelogenous leukaemia and myeloblasts, promyeiocytic, myelomonocytic, monocytic and erythroleukaemia), chronic leukaemias (such as chronic myelocytic (granulocytic) leukaemia, chronic myelogenous leukaemia, and chronic lymphocytic leukaemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myeiodysplastic syndrome, hairy cell leukaemia and myelodysplasia.
[00117] Solid tumours are abnormal masses of tissue that usually do not contain cysts or liquid areas.
Solid tumours can be benign or malignant. Different types of solid tumours are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumours, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous eel!
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumour, cervical cancer, testicular tumour, seminoma, bladder carcinoma, melanoma, and CNS tumours (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiformc) astrocytoma, CNS lymphoma, gcrminoma, mcdu!loblastoma, Schwannoma craniopharyogioma, ependymoma, pineaioma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).
[00118] AUTOIMMUNE DISEASES FOR TREATMENT OR PREVENTION
= Acute Disseminated Encephalomyelitis (ADEM) = Acute necrotizing hemorrhagic leukoencephalitis = Addison's disease = Agammaglobulinemia = Alopecia areata = Amyloidosis = Ankylosing spondylitis = Anti-GBIVI/Anti-TBM nephritis = Antiphospholipid syndrome (APS) = Autoimmune angioedema = Autoimmune aplastic anemia = Autoimmune dysautonomia = Autoimmune hepatitis = Autoimmune hyperlipidemia = Autoimmune immunodeficiency = Autoimmune inner ear disease (AIED) = Autoimmune myocarditis = Autoimmune oophoritis = Autoimmune pancreatitis = Autoimmune retinopathy = Autoimmune thrombocytopenic purpura (ATP) = Autoimmune thyroid disease = Autoimmune urticaria = Axonal & neuronal neuropathies = Balo disease = Behcet's disease = Buttons pemphigoid = Cardiomyopathy = Castleman disease = Celiac disease = Chagas disease = Chronic fatigue syndrome = Chronic inflammatory dcmyclinating polyncuropathy (CIDP) = Chronic recurrent multifocal ostomyelitis (CRMO) = Churg-Strauss syndrome = Cicatricial pemphigoid/benign mucosal pemphigoid = Crohn's disease = Cogans syndrome = Cold agglutinin disease = Congenital heart block = Coxsackie myocarditis = CREST disease = Essential mixed cryoglobulinemia = Demyelinating neuropathies = Dermatitis herpetiformis = Dermatomyositis = Devic's disease (neuromyelitis optica) = Discoid lupus = Dressler's syndrome = Endometriosis = Eosinophilic esophagitis = Eosinophilic fasciitis = Erythema nodosum = Experimental allergic encephalomyelitis = Evans syndrome = Fibromyalgia = Fibrosing alveolitis = Giant cell arteritis (temporal arteritis) = Giant cell myocarditis = Glomerulonephritis = Goodpasture's syndrome = Granulomatosis with Polyangiitis (GPA) (formerly called Wegener's Granulomatosis) = Graves' disease = Guillain-Barre syndrome = Hashimoto's encephalitis = Hashimoto's thyroiditis = Hemolytic anemia = Henoch-Schonlein puipura = Herpes gestationis = Hypogammaglobulinemia = Idiopathic thrombocytopenic purpura (ITP) = IgA nephropathy = IgG4-related sclerosing disease = Immunoregulatory lipoproteins = Inclusion body myosins = Interstitial cystitis = Juvenile arthritis = Juvenile diabetes (Type 1 diabetes) = Juvenile myositis = Kawasaki syndrome = Lambert-Eaton syndrome = Leukocytoclastic vasculitis = Lichen planus = Lichen sclerosus = Ligneous conjunctivitis = Linear IgA disease (LAD) = Lupus (SLE) = Lyme disease, chronic = Meniere's disease = Microscopic polyangiitis = Mixed connective tissue disease (MCTD) = Mooren's ulcer = Mucha-Habermann disease = Multiple sclerosis = Myasthenia gravis = Myositis = Narcolepsy = Neuromyelitis optica (Devic's) = Neutropenia = Ocular cicatricial pemphigoid = Optic neuritis = Palindromic rheumatism = PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus) = Paraneoplastic cerebellar degeneration = Paroxysmal nocturnal hemoglobinuria (PNH) = Parry Romberg syndrome = Parsonnage-Turner syndrome = Pars planitis (peripheral uveitis) = Pemphigus = Peripheral neuropathy = Perivenous encephalomyelitis = Pernicious anemia = POEMS syndrome = Polyarteritis nodosa = Type I, II, & III autoimmune polyglandular syndromes = Polymyalgia rheumatica = Polymyositis = Postmyocardial infarction syndrome = Postpericardiotomy syndrome = Progesterone dermatitis = Primary biliary cirrhosis = Primary sclerosing cholangitis = Psoriasis = Psoriatic arthritis = Idiopathic pulmonary fibrosis = Pyoderma gangrenosum = Pure red cell aplasia = Raynauds phenomenon = Reactive Arthritis = Reflex sympathetic dystrophy = Reiter's syndrome = Relapsing polychondritis = Restless legs syndrome = Retroperitoneal fibrosis = Rheumatic fever = Rheumatoid arthritis = Sarcoidosis = Schmidt syndrome = Scleritis = Scleroderma = Sjogren's syndrome = Sperm & testicular autoimmunity = Stiff person syndrome = Subacute bacterial endocarditis (SBE) = Susac's syndrome = Sympathetic ophthalmia = Takayasu's arteritis = Temporal arteritis/Giant cell arteritis = Thrombocytopcnic purpura (TTP) = Tolosa-Hunt syndrome = Transverse myelitis = Type I diabetes = Ulcerative colitis = Undifferentiated connective tissue disease (UCTD) = Uveitis = Vasculitis = Vesiculobullous dennatosis = Vitiligo = Wegener's granulomatosis (now termed Granulomatosis with Polyangiitis (GPA).
[00119] INFLAMMATORY DISEASES FOR TREATMENT OR PREVENTION
= Alzheimer's = ankylosing spondylitis = arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic arthritis) = asthma = atherosclerosis = Crohn's disease = colitis = dermatitis = diverticulitis = fibromyalgia = hepatitis = irritable bowel syndrome (IBS) = systemic lupus erythematous (SLE) = nephritis = Parkinson's disease = ulcerative colitis.
[000120] Optionally, the cells are C dificile, P aeruginosa, K
pneumoniae (eg, carbapenem-resistant Klebsiella pneumoniae or Extended-Spectrum Beta-Lactamase (ESBL)-producing K
pneumonicte), E coli (eg, ESBL-producing E. coli, or E. coli ST131-025b:H4), H
pylori, S
pneumoniae or S aureus cells.
[000121] A vector herein may be a high copy number plasmid or phagemid comprising a constitutive promoter for controlling the expression of crRNAs and optionally one or more Cas proteins [000122] In an example, promoter is a medium strength promoter. In another example, the promoter is a repressible promoter or an inducible promoter cell. Examples of suitable repressible promoters are Ptac (repressed by lad) and the Leftward promoter (pL) of phage lambda (which repressed by the ),,cI repressor). In an example, the promoter comprises a repressible operator (eg, tet0 or lac0) fused to a promoter sequence. Optionally, the promoter has an Anderson Score (AS) of 0.5>AS >0.1.
GENERALLY APPLICABLE FEATURES:
[000123] Any cell herein may be a bacterial cell, archaeal cell, algal cell, fungal cell, protozoan cell, invertebrate cell, vertebrate cell, fish cell, bird cell, mammal cell, companion animal cell, dog cell, cat cell, horse cell, mouse cell, rat cell, rabbit cell, eukaryotic cell, prokaryotic cell, human cell, animal cell, rodent cell, insect cell or plant cell. Preferably, the cell is a bacterial cell. Alternatively, the cell is a human cell.
10001241 Optionally, Cl and C2 is any Cas (eg, a Cas2, 3, 4, 5, or

6) of a Type I system. In this example, in an embodiment_ the Cas may be fused or conjugated to a moiety that is operable to increase or reduce transcription of a gene comprising the target protospacer sequence. For example the nucleic acid encoding the Cas that is introduced into a cell may comprise a nucleotide sequence encoding the moiety, wherein the Cas and moiety are expressed in the host cell as a fusion protein. In one embodiment, the Cas is N-terminal of the moiety; in another embodiment it is C-terminal to the moiety.
[000125] In an example, a vector herein is a DNA vector, eg, ssDNA
vector or dsDNA vector.
Optionally, the vector comprises a second nucleotide sequence encoding one or more Cascade proteins. For example, the Cascade protein(s) are cognate with the Cl or C2, which is a Cas3.
[000126] In an example, Casl or Cas2 is a Cas3 that is cognate with Cascade proteins encoded by the cell.
[000127] Optionally, the Cas3 is a Cas3 encoded by a CRISPR/Cas locus of a first bacterial or archaeal species, wherein in the locus the Cas3-encoding sequence is 3' of Cascade protein-encoding sequences (ie, the latter are between the Cas3 and the 5'-most promoter of the locus). Optionally, the Cas3 is a ygcB protein.
[000128] Optionally, the Cascade proteins comprise or consist of cas5 (casD, csy2), cas6 (cas6f, cse3, casE), cas7 (csc2, csy3, cse4, casC) and cas8 (casA, cas8al, cas8b1, cas8c, caslOd, cas8e, csel, cas8f, csy 1).
[000129] Optionally herein the promoter and the Cas3-encoding or crRNA-encoding sequence are spaced no more than 150, 100, 50, 40, 30, 20 or 10bp apart, eg, from 30-45, or 30-40, or 39 or around 39bp apart. Optionally herein a ribosome binding site and the Cas3-encoding or crRNA-encoding sequence are spaced no more than 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 4 or 3bp apart, cg, from 10-5, 6 or around 6bp apart.

[000130] In an example, a promoter herein is in combination with a Shine-Dalgarno sequence comprising the sequence 5'- anagaggagaaa 3' (SEQ ID NO: 5) or a ribosome binding site homologue thereof. Optionally the promoter has an Anderson Score (AS) of AS >0.5; or an Anderson Score (AS) of 0.5>AS >0.1; or an Anderson Score (AS) of <0.1.
[000131] Optionally, the first crRNA-encoding nucleic acid sequence, the second crRNA-encoding nucleic acid sequence or operon is comprised by a mobile genetic element. Suitable mobile genetic elements, eg, transposons, are disclosed in W02016177682 and US20170246221, the disclosures of which are explicitly incorporated herein for possible use in the invention and for providing one or more features for the claims herein.
[000132] Optionally, the vector is devoid of nucleotide sequence encoding one, more or all of a Casl, Cas2, Cas4, Cas6 (optionally Cas6f), Cas7 and Cas 8 (optionaly Cas8f).
Optionally, the vector is devoid of a sequence encoding a Cas6 (optionally a Cas6f). Optionally, tlhe vector comprises (optionally in 5' to 3- direction) nucleotide sequence encoding one, more or all of Cas 11, Cas7 and Cas8a1. Optionally, the vector comprises nucleotide sequence encoding Cas3' and/or Cas3". In one embodiment, the vector comprises nucleotide sequences (in 5' to 3' direction) that encode a Cas3 (eg, Cas3' and/or Cas3"), Cash, Cas7 and Cas8a1.
[000133] Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Cas 11 sequence. Optionally, the vector comprises a Type IA CRISPR
array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA
comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a host cell when the vector has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell. Similarly, the single guide RNAs encoded by the vector in one embodiment are operable with the Cas and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell.
[000134] Optionally, each cell comprises a Type IA CRISPR array that is cognate with the Cas3 (Cl or C2). Optionally, each cell comprises an endogenous Type IB, C, U, D, E or F
CRISPR/Cas system. Optionally, the vector comprises (optionally in 5' to 3' direction) nucleotide sequence encoding one, more or all of Cas8b1, Cas7 and Cas5. In one embodiment, the vector comprises nucleotide sequences (in 5' to 3' direction) that encode a Cas3, Cas8b1, Cas7 and Cas5.
Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Cas8b1 sequence. Optionally, the vector comprises a Type TB CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a host cell when the vector has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell. Similarly, the single guide RNAs encoded by the vector in one embodiment are operable with the Cos and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell.
[000135] Optionally, the cell comprises a Type TB CRISPR array that is cognate with the Cas3.
Optionally, the cell comprises an endogenous Type IA, C, U, D, E or F
CRISPR/Cas system.
Optionally, the vector comprises (optionally in 5' to 3' direction) nucleotide sequence encoding one, more or all of Cas5, Cas8c and Cas7. In one embodiment, the vector comprises nucleotide sequences (in 5' to 3' direction) that encode a Cas3, Cas5, Cas8c and Cas7. Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Cas5 sequence.
Optionally, the vector comprises a Type IC CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a host cell when the vector has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cos and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell. Similarly, the single guide RNAs encoded by the vector in one embodiment are operable with the Cas and Cascade proteins to target and modify (cg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell.
[000136] Optionally, the host cell comprises a Type IC CRISPR
array that is cognate with the Cas3. Optionally, the host cell comprises an endogenous Type IA, B, U, D, E or F CRISPR/Cas system. Optionally, the vector comprises (optionally in 5' to 3' direction) nucleotide sequence encoding one, more or all of Cas8U2, Cas7, Cas5 and Cas6. In one embodiment, the vector comprises nucleotide sequences (in 5' to 3' direction) that encode a Cas3, Cas8U2, Cas7, Cas5 and Cas6. Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Cas8U2 sequence.
[000137] Optionally, the vector comprises a Type IU CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a host cell when the vector has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell. Similarly, the single guide RNAs encoded by the vector in one embodiment are operable with the Cos and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell.
[000138] Optionally, the host cell comprises a Type IU CRISPR
array that is cognate with the Cas3. Optionally, the host cell comprises an endogenous Type IA, B, C, D, E or F CRISPR/Cas system. Optionally, the vector comprises (optionally in 5' to 3' direction) nucleotide sequence encoding one, more or all of Cas lOd, Cas7 and Cas5. Optionally, the vector comprises a nucleotide sequence encoding Cas3' and/or Cas3¨. In one embodiment, the vector comprises nucleotide sequences (in 5' to 3' direction) that encode a Cas3, CaslOd, Cas7 and Cas5.
Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the CaslOd sequence. Optionally, the vector comprises a Type ID CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a host cell when the vector has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cos and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell. Similarly, the single guide RNAs encoded by the vector in one embodiment are operable with the Cas and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell.
[000139] Optionally, the host cell comprises a Type ID CRISPR
array that is cognate with the Cas3.
[000140] Optionally, the host cell comprises an endogenous Type IA, B, C, U, E or F
CRISPR/Cas system.
[000141] Optionally, the vector comprises (optionally in 5' to 3' direction) nucleotide sequence encoding one, more or all of Cas8e, Os' I, Cas7, Cas5 and Cas6. In one embodiment, the vector comprises nucleotide sequences (in 5' to 3' direction) that encode a Cas3, Cas8e, Casll, Cas7, Cas5 and Cas6. Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Casll sequence. Optionally, the vector comprises a Type IE CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a host cell when the vector has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell. Similarly, the single guide RNAs encoded by the vector in one embodiment are operable with the Cas and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell.
[000142] Optionally, the host cell comprises a Type IE CRISPR
array that is cognate with the Cas3.
[000143] Optionally, the host cell comprises an endogenous Type IA, B, C, D, U or F
CRISPR/Cas system.
10001441 Optionally, the vector comprises (optionally in 5' to 3' direction) nucleotide sequence encoding one, more or all of Cas8f, Cas5, Cas7 and Cas6f. In one embodiment, the vector comprises nucleotide sequences (in 5' to 3' direction) that encode a Cas3, Cas8f, Cas5, Cas7 and Cas6f.
Optionally, a nucleotide sequence encoding Cas6 is between the Cas3 sequence(s) and the Cas8f sequence. Optionally, the vector comprises a Type IF CRISPR array or one or more nucleotide sequences encoding single guide RNA(s) (gRNA(s)), wherein the array and each gRNA comprises repeat sequence that is cognate with the Cas3. Thus, the array is operable in a host cell when the vector has been introduced into the cell for production of guide RNAs, wherein the guide RNAs are operable with the Cas and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell. Similarly, the single guide RNAs encoded by the vector in one embodiment are operable with the Cos and Cascade proteins to target and modify (eg, cut) a target nucleotide sequence in the host cell, optionally thereby killing the host cell.
[000145] Optionally, the host cell comprises a Type IF CRISPR
array that is cognate with the Cas3.
[000146] Optionally, the host cell comprises an endogenous Type IA, B, C, D, U or E
CRISPR/Cas system.
[000147] Optionally, the Cas and Cascade are Type IA Cas and Cascade proteins.
[000148] Optionally, the Cas and Cascade are Type TB Cas and Cascade proteins.
[000149] Optionally, the Cas and Cascade are Type IC Cas and Cascade proteins.
[000150] Optionally, the Cas and Cascade are Type ID Cas and Cascade proteins.
10001511 Optionally, the Cas and Cascade are Type IE Cas and Cascade proteins.
[000152] Optionally, the Cas and Cascade are Type IF Cas and Cascade proteins.
10001531 Optionally, the Cas and Cascade are Type 1U Cas and Cascade proteins.
[000154] Optionally, the Cas and Cascade are E coil (optionally Type IE or IF) Cas and Cascade proteins, optionally wherein the E coil is ESBL-producing E. coil or E
coil ST13 1-025b:H4.
[000155] Optionally, the Cas and Cascade are Clostridium (eg, C
dificile) Cas and Cascade proteins, optionally C dificile resistant to one or more antibiotics selected from aminoglycosides, lincomycin, tetracyclines, erythromycin, clindamycin, penicillins, cephalosporins and fluoroquinolones.
10001561 Optionally, the Cas and Cascade are Pseudomonas aeruginosa Cas and Cascade proteins, optionally P aeruginosa resistant to one or more antibiotics selected from carbapenems, aminoglycosides, cefepime, ceftazidime, fluoroquinolones, piperacillin and tazobactam.
[000157] Optionally, the Cas and Cascade are Klebsiella pneumoniae (eg, carbapenem-resistant Klebsiella pneumoniae or Extended-Spectrum Beta-Lactamase (ESBL)-producing K
pneumoniae) Cas and Cascade proteins.
[000158] Optionally, the Cas and Cascade are E coli, C difficile, P aeruginosa, K pneumoniae, P furiosus or B halodurans Cos and Cascade proteins.
10001591 Optionally, each crRNAs or gRNAs comprises a spacer sequence that is capable of hybridising to a protospacer nucleotide sequence of the cell, wherein the protospacer sequence is adjacent a PAM, the PAM being cognate to the CI or C2, wherein CI or C2 is a Cas nuclease, eg, a Cas3. Thus, the spacer hybridises to the protospacer to guide the Cas3 to the protospacer. Optionally, the Cas3 cuts the protospacer, eg, using exo- and/or endonuclease activity of the Cas3. Optionally, the Cas3 removes a plurality (cg, at least 2, 3,4, 5, 6, 7, 8, 9 or 10) nucleotides from the protospacer.

[000160] Optionally, the vector is a phage or non-replicative transduction particle. The phage or particles comprise phage coat proteins encapsidating DNA, wherein the DNA
comprises the vector.
Suitable examples of phage and particles are disclosed in U52019/0160120 the disclosures of which are incorporated herein by reference for possible use in the invention and for providing one or more features that may be included in gthe claims herein. Phage or particle is capable of infecting the cell, thereby introducing the vector into the cell.
[000161] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications and all US equivalent patent applications and patents are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Reference is made to the publications mentioned herein and equivalent publications by the US Patent and Trademark Office (USPTO) or WIPO, the disclosures of which are incorporated herein by reference for providing disclosure that may be used in the present invention and/or to provide one or more features (eg, of a vector) that may be included in one or more claims herein.
[000162] The use of the word "a" or "an" when used in conjunction with the term "comprising"
in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[000163] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have"
and "has"), "including" (and any form of including, such as "includes" and "include") or "containing"
(and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
[000164] The term "or combinations thereof' or similar as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.

Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
[000165] Any part of this disclosure may be read in combination with any other part of the disclosure, unless otherwise apparent from the context.
[000166] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.
While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
[000167] The present invention is described in more detail in the following non-limiting Examples.
EXAMPLES
EXAMPLE 1. Combination of Type I CRISPR-Cas systems to synergistically target multiple genomic protospacers [000168] A plasmid (which we call a CRISPR Guided Vector, CGV) was constructed comprising an operon with nucleotide sequences encoding a type 1 Cas3 and Cascade proteins under the control of a constitutive promoter. E. coli type IE Cas3 and Cascade was used. A cognate CRISPR
array comprising E. coli direct repeat sequences and spacers for targeting an E. coli host cell chromosome was also cloned in the vector. An adaptation module containing Casl and Cas2 was omitted in the vector (see Figure 1A).
[000169] A plasmid was constructed comprising an operon with nucleotide sequences encoding a type I Cas3 and Cascade proteins under the control of constitutive promoters. C. difficile type IB
Cas3 and Cascade was used. An adaptation module containing Casl, Cas2 and Cas4 was omitted in the vector (see Figure 1B). A cognate CRISPR array comprising C. difficile repeat sequences and spacers for targeting an E. coli host cell chromosome was cloned in a second vector, under the control of constitutive promoters (see Figure 1B).
[000170] The CGV encoding C. difficile type TB Cas3 and Cascade was transformed into E.
coli MG1655. Subsequently, the vector encoding C. difficile array and the CGV
encoding E. coli type IE CRISPR-Cas system were transformed into the cells. CFU assays arc shown in Figure 2. In Figure 2 there is shown CRISPR killing of -target strain E. coli MG1655 by C.
difficile CRISPR-Cas system in combination with E. coli CRISPR-Cas system. Killing of essentially 100% of the population was achieved when combining both systems (a bit more than 7- logioreduction in viable cells of E. coli MG1655). However, transformation of E. coli CRISPR-Cas system alone resulted in ¨4-logio reduction in the bacterial population, and transformation of C. difficile array in a bit less than 3-logio reduction. These results indicate that E. coil CRISPR-Cas system and C.
difficile CRISPR-Cas system are compatible, and their combination synergistically improves the killing efficiency of target strain greatly, hampering the growth of escapers.
Materials and methods [000171] E. coli MG1655 was grown in lysogeny broth (LB) with shaking (250 rpm) at 37 C.
When necessary, cultures were supplemented with tetracycline (10 m/mL), kanamycin (50 Kg/mL), and spectinomycin (100 lig/mL).
[000172] To construct a plasmid containing E. coli CRISPR-Cas system under a constitutive promoter, cas3, cas8e, cash, cas7, cas5, and cas6 genes from E. coli were amplified and cloned in a ColEl-type plasmid, pZE21 (Lutz and Bujard, 1997. Nucleic Acids Research, 25, 1203-1210) under the control of a promoter. cas3 was located in the beginning of the operon followed by cas8e, cash, cas7, cas5, and cas6. The adaptation module (consisting of earl and cas2) was omitted in the vector.
Additionally, a 3-spacer array targeting 3 chromosomal intergenic regions in E. coli MG1655 was included in the CGV under the control of a promoter. It contained 32 nucleotides from the genome of E. coli MG1655 per target locus (TGATTGACGGCTACGGTAAACCGGCAACGTTC;
GCTGTTAACGTACGTACCGCGCCGCATCCGGC; and CGGACTTAGTGCCAAAACATGGCATCGAAATT) separated by 29 bp direct repeats (each repeat was GAGTTCCCCGCGCCAGCGGGGATAAACCG). Additionally, the 3'-AAG protospacer adjacent motif (PAM) is located adjacent to the selected target sequences in the genome of E. coli MG1655 (Figure 1A).
[000173] C. difficile CRISPR-Cas system was constructed in a two-plasmid system. To construct a plasmid containing C. difficile cas genes, cas3, cas6, cas8b, cas7, and cas5 genes from C.
difficile were amplified and cloned in a pSC101 backbonep under the control of a promoter. The cas3 was located in the beginning of the operon followed by cas6, cas8b, cas7, and cas5. The adaptation module (consisting of casl, cas2, and cas4) was omitted in the vector (Figure 1B). A
second plasmid containing a 5-spacer array was cloned in a CloDF13 on backbone under the control of a promoter J23100. It contained 37 nucleotides from the genome of E. coli MG1655 per target locus (GCCATAATCTGGATCAGGAAGTCTTCCTTATCCATAT;
GGCTTTACGCCAGCGACGTATTGCCACAGGAATAACT;
GGGGATAGCGCGCCTGGAGCGTGCGATAGAGACTTTG;
GGCATTTACCGACCAGCCCATCAGCAGTACAGCAAAC; and TCCTGAATCAAATCCGCCTGTGGCAGGCCATAGCCCG) separated by 29 bp direct repeats (each repeat was GTTTTATATTAACTAAGTGGTATGTAAAT). Additionally, the 3'-CCT
protospacer adjacent motif (PAM) is located adjacent to the selected target sequences in the genome of E. coil MG1655 (Figure 1B).
[000174] To perform killing assays, the plasmid harboring cas3 and cascade genes of C.
difficile was transformed into E. coli MG1655 by electroporation.
Transformants were grown in liquid LB with the antibiotic to mid-log phase, and further electroporated with a plasmid harboring C.
difficile array and a plasmid with E. coil CRISPR-Cas system. Controls with empty vectors, and with each CGV separately were performed. Killing efficiency was determined by plating the transformations onto LB with antibiotics. Viability was calculated by counting colony forming units (CFUs) on the plates and data were calculated as viable cell concentration (CFU/ml).

a .

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. TABLE 1: Example Bacteria Optionally, the cell or cells are cell(s) of a genus or species selected from this Table. 0 t..) Abiotrophia Acidocella Actinomyces Alkalilimnicola Aquaspirillum is.) ,-, , k..) Abiotrophia defectiva Acidocella aminolytica Actinomyces bovis Alkalilimnicola ehrlichii Aquaspirillum polyntorphum -.1 Acaricomes Acidocella facilis Actinomyces denticolens Alkaliphilus Aquaspirillum x Acaricomes phytoseiuli Acidomonas Actinomyces europaetts Alkaliphilus oremlandii putridiconchyliwn Acetitomaculum Acidomonas methanolica Actinomyces georgiae Alkaliphilus transvaalensis Aquaspirillum serpens Acetitomaculum ruminis Acidothermus Actinomyces gerencseriae Allochromatium Aquimarina Acetivibrio Acidothermus cellulolyticus Actinomyces Allochromatium vinosum Aquimarina latercula Acetivibrio cellulolyficus Acidovorax horcleovulneris Alloiococcus Arcanobacterium Acetivibrio ethanolgignens Acidovorax anthurii Actinomyces howellii Alloiococcus otitis Arcanobacterium Acetivibrio multivorans Acidovorax caeni Actinomyces hyovaginalis Allokutzneria haemolyticum ci.) oe Acetoanaerobium Acidovorax cattle yae Actinomyces israelii Allokutzneria albata Arcanobacterittm pyo genes Acetoanaerobium note rae Acidovorax citrulli Actinomyces johnsonii Altererythrobacter Archangium Acetobacter Acidovorax defluvii Actinomyces meyeri Altererythrobacter Archangium gephyra Acetobacter aceti Acidovorax delafieldii Actinomyces naeslundii ishigakiensis Arcobacter Acetobacter cerevisiae Acidovorax facilis Actinomyces neuii Altermonas Arcobacter butzleri Acetobacter cibinongensis Acidovorax konjaci Actinomyces odontolyticus Altennonas haloplanktis Arcobacter cryaerophilus Acetobacter estunensis Acidovorax temperans Actinomyces oris Altennonas macleodii Arcobacter halophilus od r) Acetobacter fabarum Acidovorax valerianellae Actinomyces radingae Alysiella Arcobacter nitrofigilis ....1 t=i ot Acetobacter ghanensis Acinetobacter Actinomyces slackii Alysiella crassa Arcobacter skirrowii t..) o w 1-, Acetobacter indonesiensis Acinetobacter baumannii Actinomyces turicensis Alysiella filifortnis Arhodomonas -6-c, w Acetobacter lovaniensis Acinetobacter baylyi Actinomyces viscosus Arhodomonas aquaeolei ul .6.

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Lo Acetobacter motor= Acinetobacter bouvetii Actinoplanes Aminobacter Arsenophonus Acetobacter nitrogenifigens Acinetobacter calcoaceficus Actinoplanes auranticolor Aminobacter aganoensis Arsenophonus nasoniae 0 t..) o Acetobacter oeni Acinetobacter gemeri Actinoplanes brasiliensis Aminobacter aminovorans ts.) 1¨
, k..) Acetobacter orientalis Acinetobacter haemolyticus Actinoplanes consettensis Aminobacter niigataensis Arthrobacter w -.1 Acetobacter orleanensis Acinetobacter johnsonii Actinoplanes deccanensis Aminobacterium Arthrobacter agilis w Acetobacter pasteuri anus Acinetobacter junii Actinoplanes denventensis Aminobacterium mobile Arthrobacter albus Acetobacter pomorum Acinetobacter lwoffi Actinoplanes digitatis Aminomonas Arthrobacter aurescens Acetobacter senegalensis Acinetobacter parvus Actinoplanes durhamensis Aminomonas paucivorans Arthrobacter Acetobacter xylinus Acinetobacter radioresistens Actinoplanes ferrugineus Ammoniphilus chloroplienolicus Acetobacterium Acinetobacter schindleri Actinoplanes globisporus Ammoniphilus oxalaticus Arthrobacter citreus Acetobacterium bakii Acinetobacter soli Actinoplanes humidus Ammoniphilus oxalivorans Arthrobacter crystallopoietes Acetobacterium carbinolicum Acinetobacter tancloii Actinoplanes italicus Amphibacillus Arthrobacter cumminsii w v:
Acetobacterium dehalogenans Acinetobacter tjembergiae Actinoplanes liguriensis Arnphibacillus xylanus Arthrobacter globiformis Acetobacterium fimetarium Acinetobacter towneri Actinoplanes lobatus Amphritea Arthrobacter Acetobacterium malicum Acinetobacter ursingii Actinoplanes missouriensis Amphritea balenae histidinolovorans Acetobacterium paludosum Acinetobacter veneti anus Actinoplanes palleronii Amphritea japonica Arthrobacter ilicis Acetobacterium tundrae Acrocarpospora Actinoplanes philippinensis Amycolatopsis Arthrobacter luteus Acetobacterium wieringae Acrocarpospora corrugata Actinoplanes rectilineatus Amycolatopsis alba Arthrobacter methylotrophus Acetobacterium woodii Acrocarpospora Actinoplanes regularis Amycolatopsis albidoflavus Arthrobacter mysorens od r) Acetofilamentum macrocephala Actinoplanes Amycolatopsis azurea Arthrobacter nicotianae ....1 t=i ot Acetofitamentum rigidum Acrocarpospora teichomyceticus Amycolatopsis coloradensis Arthrobacter nicotinovorans t..) o w 1-, Acetohalobium pleiomorpha Actinoplanes utahensis Amycolatopsis lurida Arthrobacter oxydans -O-w Acetohalobium arabaticum Amycolatopsis mediterranei Arthrobacter pascens v:
ul .6.

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Lo Acetomicrobium Actibacter Actinopolyspora Amycolatopsis rifamycinica Arthrobacter Acetoinicrobium faecale Actibacter sediminis Actinopolyspora halophila Amycolatopsis rubido phenanthrenivorans 0 t..) o Acetomicrobium flovidum Actinoalloteichus Actinopolyspora Amycolatopsis sulphurea Arthrobacter ts.) 1.-, k..) Acetonema Actinoalloteichus 171Ortivallis Amycolatopsis tolypomycina polychromogenes w -.1 Acetonema longum cyanogriseus Actinosynnema Anabaena Atrhrobacter protophormiae x Acetothermus Actinoalloteichus Actinosynnema mirum Anctbaena cylindrica Arthrobacter Acetothermtts paticivorans hymeniacidonis Actinotalea Anctbaena flos -aquae psychrolactophilus Acholeplasma Actinoalloteichus spitiensis Actinotalea fermentans Anabctena variabilis Arthrobacter ramosus Acholeplasma axanthum Actinobaccillus Aerococcus Anaeroarcus Arthrobacter sulfonivorans Acholeplasma brassicae Actinobacillus capsulatus Aerococcus sanguinicola Anaeroarcus burkinensis Arthrobacter sulfureus Acholeplasma cavigenitalium Actinobacillus delphinicola Aerococcus urinae Anaerobaculum Arthrobacter uratoxydans Acholeplasma equiletale Actinobacillus hoininis Aerococcus urinaeequi Ancterobaculum mobile Arthrobacter ureafaciens 4, o Acholeplasma granulartun Actinobacillus indolicus Aerococcus urinaehominis Anaerobiospirillum Arthrobacter viscosus Acholeplasma hippikon Actinobacillus lignieresii Aerococcus viridans Ancterobiospirillum Arthrobacter woluwensis Acholeplasma laidlawii Actinobacillus minor Aeromicrobium succiniciproducens Asaia Acholeplasma modicum Actinobacillus muris Aeromicrobium erythrewn Ancterobiospirillum thomasii Asaia bogorensis Acholeplasma morum Actinobacillus Aeromonas Anaerococcus Asanoa Acholeplasma multilocale pleuropneumoniae Aeromonas Anaerococcus hydrogenalis Asanoa ferruginea Acholeplasma oculi Actinobacillus porcinus allosaccharophila Anaerococcus lactolyticus Asticcacaulis It r) Acholeplasma palmae Actinobacillus rossii Aeromonas bestiarum Anaerococcus prevotii Asticcacaulis biprosthecium ....1 t=i ot t..) Acholeplasma parvum Actinobacillus scotiae Aeromonas caviae Anaerococcus tetradius Asticcacaulis excentricus o w 1-, Acholeplasma pleciae Actinobacillus seminis Aeromonas encheleia Anaerococcus vaginalis Atopobacter -6-c, w Acholeplasma vituli Actinobacillus succinogenes Aeromonas Atopobacter phocae ul a .
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. Achromobacter Actinobaccillus suis enteropelo genes Anaerofustis Atopobium Achromobacter denitrift cans Actinobacillus ureae Aeromonas eucrenophila Ancterofustis stercorihomints Atopobiwn fossor t..) o Achromobacter insolitus Actinobaculum Aeromonas ichthiosmia Anaeromusa Atopobium minutum ts.) 1.-, k..) w Achroznobacter piechaudii Actinobaculum massiliense Aeromonas jandaei Ancteromusa acidaminophila Atopobiwn parvulwn -.1 Achromobacter ruhlandii Actinobaculum schaalii Aeromonas media Anaeromyxobacter Atopobium rimae Achromobacter spanius Actinobaculum suis Aeromonas pop offii Ancteromyxobacter Atopobiwn vaginae Acidaminobacter Actinonzyces urinate Aeromonas sobria dehalogenans Aureobacterium Acidaminobacter Actinocatenispora Aeromonas veronii Anaerorhabdus Aureobacterium barkeri hydrogenoformans Actinocatenispora rupis Agrobacterium Ancterorhabdus furcosa Aurobacterium Acidaminococcus Actinocatenispora Agrobacterium Anaerosinus Aurobacterium liquefaciens Acidaminococcus fermen tans thailandica gelatinovo rum Ancterosinus glycerini Avibacterium Acidaminococcus intestini Actinocatenispora sera Agrococcus Anaerovirgula Avibacterium avium 4, 1-, Acidicaldus Actinocorallia Agrococcus citreus Anaerovirgula multivorans Avibacterium gallinarum Acidicaldus organivorans Actinocorallia aurantiaca Agrococcus jenensis Ancalomicrobium Avibacterium paragallinarum Acidimicrobium Actinocorallia aurea Agromonas Ancalomicrobium adetum Avibacterium volantium Acidimicrobium ferrooxidans Actinocorallia cavernae Agromonas oligotrophica Ancylobacter Azoarcus Acidiphilium Actinocorallia glomerata Agromyces Ancylobacter aquaticus Azoarcus indigens Acidiphilium acidophilum Actinocorallia herbida Agromyces fucosus Aneurinibacillus Azoarcus tolulyticus Acidiphilium angustum Actinocorallia libanotica Agromyces hippuratus Aneurinibacillus Azoarcus toluvorans od r) Acidiphilium crypt= Actinocorallia longicatena Agromyces luteolus aneurinilyticus Azohydromonas ....1 t=i ot Acidiphilium multivorum Actinomadura Agromyces mediolanus Aneurinibacillus migulanus Azohydromonas attstralica t..) o w 1-, Acidiphilium organovorwn Actinomadttra alba Agromyces ramosus Aneurinibacillus Azohydromonas lata -O-w Acidiphilium rubrum Actinomadura atramentaria Agromyces rhizospherae thermoaerophilus v:
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. Acidisoma Actinomadura Akkermansia Angiococcus Azomonas Acidisoma sibiricum bangladeshensis Akkermansia nzuciniphila Angiococcus discifonnis Azomonas agilis 0 t..) Acidisoma tundrae Actinomadura catellafispora Albidiferax Angulomicrobium Azomonas insignis ts.) 1.-, k..) Acidisphaera Actinomadura chibensis Albidiferax ferriredttcens Angtdomicrobium tetraedrale Azomonas nzacrocytogenes w -.1 Acidisphaera rttbrifaciens Actinomadura chokoriensis Albidovulum Anoxybacillus Azorhizobium x Acidithiobacillus Actinomadura citrea Albidovulum inexpectatum Anoxybctcillus pushchinoensis Azorhizobium caulinodans Acidithiobacillus albertensis Actinomadura coendea Alcaligenes Aquabacterium Azorhizophilus Acidithiobacillus caldus Actinomadura echinospora Alculigenes denitrificans Aquabacterium commune Azorhizophilus paspuli Acidithiobacillus ferrooxidans Actinomadura fibrosa Alcaligenes faecalis Aquabacterium parvum Azospirillum Acidithiobacillus thiooxidans Ac finomadura formosensis Alcanivorax Azospirdlum brasdense Acidobacterium Actinomadura hibisca Alcanivorax borkumensis Azospirdlum halopraeferens Acidobacterium capsulatum Actinomadura kijaniata Alcanivorax jadensis Azospirdlum irakense 4, t.) Actinomadura lafina Algicola Azotobacter Actinomadura livida Algicola bacteriolytica Azotobacter beijerinckii Actinomadura Alicyclobacillus Azotobacter chroococcum luteofluorescens Alicyclobacillus Azotobacter nigri cans Actinomadura macra disulfidooxidans Azotobacter salinestris Actinomadura madurae Alicyclobacillus Azotobacter vinelandii Actinomadura oligospora sendaiensis od r) Actinomadura pelletieri Alicyclobacillus vulcanalis ....1 t=i ot Actinomadura rubrobrunea Alishewanella t..) o w 1-, Actinomadura rugatobispora Alishewanella fetalis w Actinomadura umbrina v:
ul .0 Actinomadara Alkalibacillus verrucosospora Alkalibacillus Actinomadttra vinacea haloalkaliphilus ts.) Actinonzadara viridilutea rJI
Actinomadttra viridis Actinomadttra yumaensis Bacillus Bacteroides Bibersteinia Borrelia Brevinema [see below] Bacteroides caccae Bibersteinia trehalosi Borrelia afzelii Brevinema andersonii Bacteroides coagulans Bifidobacterium Borrelia americana Brevundimonas Bacteriovorax Bacteroides eggerthii Bifidobacterium adolescentis Borrelia burgdorferi Brevundimonas alba Bactenovorax stolpii Bacteroides fragilis Bifidobacterium angulatum Borrelia carolinensis Brevundimonas aurantiaca Bacteroides galacturonicus Bifidobacterium animalis Borrelia coriaceae Brevundimonas diminuta Bacteroides helco genes Bifidobacteriutn asteroides Borrelia garinii Brevundimonas intermedia Bacteroides ovatus Bifidobacterium bifidum Borrelia japonica Brevundimonas subvibrio ides Bacteroides pectinophilus Bifidobacterium bourn Bosea Brevundimonas vancanneytii Bacteroides pyo genes Bifidobacterium breve Bosea minatitlanensis Brevundimonas variabilis Bacteroides salyersiae Bifidobacteritun catenulatum Bosea thiooxidans Brevundimonas vesicularis Bacteroides stercoris Bifidobacterium choerinum Brachybacterium Brochothrix Bacteroides suis Bifidobacterium corynefonne Brachybacterium Brochothrix campestris Bacteroides tectus Bifidobacteritun cuniculi alimentarium Brochothrix thermosphacta Bacteroides thetaiotaomicron Bifidobacterium dentium Brachybacteriwn faeclum a .

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Lo Bacteroides uniformis Bifidobacteriutn gallicutn Brachybacterium Brucella Bacteroides ureolyticus Bifidobacterium gallinartun paraconglomeratum Brucella cards 0 t..) o Bacteroides vulgatus Bifidobacterium indicum Brachybacterium rhamnosum Brucella neotomae ts.) 1.., -, k..) Balnearium Bifidobacterium longum Brachybacterium Bryobacter w -.1 Balneariufn lithotrophicum Bifidobacterium tyrofermentans Bryobacter aggregatus x Balneatrix magnumBifidobacterium Brachyspira Burkholderia Balneatrix alp ica rnerycicum Brachyspira alvinipulli Burkholderia ambifaria Balneola Bifidobucterium minimum Brachyspira hyodysenteriae Burkholderia andropogonis Balneola vulgaris Bifidobacterium Brachyspira innocens Burkholderia anthina Barnesiella pseualocatentdatunt Brachyspira murdochii Burkholderia adedonica Barnesiella viscericola Bifidobacterium Brachyspira pilosicoli Burkholderia caryophylli Bartonella pseudolongum Burkholderia cenocepacia 4, 4, Bartonella alsatica Bifidobacterhtm pullorum Bradyrhizobium Burkholderia cepacia Bartonella bacillifonnis Bifidobacterium ruminantium Bradyrhizobium canariense Burkholderia cocovenenans Bartonella clarridgeiae Bifidobacterium saeculare Bradyrhizobium elkanii Burkholderia dolosa Bartonella doshiae Bifidobacterium subtile Bradyrhizobium japonicum Burkholderia fungortun Bartonella elizabethae Bifidobacterium Bradyrhizobium liaoningense Burkholderia glathei Bartonella grahamii thermophilum Brenneria Burkholderia glumae Bartonella henselae Bilophila Brenneria alni Burkholderia graminis od r) Bartonella rochalimae Bilophila wadsworthia Brenneria nigrifluens Burkholderia kururiensis ....1 t=i ot Bartonella vinsonii Biostraticola Brenneria quercina Burkholderia multivorans t..) o 1.., Bavariicoccus Biostraticola tofi Brenneria quercina Burkholderia phenazinium -O-w Bavariicoccus seileri Brenneria salicis Burkholderia plantarii fil 4.

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Lo Bdellovibrio Bizionia Brevibacillus Burkholderia pyrrocinia Bdellovibrio bacteriovorus Bizionia argentinensis Brevibacillus agri Burkholderia silvatlantica 0 t..) o Bdellovibrio exovorus Blastobacter Brevibacillus borstelensis Burkholderia stabilis ts.) , k..) Beggiatoa Blastobacter capsulatus Brevibacillus brevis Burkholderia thailandensis w -.1 Beggiatoa alba Blastobacter denitrificans Brevibacillus centrosportts Burkholderia tropica oo Beijerinckia Blastococcus Brevibacillus choshinensis Burkholderia unamae Beijerinckia derxii Blastococcus aggregatus Brevibacillus invocatus Burkholderia vietnamiensis Beijerinckia fluminensis Blastococcus suxobsidens Brevibucillus luterosporus Buttiauxella Beijerinckia indica Blastochloris Brevibacillus parabrevis Buttiauxella agrestis Beijerinckia mobilis Blastochloris viridis Brevibacillus reuszeri Buttiauxella brennerae Belliella Blastomonas Brevibacterium Buttiauxella ferragutiae Belliella baltica Blastornonas natatoria Brevibacterium abidum Buttiauxella gaviniae 4, Bellilinea Blastopirellula Brevibacterium album Buttiauxella izardii Bellilinea caldifistulae Blastopirellula marina Brevibacterium aurantiacum Buttiauxella noackiae Belnapia Blautia Brevibacterium celere Buttiauxella wannboldiae Belnapia moabensis Blautia coccoides Brevibacterium epidermidis Butyrivibrio Bergeriella Blautia hansenii Brevibacterium Butyrivibrio fibrisolvens Bergeriella denitrificans Blautia producta frigoritolerans Butyrivibrio hungatei Beutenbergia Blautia wexlerae Brevibacterium halotolerans Butyrivibrio proteoclasticus od r) Beutenbergia cavernae Bogoriella Brevibacterium iodinum ....1 t=i It Bogoriella caseilytica Brevibacterium linens t..) o w 1-, Bordetella Brevibacterium lyticum -O-w Bordetella avi urn Brevibacterium mcbrellneri ul a ,-.
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Lo Bordetella bronchiseptica Brevibacterium otitidis Bordetella hinzii Brevibacterium oxydans 0 t..) o Bordetella holmesii Brevibacterium paucivorans ts.) 1.-, k..) w Bordetella parapertussis Brevibacterium stationis -.1 Bordetella pertussis oo Bordetella petrii Bordetella trematum Bacillus B. acidiceler B. cuninovorans B. glucanolyticus B. taeanensis B. lautus B. acidicola B. cunylolyticus B. gordonae B. tequilensis B. lehensis 4, o B. acidiproducens B. andreesenii B. gottheilii B. the nnantarcticus B. lentimorbus B. acidocaldarius B. aneurinilyticus B. graminis B. thennoaerophilus B. lentus B. acidoterrestris B. anthracis B. halmapalus B. the nnoamylovorans B. lichen iformis B. aeolius B. aquimaris B. haloalkaliphilus B. thennocatenulatus B. ligniniphilus B. aerius B. arenosi B. halochares B. thennocloacae B. litoralis B. aerophilus B. arseniciselenatis B. halodenitrifi cans B. thennocopriae B. locisalis B. agaradhaerens B. arsenicus B. halodurctns B. thennodenitrificans B. luciferensis od B. agri B. aurantiacus B. halophilus B. thennoglucosidasius B. luteolus r) ....1 t=i B. aidingensis B. arvi B. halosaccharovorans B. thennolactis B. luteus ot t..) o B. akibai B. cuyabhattai B. hemicellulosilyticus B. the nnoleovorans B. macauensis w 1-, -O-B. alcalophilus B. asahii B. hemicentroti B. thennophilus B. macerans w ul a ,-.
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Lo B. algicola B. atrophaeus B. herbersteinensis B. thennontber B. rnacquariensis B. alginolyticus B. axarquiensis B. horikoshii B. thennosphaericus B. rnacyae 0 t..) o B. alkalidiazotrophicus B. azotofixans B. horneckiae B. thiaminolyticus B. malachensis ts.) 1.-, k..) B. alkalinitrilicus B. azotofonnans B. horti B. thioparans B. mannanilyticus w -.1 B. alkalisediminis B. badius B. huizhouensis B. thuringiensis B. marisflavi B. alkalitelluris B. barbaricus B. humi B. tianshenii B. marismortui B. altitudinis B. bataviensis B. hwajinpoensis B. trypoxylicola B. mannarensis B. alveayuensis B. beijingensis B. idriensis B. tusciae B. massiliensis B. alvei B. benzoevorans B. indicus B. validus B. megateriuin B. amvloliquefaciens B. beringensis B. infantis B. vallismortis B. mesonae . B. B. berkeleyi B. infernus B. vedderi B. methanolicus a. subsp. amyloliquefaciens B. beveridgei B. insolitus B. velezensis B. methylotrophicus 4, = B. a. subsp. plantanun B. bogoriensis B. invictae B. vietnamensis B. migulanus B. boroniphilus B. iranensis B. vireti B. mojavensis B. dipsosauri B. borstelensis B. isabeliae B. vulcani B. mucilaginosus B. drentensis B. brevis Migula B. isronensis B. wakoensis B. =rails B. edaphicus B. butanolivorans B. jeotg,ali B. weihenstephanensis B. murimartini B. ehimensis B. canaverahus B. kaustophilus B. xiamenensis B. mvco ides B. eiseniae B. carboniphilus B. kobensis B. xiaoxiensis B. naganoensis od r) B. enclensis B. cecembensis B. kochii B. zhanjiangensis B. nanhaiensis ....1 t=i ot B. endophyticus B. celhdosilyticus B. kokeshiiformis B. peoriae B. nanhaiisediminis t..) o w 1-, B. endoradicis B. centrosporus B. koreensis B. persepolensis B. nealsonii -O-w B. farraginis B. cereus B. korlensis B. persicus B. neidei ul a '.' P
Lo B. fastidiosus B. chagannorensis B. kribbensis B. pervagus B. neizhouensis B. fengqiuensis B. chitinolyticus B. krulwichiae B. plakortidis B. niabensis 0 r..) o B. firmus B. chondroitinus B. laevolacticus B. pocheonensis B. niacini ts.) 1.-, k..) B. flexus B. choshinensis B. larvae B. polvgoni B. novalis w -.1 B. foraminis B. chungangensis B. laterosporus B. polvmyxa B. oceanisediminis B. fordii B. cibi B. salexigens B. popilliae B. odysseyi B. formosus B. cirettlans B. saliphilus B. pseudalealophilus B. okhensis B. fortis B. clarkii B. schlegelii B. pseudofinnus B. okuhidensis B. fumarioli B. clausii B. sediminis B. pseudomycoides B. oleronius B. fun iculus B. coagulans B. selenatarsenatis B. psvchrodurans B. oryzaecorticis B. fusiformis B. coahuilensis B. selenitireducens B. psychrophilus B. oshimensis B. galactophilus B. cohnii B. seohaeanensis B. psvchrosaccharolyticus B. pabuli 4, oe B. galactosidilyticus B. composti B. shacheensis B. psychrotolerans B. pakistctnensis B. galliciensis B. curdlanolyticus B. shackletonii B. pulvifaciens B. pallidus B. gelatini B. cycloheptanicus B. siamensis B. pumilus B. pallidus B. gibsonii B. cytotoxicus B. silvestris B. purgationiresistens B. panacisoli B. ginsengi B. daliensis B. simplex B. pycnus B. panaciterrae B. ginsengihumi B. decisifrondis B. siralis B. qingdaonensis B. pantothenticus B. ginsengisoli B. decolorationis B. smithii B. qingshengii B. parctbrevis od r) B. globisporus (eg, B. B. deserti B. soli B. reuszeri B. paraflexus ....1 t=i ot g. subsp. Globisportts; or B. B. solimangrovi B. rhizosphaerae B. pasteurii r..) o w 1-, g. subsp. Marinus) B. solisalsi B. rigui B. patagoniensis -O-w B. songklensis B. runs ul B. sonorensis B. safensis B. sphaericus B. salarius B. sporothermodurans ts.) B. stearothermophdus B. stratosphericus B. subterraneus B. subtilis (eg, B.
s. subsp. Inaquosorum, or B.
s. subsp. Spizizeni, or B.
s. subsp. Subtilis) Caenimonas Campylobacter Cardiobacterium Catenuloplanes Curtobacterium Caenhnonas koreensis Campylobacter coli Cardiobacterium hominis Catenuloplanes atrovinosus Curtobacterium Caldalkalibacillus Campylobacter concisus Carnimonas Catenuloplanes castoneus albidum Caldalkalibacillus uzonensis Campylobacter curvus Carnimonas nigrificans Catenuloplanes crispus Curtobacterium citretts Caldanaerobacter Campylobacter fetus Carnobacterium Catenuloplanes indicus Caldanaerobacter subterraneus Campylobacter gracilis Carnobacterium Catenuloplanes japonicus Caldanaerobius Campylobacter helveticus alterfunditum Catenuloplanes nepalensis Caldaraterobius fijiensis Campylobacter hominis Carnobacte riurn dive rgetts Catenuloplanes niger Caldanaerobius Campylobacter hyointestinatis Carnobacterium funditum Chryseobacterium polysaccharolyticus Campylobacter jejttni Carnobacterium gallinarum Chtyseobacterium Caldanaerobius zeae Campylobacter (art Carnobacterium balustinum Campylobacter mucosalis maltaromaticum Caldanaerovirga Campylobacter rectus Carnobacterium mobile Citrobacter Caldanaerovirga acetigignens Campylobacter showae Carnobacterium viridans C. amalonaticus Caldicellulosiruptor Campylobacter sputorum Caryophanon C.
braakii Caldicellulosiruptor bescii Campylobacter upsaliensis Caryophanon latum C. diversus Caldicellulosiruptor kristjanssonii Capnocytophaga Caryophanon tenue C. fartneri Caldicellulosiruptor owensensis Capnocytophaga canimorsus Catellatospora C. freundii Capnocytophaga cynodegmi Catellatospora citrea C.
gillenii Capnocytophaga gingivalis Catellatospora C. koseri Capnocytophaga granulosa methionotrophica C.
murliniae Capnocytophaga haemolytica Catenococcus C.
pasteurii[11 Capnocytophaga ochracea Catenococcus thiocycli .. C.
rodentium Capnocytophaga sputigena C.
sedlakii C. werkmanii C. youngae Clostridium (see below) Coccochloris Coccochloris elabens Corynebacterium Cmynebacterium flavescens Cotynebacterium variabile Clostridium .0 Clostridium absonum, Clostridium aceticum, Clostridium acetireducens, Clostridium ace tobutylicum, Clostridium acidisoli, Clostridium aciditolerans, Clostridium acidurici, Clostridium aerotolerans, Clostridium aestuarii, Clostridium akagii, Clostridium aldenense, Clostridium aldrichii, Clostridium algidicami, Clostridium algidixylanolyticum, Clostridium algifaecis, Clostridium algoriphilum, Clostridium alkalicellulosi, Clostridium arninophilum, ts.) Clostridium aminovalericum, Clostridium amygdalinum, Clostridium amylolyticum, Clostridium arbusti, Clostridium arcticum, Clostridium argentinense, Clostridium asparagifonne, Clostridium aurantibutyricum, Clostridium autoethanogenum, Clostridium baratii, Clostridium barkeri, Clostridium bartlettii, Clostridium beijerinckii, Clostridium bifermentans, Clostridium bolteae, Clostridium bornimense, Clostridium botulinttm, Clostridium bowmanii, Clostridium bryantii, Clostridium butyricum, Clostridium cadaveris, Clostridium caenicola, Clostridium caminithermale, Clostridium carboxidivorans, Clostridium carnis, Clostridium cavendishii, Clostridium celatum, Clostridium celerecrescens, Clostridium cellobioparum, Clostridium cellulofermentans, Clostridium cellulolyticum, Clostridium cellulosi, Clostridium cellulovorans, Clostridium chartatabidum, Clostridium chauvoei, Clostridium chromiireducens, Clostridium citron iae, Clostridium clariflavum, Clostridium clostridioforme, Clostridium cocco ides, Clostridium cochlearium, Clostridium colletant, Clostridium colicanis, Clostridium colinum, Clostridium collagenovorans, Clostridium cylindrospo rum, Clostridium difficile, Clostridium diolis, Clostridium disporicum, Clostridium drakei, Clostridium durum, Clostridium estertheticum, Clostridium estertheticum estertheticum, Clostridium estertheticum laramiense, Clostridium fallax, Clostridium felsineum, Clostridium fervidum, Clostridium fimetarium, Clostridium fonnicaceticurn, Clostridium fi-igidicarnis, Clostridium frigoris, Clostridium ganghwense, Clostridium gasigenes, Clostridium ghonii, Clostridium glycolicum, Clostridium glycyrrhizinilyticum, Clostridium grantii, Clostridium haemolyticum, Clostridium halophilum, Clostridium hastiforme, Clostridium hathewayi, Clostridium herbivorans, Clostridium hiranonis, Clostridium histolyticum, Clostridium homopropionicum, Clostridium huakuii, Clostridium hungatei, Clostridium hydrogeniformans, Clostridium hydroxybenzoicwn, Clostridium hylemonae, Clostridium jejuense, Clostridium indolis, Clostridium innocuum, Clostridium intestinale, Clostridium irregulare, Clostridium isatidis, Clostridium josui, Clostridium kluyveri, Clostridium lactatifermentans, Clostridium lacusflyxellense, Clostridium laramiense, Clostridium lavalense, Clostridium lentocelluin, Clostridium lentoputrescens, Clostridium leptum, Clostridium limosum, Clostridium litorale, Clostridium lituseburense, Clostridium ljungdahlii, Clostridium lortetii, Clostridium lundense, Clostridium magnum, Clostridium malenominatum, Clostridium man genotii, Clostridium mayombei, Clostridium methoxybenzovorans, Clostridium methylpentosum, Clostridium neopropionicum, Clostridium flexile, Clostridium nitrophenolicum, Clostridium novyi, Clostridium oceanicwn, Clostridium orbiscindens, Clostridium oroticum, Clostridium oxalicum, Clostridium papyrosolvens, Clostridium paradoxum, Clostridium paraperfringens (Alias: C. welchii), Clostridium paraputrificum, Clostridium pascui, Clostridium pasteurianum, Clostridium .0 peptidivorans, Clostridium perenne, Clostridium perfringens, Clostridium pfennigii, Clostridium phytofermentans, Clostridium pilifonne, Clostridium polysaccharolyticum, Clostridium populeti, Clostridium propionicum, Clostridium proteoclasticum, Clostridium proteolyticum, Clostridium psychrophilum, Clostridium puniceum, Clostridium purinilyticum, Clostridium putrefaciens, Clostridium putrificum, Clostridium quercicolum, Clostridium quinii, Clostridium ,t2, ramosum, Clostridium rectum, Clostridium roseum, Clostridium saccharobutylicum, Clostridium saccharogumia, Clostridium sacchctrolvticum, Clostridium saccharoperbutylacetonicum, Clostridium sardiniense, Clostridium sartagoforme, Clostridium scatolo genes, Clostridium schirmacherense, Clostridium scindens, Clostridium septicum, Clostridium sordellii, Clostridium sphenoides, Clostridium spiroforme, Clostridium sporo genes, Clostridium sporosphaeroides, Clostridium stercorarium, Clostridium stercorarium leptospartum, Clostridium stercorarium stercorarium, Clostridium stercorarium thermolacticum, Clostridium sticklandii, Clostridium stratninisolvens, Clostridium subtenninale, Clostridium sufflavum, Clostridium sttlfidigenes, Clostridium symbiosum, Clostridium tagluense, Clostridium tepidiprofundi, Clostridium tennitidis, Clostridium tertium, Clostridium tetctni, Clostridium tetanomorp hum, Clostridium thermaceticum, Clostridium thennautotrophicum, Clostridium thermoalcaliphilum, Clostridium thermobutyricum, Clostridium thermocellum, Clostridium thermocopriae, Clostridittin thennohydrosulfuricum, Clostridium thermolacticum, Clostridium thermopalinarium, Clostridium thennopapyrolyticum, Clostridium thermosaccharolyticum, Clostridium thermosuccinogenes, Clostridium thermosulfurigenes, Clostridium thiosulfatireducens, Clostridium tyrobutyricum, Clostridium uliginosum, Clostridium ultunense, Clostridium villosum, Clostridium vincentii, Clostridium viride, Clostridiwn xylanolyticwn, Clostridium xylanovorans Dactylosporangium Deinococcus Delftia Echinicola Dactvlosporangium aurantiacum Deinococcus aeritts Delftia acidovorans Echinicola pacifica Dactvlosporangium fulvum Deinococcus apachensis Desulfovibrio Echinicola vietnamensis Dactvlosporangium matsuzakiense Deinococcus aqua ticus Desulfovibrio desulfuricans Dactvlosporangium roseum Deinococcus aquatilis Diplococcus Dactvlosporangium thailandense Deinococcus caeni Diplococcus pneumoniae Dactvlosporangium vinaceum Deinococcus radiodurans Deinococcus radiophilus Enterobacter Enterobacter kobei Faecalibacterium Flavobacterium E. aerogenes E. ludwigil Faecalibacterittm prausnitzii Flavobacterium antarcticum E. amnigenus E. mori Fangia Flavobacterium aquatile ts.) E. agglomerans E. nimipressuralis Fangio hongkongensis Flavobacterium E. arachidis E. olyzae Fastidiosipila aquidurense E. asburiae E. pulveris Fastidiosipila sanguinis Flavobacterium balustinum E. cancerogenous E. pyrinus Fusobacterium Flavobacterium croceum E. cloacae E. radicincitans Fttsobacterium nucleatum Flavobacterium cucumis E. cowanii E. taylorae Flavobacterium E. dissolvens E. turicensis daej eon ense E. gergoviae E. sakazakii Enterobacter soli Flavobacteriwn defluvii E. helveticus Enterococcus Flavobacterium degerlachei E. hormaechei Enterococcus durans Flavobacterium E. intermedius Enterococcus faecalis denitrificans Enterococcus faecium Flavobacterium filum Erwinia Flavobacterium flevense Erwinia hapontici Flavobacterium frigidarium Escherichia Flavobacterium mizutaii Escherichia coli Flavobacterium okeanokoites a .
.

,.., P
. Gaetbulibacter Haemophilus Ideonella Janibacter Gaetbulibacter saemankumensis Haemophilus aegyptius Ideonella azotifigens Janibacter anophelis 0 t..) o Gallibacterium Haemophilus aphrophilus Idiomarina Janibacter corallicola ts.) 1.-, k..) Gallibacterium anatis Haemophilus felts Idiomarina abyssalis Janibacter limosus w -.1 Gallicola Haemophilus gallinarum Idiontarina baltica Janibacter melonis Gallicola barnesae Haemophilus haemolyticus Idiomarina fontislapidosi Janibacter terrae Garciella Haemophilus influenzae Idiomarina loihiensis Jannaschia Garciella nitratireducens Haemophilus paracuniculus Idiomarina ramblicola Jannaschia cystaugens Geobacillus Haemophilus parahaemolyticus Idiomarina seosinensis Jannaschia helgolandensis Geobacillus the rmoglucosidasius Haemophilus parainfluenzae Idiomarina zobellii Jannaschia pohangensis Geobacillus stearothermophilus Haemophilus Ignatzschineria Jannaschia rubra Geobacter paraphrohaemolytictts Ignatzschineria larvae vi 4, Geobacter bemidjiensis Haemophilus parasuis Janthinobacterium Geobacter bremensis Haemophilus pittmaniae Ignavigranum Janthinobacterium Geobacter chapellei Hafnia Ignavigranum ruoffiae agaricidamnosum Geobacter grbiciae Hafitia alvei llumatobacter Janthinobacterium lividum Geobacter hydrogenophilus Hahella Ilumatobacter fluminis Jejuia Geobacter lovleyi Hahella ganghwensis Ilyobacter Jejuia pallidilutea Geobacter metallireducens Halalkalibacillus Ilyobacter delafieldii Jeotgalibacillus od r) Geobacter pelophilus Halalkalibacillus hcdophilus Ilyobacter insuetus Jeotgalibacillus ....1 t=i ot Geobacter pickeringii Helicobacter Ilyobacter polytropus alimentarius t..) o w 1-, Geobacter sulfurreducens Helicobacter pylori Ilyobacter tartaricus Jeotgalicoccus -6-w Jeotgalicoccus halotolerans v:
ul Geodermatophilus Geodermatophilus obscurus Gluconacetobacter Gluconacetobacter xylinus Gordonia Gordonia rubripertincta Kaistia Labedella Listeria ivanovii Micrococcus Nesterenkonia Kaistia adipata Labedella gwakjiensis L. marthii Micrococcus luteus Nesterenkonia ho labia Kaistia soli Labrenzia L. monocyto genes Micrococcus lylae Nocardia Kangiella Labrenzia aggregata L. newyorkensis Moraxella Nocardia argentinensis Kangiella aquimarina Labrenzia alba L. riparia Moraxella bovis Nocardia corallina Kangiella koreensis Labrenzia alexandrii L. rocourtiae Moraxella nonliquefaciens Nocardia Labrenzia marina L. seeligeri Moraxella osloensis otitidiscaviarum Kerstersia Labrys L. weihenstephanensis Nakamurella Kerstersia gviorum Labrys methylaminiphilus L.
welshimeri Nakamurella multipartita Kiloniella Labrys miyagiensis Listonella Nannocystis Kiloniella laminariae Labrys monachus Listonella anguillarum Nannocystis pusilla Klebsiella Labrys okinawensis Macrococcus Natranaerobius K. granulomatis Labrys portucalensis Macrococcus bovicus Natranaerobius K. oxytoca Marinobacter the rmophilus K. pneumoniae Lactobacillus Marinobacter algicola Natranaerobius trueperi K. terrigena [see below] Marinobacter brvozoorum Naxibacter K. variicola Laceyella Marinobacter flavimaris Naxibacter alkalitolerans Kluyver a Laceyella putido Meiothermus Neisseria ts.) Kluyvera ascorbata Lechevalieria Meiothennus ruber Neisseria cinerea Kocuria Lechevalieria aerocolonigenes Methylophilus Neisseria denitrificans Kocuria roasea Legionella Methylophilus Neisseria gonorrhoeae Kocuria varians [see below] methylotrophus Neisseria lactamica Kurthia Listeria Microbacterium Neisseria mucosa Kurthia zopfii L. aquatica Microbacterium Neisseria sicca L. booriae ammoniaphilum Neisseria subflava L. cornellensis Microbacterium arborescens Neptunomonas L. fleischmannii Microbacterium liquefaciens Neptunomonas japonica L. floridensis Microbacterium oxydans L. grandensis L. grayi L. innocua Lactobacillus L. acetotolerans L. catenaformis L. mall L. parakefiri L. sakei L. acidifarinae L. ceti L. rnanihotivorans L. paralimentarius L. sativarius L. acidipiscis L. coleohominis L. mindensis L. paraplantarum L. sanfranciscensis L. acidophilus L. collinoides L. mucosae L. pentosus L. satsumensis a .
' '6-.

,.., P
. Lactobacillus agilis L. composti L. murinus L. perolens L. secaliphilus L. algidus L. conatvus L. nagelii L. plantarum L. sharpeae 0 t..) o L. alimentarins L. coryniformis L. namurensis L. pontis L. siliginis ts.) 1¨

, k..) L. amylolyticus L. crispatus L. nantensis L. protectus L. spicheri w -.1 L. amylophilus L. crustorum L. oligofennentans L. psittaci L. suebicus L. amylotrophicus L. curvatus L. oris L. rennini L. thailandensis L. amylovorus L. delbrueckii subsp. L. panis L. reuteri L. ultunensis L. animalis bulgaricus L. pantheris L. rhamnosus L. vaccinostercus L. antri L. delbrueckii subsp. L. parabrevis L. rimae L. vaginalis L. apodeini delbrueckii L. parabuchneri L. rogosae L. versmoldensis L. aviarius L. delbrueckii subsp. lactis L. paracasei L. rossiae L. vini L. bifennentans L. dextrinicus L. paracollino ides L. ruminis L. vitulinus un L. brevis L. diolivorans L. parafarraginis L. saerimneri L. zeae L. buchneri L. equi L. homohiochii L. jensenii L. zymae L. camelliae L. equigenerosi L. iners L. johnsonii L. gastricus L. casei L. farraginis L. ingluviei L. kalixensis L. ghanensis L. kitasatonis L. farciminis L. intestinalis L. kefiranofaciens L. graminis L. kunkeei L. fermentum L. fuchuensis L. kefiri L. hammesii L. leichmannii L. fomicalis L. gallinarum L. kimchli L. hamsteri od r) L. lindneri L. fructivorans L. gasseri L. helvetictts L. harbinensis ....1 t=i ot L. malefermentans L. frumenti L. hilgardii L. hayakitensis t..) o w 1-, -O-w ul .6.

.0 Legionella Legionella adelaidensis Legionella drancourtit Candidatus Legionella jconii Legionella quinlivanii Legionella anisa Legionella dresdenensis Legionella jordanis Legionella rowbothamii ts.) Legionella beliardensis Legionella drozanskii Legionella lansingensis Legionella rubrilucens Legionella binninghamensis Legionella dumoffii Legionella londiniensis Legionella sainthelensi Legionella bozemanae Legionella erythra Legionella Ion gbeachae Legionella santicrttcis Legionella brunensis Legionella fairfieldensis Legionella lytica Legionella shakespearei Legionella busanensis Legionella fallonii Legionella maceachenni Legionella spiritensis Legionella cardiaca Legionella feeleii Legionella massiliensis Legionella steelei Legionella cherrii Legionella geestiana Legionella micdadei Legionella steigenvalth Legionella cincinnatiensis Legionella gcnomospccics Legionella monrovica Legionella taurinensis Legionella clemsonensis Legionella gonnanii Legionella moravica Legionella tucsonensis oe Legionella donaldconti Legionella gratiana Legionella nctga,sakiensis Legionella tunisiensis Legionella gresilensis Legionella nautarum Legionella wadsworthii Legionella hackeliae Legionella norrlandica Legionella waltersii Legionella impletisoli Legionella oakridgensis .. Legionella worsleiensis Legionella israelensis Legionella parisiensis Legionella yabuuchiae Legionella jamestowniensis Legionella pittsburghensis Legionella pneumophila Legionella quateirensis a .
.

,.., P
. Oceanibulbus Paenibacillus Prevotella Quadrisphaera Oceanibulbus inclolifex Paenibacillus thiaminolvticus Prevotella albensis Quadrisphaera granulorum 0 t..) o Oceanicaulis Pantoea Prevotella amnii Quatrionicoccus ts.) 0.
, k..) Oceanicaulis alexandrii Pantom agglomerans Prevotella bergensis Quatrionicoccus w -.1 Oceanicola Prevotella bivia australiensis Oceanicola batsensis Paracoccus Prevotella brevis Oceanicola granulosus Paracoccus alcaliphilus Prevotella buantii Quinella Oceanicola nanhaiensis Paucimonas Prevotella buccae Quinella ovalis Oceanimonas Pauchnonas lemoignei Prevotella buccalis Ocean imonas baumannh Pectobacterium Prevotella copri Ralstonia Oceaniserpentilla Pectobacterium aroidearum Prevotella dentalis Ralstonia eutropha Oceaniserpentilla haliotis Pectobacterium atrosepticum Prevotella denticola Ralstonia insidiosa vi v:
Oceanisphaera Pectobacterium Prevotella disienc Ralstonia mannitolllytica Oceanisphaera don ghaensis betavasculorum Prevotella histicola Ralstonia pickettii Oceanisphaera litoralis Pectobacterium cacticida Prevotella intermedia Ralstonia Oceanithermus Pectobacterium carnegieana Prevotella maculosa pseudosolanacearum Oceanithermus desulfurans Pectobacterium carotovorum Prevotella marshii Ralstonia syzygii Oceanithermus profundus Pectobacterium chrysanthemi Prevotella tnelaninogenica Ralstonia solanacearum Oceanobacillus Pectobacterium cypripedu Prevotella micans Ramlibacter od r) Oceanobacillus caeni Pectobacterium rhapontici Prevotella multifonnis Ramlibacter henchirensis ....1 t=i ot Oceanospirillum Pectobacterium wasabiae Prevotella nigrescens Ramlibacter tataouinensis t..) o w 1-, Oceanospirilluin linum Planococcus Prevotella oralis -O-w Planococcus citreus Prevotella oris v:
ul .6.

Planomicrobium Prevotella oulorum Raoultella Planomicrobium okeanokoites Prevotella pallens Raoultella ornithinolvtica Plesiomonas Prevotella salivae Raoultella planticola ts.) Plesiomonas shigelloides Prevotella stercorea Raoultella terrigena Proteus Prevotella tannerae Rathayibacter Proteus vulgaris Prevotella timonensis Rathavibacter caricis Prevotella veroralis Rathayibacter festucae Providencia Rathavibacter iranicus Providencia stuartii Rathavibacter rathayi Pseudomonas Rathavibacter toxicus Pseudomonas aeruginosa Rathavibacter tritici Pseudomonas alcaligenes Rhodobacter Pseudomonas anguillispetica Rhodohacter sphaero ides Pseudomonas fluorescens Ruegeria Pseudoalteromonas Rue geria gelatinovorans haloplanktis Pseudomonas mendocina Pseudomonas pseudoalcaligenes Pseudomonas putida Pseudomonas tutzeri Pseudomonas syringae a .
.0 '.' P
. Psychrobacter Psychrobacter faecalis t..) o Psychrobacter ts.) 1¨

, k..) phenylpyrttvicus w ¨1 cii oo Saccharococcus Sagittula Sanguibacter Stenotrophomonas Tatlockia Saccharococcus thermophilus Sagittala stellata Sanguibacter keddieii Stenotrophomonas Tatlockia maceachemii Saccharomonospora Salegentibacter Sanguibacter sttarezii maltophilia Tatlockia micdadei Saccharomonospora azurea Salegentibacter salegens Saprospira Streptococcus Tenacibaculum Saccharomonospora cyanea Salimicrobium Saprospira grandis Tenacibaculum Saccharomonospora viridis Salimicrobium album Sarcina [also see below] amylolyticum o 1¨, Saccharophagus Salinibacter Sarcina maxima Streptomyces Tenacibaculum discolor Saccharophagus degradans Salinibacter ruber Sarcina ventriculi Streptomyces Tenacibaculum Saccharopolyspora Salinicoccus Sebaldella achromogenes gallaicum Saccharopolyspora erythraeu Salinicoccus alkaliphilus Sebaldella termitidis Streptomyces cesalbus Tenacibaculum Saccharopolyspora gregorii Salinicoccus hispanicus Streptomyces cescaepitosus lutimaris Saccharopolyspora hirsuta Salinicoccus roseus Serratia Streptomyces cesdiastaticus Tenacibaculum Saccharopolyspora hordei Salinispora Serratia fonticola Streptomyces cesexfoliatus mesophilum od r) Saccharopolyspora rectivirgula Salinispora arenicola Serratia marcescens Streptomyces fitnbriatus Tenacibaculum ....1 t=i ot Saccharopolyspora spin osa Salinispora tropica Sphaerotilus Streptomyces fradiae skagerrakense t.) o w Saccharopolyspora taberi Salinivibrio Sphaerotilus natans Streptontyces fulvissimus -O-w Salinivibrio costicola Streptomyces griseorttber ul .6.

Saccharothrix Salmonella Sphingobacterium Streptomyces griseus Tepidanaerobacter Saccharothrix australiensis Salmonella bongori Sphingobacterium multivorttm Streptomyces lavendulae Tepidanaerobacter Saccharothrix coeruleofitsca Salmonella enterica Staphylococcus Streptornyces syntrophicus ts.) Saccharothrix espanaensis Salmonella subterranea [see below] phae ochromo genes Tepidibacter Saccharothrix longispora Salmonella typhi Streptomyces Tepidibacter Saccharothrix mutabilis the rmodiastaticus fomticigenes Saccharothrix syringae Streptomyces tube rcidicus Tepidibacter Saccharothrix tangerinus thalassicus Saccharothrix texasensis Thermus Thermus aquaticus Themws filiformis Therms thermophilus Staphylococcus S. arlettae S. equorurn S. micron S.
schleiferi S. agnetis S. fells S. muscae S.
sciuri S. aureus S. fleurettii S. nepalensis S.
sintiae S. auricularis S. gallinarum S. pasteuri S.
simulans S. cap itis S. haemolyticus S. petrasii S.
stepanovicii S. caprae S. hominis S. pettenkoferi S.
succinus S. carnosus S. hyicus S. pisciferrnentans S.
vitulinus S. caseolyticus S. intermedius S. pseudintermedius S.
warneri S. chromo genes S. kloosii S. pseudolugdunensis S.
xylosus .0 S. cohnii S. leei S. pulvereri S. condimenti S. lentus S. rostri S. delphini S. lugdunensis S. saccharolyticus S. devriesei S. lutrae S. saprophyticus S. epideMlidis S. lyticans S. massiliensis Streptococcus Streptococcus agalactiae Streptococcus infantarius Streptococcus orisratti Streptococcus thermophilus Streptococcus anginosus Streptococcus in/ac Streptococcus parasanguinis Streptococcus sanguinis Streptococcus bovis Streptococcus intennedius Streptococcus peroris Streptococcus sobrinus Streptococcus canis Streptococcus lactarius Streptococcus pneumoniae Streptococcus suis Streptococcus constellatus Streptococcus milleri Streptococcus Streptococcus uberis Streptococcus downei Streptococcus mitis pseudopneumoniae Streptococcus vestibularis Streptococcus dysgalactiae Streptococcus mutans Streptococcus pyo genes Streptococcus viridans Streptococcus equines Streptococcus rails Streptococcus ratti Streptococcus Streptococcus faecalis Streptococcus tigurinus Streptococcus salivariu zooepidemicus Streptococcus ferus Uliginosibacterium Vagococcus Vibrio Virgibacillus Xanthobacter Vagococcus carniphdus Vibrio aerogenes Virgibacillus Xanthobacter agilis Uliginosibacterium gangwonense Vagococcus elongatus Vibrio aestuarianus halodenitrificans Xanthobacter a .
.0 '.' P
. Ulvibacter Vagococcus fessus Vibrio albensis Virgibacillus aminoxidans Ulvibacter litoralis Vagococcus fluvialis Vibrio alginolyticus pantothenticus Xanthobacter 0 t..) o Umezawaea Vagococcus lutrae Vibrio cainpbellii Weissella autotrophicus ts.) , k..) Umezawaea tan gerina Vagococcus salmoninarum Vibrio cholerae Weissella cibaria Xanthobacter flavus w -.1 Undibacterium Variovorax Vibrio cincinnatiensis Weissella confusa Xanthobacter tagetidis Undibacterium pigrum Variovorax boronicumulans Vibrio coralliilvticus Weissella halotolerans Xanthobacter viscosus Ureaplasma Variovorax dokdonensis Vibrio cyclitrophicus Weissella hellenica Xanthomonas Ureaplasma urealyticum Variovorax paradoxus Vibrio diazotrophicus Weissella kandleri Xanthomonas Variovorax soli Vibrio fhivialis Weissella koreensis albilinecins Ureibacillus Veillonella Vibrio furnissii Weissella minor Xanthomonas alfalfae Ureibacillus composti Veillonella atypica Vibrio gazo genes Weissella Xanthomonas Ureibacillus stnvonensis Veillonella caviae Vibrio halioticoli paramesenteroides arboricola o 4, Ureibacillus terrenus Veillonella criceti Vibrio harveyi Weissella soli Xanthomonas Ureibacillus thermophilus Veillonella dispar Vibrio ichthyoenteri Weissella thailandensis axonopodis Ureibacillus thermosphaericus Veillonella montpellierensis Vibrio mediterranei Weissella yin' descens Xanthomonas Veillonella parvula Vibrio metschnikovii Williamsia cainpestris Veillonella ratti Vibrio mytili Williamsia marianensis Xanthomonas citri Veillonella rodentium Vibrio natriegens Williamsia mans Xanthomonas codiaei Venenivibrio Vibrio navarrensis Williamsia serinedens Xanthomonas od r) Venenivibrio stagnispumantis Vibrio nereis Winogradskyella cucurbitae ....1 t=i It Vibrio nigripulchritudo Wino gradskyella Xanthomonas t..) o w 1-, Verminephrobacter Vibrio ordalii thalassocola euvesicatoria -O-w Verminephrobacter eiseniae Vibrio orientalis Xanthomonas fragariae Vibrio parahaemolyticus Wolbachia Xanthomonas fuscans Verrucomicrobium Vibrio pectenicida Wolbachia persica Xanthomonas gardneri Verrucomicrobium spinosurn Vibrio penaeicida Xanthornonas hortorum Vibrio proteolyticus Wolinella Xanthomonas hyacinthi ;=,s' Vibrio shilonii Wolinella succino genes Xanthomonas perforans Vibrio splendidus Xanthomonas phaseoli Vibrio tubiashii Zobellia Xanthomonas pisi Vibrio vulmficus Zobellia galactanivorans Xanthomonas populi Zobellia uliginosa Xanthomonas theicola Zoogloea Xanthomonas Zoo gioea ramigera translucens Zoogloea resiniphila Xanthomonas vesicatoria Xylella Xylella fastidiosa Xylophilus Xylophilus ampelinus Xenophilus Yangia Yersinia mollaretii Zooshikella Zobellella Xenophilus azovorans Yangia pacifica Yersinia philomiragia Zooshikella ganghwensis Zobellella denitrificans Yersinia pestis Zobellella taiwanensis ,õ`s"

Xenorhabdus Yaniella Yersinia pseudotuberculosis Zunongwangia Xenorhabdus bedding ii Yaniellct flava Yersinia rohdei Zunongwangia pro funda Zeaxanthinibacter Xenorhabdus bovienii Yaniella halototerans Yersinia ruckeri Zymobacter Zeaxanthinibacter ts.) Xenorhabdus cabanillash Yeosuana Yokenella Zvmobacter palmae enoshimensis Xenorhabdus doucetiae Yeosuana aromativorans Yokenella regensburgei Zymomonas Zhihengliuella Xenorhabdus grfffiniae Yersinia Yonghaparkia Zvmomonas mobilis Zhihengliuella Xenorhabdus hominickh Yersinia aldovae Yonghaparkia alkaliphila Zymophilus halotolerans Xenorhabdus koppenhoeferi Yersinia bercovieri Zavarzinia Zvmophilus paucivorans Xylanibacterium Xenorhabdus nematophila Yersinia enterocolitica Zavarzinia compransoris Zvmophilus raffinosivorans Xvlanibacterium ulmi Xenorhabdus poinarii Yersinia entomophaga Xylanibacter Yersinia frederiksenh Xylanibacter oryzae Yersinia intermedia Yersinia kristensenii Table 2: Example Cos Cl may be a Cas (eg, a Cas3 or a Cascade Cos) selected from the following types. Additionally or alternatively, C2 may be a Cas (eg, a Cas3 or a Cascade Cas) selected from the following types.
Cascade Cas may be selected from the following types.
Bacteria / Phage CRISPR-Cas Type Clostridium botulinum Type I-B
Clostridium tetani Type I-B
Eggerthella lenta Type I-C
Moraxella bovoculi Type I-C
Streptococcus mutans Type I-C
Streptococcus mutans Type I-C
Streptococcus pyogenes Type I-C
Bacillus halodurans Type I-C
Prevotella enoeca Type I-C
Bacteroides fragilis Type I-C
Pseudomonas aeniginosa Type I-C
Nostoc sp. CENA543 Type I-D
Escherichia coli Type I-E
Vibrio cholerae Type I-E
Citrobacter freundii Type I-E
Salmonella enterica Type I-E
Klebsiella pneumoniae Type I-E
Streptococcus mutans Type I-E
Pseudomonas aeruginosa Type I-F

Yersinia pestis Type 1-F
Serralia marcescens Type I-F
Geobacter sulfurreducens Type I-U
Salinispora arenicola Type I-U
Vibrio phage ICP1 Type I-F
Table 3: Example Cas, Types and Classes Type Cas Nuclease Target Cas3 DNA
Class 1 III Cas 10 DNA or RNA
IV
II Cas9 DNA
Class 2 V Cas 12 DNA
VI Cas 13 RNA

Claims (53)

CLAIMS:

1. A method of modifying the genome of one or more cells, the method comprising introducing into each cell components (a), (b), (c) or (d):-(a) first and second crRNAs;
(b) nucleic acid encoding first and second crRNAs, wherein the nucleic acid is expressed in the cell for producing the crRNAs;
(c) a first nucleic acid encoding a first crRNA, and a second nucleic acid encoding a second crRNA, wherein the nucleic acids are expressed in the cell for producing the crRNAs; or (d) a first crRNA and a nucleic acid encoding a second crRNA, wherein the nucleic acid is expressed in the cell for producing the second crRNA;
wherein for each cell (e) the first crRNA (crRNA 1) is capable of guiding a first Cas (C1) to a protospacer sequence (PS1) comprised by the cell genome to modify PS1; and (f) the second crRNA (crRNA2) is capable of guiding a second Cas (C2) to a protospacer sequence (PS2) comprised by the cell genome to modify PS2;
(g) C 1 and C2 are different;
(h) PS1 and PS2 are different; and (i) crRNA1, crRNA2, C 1 and C2 are provided in the cell, whereby the genome of the cell is subjected to Cas modification.

2. The method of claim 1, wherein (a) C 1 is a Class 1 Cas and C2 is a Class 1 Cas;
(b) C 1 is a Class 1 Cas and C2 is a Class 2 Cas;
(c) C 1 is a Class 2 Cas and C2 is a Class 2 Cas;
(d) C 1 is a Type I Cas (optionally Type I-A, B, C, D, E, F or U) and C2 is a Type I Cas (optionally Type I-A, B, C, D, E, F or U);
(e) C 1 is a Type I (optionally Type I-A, B, C, D, E, F or U) or II Cas and C2 is a Type II Cas;
(f) C 1 is a Type I (optionally Type I-A, B, C, D, E, F or U) or II Cas and C2 is a Type III
Cas (optionally Type I-A or B);
(g) C 1 is a Type I (optionally Type I-A, B, C, D, E, F or U) or II Cas and C2 is a Type IV
Cas;
(h) C 1 is a Type I (optionally Type I-A, B, C, D, E, F or IJ) or II Cas and C2 is a Type V
Cas; or (i) C 1 is a Type I or II Cas and C2 is a Type VI Cas.

3. The method of any preceding claim, wherein (a) Cl is a Type IB or C Cas and C2 is a Type 1-E or F Cas (optionally Cl is a Type IB Cas3 and C2 is a Type IE Cas);
(b) Cl is a Type IC or C Cas and C2 is a Type I-E or F Cas (optionally Cl is a Type IC Cas3 and C2 is a Type IE Cas3); or (c) Cl is a Type II Cas9 and C2 is a Type I Cas3 (optionally C2 is an E colt Type IE or F
Cas3; or a C difficile Cas IB).

4. The method of any preceding claim, wherein (a) Cl is a Cas3 (optionally a Type 1-A, B, C, D, E, F or U Cas3) and C2 is a Cas3 (optionally a Type I-A, B. C, D, E, F or U Cas3);
(b) Cl is a Cas9 and C2 is a Cas3 (optionally a Type I-A, B, C, D, E, F or U
Cas3);
(c) Cl is a Cas3 (optionally a Type I-A, B, C, D, E, F or U Cas3) and C2 is a Cas10 (optionally Cas10 subtype A, B, C or D);
(d) Cl is a Cas9 and C2 is a Cas10 (optionally Cas10 subtype A, B, C or D);
(e) CI is a Cas9 and C2 is a Cas12 (optionally Cas12a):
(f) Cl is a Cas3 (optionally a Type I-A, B, C, D, E, F or U Cas3) and C2 is a Cas12 (optionally Cas12a);
(g) Cl is a Cas9 and C2 is a Cas13 (optionally Cas13a, Cas13b, Cas13c or Cas13d): or (h) Cl is a Cas3 (optionally a Type I-A, B, C, D, E, F or U Cas3) and C2 is a Cas13 (optionally Cas13a, Cas13b, Cas13c or Cas13d).

5. The method of any preceding claim, wherein PS1 and PS2 are protospacers comprised by (a) RNA and RNA respectively;
(b) DNA and RNA respectively;
(c) RNA and DNA respectively; or (d) DNA and DNA respectively.

6. The method of any preceding claim, wherein Cl is a Clostridiaceae Cas3 (optionally a C dtfficile Cas3, such as a Type I-B Cas3) arid C2 is an Enterobacteriaceae Cas3 (optionally an E coli Cas3, such as a Type I-E Cas3).

7. The method of any preceding claim, wherein Cl is a spCas9 or saCas9 and C2 is a Type I Cas3 (optionally C2 is an E coli Type 1-E or F Cas3).

8. The method of any preceding claim, wherein PS1 and PS2 are subjected to Cas modification by Cl and C2.

9. The method of any preceding claim, wherein the modification is cutting of the genorne.

10. The method of any preceding claim, wherein PS1 is a chromosomal sequence of the cell.

11. The method of any preceding claim, wherein PS2 is a chromosomal sequence of the cell.

12. The method of any preceding claim, wherein each cell is a bacterial or archacal cell, optionally an E coli cell or C difficile cell.

13. The method of any preceding claim, wherein the step of introducing comprises infecting the cell with a virus (optionally a bacteriophage wherein the cell is a bacterial cell) or introducing a plasmid (optionally a conjugative plasmid) or introducing a phagemid into the cell, wherein the virus, plasmid or phagemid encodes the crRNAs.

14. The method of claim 13, wherein the virus, plasmid or phagemid encodes CI
and/or C2.

15. The method of claim 13, wherein the virus, plasmid or phagemid encodes one of said Cl and C2, and the other Cas is an endogenous Cas encoded by the genome of the cell.

16. The method of claiin 13, wherein the each of Cl and C2 is an endogenous Cas encoded by the genome of the cell.

17. The method of any preceding claim when dependent on claim 1(b) or (c), wherein each crRNA is expressed under the control of a constitutive promoter.

18. The method of any preceding claim wherein each Cas is expressed under the control of a constitutive promoter.

19. The method of any preceding claim when dependent on claim 1(b) or (c), wherein each crRNA is expressed under the control of a strong promoter.

20. The method of any preceding claim, wherein a first plurality of different crRNAs are expressed in one or more of the cells wherein each crRNA is operable with CS1 and the plurality targets at least 2 different protospacers comprised by the genome of the cell; and/or a second plurality of different crRNAs are expressed in one or more of the cells wherein each crRNA
is operable with CS2 and the second plurality targets at least 2 different protospaccrs comprised by thc genomc of the cell.

21. The method of any preceding claim, wherein the one or more cells are killed by the method.

22. The method of any preceding claim wherein the first crRNA is comprised by a guide RNA
wherein the guide RNA further comprises a tracrRNA and/or the second crRNA is comprised by a guide RNA wherein the guide RNA further comprises a traerRNA.

23. A method of killing a plurality of cells (optionally prokaryotic cells) of a first species or strain, the method comprising carrying out the method of any preceding claim using the cells, wherein Cl and/or C2 is a Cas nuclease and the genomes of the cells are cut by Cas nuclease cutting and the cells are killed.

24. The method of claim 23, the method reduces the number of cells of said plurality at least 105-fold.

25. The method of claim 23, the method kills at least 99.999% cells of said plurality.

26. The method of any one of claims 23 to 25, wherein the species is E coli or C

27. A method of editing the genome of one or more cells, the method comprising (a) modifying the genome of each cell by carrying out the method of any one of claims 1 to 22, wherein the genome is subjected to Cas cutting; and (b) inserting a nucleic acid at or adjacent to a Cas cut site in thc genome and/or deleting a nucleic acid sequence from the genome at or adjacent to a Cas cut site in the genome, wherein a cell with an edited genuine is produced; and (c) optionally isolating from the cell a nucleic acid comprising the insertion or the deletion;
or sequencing a nucleic acid sequence of the cell wherein the nucleic acid sequence comprises the insertion or the deletion.

28. The method of claim 27 further comprising (a) culturing the modified cell(s) to produce progeny thereof; and optionally isolating the progeny cells; or (h) inserting a sequence obtained from a cell in step (c) into a recipient cell and growing a cell line therefrom.

29. The method of claim 28, wherein the progeny cells or cell line expresses a protein, wherein the protein is encoded by a nucleotide sequence that comprises the inserted nucleic acid sequence, the method further comprising obtaining the expressed protein or isolating the expressed protein from the cells or cell line.

30. The method of claim 28 or 29 further combining the progeny cells, cell line or protein with a pharmaceutically acceptable carrier, diluent or excipient, thereby producing a pharmaceutical composition.

31. A method of treating or preventing a disease or condition in a human or animal subject, the method comprising (i) administering to the subject a pharmaceutical composition obtained by claim 30 wherein the composition comprises said protein, wherein the protein mediates treatment or prevention of the disease or condition; or (ii) administering to the subject a pharmaceutical composition obtained by claim 30, wherein when the composition comprises said progeny cells or cell line, the cells or cell line expresses a protein or RNA in the subject, wherein the protein or RNA mediates treatment or prevention of the disease or condition.

32. The method of claim 29, 30 or 31, wherein the protein is an antibiotic, antibacterial agent, enzyme, growth factor, antibody or fragment thereof, hormone, blood component, cytokine, immune checkpoint modulator (eg, inhibitor), analgesic, neurotransmitter, anti-inflammatory agent or anti-neoplastic agent.

33. The method of any one of claims 23 to 26, wherein the plurality of cells is comprised by a microbiome sample and produces a modified cell sample in which cells of the first species or strain have been killed, thc mcthod further comprising combining thc modified sample with a pharmaceutically acceptable carrier, diluent or excipient, thereby producing a pharmaceutical com p ositi on comprising a cell transplant

34. A method of treating or preventing a disease or condition in a human or animal subject, the method comprising administering to the subject a pharmaceutical composition obtained by claim 33.

35. The method of any one of claims 23 to 26, wherein the plurality of cells is comprised by an environmental sample (eg, an aqueous, water, oil, petroleum, soil or fluid sample).

36. A composition for use in a method treating or preventing a disease or condition in a human or animal subject that is mediated by target cells, the composition comprising components (a), (b) or (d): -(a) first and second crRNAs;

(b) nucleic acid encoding first and second crRNAs, wherein the nucleic acid is expressible in a target cell for producing the crRNAs;
(c) a first nucleic acid encoding a first crRNA, and a second nucleic acid encoding a second crRNA, wherein the nucleic acids are expressible in a target cell for producing the crRNAs; or (d) a first crRNA and a nucleic acid encoding a second crRNA, wherein the nucleic acid is expressible in a target cell for producing the sccond crRNA;
wherein (e) the first crRNA (crRNA1) is capable of guiding a first Cas (C1) to a protospacer sequence (PS1) comprised by a target cell genome to modify PS 1; and (f) the second crRNA (crRNA2) is capable of guiding a second Cas (C2) to a protospacer sequence (PS2) comprised by the target cell genome to modify PS2;
(g) Cl and C2 are different;
(h) PS1 and PS2 are different; and wherein the method comprises administering the composition to the subject whereby said components of the composition are introduced into target cells wherein crRNA1, crRNAZ Cl and C2 are provided in each cell and the genome of each cell is subjected to Cas modification and the disease or condition is treated or prevented.

37. The composition of claim 36, wherein the treating or preventing comprises carrying out the method of any one of claims 1 to 35 on the cells.

38. The composition of claim 36 or 37, wherein the method is for reducing an infection of the subject by target cells (optionally wherein the target cells are pathogenic cells).

39. A composition comprising components (a), (b) or (d):-(a) first and second crRNAs;
(b) nucleic acid encoding first and second crRNAs, wherein the nucleic acid is expressible in a target cell for producing the crRNAs;
(c) a first nucleic acid encoding a first crRNA, and a second nucleic acid encoding a second crRNA, wherein the nucleic acids are expressible in a target cell for producing the crRNAs; or (d) a first crRNA and a nucleic acid encoding a second crRNA, wherein the nucleic acid is expressible in a target cell for producing the second crRNA;
whcrcin (e) the first crRNA (crRNA1) is capable of guiding a first Cas (C1) to a protospacer sequence (PS1) comprised by a target cell genome to modify PS1; and (f) the second crRNA (crRNA2) is capable of guiding a second Cas (C2) to a protospacer sequence (PS2) comprised by the target cell genome to modify PS2;
(g) Cl and C2 are different;
(h) PSI and PS2 are different; and whcrcin when said components of thc composition arc introduced into a target cell whereby crRNA1, crRNA2, Cl and C2 are provided in the cell, the genome of the cell is subjected to Cas modification.

40. The composition of any one of claims 36 to 39, wherein the genome of each cell is edited or the cell is killed.

41. The composition of any one of claims 36 to 40, wherein each cell is a prokaryotic cell (optionally bacterial or archaeal cell).

42. The composition or method of any preceding claim, wherein said nucleic acid(s) is(are) comprised by a virus, phage, plasmid (optionally a conjugative plasmid), nanoparticle or phagemid.

43. The composition or method of any preceding claim, wherein said nucleic acid(s) encode Cl and/or C2.

44. The composition or method of any preceding claim, wherein Cl is a Type I
Cas and said nucleic acid(s) encode one or more Cascade Cas that are operable with Cl and/or wherein C2 is a Type Cas and said nucleic acid(s) encode one or more Cascade Cas that are operable with C2.

45. A pharmaceutical composition which is a composition according to any one of claims 36 to 43, wherein the composition comprises a pharmaceutically acceptable excipient, diluent or carrier.

46. The composition of any one of claims 36 to 45, wherein the composition is comprised by a sterile medicament administration device, optionally a syringe, IV bag, intranasal delivery device, inhaler, nebuliser or rectal administration device).

47. The composition or method of any preceding claim, wherein the cells are comprise by a gut, lung, kidney, urethral, bladder, blood, vaginal or skin microbiome of the subject.

48. The composition or method of any preceding claim, wherein the method is carried out on a human or animal subject, wherein the cells are killed by the method and the killing upregulates or downregulates immune cells (optionally (i) upregulating CD8-, CD4+,TH1, TH2, TH17, T
regulatory or T effector cells; or (ii) downregulating CD8+, CD4+,TH1, TH2, TH17, T regulatory or T effector cells) in the subject, thereby treating or preventing a disease or condition in the subject.

49. The composition or method of any preceding claim, wherein the method comprises introducing into each cell or expressing in each cell at least 3 different types of crRNAs wherein the different types target different protospacer sequences comprised by the cell genome; and optionally wherein Cl and C2 are Class 1 Cas nucleases.

50. The composition or method of any preceding claim, wherein the method comprises introducing into each cell a nucleic acid encoding a Cas3, Cas8e, Casll, Cas7, Cas5, and Cas6 and/or a nucleic acid encoding a Cas3, Cas6, Cas8b, Cas7, and Cas5.

51. A method of modifying the genome of a cell, the method comprising (a) using a first CRISPR/Cas system to modify a first protospacer of the genome; and (b) using a second CRISPR/Cas system to modify a second protospacer of the genome, wherein the second protospacer is different to the first protospacer;
wherein the systems comprise different Cas and are provided simultaneously in the cell.

52. The method of claim 51, wherein the method is according to any one of claims 1 to 35.

53. A method of (a) producing synergistic Cas nuclease cutting of a cell genome;
(b) reducing a population of cells of a first species or strain by at least 100,000, 1,000,000 or 10,000,000-fold;
(c) killing at least at least 99%. 99.9%. 99.99%, 99.999%, 99.9999% or 99.99999% cells of a first species or strain comprised by a microbiome;
(d) producing synergistic Class 1 Cas modification of a cell genome; or (e) reducing bacterial cells of a first species or strain (eg, E coli cells) in a cell population by at least 105, 106 or 107-fold, wherein the population comprises at least 100,000;
1,000,000; or 10,000,000 cells respectively;
wherein the method is according to any one of claims 1 to 35, 51 and 52.