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WO2025046062A1 - Nouveau type de système crispr/cas - Google Patents

Nouveau type de système crispr/cas Download PDF

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Publication number
WO2025046062A1
WO2025046062A1 PCT/EP2024/074264 EP2024074264W WO2025046062A1 WO 2025046062 A1 WO2025046062 A1 WO 2025046062A1 EP 2024074264 W EP2024074264 W EP 2024074264W WO 2025046062 A1 WO2025046062 A1 WO 2025046062A1
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sequence
seq
cell
concept
nucleic acid
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Szabolcs SEMSEY
Ioannis MOUGIAKOS
Emilie SØNDBERG
Jasper Clube
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SNIPR Biome ApS
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SNIPR Biome ApS
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Priority claimed from PCT/EP2023/073881 external-priority patent/WO2024047151A1/fr
Priority claimed from GBGB2402011.7A external-priority patent/GB202402011D0/en
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Publication of WO2025046062A1 publication Critical patent/WO2025046062A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the technology described herein relates to new and synthetic CRISPR/Cas systems and components thereof, vectors, proteins, ribonucleoprotein complexes and methods of using the new system and components.
  • Types I-VI CRISPR/Cas systems are known.
  • Type-S CRISPR/Cas.
  • the base components of this system do not rely on the signature genes of any of the Type I-VI systems that have already been elucidated.
  • Type-S does not use a Cas3 (the signature protein of Type I systems), a Cas9 (the signature protein of Type II systems), a CaslO (the signature protein of Type III systems), a Casl2 (the signature protein of Type V systems) or a Casl3 (the signature protein of Type VI systems).
  • Cas3 the signature protein of Type I systems
  • Cas9 the signature protein of Type II systems
  • CaslO the signature protein of Type III systems
  • Casl2 the signature protein of Type V systems
  • Casl3 the signature protein of Type VI systems
  • the base components do not comprise an RNA-guided DNA nuclease. Nevertheless, we surprisingly found that a ribonucleoprotein complex of these components with a crRNA is able to effectively and specifically target a predetermined site on DNA. Using this system, it was also surprisingly possible to modify a cell without inducing dsDNA breaks. We could also surprisingly use the system to inhibit growth of bacterial cells. We fused several base components of the system to a base editor and found that it was able to perform base editing in an effective manner.
  • the synthetic system When fused to a base editor or DNA nuclease, again, surprisingly, the synthetic system does not require the presence of all of the base components (Cas-Sl, Cas-S2, Cas-S3, Cas-S4 and Cas-S5) of the Type-S system to effective perform the functions of the effector domains.
  • the system and its components therefore, provide new means to carry out nucleic acid editing and cell targeting in environmental, medical and other settings.
  • a Cas-Sl protein or a nucleic acid encoding a Cas-Sl is provided.
  • One or more nucleic acids encoding a Cas-Sl, S2, S3, S4 and S5 protein are provided.
  • One or more nucleic acids encoding a Cas-S4 and S5 protein are provided.
  • One or more nucleic acids encoding a Cas-S3, S4 and S5 protein are provided.
  • One or more nucleic acids encoding a Cas-S2, S3, S4 and S5 protein are provided.
  • One or more nucleic acids encoding a Cas-Sl and S2 protein are provided.
  • One or more nucleic acids encoding a Cas-Sl, S2 and S3 protein are provided.
  • One or more nucleic acids encoding a Cas-Sl, S2, S3 and S4 protein are provided.
  • One or more nucleic acids comprising SEQ ID Nos:7-ll.
  • One or more nucleic acids comprising SEQ ID Nos:8-ll.
  • One or more nucleic acids comprising SEQ ID Nos:9-ll.
  • One or more nucleic acids comprising SEQ ID Nos: 10 and 11.
  • One or more nucleic acids comprising SEQ ID Nos:7-10.
  • One or more nucleic acids comprising SEQ ID NOs:7-9.
  • One or more nucleic acids comprising SEQ ID Nos:7 and 8.
  • a nucleic acid comprising SEQ ID NO:7.
  • a nucleic acid comprising SEQ ID NO:8.
  • a nucleic acid comprising SEQ ID NO: 11.
  • each said sequence is operably connected to a heterologous promoter (i.e. a promoter that is not naturally operably connected to the sequence).
  • each said sequence is operably connected to a promoter that is a eukaryotic promoter, or virus (e.g. phage) promoter.
  • a protein or ribonucleoprotein complex comprising 1, 2, 3, 4 or all of a Cas-Sl, S2, S3, S4 and S5 protein.
  • a protein or ribonucleoprotein complex comprising a Cas-Sl, S2, S3, S4 and S5 protein.
  • a protein or ribonucleoprotein complex comprising a Cas-S4 and S5 protein.
  • a protein or ribonucleoprotein complex comprising a Cas-S3, S4 and S5 protein.
  • a protein or ribonucleoprotein complex comprising a Cas-S2, S3, S4 and S5 protein.
  • nucleic acid vector or a plurality of nucleic acid vectors or (ii) a nucleic acid or a plurality of nucleic acids comprising expressible nucleotide sequences, wherein the sequences comprise a) a nucleotide sequence that encodes a polypeptide which comprises an amino acid that is at least 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO: l;and/or b) a nucleotide sequence that encodes a polypeptide which comprises an amino acid that is at least 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to SEQ ID NO:2; and/or c) a nucleotide sequence that encodes a polypeptide which comprises an amino acid that is at least 98 or 99% identical to SEQ ID NO:3; and/or d) a nucleotide sequence that encodes a polypeptide which comprises an amino
  • PAM protospacer adjacent motif
  • the crRNA may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • the PAM may alternatively be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a kit comprising a) One or more polypeptides described herein, or one, or more nucleic acids encoding such polypeptide(s); and b) A crRNA, or one or more nucleic acids encoding a crRNA), wherein the crRNA is cognate to PAM having the sequence 5'-AAG-3'; wherein said polypeptide(s) are operable for use with the crRNA to form a ribonucleoprotein complex for protospacer targeting in a polynucleotide.
  • the polypeptides may comprise Cas-Sl, S2, S3, S4 and S5.
  • the polypeptides may be any polypeptide, protein, fusion protein or complex described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • the crRNA may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • the PAM may alternatively be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a ribonucleoprotein complex comprising a) One or more polypeptides described herein, or one, or more nucleic acids encoding such polypeptide(s); and b) A crRNA, or one or more nucleic acids encoding a crRNA), wherein the crRNA is cognate to PAM having the sequence 5'-AAG-3'.
  • the polypeptides may be any polypeptide, protein, fusion protein or complex described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • the crRNA may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • the PAM may alternatively be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • nucleic acid vectors or one or more nucleic acids comprising at least one nucleotide sequence selected from SEQ ID NOs:7-ll, wherein a said nucleotide sequence is a) operably connected to a heterologous promoter, synthetic promoter, eukaryotic promoter or non-bacterial promoter; and/or b) comprised by a cell that is a eukaryotic cell, a non-bacterial cell, or a cell that is not a bacterial cell (e.g. an Ecoli, Psurdomonasov Klebsiella cell) that comprises endogenous nucleotide sequence comprising SEQ ID NOs:7-ll.
  • a bacterial cell e.g. an Ecoli, Psurdomonasov Klebsiella cell
  • a fusion protein comprising a polypeptide (Px), wherein Px a) comprises an amino acid sequence that is at least (about) 80% identical to a sequence selected from SEQ ID Nos: 1-5; and b) is fused to a heterologous polypeptide (Py).
  • Py may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a protein or plurality of proteins operable for use with a crRNA to form a ribonucleoprotein complex for protospacer targeting in a polynucleotide comprising one, more or all polypeptides selected from a) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:1; b) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:2; c) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:3; d) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:4; and e) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:5; wherein the complex is operable with a protospacer adjacent motif (PAM) having the sequence 5'-AAG-3'.
  • the crRNA may be as described
  • a cell wherein the cell is a) a eukaryotic cell comprising the vector(s) or protein of the first, second or third configuration; b) an animal, plant, insect or fungus cell comprising the vector(s) or protein of the first, second or third configuration; or c) a prokaryotic cell comprising the vector(s) or protein of the first, second or third configuration, wherein the cell is not a bacterial cell (e.g. an E coli, Pseudomonas or Klebsiella cell) that comprises endogenous nucleotide sequences that encode the polypeptides of part a) to e) as recited in the second Configuration.
  • a bacterial cell e.g. an E coli, Pseudomonas or Klebsiella cell
  • a composition (optionally an in vitro composition or wherein the composition is comprised by a medical container) comprising a crRNA or nucleic acid encoding the crRNA or guide RNA; and one, more or all of a) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:1; b) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:2; c) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:3; d) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:4; and e) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO: 5 wherein
  • the crRNA comprises a spacer that is cognate to a first protospacer, wherein the protospacer is f) not found in E coir, g) is a eukaryotic cell protospacer; or h) an animal (optionally mammal or human), plant, fungus cell protospacer; or
  • E coii xa a protospacer of an E coii xa is devoid of endogenous nucleotide sequences that encode the polypeptides of a) to e).
  • the crRNA may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a composition (optionally an in vitro composition or wherein the composition is comprised by a medical container) comprising a crRNA or nucleic acid encoding the crRNA; and nucleic acid encoding one, more or all of a) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:1; b) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:2; c) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:3; d) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:4; and e) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO: 5 wherein
  • the crRNA comprises a spacer that is cognate to a first protospacer, wherein the protospacer is f) not found in E coir, g) is a eukaryotic cell protospacer; or h) an animal (optionally mammal or human), plant, fungus cell protospacer; or
  • E coii xa a protospacer of an E coii xa is devoid of endogenous nucleotide sequences that encode the polypeptides of a) to e).
  • the crRNA may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein. In a Sixth Configuration
  • a method of modifying a nucleic acid target site in a cell comprising a) contacting the cell with the vector(s) wherein the vector(s) comprise(s) one or more nucleotide sequences for producing a crRNA, wherein the crRNA comprises a spacer that is cognate to a first protospacer, wherein the protospacer is: not found in E. coir, a eukaryotic cell protospacer; or an animal (optionally human), plant, insect, fungus cell protospacer; b) allowing introduction into the cell of
  • the cRNA of the composition (or nucleic acids encoding the crRNA or guide RNA whereby the cRNA are expressed in the cell) and the nucleic acid of the composition encoding the polypeptide(s), whereby the polypeptide(s) are expressed in the cell; c) wherein the crRNA guide form a complex with the polypeptide(s) to guide the complex to the target site.
  • the vector(s) and crRNA may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • the polypeptides may be any polypeptide, protein, fusion protein or complex described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method of treating or preventing a disease or condition that is mediated by target cells in a subject comprising carrying out any of the methods described herein to modify the target cells, wherein said contacting comprises administering the vector(s) or composition to the subject and wherein the modification treats or prevents the disease or condition.
  • composition(s) may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method of introducing a targeted edit in a target polynucleotide comprising contacting the target polynucleotide with a CRISPR/Cas system comprising crRNA and a protein complex comprising one, more or all polypeptides selected from a) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:1; b) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:2; c) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:3; d) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:4; and e) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO: 5 wherein the crRNA comprises a spacer that is cognate to a first protospacer comprised by the polynucleot
  • a method of targeting a polynucleotide (optionally in a cell or in vitro), the method comprising a) contacting the polynucleotide with the protein(s) and crRNA of the Second Aspect of the Third Configuration; b) allowing the formation of a ribonucleoprotein complex comprising the protein(s) and crRNA, wherein the complex is guided to a target site comprised by the polynucleotide to modify the polynucleotide or replication thereof.
  • a method of transcriptional control or replication of a target DNA comprising contacting the target DNA with the complex, wherein the complex is devoid of a DNA nuclease and the complex binds to the target DNA, thereby controlling the transcription or replication of the target DNA.
  • a method of controlling replication of a target RNA comprising contacting the target RNA or a DNA encoding the RNA with the complex, wherein the complex binds to the target RNA or DNA, thereby controlling the transcription of the target RNA.
  • a method of editing a target DNA comprising contacting the target DNA with the complex, wherein the complex binds to the target DNA, thereby editing the target DNA.
  • a method for cleaving a double stranded DNA comprising contacting said dsDNA with the complex, wherein the complex comprises a nuclease, wherein the dsDNA comprises a protospacer sequence flanked at its 5' by a PAM having the sequence 5'-AAG-3' or a PAM that is identical except for one base change, whereby the nuclease cleaves the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer.
  • the PAM may alternatively be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method for cleaving a single stranded DNA comprising contacting said DNA with the complex, wherein the complex comprises a nickase, wherein the DNA comprises a protospacer sequence flanked at its 5' by a PAM having the sequence 5'-AAG-3' or a PAM that is identical except for one base change, whereby the nuclease cleaves a single strand of the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer.
  • the PAM may alternatively be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method of marking or identifying a region of a DNA comprising contacting said DNA with the complex, wherein the DNA comprises a protospacer sequence flanked at its 5' by a PAM having the sequence 5'-AAG-3' or a PAM that is identical except for one base change, whereby the complex binds to the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer, and optionally wherein the complex comprises a detectable label.
  • the PAM may alternatively be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method of modifying transcription of a region of a DNA comprising contacting said DNA with the complex, wherein the DNA comprises a protospacer sequence flanked at its 5' by a PAM having the sequence 5'-AAG-3' or a PAM that is identical except for one base change, whereby the complex binds to the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer, whereby the complex up- or down-regulates transcription of the region of DNA or an adjacent gene.
  • the PAM is not CGG.
  • the PAM can be AAN, ANG, NAG; or
  • the PAM can be AAG with no or one nucleotide change.
  • the PAM may alternatively be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method of modifying a target dsDNA of a cell without introducing dsDNA breaks comprising producing in the cell the complex, wherein the complex targets a target DNA comprised by the cell, the target DNA comprising a protospacer sequence flanked at its 5' by a PAM having the sequence 5'-AAG-3' or a PAM that is identical except for one base change, whereby the complex binds to the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer, whereby the DNA is modified without introducing breaks in the DNA.
  • the PAM may alternatively be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method of inhibiting the growth or proliferation of a cell without introducing lethal dsDNA breaks comprising producing in the cell the complex, wherein the complex targets a target DNA comprised by the cell, the target DNA comprising a protospacer sequence flanked at its 5' by a PAM having the sequence 5'-AAG-3' or a PAM that is identical except for one base change, whereby the complex binds to the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer, whereby replication of the DNA is inhibited without introducing breaks in the DNA, thereby inhibiting the growth or proliferation of a cell.
  • the PAM may alternatively be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method of treating or preventing a disease or condition in a human, animal, plant or fungus subject comprising carrying out the method of any of the Fifth to Thirteenth Aspects, wherein cells of the subject comprise said DNA or RNA and the cells mediate the disease or condition.
  • PAM protospacer adjacent motif
  • the PAM may alternatively be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a ribonucleoprotein CRISPR/Cas complex comprising a plurality of Cas proteins and a crRNA, wherein the RNA is capable of guiding the complex to a protospacer comprised by a target DNA, wherein the 5'end of the protospacer is flanked by a protospacer adjacent motif (PAM) having the sequence 5'-AAG-3', wherein a) the complex is devoid of a DNA nuclease and is capable of modifying DNA without introducing a double stranded DNA break; b) the complex is devoid of a DinG, a Cas3 and a CaslO; and c) the complex does not comprise all of the Cas proteins of a Type I, II, III, V or VI CRISPR/Cas complex.
  • PAM protospacer adjacent motif
  • the PAM may alternatively be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a cell comprising the nucleic acid(s) or complex described herein, wherein the DNA is comprised by a chromosome of the cell.
  • a cell comprising the nucleic acid(s) or complex described herein, wherein the DNA is comprised by a plasmid in the cell.
  • a method of modifying a DNA comprising a) contacting the DNA with the nucleic acid(s) or vector(s) described herein; b) allowing the formation of a ribonucleoprotein complex comprising the Cas protein(s) and crRNA, wherein the complex is guided to a target site comprised by the DNA to modify the DNA.
  • the crRNA and Cas protein(s) may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method of inhibiting the replication of a plasmid comprising DNA comprising a) contacting the DNA with the nucleic acid(s) or vector(s) described herein; b) allowing the formation of a ribonucleoprotein complex comprising the Cas protein(s) and crRNA, wherein the complex is guided to a target site comprised by the DNA to inhibit replication of the plasmid.
  • the crRNA and Cas protein(s) may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method of inhibiting the transcription of a nucleotide sequence comprised by a DNA comprising a) contacting the DNA with the nucleic acid(s) or vector(s) described herein; b) allowing the formation of a ribonucleoprotein complex comprising the Cas protein(s) and crRNA, wherein the complex is guided to a target site comprised by the nucleotide sequence to inhibit transcription thereof.
  • the crRNA and Cas protein(s) may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method of inhibiting the growth or proliferation of a cell comprising DNA, the method comprising a) contacting the DNA with the nucleic acid(s) or vector(s) described herein; b) allowing the formation of a ribonucleoprotein complex comprising the Cas protein(s) and crRNA, wherein the complex is guided to a target site comprised by the DNA to inhibit growth or proliferation of the cell.
  • the crRNA and Cas protein(s) may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • a method of treating or preventing a disease or condition that is mediated by target cells in a human or animal subject comprising carrying out any of the methods described herein to modify the target cells, wherein said contacting comprises administering the nucleic acid(s) to the subject and wherein the modification treats or prevents the disease or condition.
  • One or more nucleic acid(s) of the First Aspect for use in a method of treating or preventing a disease or condition that is mediated by target cells in a human or animal subject, the method comprising carrying out any of the methods described hereinn to modify the target cells, wherein said contacting comprises administering the nucleic acid(s) to the subject and wherein the modification treats or prevents the disease or condition.
  • Figure 1 Cloning components of a Type-S CRISPR system.
  • a plasmid (p 1624) was constructed carrying a pSClOl replication origin and a tetracycline resistance marker as well as DNA sequences encoding an RNA guided endonuclease (rge), two ORFs downstream of the rge gene, a cognate CRISPR array and four ORFs immediately upstream of the CRISPR array.
  • ORFs 140 and 423 were predicted to code for an error prone DNA polymerase, while ORFs 624 and 188 were predicted to code for a helicase and an RNA endonuclease respectively.
  • the target plasmid pl631 was constructed by first creating a synthetic DNA fragment containing spacer like sequences from the Type-S CRISPR/Cas system separated by the predicted PAM sequence (AAG), and then inserting this sequence into a plasmid containing a pl5A replication origin, a chloramphenicol resistance marker (CmR), and the amilCP gene producing a purple colour.
  • AAG predicted PAM sequence
  • CmR chloramphenicol resistance marker
  • FIG. 2 CRISPR-Cas system inhibits the growth of colonies transformed with the pl631 target plasmid.
  • Strain bSNP3127 was transformed with either pl760 (right, see Figure 3 for content), or pl624 (middle, see Figure 1 for content) or a no plasmid control (left).
  • Each of the resulting strains was transformed with a 1: 1 mixture of target (purple) and non-target (white) plasmids and streaked on LB plates supplemented with the appropriate antibiotics for selection. No purple colonies were observed in the presence of pl760.
  • Figure 3 Schematic representation of the plasmid library constructed upon deletion of a series of genes in pl760 (see Figure 3 for content). Boxes indicate gene deletions. The constructs that showed plasmid inhibition are indicated with *.
  • Figure 4 Five genes and a CRISPR array comprise the Type-S CRISPR-Cas system. The bSNP3127 strains carrying some of the plasmids shown in Figure 3 are transformed with a 1: 1 mixture of target pl631 (purple) and non-target (colourless) plasmids. Purple colonies with the tested plasmids were observed only in the absence of the CRISPR array.
  • FIG. 5 The CRISPR-Cas system uses all five genes (S1-S5) and a CRISPR array for plasmid inhibition.
  • Strains b5700 ACas-Sl
  • b5702 ACas-S3
  • b5703 ACas-S4
  • b5704 ACas-S5
  • b4816 negative control, no Type-S CRISPR-Cas system
  • b5408 positive control, full Type-S CRISR-Cas system present
  • Figure 7 Plasmid clearance assays for evaluation of the targeting activity of each spacer in the Type-S CRISPR array. All four tested spacers display similar targeting efficiency.
  • Figure 8 The Type-S CRISPR-Cas system does not induce lethal dsDNA breaks in the chromosome of E. coli.
  • Figure 9 Schematic representation of the positions of the protospacers employed for the Type-S CRISPR-Cas binding assays.
  • Figure 10 Type-S CRISPR-Cas binds to chromosomal DNA. Growth curves of the bSNP5810 derivative strains in the presence of increasing chloramphenicol concentrations.
  • Figure 11 Graphical representation of the Type-S CRISPR-Cas PAM preferences in a 'PAM wheel' form (see Leenay et al, Technology, 62(1), 137-147, 2016, doi:https://doi.org/10.1016/j.molcel.2016.02.031).
  • the inner ring corresponds to the 3 rd nucleotide position upstream (5') of the protospacer
  • the middle ring corresponds to the 2 nd nucleotide position upstream (5') of the protospacer
  • the outer ring corresponds to the 1st nucleotide position upstream (5Q of the protospacer.
  • every sequence occupies a sector, with an area proportional to the relative enrichment of the sequence.
  • Figure 12 Graphical representation of the Type-S CRISPR/Cas targeting efficiency on the most preferred PAMs.
  • the x-axis notes the PAMs of the most targeted p2259 library members, according to the generated NGS data.
  • the y-axis depicts the fold reduction of sequencing reads for each PAM, when comparing the NGS data generated by strain b5408 (Cas- S targeting) to the NGS data generated by strain b6259 (control Cas-S).
  • Figure 13 Graphical representation of the b5408 (wt Type-S) transformation efficiency with each member of the pl935 plasmid library compared to its transformation efficiency with the negative control p2361 plasmid (No PAM).
  • the wild type protospacer is depicted at the top of each graph.
  • the complementary spacer-protospacer sections for each tested protospacer are indicated with dots.
  • the mutated protospacer sections are indicated with the corresponding single letter nucleotide codes. Its tested protospacer has a unique code that is denoted at the left side of the graphs.
  • A PAM proximal mismatches (stretches);
  • B PAM distal mismatches (stretches);
  • C PAM proximal single mismatches;
  • D PAM proximal double mismatches;
  • E PAM proximal triple mismatches;
  • F PAM proximal quadruple mismatches.
  • the 'cross(es)' symbols indicate presence of low (single cross) or high (double cross) number of -not possible to count - "micro-colonies" at the corresponding transformation plates.
  • the micro-colonies possibly indicate inefficient Type-S CRISPR/Cas targeting that does not completely stop plasmid replication. As a result, colonies grow with a significantly slower rate on selection medium.
  • Figure 14 Graphical representation of the plasmids used in Examples 4 and 5.
  • the Type-S variant that each plasmid expresses is noted at the left side of the figure.
  • the name of each plasmid is noted at the bottom left side of each plasmid.
  • the Type-S genes are presented as boxes with solid fills and the corresponding gene names are noted above each box. Dotted lines correspond to deleted Type-S genes.
  • the expression of the Type-S genes was driven by the pBolA promoter, which is presented as an arrow that points to the direction of the transcription.
  • the expression of the crRNA module was driven by the 'Leader' sequence, presented as a box filled with diagonal stripes and is located right upstream (5Q of the crRNA expressing module.
  • the CRISPR repeats (SEQ ID No:6) of the crRNA expressing module are presented as triangles and the spacer is presented as a circle. The locations of the BsmBI recognition sites are indicated with asterisk (*) signs.
  • the tetracycline resistance marker gene, the SC101 origin of replication and the repAlOl replication protein gene are presented as boxes with checker, brick and grid fills respectively.
  • Figure 15 Schematic representation of the positions of the protospacers employed for the Type-S CRISPRi assays for downregulation of the GFP expression from strain b5815 that contains chromosomally integrated gfp gene. Each protospacer position is indicated with a triangle and the ID of each protospacer is noted above the triangle.
  • Figure 16 Graphical representation of the chromosomal CRISPRi activity of the wild type Type-S system on the GFP expression of strain b5815 over a 24-hour period.
  • the targeted protospacers were located A) at the beginning of the gfp orf and in the p70a promoter region (Protospacers indicated in brackets: S1F, SIR, S2F, S2R, S3F, S3R), B) at the end of the gfp orf and the 200bp regions upstream and downstream of gfp (Protospacers indicated in brackets S4F, S4R, S5F, S6F, S6R) C) 1 kb upstream (Protospacers indicated in brackets: UlkF, UlkR) and 2 kb upstream (Protospacers indicated in brackets: U2kF, U2kR) of the gfp orf.
  • Figure 17 Graphical representation of the chromosomal CRISPRi activity of the AcasS3 Type-S system on the GFP expression of strain b5815 over a 24-hour period.
  • the targeted protospacers were located A) at the beginning of the gfp orf and in the p70a promoter region (Protospacers S1F, SIR, S2F, S2R, S3F, S3R), B) at the end of the gfp orf and the 200 bp regions upstream and downstream of gfp (Protospacers S4F, S4R, S5F, S6F, S6R).
  • Figure 18 Graphical representation of the chromosomal CRISPRi activity of the A) AcasS3 Type-S system, B) AcasSlAcasS3 Type-S system and C) AcasSl Type-S system on the GFP expression of strain b5815 over a 24-hour period.
  • the targeted protospacers were located at the beginning of the gfp orf and in the p70a promoter region (Protospacers S1F, SIR, S2F, S2R, S3F, S3R).
  • the plasmids employed for the transformation of b5815 strain is denoted with (:).
  • Figure 19 Graphical representation of on-plasmid CRISPRi activity of the A) AcasS3 Type-S system, B) AcasS4 Type-S system, C) AcasS4 Type-S system and D) AcasSlAcasS3 Type-S system on the GFP expression of strain b6386 over a 24-hour period.
  • the targeted protospacers were located at the beginning of the gfp orf and in the p70a promoter region (Protospacers S1F, SIR, S2F, S2R, S3F, S3R).
  • Figure 20 Schematic representation of the plasmid combinations employed for the A) CasSl, B) CasSl (ACasS3), C) CasS3 and D) CasS4 base editing assays.
  • the asterix (*) indicates that the spacer is directed against one of: a non-targeting control, S1F, SIR, S2F, S2R, S3F or S3R.
  • Figure 21 Heatmap based visualization of representative sequencing results from the Type-S CRISPR/Cas base editing assays.
  • the PmCDAl cytidine deaminase is fused to the C-terminus of A) the Cas-Sl subunit of the wild type Type-S CRISPR/Cas system, B) the Cas-Sl subunit of the ACas-S3 Type-S CRISPR/Cas system, C) the Cas-S3 subunit of the wild type Type-S CRISPR/Cas system, D) the Cas-S2 subunit of the wild type Type-S CRISPR/Cas system.
  • each box On the top side of each box are noted i) the code of the tested protospacer and ii) the nucleotide sequence of the protospacer (highlighted with a boxy arrow that additionally shows the direction of the protospacer) together with the sequence of the immediately neighboring genomic regions where base-editing modifications were detected.
  • the codes of unique Sanger sequencing results each from a distinct colony
  • each heatmap cell represents the relative C to T (or G to A) abundance for each position, according to the height ratio of the corresponding C and T peaks from the Sanger sequencing results.
  • black boxes represent colonies with 100% C to T mutated genotype for the corresponding positions, whereas off-white boxes correspond to 95% to 5% (reliable limit of detection for Sanger sequencing) C to T mixed mutant genotypes for the corresponding positions.
  • Figure 22 Schematic representation of the plasmid combinations employed for the TevCasS plasmid targeting assays.
  • Figure 23 Graphical representation of the results from the TevCasS plasmid targeting assays.
  • the x-axis corresponds to the drop in the transformation efficiency of a MG1655 strain that expresses TevCasSl and targets the target sequence on a TevCasS target plasmid relative to the transformation efficiency of a MG1655 strain that expresses TevCasSl and does not target the target sequence on a TevCasS target plasmid.
  • the y-axis corresponds to the size of the I-TevI spacer sequence in the target sequence of a TevCasS target plasmid.
  • Figure 24 Graphical representation of the results from the TevCasS chromosomal targeting assays.
  • the x-axis corresponds to the drop in the transformation efficiency of a MG1655 strain that expresses TevCasSl and targets a chromosomal TevCasS target sequence relative to the transformation efficiency of a MG1655 strain that expresses TevCasSl and does not target a chromosomal TevCasS target sequence.
  • the y-axis corresponds to the size of the I-TevI spacer sequence in the target sequence of a chromosomal TevCasS target sequence.
  • Figure 25 Graphical representation of the TevCasS target cleavage site preferences in a 'Krona plot' form.
  • the sequence of the TevCasS target cleavage site is S'CNiNzNsG-S'.
  • the inner ring corresponds to Ni
  • the middle ring corresponds to N2 nucleotide
  • the outer ring corresponds to N3.
  • every sequence occupies a sector, with an area proportional to the relative enrichment of the sequence.
  • the technology described herein relates to new and synthetic CRISPR/Cas systems and components thereof, vectors, proteins, ribonucleoprotein complexes and methods of using the new system and components.
  • CRISPR-S CRISPR-S
  • Type-S CRISPR/Cas CRISPR/Cas
  • Type-S does not use a Cas3 (the signature protein of Type I systems), a Cas9 (the signature protein of Type II systems), a CaslO (the signature protein of Type III systems), a Casl2 (the signature protein of Type V systems) or a Casl3 (the signature protein of Type VI systems).
  • the basic components do not comprise a IscB, IsrB or IshB protein (IscBs and IsrBs share a common evolutionary history with Cas9). We refer to the new system as Type-S.
  • the vector(s) herein does not encode a Cas3, 9, 10, 12 and/or 13.
  • the nucleic acid herein may not encode a Cas3, 9, 10, 12 and/or 13.
  • the complex herein may not comprise a Cas3, 9, 10, 12 and/or 13.
  • the method herein may not use a Cas3, 9, 10, 12 and/or 13.
  • the vector(s) or nucleic acid(s) herein do not encode a TnpB.
  • the complexes described herein do not comprise a TnpB.
  • the vector(s) or nucleic acid(s) herein do not encode a HD-nuclease domain.
  • the complexes herein do not comprise a HD-nuclease domain.
  • the vector(s) or nucleic acid(s) herein encodes a Cas8.
  • the complexes herein comprise a Cas8.
  • the complexes may comprise a protein for PAM identification, a protein for interaction with a Cas8, a protein for processing pre-crRNA, and a scaffolding protein, wherein the complexes comprises a Cas-Sl to Cas-S5 protein.
  • the complexes may comprise a protein for PAM identification, a protein for interaction with a Cas8, and a scaffolding protein, wherein the complex comprises a Cas-Sl, Cas-S2, Cas-S4 and Cas-S5 protein.
  • the vector(s) or nucleic acid(s) comprise a CRISPR array for producing the crRNA.
  • the array may comprise one or more repeat sequences and one or more spacer sequences.
  • the spacer sequences may be complementary (as defined elsewhere herein) to a first protospacer sequence.
  • the spacer sequences of the array may be complementary to a second, third, fourth... etc protospacer in a target sequence, for example if multiplex editing or modification is desired.
  • the repeat sequence is SEQ ID NO:6 or SEQ ID No:70 or a sequence that is identical except for 1, 2, 3, 4, or 5 changes, in particular 1 or 2, e.g. 1 change.
  • the vector(s) or nucleic acid(s) herein may comprise a Type-S array, i.e. an array that encodes a crRNA that is operable with at least Cas-Sl, 2, 3, 4 and 5 to target a first protospacer (or e.g. a first and second protospacer) of a nucleic acid or target sequence.
  • the vector(s) or nucleic acid(s) herein may comprise a Type-S array, i.e. an array that encodes a crRNA that is operable with at least Cas-Sl, 2, 4 and 5 to target a first protospacer (or e.g. a first and second protospacer) of a nucleic acid or target sequence.
  • the vector(s) or nucleic acid(s) herein may comprise a Type-S array, i.e. an array that encodes a crRNA that is operable with at least Cas-S2, 3, 4 and 5 to target a first protospacer (or e.g. a first and second protospacer) of a nucleic acid or target sequence.
  • the vector(s) or nucleic acid(s) herein may comprise a Type-S array, i.e. an array that encodes a crRNA that is operable with at least Cas-S2, 4 and 5 to target a first protospacer (or e.g. a first and second protospacer) of a nucleic acid or target sequence.
  • the protospacer is downstream of a 5'-AAG-3' PAM in the nucleic acid or target sequence.
  • the PAM can be AAN, ANG, NAG; or the PAM can be AAG with no or one nucleotide change.
  • the PAM is not CGG.
  • the PAM can be AHN, KAG, AGG, GAC or GTG.
  • the PAM is selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG- 3', 5'-AAT-3', 5'-ACA-3', 5'-ACT-3', 5'-ATC-3', 5'-ATA-3', 5'-GAG-3', 5'-TAG-3', 5'-ACC-3', 5'-AGG-3', 5'-ATT-3', 5'-GAC-3' and 5'-GTG-3'.
  • the PAM is selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG-3', 5'-AAT-3', 5'-ACA-3', 5'-ACT-3', 5'-ATC-3', 5'-ATA-3', 5'-GAG-3'and 5'-TAG-3'.
  • the PAM is selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'- ACG-3', 5'-AAT-3' and 5'-ACA-3', e.g. 5'-AAC-3' and 5'-ATG-3'.
  • the PAM is 5'-AAC-3'.
  • Type-S system components (Cas-Sl, Cas-S2, Cas-S3, Cas-S4 and Cas-S5) do not comprise an RNA-guided DNA nuclease. Nevertheless, we surprisingly found that a ribonucleoprotein complex of these components with a crRNA is able to effectively and specifically target a predetermined site on DNA. Using this system, it was also surprisingly possible to modify a cell without inducing dsDNA breaks, see Example 1.3.4 hereinbelow. Armed with this knowledge, we can see many applications of the new system. It is possible, for example, to provide one or more proteins or domains with effector activity (e.g.
  • nuclease see Example 5 hereinbelow
  • base editing see Example 4 hereinbelow
  • prime editing activity together with one or more the components of the system to produce synthetic systems that can modify cells and nucleic acid in a RNA-directed or DNA-directed manner.
  • the Type-S system and its components therefore, provide new means to carry out nucleic acid editing and cell targeting in environmental, medical and other settings.
  • a Cas-Sl protein or a nucleic acid encoding a Cas-Sl is provided.
  • One or more nucleic acids encoding a Cas-Sl, S2, S3, S4 and S5 protein are provided.
  • One or more nucleic acids encoding a Cas-S4 and S5 protein are provided.
  • One or more nucleic acids encoding a Cas-S3, S4 and S5 protein are provided.
  • One or more nucleic acids encoding a Cas-S2, S3, S4 and S5 protein are provided.
  • One or more nucleic acids encoding a Cas-Sl, S2, S4 and S5 protein are provided.
  • One or more nucleic acids encoding a Cas-S2, S3, S4 and S5 protein are provided.
  • One or more nucleic acids encoding a Cas-S2, S4 and S5 protein are provided.
  • each said sequence is operably connected to a heterologous promoter (i.e. a promoter that is not naturally operably connected to the sequence).
  • each said sequence is operably connected to a promoter that is a eukaryotic promoter, or virus (e.g. phage) promoter.
  • the promoter may be a constitutive promoter.
  • the promoter may be an inducible promoter. Promoters may be as described in any Configuration, Concept, aspect, example, embodiment, option or other feature described herein.
  • nucleic acid(s) or vector(s) encode a Cas nuclease.
  • the nucleic acid(s) may encode a non-Cas effector protein or domain, such as a nuclease that is not a Cas nuclease.
  • a protein or ribonucleoprotein complex comprising 1, 2, 3, 4 or all of a Cas-Sl, S2, S3, S4 and S5 protein.
  • a protein or ribonucleoprotein complex comprising a Cas-Sl, S2, S3, S4 and S5 protein.
  • a protein or ribonucleoprotein complex comprising a Cas-S4 and S5 protein.
  • a protein or ribonucleoprotein complex comprising a Cas-S3, S4 and S5 protein.
  • a protein or ribonucleoprotein complex comprising a Cas-S2, S3, S4 and S5 protein.
  • a protein or ribonucleoprotein complex comprising a Cas-Sl, S2, S4 and S5 protein.
  • a protein or ribonucleoprotein complex comprising a Cas-S2, S3, S4 and S5 protein.
  • a protein or ribonucleoprotein complex comprising a Cas-S2, S4 and S5 protein.
  • the complex does not comprise a Cas nuclease.
  • the complex may comprise a non-Cas effector protein or domain, such as a nuclease that is not a Cas nuclease.
  • Type-S proteins are capable of RNA-directed modification of a DNA sequence, optionally without creating a break in the DNA.
  • RNA-directed modification of a DNA sequence optionally without creating a break in the DNA.
  • Cas-Sl is a Cas protein that comprises an amino acid that is at least (about) 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO:1.
  • the identity is at least (about) 80%. Even more preferably, the identity is at least (about) 90 or 95%. In another embodiment, the identity is at least about 96%. In another embodiment, the identity is at least about 97%. In another embodiment, the identity is at least about 98%.
  • the identity is at least about 99%.
  • the Cas-Sl is Cas-Sl.1.
  • Cas-Sl.1 is a Cas protein that comprises the amino acid of SEQ ID NO: 1.
  • Cas-S2 is a Cas protein that comprises an amino acid that is at least (about) 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO:2.
  • the identity is at least (about) 80%.
  • the identity is at least (about) 90 or 95%. In another embodiment, the identity is at least about 96%. In another embodiment, the identity is at least about 97%. In another embodiment, the identity is at least about 98%. In another embodiment, the identity is at least about 99%.
  • the Cas-S2 is Cas-S2.1.
  • Cas-S2.1 is a Cas protein that comprises the amino acid of SEQ ID NO:2.
  • Cas-S3 is a Cas protein that comprises an amino acid that is at least (about) 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO:3.
  • the identity is at least (about) 80%. Even more preferably, the identity is at least (about) 90 or 95%.
  • the identity is at least about 96%.
  • the identity is at least about 97%.
  • the identity is at least about 98%.
  • the identity is at least about 99%.
  • the Cas-S3 is Cas-S3.1.
  • Cas-S3.1 is a Cas protein that comprises the amino acid of SEQ ID NO:3.
  • Cas-S4 is a Cas protein that comprises an amino acid that is at least (about) 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO:4.
  • the identity is at least (about) 80%. Even more preferably, the identity is at least (about) 90 or 95%. In another embodiment, the identity is at least about 96%. In another embodiment, the identity is at least about 97%. In another embodiment, the identity is at least about 98%. In another embodiment, the identity is at least about 99%.
  • the Cas-S4 is Cas-S4.1.
  • Cas-S4.1 is a Cas protein that comprises the amino acid of SEQ ID NO:4.
  • Cas-S5 is a Cas protein that comprises an amino acid that is at least (about) 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID NO: 5.
  • the identity is at least (about) 80%. Even more preferably, the identity is at least (about) 90 or 95%.
  • the identity is at least about 96%.
  • the identity is at least about 97%.
  • the identity is at least about 98%.
  • the identity is at least about 99%.
  • the Cas-S5 is Cas-S5.1.
  • Cas-S5.1 is a Cas protein that comprises the amino acid of SEQ ID NO: 5.
  • any percentage identity herein is at least (about) 70%.
  • any percentage identity herein is at least (about) 80%.
  • any percentage identity herein is at least (about) 90%.
  • any percentage identity herein is at least 95%.
  • any percentage identity herein is at least about 96%.
  • any percentage identity herein is at least about 97%.
  • any percentage identity is at least about 98%.
  • any percentage identity herein is at least about 99%.
  • Percent identity for amino acid sequences are determined using the blastP algorithm with the following para meters :-
  • the default parameters are adjusted for short input sequences, the expect threshold is set at 0.05 and the length of the seed sequence that initiates an alignment is set at 6. Regions of low compositional complexity are masked.
  • the employed scoring matrix is 'BLOSUM62', with scoring costs to create and extend a gap being 11 and 1 respectively.
  • Conditional compositional score matrix adjustment is employed to compensate for amino acid composition of compared sequences.
  • Percent identity for nucleotide sequences are determined using the blastn algorithm with the following para meters :-
  • the default parameters are automatically adjusted for short input sequences, the expect threshold is set at 0.05 and the length of the seed sequence that initiates an alignment is set at 28. Regions of low compositional complexity are masked.
  • the query sequence is masked while producing seed sequences used to scan databases, but not masked for extensions. Matches are scored as +1 and mismatches are scored as -2.
  • an amino acid sequence described herein is identical to the reference SEQ ID NO except for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, in particular 1 to 5, for example 1 to 3, such as 1 or 2, e.g. 1 amino acid change.
  • a nucleic acid sequence described herein is identical to the reference SEQ ID NO except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotide changes.
  • an amino acid sequence of the polypeptide is identical to SEQ ID NO:1 except for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, in particular 1 to 5, for example 1 to 3, such as 1 or 2, e.g. 1 amino acid change.
  • an amino acid sequence of the polypeptide is identical to SEQ ID NO: 2 except for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, in particular 1 to 5, for example 1 to 3, such as 1 or 2, e.g. 1 amino acid change.
  • an amino acid sequence of the polypeptide is identical to SEQ ID NO: 3 except for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, in particular 1 to 5, for example 1 to 3, such as 1 or 2, e.g. 1 amino acid change.
  • an amino acid sequence of the polypeptide is identical to SEQ ID NO:4 except for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, in particular 1 to 5, for example 1 to 3, such as 1 or 2, e.g. 1 amino acid change.
  • an amino acid sequence of the polypeptide is identical to SEQ ID NO: 5 except for 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes, in particular 1 to 5, for example 1 to 3, such as 1 or 2, e.g. 1 amino acid change.
  • an amino acid sequence described herein is identical to the reference SEQ ID NO except for a total number of amino acid changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of amino acids in the reference sequence.
  • a nucleotide sequence described herein is identical to the reference SEQ ID NO except for a total number of nucleotide changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of nucleotides in the reference sequence.
  • the amino acid sequence of the polypeptide is SEQ ID NO: 1 or an amino acid sequence that is identical to SEQ ID NO: 1 except for a total number of amino acid changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of amino acids in SEQ ID NO:1, in particular no more than (about) 20%, such as no more than (about) 10% of the number of amino acids in SEQ ID NO: 1.
  • the amino acid sequence of the polypeptide is SEQ ID NO: 3 or an amino acid sequence that is identical to SEQ ID NO: 3 except for a total number of amino acid changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of amino acids in SEQ ID NO:3, in particular no more than (about) 20%, such as no more than (about) 10% of the number of amino acids in SEQ ID NO:3.
  • the amino acid sequence of the polypeptide is SEQ ID NO:4 or an amino acid sequence that is identical to SEQ ID NO:4 except for a total number of amino acid changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of amino acids in SEQ ID NO:4, in particular no more than (about) 20%, such as no more than (about) 10% of the number of amino acids in SEQ ID NO:4.
  • the amino acid sequence of the polypeptide is SEQ ID NO: 5 or an amino acid sequence that is identical to SEQ ID NO: 5 except for a total number of amino acid changes wherein the total number is no more than (about) 5, 10, 15, 20, 25 or 30% of the number of amino acids in SEQ ID NO: 5, in particular no more than (about) 20%, such as no more than (about) 10% of the number of amino acids in SEQ ID NO:5.
  • the vector(s) herein encode Cas-Sl, 2, 3, 4 and 5.
  • the vector(s) herein encode Cas-S4 and 5.
  • the vector(s) herein encode Cas-S3, 4 and 5.
  • the vector(s) herein encode Cas-S2, 3, 4 and 5.
  • the vector(s) herein encode Cas-Sl and 2.
  • the vector(s) herein encode Cas-Sl, 2 and 3.
  • the vector(s) herein encode Cas-Sl, 2, 3 and 4.
  • the vector(s) herein encode Cas-Sl, 2, 4 and 5. In an embodiment, the vector(s) herein encode Cas-S2, 4 and 5. In an embodiment, the vector(s) herein encode Cas-Sl.l, 2.1, 3.1, 4.1 and 5.1.
  • the vector(s) herein encode Cas-S4.1 and 5.1.
  • the vector(s) herein encode Cas-S3.1, 4.1 and 5.1.
  • the vector(s) herein encode Cas-S2.1, 3.1, 4.1 and 5.1.
  • the vector(s) herein encode Cas-Sl.l and 2.1.
  • the vector(s) herein encode Cas-Sl.l, 2.1 and 3.1.
  • the vector(s) herein encode Cas-Sl.l, 2.1, 3.2 and 4.1.
  • the vector(s) herein encode Cas-Sl.l, 2.1, 4.1 and 5.1.
  • the vector(s) herein encode Cas-S2.1, 4.1 and 5.1.
  • the complex herein comprises Cas-Sl, 2, 3, 4 and 5.
  • the complex herein comprises Cas-S4 and 5.
  • the complex herein comprises Cas-S3, 4 and 5.
  • the complex herein comprises Cas-S2, 3, 4 and 5.
  • the complex herein comprises Cas-Sl and 2.
  • the complex herein comprises Cas-Sl, 2 and 3.
  • the complex herein comprises Cas-Sl, 2, 3 and 4.
  • the complex herein comprises Cas-Sl, 2, 4 and 5.
  • the complex herein comprises Cas-S2, 4 and 5.
  • the complex herein comprises Cas-Sl.l, 2.1, 3.1, 4.1 and 5.1.
  • the complex herein comprises Cas-S4.1 and 5.1.
  • the complex herein comprises Cas-S3.1, 4.1 and 5.1.
  • the complex herein comprises Cas-S2.1, 3.1, 4.1 and 5.1.
  • the complex herein comprises Cas-Sl.l and 2.1.
  • the complex herein comprises Cas-Sl.l, 2.1 and 3.1.
  • the complex herein comprises Cas-Sl.l, 2.1, 3.1 and 4.1.
  • the complex herein comprises Cas-Sl.l, 2.1, 4.1 and 5.1.
  • the complex herein comprises Cas-S2.1, 4.1 and 5.1.
  • the fusion protein herein comprises Cas-Sl.l, 2.1, 3.1, 4.1 or 5.1.
  • the fusion protein herein comprises Cas-Sl.l, 2.1, 3.1, 4.1 and 5.1.
  • the fusion protein herein comprises Cas-S4.1 and 5.1.
  • the fusion protein herein comprises Cas-S3.1, 4.1 and 5.1. In an embodiment, the fusion protein herein comprises Cas-S2.1, 3.1, 4.1 and 5.1.
  • the method herein uses Cas-Sl.l, 2.1, 3.1, 4.1 and 5.1.
  • the method herein uses Cas-Sl.l, 2.1, 4.1 and 5.1.
  • the method herein uses Cas-S2.1, 4.1 and 5.1.
  • one or more vectors and nucleic acids comprising one or more nucleotide sequences selected from SEQ ID NOs:7-ll.
  • One or more nucleic acids comprising SEQ ID Nos:7-ll.
  • One or more nucleic acids comprising SEQ ID Nos:8-ll.
  • One or more nucleic acids comprising SEQ ID Nos:9-ll.
  • One or more nucleic acids comprising SEQ ID Nos: 10 and 11.
  • One or more nucleic acids comprising SEQ ID Nos:7, 8, 10 and 11.
  • One or more nucleic acids comprising SEQ ID Nos:8, 9, 10 and 11.
  • One or more nucleic acids comprising SEQ ID Nos:8, 10 and 11.
  • a nucleic acid comprising SEQ ID NO:7.
  • a nucleic acid comprising SEQ ID NO:8.
  • a nucleic acid comprising SEQ ID NO:9.
  • a nucleic acid comprising SEQ ID NO: 10.
  • a nucleic acid comprising SEQ ID NO: 11.
  • a nucleic acid comprising SEQ ID Nos:8, 9, 10 and 11.
  • a nucleic acid comprising SEQ ID Nos:8, 10 and 11.
  • One or more nucleic acid vectors comprising SEQ ID NOs:7-ll.
  • One or more nucleic acid vectors comprising SEQ ID NOs:8-ll.
  • One or more nucleic acid vectors comprising SEQ ID NOs:9-ll.
  • One or more nucleic acid vectors comprising SEQ ID Nos: 10 and 11.
  • One or more nucleic acid vectors comprising SEQ ID Nos:7, 8, 10 and 11.
  • One or more nucleic acid vectors comprising SEQ ID Nos:8, 9, 10 and 11.
  • One or more nucleic acid vectors comprising SEQ ID Nos:8, 10 and 11.
  • a nucleic acid vector comprising SEQ ID NO: 7.
  • a nucleic acid vector comprising SEQ ID NO: 8.
  • a nucleic acid vector comprising SEQ ID NO:9.
  • a nucleic acid vector comprising SEQ ID NO: 10.
  • a nucleic acid vector comprising SEQ ID NO: 11.
  • a nucleic acid vector comprising SEQ ID Nos:7-ll.
  • a nucleic acid vector comprising SEQ ID Nos:7, 8, 10 and 11.
  • a nucleic acid vector comprising SEQ ID Nos:8, 9, 10 and 11.
  • a nucleic acid vector comprising SEQ ID Nos:8, 10 and 11.
  • a vector comprising SEQ ID NO: 12.
  • a nucleic acid comprising SEQ ID NO: 12.
  • the vector(s) or nucleic acid(s) is comprised by a cell that is not an E. coh'ov Klebsiella (e.g. not in a K. pneumoniae) cell.
  • the vector(s) or nucleic acid(s) is comprised by a cell that is not a Pseudomonas ze ⁇ .
  • the vector(s) or nucleic acid(s) is comprised by a cell that is not an E. coii or Klebsiella cell that comprises endogenous nucleotide sequence comprising SEQ ID NOs:7-ll.
  • the vector(s) or nucleic acid(s) is comprised by a cell (e.g.
  • the vector(s) or nucleic acid(s) is comprised by a eukaryotic cell. In an embodiment, the vector(s) or nucleic acid(s) is comprised by a human cell. In an embodiment, the vector(s) or nucleic acid(s) is comprised by an animal cell. In an embodiment, the vector(s) or nucleic acid(s) is comprised by a plant cell. In an embodiment, the vector(s) or nucleic acid(s) is comprised by a fungal cell.
  • nucleic acid vectors or one or more nucleic acids comprising at least one nucleotide sequence selected from SEQ ID NOs:7-ll, wherein a said nucleotide sequence is a) operably connected to a heterologous promoter, synthetic promoter, eukaryotic promoter or non-bacterial promoter; and/or b) comprised by a cell that is a eukaryotic cell, a non-bacterial cell, or a cell that is not a bacterial cell (e.g. an E. coll, Pseudomonas or Klebsiella cell) that comprises endogenous nucleotide sequence comprising SEQ ID NOs:7-ll.
  • a bacterial cell e.g. an E. coll, Pseudomonas or Klebsiella cell
  • nucleic acid vectors or one or more nucleic acids comprising at least one nucleotide sequence selected from SEQ ID NOs:7-ll, wherein a said nucleotide sequence is comprised by a cell (e.g. a bacterial cell) that does not comprise endogenous nucleotide sequence comprising SEQ ID NOs:7-ll.
  • a cell e.g. a bacterial cell
  • any vector or nucleic acid or nucleotide sequence herein is codon optimized for use in a human, animal (e.g. mammalian, rodent, mouse or rat), plant or fungus cell.
  • any vector or nucleic acid or nucleotide sequence herein is codon optimized for use in a eukaryotic cell.
  • Vectors and nucleic acid sequences for use in eukaryotic cells may comprise nuclear localisation sequences (NLSs). The NLSs facilitate entry of the proteins and polypeptides described herein into eukaryotic cells.
  • NLSs nuclear localisation sequences
  • any vector or nucleic acid or nucleotide sequence herein is codon optimized for use in a prokaryotic cell, e.g. a bacterial or archaeal cell.
  • the bacterial cell is not an E. coliceW.
  • the bacterial cell is not a Klebsiella e. . K. pneumoniae) cell.
  • the bacterial cell is not a Pseudomonas cell.
  • the cell comprises a DNA or polynucleotide that is modified or targeted by the methods described herein.
  • the cell herein is a human, animal (e.g. mammalian, rodent, mouse or rat), plant or fungus cell.
  • the cell herein is a Homo sapiens, Drosophila meianogaster, Mus muscuius, Ratus norvegicus, Caenorhabditis eiegans, or Arabidopsis thaiiana cell.
  • the cell herein is a prokaryotic cell, e.g. a bacterial or archaeal cell.
  • the bacterial cell is not an E. co// cell.
  • the bacterial cell is not a Klebsiella cell.
  • the bacterial cell is not a Pseudomonas oe ⁇ .
  • an animal or mammal herein is a vertebrate.
  • the crRNA disclosed herein is 15-100 nucleotides long and comprises a sequence (e.g. a spacer sequence) of at least 10 contiguous nucleotides that is complementary to the target sequence.
  • the crRNA is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long.
  • the crRNA comprises a sequence (e.g. a spacer sequence) of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides that is complementary to the target sequence.
  • the crRNA is as described elsewhere herein.
  • the crRNA may comprise two repeat sequences.
  • the crRNA may comprise at least one repeat sequence having the nucleotide sequence of SEQ ID No:6.
  • the crRNA may comprise two repeat sequences (e.g. having the nucleotide sequence of SEQ ID No:6) and one spacer sequence which hybridizes to a first protospacer sequence in a target sequence.
  • the crRNA may comprise at least one repeat sequence having the nucleotide sequence of SEQ ID No: 70.
  • the crRNA may comprise two repeat sequences (e.g. having the nucleotide sequence of SEQ ID No:70) and one spacer sequence which hybridizes to a first protospacer sequence in a target sequence.
  • the spacer sequence may be from about 25 to 39 nucleotides in length.
  • the spacer sequence may be from about 29 to 35 (e.g. from about 30 to 34, or from about 31 to 33) nucleotides in length.
  • the spacer sequence may be about 32 nucleotides in length.
  • the spacer sequence may be 32 nucleotides in length.
  • the spacer may be (about) 70% (such as (about) 80%, or (about) 90%, or (about) 95%) complementary to the protospacer sequence in the target sequence.
  • the spacer may be 100% complementary to the protospacer sequence in the target sequence.
  • the spacer and/or protospacer may be as described in Concept 10 herein.
  • the target sequence may be an RNA.
  • the target sequence may be a single stranded DNA.
  • the target sequence may be a sequence of a dsDNA.
  • the target sequence may be a sequence in the genome of a mammal, e.g. a human.
  • the target sequence comprises a sequence associated with a disease or disorder in a human or animal subject.
  • the target sequence comprises a point mutation associated with a disease or disorder and the complex edits a point mutation in the target sequence.
  • the target sequence is comprised by a gene whose expression is errant (e.g. is present, or over-expressed) in a disease or disorder, and the complex introduces a point mutation (or introduces one or more mutations in a region of the target sequence) which disrupts or prevents expression of the gene.
  • the (one or more) point mutation(s) may be located between about 10 to about 20 nucleotides upstream (5Q of a 5'-AAG-3' PAM in the target sequence.
  • the (one or more) point mutation(s) may be located between about 10 to about 400 nucleotides, or between about 10 and 350 nucleotides, or between about 10 and 300 nucleotides, or between about 10 and 250 nucleotides, or between about 10 and 200 nucleotides, or between about 10 and 150 nucleotides upstream (5Q or downstream (3Q (e.g. downstream) of a PAM in the target sequence.
  • the (one or more) point mutation(s) may be located between about 30 and 120 nucleotides (for example between about 50 and 100 nucleotides) upstream (5Q or downstream (3Q (e.g. downstream) of a PAM in the target sequence.
  • the PAM can be AAN, ANG, NAG; or the PAM can be AAG with no or one nucleotide change.
  • the PAM can be AHN, KAG, AGG, GAC or GTG.
  • the PAM is selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG- 3', 5'-AAT-3', 5'-ACA-3', 5'-ACT-3', 5'-ATC-3', 5'-ATA-3', 5'-GAG-3', 5'-TAG-3', 5'-ACC-3', 5'-AGG-3', 5'-ATT-3', 5'-GAC-3' and 5'-GTG-3'.
  • the PAM is selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG-3', 5'-AAT-3', 5'-ACA-3', 5'-ACT-3', 5'-ATC-3', 5'-ATA-3', 5'-GAG-3'and 5'-TAG-3'.
  • the PAM is selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'- ACG-3', 5'-AAT-3' and 5'-ACA-3', e.g. 5'-AAC-3' and 5'-ATG-3'.
  • the PAM is 5'-AAC-3'.
  • the mutation is a C to T point mutation (or the one or more mutations are C-to-T point mutations), wherein the complex deaminates the target C point mutation, and wherein the deamination results in a sequence that is not associated with a disease or disorder.
  • the target C point mutation (or the one or more mutations are target C point mutations) is (are) present in the DNA strand that is not complementary to the crRNA.
  • the (one or more) C to T point mutation(s) is introduced by a cytidine deaminase (e.g. a PmCDAl cytidine deaminase as described elsewhere herein), which is fused (e.g.
  • the fusion protein may comprise all of Cas-Sl, Cas-S2, Cas-S3, Cas-S4 and Cas-S5.
  • the fusion protein comprising the cytidine deaminase fused to a Cas-Sl protein may be devoid of a Cas-S3 protein.
  • the fusion protein may further comprises a UGI protein.
  • A is a CasS protein selected from a Cas-Sl, Cas-S3 or Cas-S4 (in particular, Cas-Sl);
  • linker B is optionally present, and when present comprises a linker (for example any of the linkers as described herein, in particular an XTEN linker, for example having the amino acid sequence of SEQ ID No: 17);
  • a linker for example any of the linkers as described herein, in particular an XTEN linker, for example having the amino acid sequence of SEQ ID No: 17;
  • C is a base editor, such as a cytidine deaminase (e.g. a PmCDAl cytidine deaminase as described elsewhere herein, for example having the amino acid sequence of SEQ ID No: 19);
  • a cytidine deaminase e.g. a PmCDAl cytidine deaminase as described elsewhere herein, for example having the amino acid sequence of SEQ ID No: 19
  • D is optionally present and when present comprises a linker (for example any of the linkers as described herein, in particular the 10 amino acid linker of SEQ ID No:23);
  • E is a uracil DNA glycosylase inhibitor (UGI) protein (as described in more detail elsewhere herein, for example having the amino acid sequence of SEQ ID No: 21); and the further proteins in complex with the fusion protein comprise a Cas-S2 and a Cas-S5 protein and a Cas-Sl and/or a Cas-S4 protein as required such that the fusion protein complex comprises at least a Cas-Sl, Cas-S2, Cas-S4 and a Cas-S5 protein.
  • UMI uracil DNA glycosylase inhibitor
  • the fusion protein complex may further comprise a Cas-S3 protein.
  • a first vector which expresses the fusion protein of the formula A-B-C-D-E and a second vector which expresses the further proteins of the fusion protein complex.
  • the fusion protein complex may further comprise a crRNA as described elsewhere herein.
  • the mutation is a A to G point mutation, wherein the complex deaminates the target A point mutation, and wherein the deamination results in a sequence that is not associated with a disease or disorder.
  • the target A point mutation is present in the DNA strand that is not complementary to the crRNA.
  • isolated composition, vector(s), nucleic acid(s) or protein(s) as described herein. Isolation may, for example, mean that the composition, vector(s), nucleic acid or protein(s) are provided to the exclusion of an E. co// cell.
  • the target sequence is comprised by a gene whose expression is errant (e.g. is present, or over-expressed) in a disease or disorder, and the complex introduces a double-stranded (or single stranded) break in the target sequence thereby disrupting or preventing expression of the gene.
  • the target sequence is comprised by a pathogenic bacterium which causes a disease or disorder, and the complex introduces a double-stranded break (or single stranded break) in the chromosome of the bacterium thereby killing (e.g. selectively killing) the bacterium.
  • the target sequence is comprised by an antibiotic resistance gene comprised by a pathogenic bacterium which causes a disease or disorder, and the complex introduces a doublestranded break (or a single stranded break) in the target sequence thereby disrupting or preventing expression of the antibiotic gene and results in re-sensitisation of the bacterium to the antibiotic.
  • the double-stranded break (or single stranded break) may be located upstream (5Q of a PAM sequence (for example between about 29 to about 40 nucleotides (e.g. between 30 and 32 nucleotides) upstream (5Q of a PAM sequence) in the target sequence.
  • the PAM sequence may be any as described herein, for example selected from AHN, KAG, AGG, GAC and GTG (also see Concept 39 herein).
  • Double stranded breaks may be introduced by fusion proteins as described elsewhere herein in which Py is a nuclease (e.g. any nuclease described herein).
  • Single stranded breaks in a chromosome (or in single stranded DNA) may be introduced by fusion proteins as described elsewhere herein in which Py is a nickase (e.g. any nickase described herein).
  • the double stranded break is introduced by a nuclease.
  • a single stranded break is introduced by a nickase.
  • the double stranded break is introduced by an I-Tev nuclease (e.g. an I-TevI nuclease as described elsewhere herein), which is fused (e.g. to the N-terminus) of a Cas-Sl or Cas-S4 protein, in particular Cas-Sl.
  • the fusion protein complex may comprise all of Cas-Sl, Cas-S2, Cas-S4 and Cas-S5, and optionally may further comprise a Cas-S3.
  • fusion protein complex comprising a fusion protein in a complex with further proteins, and wherein the fusion protein comprises a protein of the formula A-B-C (in 5' to 3' orientation), wherein:
  • A is an I-Tev nuclease (for example any of the I-TevI proteins described herein, for example having the amino acid sequence of SEQ ID No: 5);
  • linker B is optionally present, and when present comprises a linker (for example any of the linkers as described herein, in particular an XTEN linker, for example having the amino acid sequence of SEQ ID No: 17);
  • a linker for example any of the linkers as described herein, in particular an XTEN linker, for example having the amino acid sequence of SEQ ID No: 17;
  • C is a CasS protein selected from Cas-Sl and Cas-S4; and the further proteins in complex with the fusion protein comprise a Cas-S2 and Cas-S5 protein and a Cas-Sl or Cas-S4 protein as required such that the fusion protein complex comprises at least a Cas-Sl, Cas-S2, Cas-S4 and a Cas-S5 protein.
  • the fusion protein complex may further comprise a Cas-S3 protein.
  • a first vector which expresses the fusion protein of the formula A-B-C and a second vector which expresses the further proteins of the fusion protein complex.
  • the fusion protein complex may further comprise a crRNA as described elsewhere herein.
  • the fusion protein complex comprising an I-Tev nuclease recognizes and cleaves a target sequence comprising, in 5' to 3' orientation,: a) an I-TevI cleavage site nucleotide sequence (as described elsewhere herein, but in particular wherein the cleavage site nucleotide sequence is 5'-CNNNG-3', such as wherein the I-TevI cleavage site nucleotide sequence has the nucleotide sequence of SEQ ID No:42); b) an I-TevI spacer nucleotide sequence (as described elsewhere herein, but in particular wherein the I-TevI spacer nucleotide sequence is about 31 nucleotides in length, for example wherein the I-TevI spacer nucleotide sequence is at least (about) 80% (e.g.
  • a PAM sequence (as described elsewhere herein, but in particular wherein the PAM is selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG-3', 5'-AAT-3' and 5'-ACA-3', e.g.
  • the fusion protein complex comprising an I-Tev nuclease introduces a double stranded break within the cleavage site nucleotide sequence.
  • the cleavage site nucleotide sequence comprises the sequence of SEQ ID No:42
  • the fusion protein introduces a break after the second C on the forward strand of the target sequence and after the first T on the reverse strand of the target sequence (as the cleavage site nucleotide sequence on the reverse strand comprises the sequence 5'-CGTTG-3')-
  • the I-TevI nuclease nicks the forward strand immediately after the second C, and immediately after the first T on the reverse strand.
  • the double nicking of the cleavage site results in a staggered double stranded DNA break and each site of the break harbours single stranded, dinucleotide overhangs; 5'-AC-3' for the forward strand and 5'-GT-3' for the reverse strand.
  • the vector(s), complexes, nucleic acid(s) or protein(s) are not comprised by a cell, wherein the cell comprises endogenous nucleotide sequences encoding Cas-Sl, S2, S3, S4 and S5 proteins.
  • the vector(s), complexes, nucleic acid(s) or protein(s) are not comprised by a cell of a species selected from the species in Table 3.
  • the vector(s), complexes, nucleic acid(s) or protein(s) are not comprised by an E coli cell.
  • the vector(s), complexes, nucleic acid(s) or protein(s) are not comprised by a Ktedonobacter, e.g.
  • the vector(s), complex, nucleic acid or protein(s) are not comprised by an AHochromatium, e.g. AHochromatium warmingii cell.
  • the vector(s), complexes, nucleic acid(s) or protein(s) are not comprised by an Ignatius, e.g. Ignatius tetrasporus cell.
  • the vector(s), complexes, nucleic acid(s) or protein(s) are not comprised by a cell, wherein the cell comprises endogenous nucleotide sequences encoding a polypeptide comprising SEQ ID NO: 1, a polypeptide comprising SEQ ID NO: 2, a polypeptide comprising SEQ ID NO: 3, a polypeptide comprising SEQ ID NO: 4 and a polypeptide comprising SEQ ID NO: 5.
  • a nucleic acid vector or a plurality of nucleic acid vectors comprising expressible nucleotide sequences, wherein the sequences comprise a) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least (about) 80% (such as about 90%) identical to SEQ ID NO: 1; b) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least (about) 80% (such as about 90%) identical to SEQ ID NO: 2; c) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least (about) 80% (such as about 90%) identical to SEQ ID NO: 3; d) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least (about) 80% (such as about 90%) identical to SEQ ID NO: 4; and e) a nucleotide sequence that encodes a polypeptide which comprises
  • a nucleic acid vector or a plurality of nucleic acid vectors comprising expressible nucleotide sequences, wherein the sequences comprise a) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 94% (such as 95%) identical to SEQ ID NO: 1; b) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 98% (such as 99%) identical to SEQ ID NO: 2; c) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 99% (such as 100%) identical to SEQ ID NO: 3; d) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 98% (such as 99%) identical to SEQ ID NO: 4; and e) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 88% (
  • a nucleic acid vector or a plurality of nucleic acid vectors comprising expressible nucleotide sequences, wherein the sequences comprise a) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 98% (such as 99%) identical to SEQ ID NO: 2; b) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 98% (such as 99%) identical to SEQ ID NO: 4; and c) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 88% (such as 89%) identical to SEQ ID NO: 5.
  • Concept 1C-2 A nucleic acid vector or a plurality of nucleic acid vectors according to Concept 1C or 1C-1, wherein the vector(s) further comprise(s) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least 99% (such as 100%) to SEQ ID NO:3.
  • Concept ID-1 A nucleic acid vector or a plurality of nucleic acid vectors according to Concept ID, wherein the vector(s) further comprise(s) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least about 80% (such as about 90%) identical to SEQ ID NO:1.
  • Concept ID-2 A nucleic acid vector or a plurality of nucleic acid vectors according to Concept ID or ID-1, wherein the vector(s) further comprise(s) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least about 80% (such as about 90%) to SEQ ID NO:3.
  • nucleic acids comprising the recited components.
  • the disclosures relating to vector(s) herein are also to be read as applying mutatis mutandis to nucleic acid(s).
  • the nucleic acid may be comprised by a chromosome of a cell, e.g. a prokaryotic cell.
  • the nucleic acid may be comprised by a plasmid.
  • the modifying may be modifying of said chromosome or plasmid, in particular the chromosome.
  • the crRNA may comprise a spacer that is capable of hybridizing to a protospacer comprised by the chromosome or plasmid, in particular the chromosome.
  • the crRNA may comprise a spacer that is cognate to a protospacer comprised by the chromosome or plasmid, in particular the chromosome.
  • Concept 2 The vector(s) of Concept 1, wherein at least one of the nucleotide sequences is operably connected to a promoter that is a) heterologous to the at least one nucleotide sequence(s); b) not an E. coli or Klebsiella promoter; c) a eukaryotic cell promoter; d) an animal promoter (optionally a mammalian or human promoter); e) a plant promoter; f) a fungus promoter (optionally a yeast promoter); g) an insect promoter; h) a virus promoter (optionally a virus, AAV or lentivirus promoter); or i) a synthetic promoter.
  • a promoter that is a) heterologous to the at least one nucleotide sequence(s); b) not an E. coli or Klebsiella promoter; c) a eukaryotic cell promoter; d) an animal promoter (optionally a mamma
  • the promoter may be a constitutive promoter.
  • the promoter may be an inducible promoter. Suitable promoters are well known to those in the art. Inducible promoters may be particularly useful when it is desired to express the Type-S protein(s) and complexes only under certain defined parameters. In other circumstances where it is desried to constantly produce the Type-S protein(s) and complexes described herein, constitutive promoters may be employed.
  • the promoter may be a BolA promoter, e.g. a pBolA promoter comprising the nucleotide sequence of SEQ ID No: 14.
  • the promoter may be a p70a promoter, e.g. a p70a promoter comprising the nucleotide sequence of SEQ ID No: 15.
  • the promoter may be an arabinose-inducible promoter, e.g.
  • a pBAD promoter comprising the nucleotide sequence of SEQ ID No: 24.
  • Concept 3 The vector(s) of Concept 1 or Concept 2, wherein at least one of the nucleotide sequences is operably connected to a constitutive promoter.
  • all of the nucleotides sequences are operably connected to a constitutive promoter. In an embodiment, all of the nucleotides are comprised by a single operon under the control of a single constitutive promoter.
  • all of the nucleotides sequences are operably connected to a inducible promoter. In an embodiment, all of the nucleotides are comprised by a single operon under the control of a single inducible promoter.
  • a nucleic acid vector comprising an expressible nucleotide sequence which comprises a) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least (about) 90% identical to SEQ ID NO:2; b) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least (about) 90% identical to SEQ ID NO:4; and c) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least (about) 90% identical to SEQ ID NO: 5.
  • a nucleic acid vector comprising an expressible nucleotide sequence selected from a) a nucleotide sequence that encodes a polypeptide, which comprises an amino acid sequence that is at least (about) 80% (such as about 90%) identical to SEQ ID NO: 1; b) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least (about) 80% (such as about 90%) identical to SEQ ID NO: 2; c) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least (about) 80% (such as about 90%) identical to SEQ ID NO: 3; d) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least (about) 80% (such as about 90%) identical to SEQ ID NO: 4; and e) a nucleotide sequence that encodes a polypeptide which comprises an amino acid sequence that is at least (about) 80% (such as
  • Concept 8B The vector of Concept 7A or 7B, wherein the vector further comprises a nucleotide sequence that encodes a Cas protein domain which is operable with the polypeptides of parts a), b) and c) to bind to a nucleotide sequence selected from double stranded DNA, single stranded DNA and RNA.
  • Such Cas proteins may be selected from Cas proteins of naturally occurring Type I, II, III, IV, V or VI CRISPR/Cas complexes, which are well known to those skilled in the art. Methods provided herein can be used to identify proteins which are operable with the Cas-S2, Cas-S4 and Cas-S5 proteins to provide additional activities.
  • the vector(s) comprise(s) one or more nucleotide sequences for producing a crRNA, wherein the crRNA comprises a spacer that is cognate to a first protospacer in a target sequence, optionally wherein the protospacer is a) not found in E coir, b) an eukaryotic cell protospacer; or c) an animal (optionally human), plant, insect, or fungus cell protospacer.
  • the protospacer may not be found in a bacterium which comprises endogenous nucleotide sequences that encode the polypeptides of a) to e) as recited in Concept 1.
  • the crRNA optionally comprises a spacer having at least 10 consecutive nucleotide sequences which are complementary to the protospacer.
  • the crRNA optionally comprises a spacer having at least 15 consecutive nucleotide sequences which are complementary to the protospacer.
  • the crRNA optionally comprises a spacer having at least 20 consecutive nucleotide sequences which are complementary to the protospacer.
  • the crRNA optionally comprises a spacer having at least 25 consecutive nucleotide sequences which are complementary to the protospacer.
  • the crRNA optionally comprises a spacer having at least 28 consecutive nucleotide sequences which are complementary to the protospacer.
  • the spacer and protospacer may be from about 10 to 40 nucleotides in length.
  • the spacer and protospacer may be between about 25 and 40 nucleotides, such as between about 25 and 38 nucleotides, such as between about 28 and 35 nucleotides.
  • the spacer and protospacer may be from about 25 to 39 nucleotides in length.
  • the spacer and protospacer may be from about 29 to 35 (e.g. from about 30 to 34, or from about 31 to 33) nucleotides in length.
  • the spacer and protospacer may be about 32 nucleotides in length.
  • the spacer and protospacer may be about 28 nucleotides in length.
  • the spacer and protospacer may be 32 nucleotides in length.
  • the spacer and protospacer may be 28 nucleotides in length.
  • the spacer and/or protospacer may have any of the features described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein.
  • the spacer may be (about) 90% complementary to the protospacer.
  • the spacer may be (about) 70% complementary to the protospacer.
  • the spacer may be (about) 80% complementary to the protospacer.
  • the spacer may be (about) 95% complementary to the protospacer.
  • the spacer may 100% complementary to the protospacer across its entire length.
  • nucleotides 1 to 28 of the spacer which are 5' of the PAM sequence are complementary to the protospacer sequence.
  • the crRNA comprises at least one repeat sequence.
  • the crRNA comprises two repeat sequences.
  • the repeat sequence may be any sequence which is capable of forming a hairpin loop and being recognised by the Type-S system.
  • the repeat sequence may be about 20 to 25 nucleotides (e.g. 22 or 23 nucleotides) in length.
  • the repeat sequence may be a nucleotide sequence which is at least about 80%, e.g. about 90% (such as at least about 95, 96, 97 or 98%) identical to the nucleotide sequence of SEQ ID No:6.
  • the repeat sequence may be a nucleotide sequence of SEQ ID No:6.
  • the repeat sequence may be a nucleotide sequence which is at least about 80%, e.g. about 90% (such as at least about 95, 96, 97 or 98%) identical to the nucleotide sequence of SEQ ID No:70.
  • the repeat sequence may be a nucleotide sequence of SEQ ID No:70.
  • the crRNA may be encoded by a CRISPR array (for example a CRISPR array as described elsewhere herein).
  • vector(s) of any preceding Concept wherein the vector(s) comprise(s) a) a nuclear localization sequence (NLS); b) a phage packaging sequence (optionally a acor rossite); c) a plasmid origin of replication; d) a plasmid origin of transfer; e) a bacterial plasmid backbone; f) a eukaryotic plasmid backbone; g) a human virus (optionally AAV or lentivirus) structural protein gene; h) a virus (optionally AAV or lentivirus) rep and/or cap sequence; i) a selection marker or sequence encoding a selectable marker; j) a eukaryotic promoter; or k) a nucleotide sequence of a human, animal, plant or fungus gene.
  • NLS nuclear localization sequence
  • b) a phage packaging sequence optionally a acor rossite
  • the NLS sequence may be from an SV40 Large T-antigen (see Kalderon et al., Cell, 39(3), 499-509, 1984, doi: https://doi.org/10.1016/0092-8674(84)90457-4, which is incorporated herein by reference in its entirety), from nucleoplamin, importin a, c-myc, EGL-13, TUS-protein, hnRNP Al or yeast transcription repressor Mata2.
  • Any of the vector(s) as described herein may be delivered to bacterial cells via a phage particle. Any of the vector(s) as described herein may be delivered to bacterial cells i//aa phagemid particle packaged within a phage particle. Any of the vector(s) as described herein may be delivered to bacterial cells waa conjugative plasmid.
  • each vector is a) a plasmid vector (optionally a conjugative plasmid) b) a transposon vector (optionally a conjugative transposon); c) a virus vector (optionally a phage, AAV or lentivirus vector); d) a phagemid (optionally a packaged phagemid); or e) a nanoparticle (optionally a lipid nanoparticle).
  • a virus vector optionally a phage, AAV or lentivirus vector
  • a phagemid optionally a packaged phagemid
  • a nanoparticle optionally a lipid nanoparticle.
  • a fusion protein comprising a polypeptide (Px), wherein Px a) comprises an amino acid sequence that is at least (about) 80% identical (e.g. (about) 90% identical) to a sequence selected from SEQ ID Nos: 1-5; and b) is fused to a heterologous polypeptide (Py).
  • a fusion protein comprising a Cas-Sl fused to a heterologous polypeptide (Py).
  • a fusion protein comprising a Cas-S2 fused to a heterologous polypeptide (Py).
  • a fusion protein comprising a Cas-S3 fused to a heterologous polypeptide (Py).
  • a fusion protein comprising a Cas-S4 fused to a heterologous polypeptide (Py).
  • a fusion protein comprising a Cas-S5 fused to a heterologous polypeptide (Py).
  • a fusion protein complex comprising:
  • Px fusion protein polypeptide
  • Px a comprises an amino acid sequence that is at least (about) 80% identical (e.g. (about) 90% identical) to a sequence selected from SEQ ID Nos: 1, 3, and 4; and b) is fused to a heterologous polypeptide (Py);
  • polypeptide which comprises an amino acid sequence that is at least (about) 80% identical (e.g. (about) 90% identical) to SEQ ID No:2;
  • polypeptide which comprises an amino acid sequence that is at least (about) 80% identical (e.g. (about) 90% identical) to SEQ ID No: 5;
  • a crRNA comprising a spacer sequence that is cognate to a first protospacer in a target sequence
  • polypeptide which is at least (about) 80% identical (e.g. (about) 90% identical) to a sequence selected from SEQ ID Nos: 1, 3, and 4 and is not based on the amino acid sequence of the polypeptide recited in part (i)a); and
  • polypeptide which is at least (about) 80% identical (e.g. (about) 90% identical) to a sequence selected from SEQ ID Nos: 1, 3, and 4 and is not based on the amino acid sequence of the polypeptide recited in part (i)a), and, if present, is not based on the amino acid sequence of the polypeptide recited in part (v).
  • a fusion protein complex comprising: (i) a fusion protein polypeptide (Px), wherein Px a) comprises a Cas-Sl, Cas-S3 or Cas-S4 protein; and b) is fused to a heterologous polypeptide (Py);
  • a crRNA comprising a spacer sequence that is cognate to a first protospacer in a target sequence
  • the fusion protein comprises 2 or more of Cas-Sl to S5, e.g. comprises Cas-Sl and S2, or S4 and S5.
  • Py may be fused (directly or indirectly) to the N-terminus of the CasS protein.
  • the Py may be fused (directly or indirectly) to the C-terminus of the CasS protein.
  • a peptide linker may be from about 10 to 20 amino acids (e.g. about 16 amino acids) in length.
  • a peptide linker may be from about 8 to 12 amino acids (e.g. about 10 amino acids) in length.
  • a peptide linker may have the amino acid sequence of SEQ ID No: 17.
  • a peptide linker may have the amino acid sequence of SEQ ID No:23. Linkers may be as described elsewhere herein (e.g. in Concepts 27 or 29 herein).
  • heterologous refers to a polypeptide that is not naturally found fused to Px.
  • Py comprises an I-Tev nuclease or mutH protein.
  • Py is an I-Tev nuclease, such as an I-TevI nuclease, for example as described elsewhere herein.
  • the I-TevI nuclease is a protein comprising the amino acid sequence of SEQ ID No:45.
  • the I-TevI nuclease is encoded by a nucleic acid sequence which encodes an amino acid sequence comprising the amino acids of SEQ ID: No:45.
  • the I- TevI nuclease is encoded by a nucleic acid sequence of SEQ ID: No:46.
  • the I-TevI nuclease is a protein which has an amino acid sequence which is at least about 80% identical (e.g. is about 85%, 90%, 95%, 96%, 97% or 98% identical) to the amino acid of SEQ ID No:45 and is capable of cleaving an I-TevI cleavage site nucleotide sequence having the nucleotide sequence of SEQ ID No:42 and is capable of recognising an I-TevI spacer nucleotide sequence having the nucleotide sequence of SEQ ID No:43.
  • the selected sequence is SEQ ID NO: 1.
  • the selected sequence is SEQ ID NO: 2.
  • the selected sequence is SEQ ID NO: 3.
  • the selected sequence is SEQ ID NO: 4.
  • the selected sequence is SEQ ID NO: 5.
  • the effector domain is a domain that comprises nuclease activity, nickase activity, recombinase activity, reverse transcriptase, helicase, deaminase activity, methyltransferase activity, methylase activity, acetylase activity, acetyltransferase activity, transcriptional activation activity, or transcriptional repression activity.
  • Py may be naturally occurring. Py may be a synthetic sequence. Py may be a synthetically mutated derivative of a naturally occurring sequence.
  • the effector domain is a nucleic acid editing domain.
  • the nucleic acid editing domain comprises a deaminase domain.
  • the deaminase domain may be a cytidine deaminase domain.
  • the cytidine deaminase domain may be an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase.
  • APOBEC apolipoprotein B mRNA-editing complex
  • the cytidine deaminase domain may be at least (about) 80%, at least (about) 85%, at least (about) 90%, at least (about) 92%, at least (about) 95%, at least (about) 96%, at least (about) 97%, at least (about) 98%, at least (about) 99%, or at least (about) 99.5% identical to the cytidine deaminase domain of any one of SEQ ID NOs:350-389 disclosed in US application number 16/976,047 or WO2019/168953, which sequences are explicitly incorporated herein by reference.
  • Py comprises a uracil glycosylase inhibitor (UGI) domain.
  • the UGI domain may comprise the amino acid sequence of SEQ ID NO:500 disclosed in US application number 16/976,047 or WO2019/168953, which sequence is explicitly incorporated herein by reference.
  • the UGI domain may have the amino acid sequence of SEQ ID No:21.
  • the UGI domain may have the nucleotide sequence of SEQ ID No:20.
  • Py comprises the amino acid sequence of SEQ ID NO: 534.
  • Py may comprise the amino acid sequence of SEQ ID NO: 538.
  • Py comprises the fusion protein further comprises a second UGI domain.
  • Py comprises the amino acid sequence of SEQ ID NO: 540.
  • Py comprises the amino acid sequence of SEQ ID NO: 541.
  • the deaminase domain is an adenosine deaminase domain.
  • the fusion protein may comprise a second adenosine deaminase domain.
  • the first adenosine deaminase domain and the second adenosine deaminase domain comprises an ecTadA domain, or variant thereof.
  • the first adenosine deaminase domain and the second adenosine deaminase domain may comprise the amino acid sequence of any one of SEQ ID NOs:400-458.
  • the first adeosine deaminase may comprise the amino acid sequence of SEQ ID NO:400.
  • the second adeosine deaminase may comprise the amino acid sequence of SEQ ID NO:458.
  • Py comprises the amino acid sequence of SEQ ID NO: 535 or 539.
  • Base editors that may be used with the Cas-S system are well known in the art.
  • Base editors include systems where a CasS protein is fused to a cytosine or adenosine deaminase domain and directed to the target sequence to make the desired modification.
  • the base editor is selected from:
  • CBEs Cytosine Base Editors
  • ABEs Adenine Base Editors that convert A:T into G:C (Gaudelli et al., Nature, 551 (7681), 464-471, 2017, incorporated herein by reference in its entirety)
  • CABEs Cytosine Adenine Base Editors
  • Adenine Cytosine Base Editors that convert A:T into C:G (W02020/181180, incorporated herein by reference in its entirety)
  • Adenine Thymine Base Editors that convert A:T into T:A (W02020/181202, incorporated herein by reference in its entirety)
  • Thymine Adenine Base Editor that convert T:A into A:T (W02020/181193 (or US2022/0170013); W02020/181178; W02020/181195, each of which is incorporated herein by reference in its entirety).
  • Base editors differ in the base modification enzymes.
  • CBEs rely on ssDNA cytidine deaminase among which: APOBEC1, rAPOBECl, APOBEC1 mutant or evolved version (evoAPOBECl), and APOBEC homologs (APOBEC3A (eA3A), Anc689), Cytidine deaminase 1 (CDA 1), evoCDA 1, FERNY, evoFERNY.
  • ABEs rely on deoxyadenosine deaminase activity of a tandem fusion TadA-TadA* where TadA* is an evolved version of TadA, an E. co/itRNA adenosine deaminase enzyme, able to convert adenosine into Inosine on ssDNA.
  • TadA* include TadA-8a-e and TadA-7.10.
  • DNA-based editor proteins include, but are not limited to BE1, BE2, BE3, BE4, BE4-GAM, HF-BE3, Sniper-BE3, Target-AID, Target-AID-NG, ABE, EE-BE3, YE1-BE3, YE2-BE3, YEE-BE3, BE-PLUS, SaBE3, SaBE4, SaBE4-GAM, Sa(KKH)-BE3, VQR-BE3, VRER-BE3, EQR-BE3, xBE3, Casl2a-BE, Ea3A-BE3, A3A-BE3, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, ABE8e, SpRY-ABE, SpRYCBE, SpG-CBE4, SpG-ABE, SpRY-CBE4, SpCas9-NG-ABE, SpCas9-NG,
  • Py is a cytidine deaminase.
  • the cytidine deaminase may be a PmCDAl cytidine deaminase.
  • the cytidine deaminase may be from sea lamprey.
  • the PmCDAl cytidine deaminase comprises the amino acid sequence of SEQ ID No: 19.
  • the PmCDAl cytidine deaminase is encoded by a nucleic acid sequence which encodes an amino acid sequence comprising the amino acids of SEQ ID No: 19.
  • the PmCDAl cytidine deaminase is encoded by a nucleic acid sequence of SEQ ID No: 18.
  • the PmCDAl cytidine deaminase is a protein which has an amino acid sequence which is at least about 80% identical (e.g. is about 85%, 90%, 95%, 96%, 97% or 98% identical) to the amino acid of SEQ ID No: 19 and is capable of making a C to T mutation in the target nucleic acid.
  • fusion protein of Concept 14 wherein the fusion protein is in the form of a fusion protein complex which comprises at least two further polypeptides, wherein the two further polypeptides each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No: 2, SEQ ID No:4 and SEQ ID No:5, and wherein the fusion protein complex comprises amino acid sequences that are at least 90% identical to each of the sequences of SEQ ID No:2, SEQ ID No:4 and SEQ ID No:5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:2, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No:4 and SEQ ID No: 5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:4, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No:2 and SEQ ID No: 5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: 5, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No: 2 and SEQ ID No:4.
  • Concept 16 The fusion protein complex of Concept 15, wherein the protein complex comprises at least 3 further polypeptides, and and wherein the fusion protein complex comprises amino acid sequences that are at least 90% identical to each of the sequences of SEQ ID No:2, SEQ ID No:3, SEQ ID No:4 and SEQ ID No:5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:2, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No: 3, SEQ ID No:4 and SEQ ID No: 5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: 3, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No:2, SEQ ID No:4 and SEQ ID No:5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:4, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No: 2, SEQ ID No: 3 and SEQ ID No: 5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:5, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No:2, SEQ ID No:3 and SEQ ID No:4.
  • Concept 17A The fusion protein complex of Concept 15, wherein the protein complex comprises at least 3 further polypeptides, and wherein the fusion protein complex comprises amino acid sequences that are at least 90% identical to each of the sequences of SEQ ID No:l, SEQ ID No:2, SEQ ID No:4 and SEQ ID No:5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:l, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No: 2, SEQ ID No:4 and SEQ ID No: 5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: 2
  • the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No: l, SEQ ID No:4 and SEQ ID No:5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:4, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No: l, SEQ ID No:2 and SEQ ID No:5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:5, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No:l, SEQ ID No: 2 and SEQ ID No:4.
  • the fusion protein complex of Concept 15 wherein the protein complex comprises at least 4 further polypeptides, and wherein the fusion protein complex comprises amino acid sequences that are at least 90% identical to each of the sequences of SEQ ID No:l, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4 and SEQ ID No:5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:l, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No: 2, SEQ ID No:3, SEQ ID No:4 and SEQ ID No:5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:2, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No:l, SEQ ID No:3, SEQ ID No:4 and SEQ ID No:5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: 3, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No: l, SEQ ID No:2, SEQ ID No:4 and SEQ ID No: 5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:4, and the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No: l, SEQ ID No:2, SEQ ID No:3 and SEQ ID No: 5.
  • Px can comprise an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: 5
  • the further polypeptides comprise an amino acid sequence that is at least 90% identical to each of the sequences of SEQ ID No:l, SEQ ID No:2, SEQ ID No:3 and SEQ ID No:4.
  • fusion protein of Concept 14 wherein Px comprises an amino acid sequence that is selected from an amino acid sequence that is at least 90% identical to the sequence selected from the sequences of SEQ ID Nos:l, 3, and 4; wherein the fusion protein is in the form of a fusion protein complex which comprises at least two further polypeptides, wherein the two further polypeptides each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:2 and Seq ID No:5, and
  • polypeptide which is at least 90% identical to a sequence selected from SEQ ID Nos: 1, 3, and 4 and is not based on the amino acid sequence of the polypeptide recited in part I;
  • polypeptide which is at least 90% identical to a sequence selected from SEQ ID Nos: 1, 3, and 4 and is not based on the amino acid sequence of the polypeptide recited in part I, and, if present, is not based on the amino acid sequence of the polypeptide recited in part (i).
  • a deaminase such as a cytosine or adenine deaminase
  • base editor such as a cytosine or adenine deaminase
  • a prime editor such as a cytosine or
  • Py is a nuclease, such as any of the nucleases disclosed herein, in particular an I-TevI nuclease.
  • Py may be a DNA nuclease.
  • Py may be an RNA nuclease.
  • Py may be a nickase or a dead nuclease.
  • Py is a base editor, such as any of the base editors disclosed herein, in particular an a PmCDAl cytidine deaminase.
  • Py is a prime editor.
  • the I-TevI nuclease is fused to the N-terminus of an amino acid sequence (e.g. any CasS protein described herein).
  • the I-TevI nuclease is fused to the N- terminus of an amino acid sequence that is at least (about) 80% (e.g. (about) 90%) identical to the sequence of SEQ ID No: l.
  • the I-TevI nuclease may be as described elsewhere herein (e.g. as in Concept 14).
  • I-TevI comprises 245 amino acids, and consists of an N-terminal catalytic domain and a C-terminal DNA-binding domain that are connected by a long, flexible linker.
  • the crystal structure of the DNA-binding domain of I-TevI (comprising residues 130 to 245) complexed with the 20-bp primary binding region of its DNA target, reveals the presence of a zinc finger (comprising residues 151 to 167) that makes backbone contacts with the DNA from the minor groove, an elongated segment containing a minor groove-binding a-helix (comprising residues 183 to 194) and a helix- turn-helix (comprising residues 204 to 245).
  • the N-terminal catalytic domain was shown to comprise residues 1-92.
  • Biochemical data have shown that the zinc finger does not contribute to the DNA-binding affinity or to the specificity of the enzyme, but rather that it has a novel function and acts as a distance determinant that controls the relative positions of the catalytic and DNA- binding domains, see for example, Roey et al, 2005, DOI: 10.1007/0-387-27421-9_7, which is incorporated herein by reference in its entirety.
  • the I-Tevl does not comprise a complete helix-turn-helix domain.
  • the I-Tevl may comprise the amino acid sequence of SEQ ID No:45.
  • the I-Tevl may not comprise a helix-turn-helix domain.
  • the I-Tevl may comprise amino acids 1 to 195-203 of SEQ ID No:45.
  • the I-Tevl may not comprise the minor groove-binding a-helix.
  • the I-Tevl may comprise amino acids 1 to 167 of SEQ ID No:45.
  • the I-Tevl may not comprise a complete zinc finger domain.
  • the I-Tevl may comprise amino acids 1 to 162 of SEQ ID No:45.
  • the I-Tevl may comprise amino acids 1 to 92 of SEQ ID No:45.
  • the I-Tevl may comprise the amino acid sequence of SEQ ID No:45.
  • the I-Tevl nuclease may be a T4 bacteriophage I-Tevl.
  • the I-Tevl nuclease may be from a T4 bacteriophage.
  • the I-Tevl nuclease may be encoded by the intron of bacteriophage T4.
  • the I- TevI nuclease may comprise amino acids 1 to 206 of a naturally occurring I-Tevl nuclease.
  • the I- TevI nuclease may comprise an amino acid sequence of SEQ ID No:45.
  • the I-Tevl nuclease may be encoded by a nucleotide sequence of SEQ ID No:44.
  • ABE adenine base editor
  • CBE cytosine base editor
  • a cytidine deaminase or an apolipoprotein B mRNA-editing complex (APOBEC) family deaminase a cytosine guanine base editor (CGBE), a cytosine adenine base editor (CABE), an adenine cytosine base editor (ACBE), an adenine thymine base editor (ATBE), a thymine adenine base editor (TABE), and a uracil DNA glycosylase inhibitor (UGI) protein.
  • CGBE cytosine guanine base editor
  • CABE cytosine adenine base editor
  • ACBE adenine cytosine base editor
  • ATBE adenine thymine base editor
  • TABE thymine adenine base editor
  • UBI uracil DNA glycosylase inhibitor
  • Py is a CBE.
  • Py is a PmCDAl cytidine deaminase (as described elsewhere herein, for example in Concept 14).
  • Py is a PmCDAl cytidine deaminase in combination with a UGI protein.
  • Py is a CBE linked wa a linker (as described elsewhere herein) to a UGI protein.
  • Py is a PmCDAl cytidine deaminase linked wa a linker (as described elsewhere herein) to a UGI protein.
  • Py is a CBE linked via a linker (as described elsewhere herein) to a UGI protein, and the CBE is linked to a CasS protein described herein via a linker (as described elsewhere herein).
  • Py is a PmCDAl cytidine deaminase linked via a linker (as described elsewhere herein) to a UGI protein, and the PmCDAl is linked to a CasS protein described herein via a linker (as described elsewhere herein).
  • Py is a CBE linked via a linker (as described elsewhere herein) to a UGI protein, and the UGI protein is linked to a CasS protein described herein via a linker (as described elsewhere herein).
  • Py is a PmCDAl cytidine deaminase linked i//aa linker (as described elsewhere herein) to a UGI protein, and the UGI protein is linked to a CasS protein described herein via a linker (as described elsewhere herein).
  • the CasS protein is selected from a CasSl protein, a CasS3 protein and a CasS4 protein.
  • the base editor is a PmCDAl cytidine deaminase having the amino acid sequence encoded by SEQ ID No: 19.
  • the base editor is from sea lamprey.
  • the base editor e.g. PmCDAl cytidine deaminase
  • the base editor is fused via a peptide linker to the amino acid sequence of Part I.
  • the peptide linker may be as described in Concept 14 or Concept 29.
  • the base editor e.g. PmCDAl cytidine deaminase
  • the linker may from (about) 10 to 20 amino acids in length.
  • the linker may be from (about) 10 to 22, from (about) 11 to 21, from (about) 12 to 20, from (about) 13 to 19, from (about) 14 to 18, from (about) 15 to 17 amino acids in length.
  • the linker may be (about) 16 amino acids in length.
  • the linker may be a linker having the amino acid sequence of SEQ ID No: 17.
  • the linker may be any linker described herein.
  • the UGI protein has an amino acid sequence of SEQ ID No:21.
  • the UGI protein may be as described elsewhere herein (e.g. in Concept 14).
  • the UGI protein may be fused directly to the base editor.
  • the UGI protein may be fused to the base editor via a linker, e.g. a peptide linker as described elsewhere herein.
  • the crRNA, spacer and/or protospacer sequence may have any of the features described elsewhere herein (e.g. in Concept 10, or in the First Aspect of the Detailed Description).
  • the protospacer is not found in a bacterium which comprises endogenous nucleotide sequences that encode the polypeptides of a) to e) as recited in Concept 1A or IB.
  • the protospacer is a eukaryotic cell protospacer.
  • the protospacer is heterologous to the source of Py.
  • the protospacer when Py is a naturally occurring polypeptide, the protospacer is from a different species (or genera) to the Py.
  • the protospacer is an animal (optionally human), plant, insect, fungus cell protospacer.
  • the fusion protein complex forms a ribonucleoprotein complex with a crRNA which comprises a spacer that is cognate to a first protospacer in a target sequence. In one embodiment, the fusion protein forms a ribonucleoprotein complex with a crRNA which comprises a spacer that is cognate to a first protospacer in a target sequence.
  • One or more nucleic acid vector(s) comprising one or more nucleotide sequence(s) encoding a fusion protein or fusion protein complex as recited in any one of Concepts 14 to 30.
  • the vector(s) may further encode a crRNA as described elsewhere herein (e.g. in the First Aspect of Detailed Description, in Concept 10 or in Concept 30).
  • a first and second vector which encode a fusion protein complex as described elsewhere herein.
  • the first vector encode the fusion protein Px and the second vector encodes the further proteins of the fusion protein complex.
  • the vector(s) may encode a second, third, fourth... etc, crRNA which comprises a spacer that is cognate to a second, third, fourth... etc, protospacer in a target sequence.
  • Expression of multiple crRNAs may be useful for multiplex editing, for example for targeting multiple genes in a cell or organism.
  • Concept 32 The vector(s) of Concept 31, wherein the fusion protein is encoded by a single first nucleotide sequence and wherein the at least two further polypeptides are encoded by a second nucleotide sequence.
  • the fusion proteins described herein are encoded by a single first nucleotide sequence comprised by a first vector, and the at least two further CasS polypeptides of the complex are encoded by a second nucleotide sequence comprised by a second vector. In one embodiment, all of the further CasS polypeptides of the fusion protein complexes described herein are encoded by a second nucleotide sequence. In one embodiment, all of the further CasS polypeptides of the complexes described herein are encoded by a second nucleotide sequence comprised by a second vector.
  • Concept 33 The vector(s) of Concept 31, wherein the fusion protein complex is encoded by: a) a first nucleotide sequence encoding Py fused to an amino acid sequence that is at least (about) 80% (e.g. (about) 90%) identical to the sequence of SEQ ID No: l, SEQ ID No:3 or Seq ID No:4; and b) a second nucleotide sequence encoding the at least two further polypeptides encoding amino acid sequences that are at least (about 80% (e.g. (about) 90%) identical to the sequences of SEQ ID Nos:l, 2, 3, 4 and/or 5.
  • the fusion protein complexes described herein comprise polypeptides comprising amino acid sequences at least (about) 80% (e.g. (about) 90%) identical to the each of the sequences of SEQ ID Nos:l, 2, 3, 4 and 5.
  • the fusion protein complexes described herein comprise polypeptides comprising amino acid sequences at least (about) 80% (e.g. (about) 90%) identical to the each of the sequences of SEQ ID Nos: 2, 4 and 5.
  • the fusion protein complexes described herein comprise polypeptides comprising amino acid sequences at least (about) 80% (e.g. (about) 90%) identical to the each of the sequences of SEQ ID Nos:2, 3, 4 and 5.
  • the fusion protein complexes described herein comprise polypeptides comprising amino acid sequences at least (about) 80% (e.g. (about) 90%) identical to the each of the sequences of SEQ ID Nos:l, 2, 4 and 5.
  • crRNAs are encoded to target multiple protospacers, they may all be encoded by the first nucleotide sequence. Where multiple crRNAs are encoded to target multiple protospacers, they may all be encoded by the second nucleotide sequence.
  • the eukaryotic cell promoter may be any of those described herein.
  • the promoter is a mammalian or human promoter.
  • the animal, mammalian or human promoter may be any of those described herein.
  • the plant promoter may be any of those described herein.
  • the promoter is a yeast promoter.
  • the fungal or yeast promoter may be any of those described herein.
  • the insect promoter may be any of those described herein.
  • the promoter is a viral, AAV or lentiviral promoter.
  • the viral, AAV or lentiviral promoter may be any of those described herein.
  • Concept 35H The vector(s) of any one of Concepts 31 to 34, wherein at least one of the nucleotide sequences is operably connected to a promoter that is a synthetic promoter.
  • the synthetic promoter may be any of those described herein.
  • the crRNA and other features of the repeat sequences may have the other features as described elsewhere herein (for example in the First Aspect of Detailed Description, or in Concept 10).
  • the protospacer may be immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence in the target sequence selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG-3', 5'-AAT-3', 5'-ACA-3', 5'-ACT-3', 5'-ATC-3', 5'-ATA-3', 5'-GAG-3' and 5'-TAG-3'.
  • PAM Protospacer Adjacent Motif
  • the protospacer may be immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence in the target sequence selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG-3', 5'-AAT-3' and 5'-ACA-3'.
  • PAM Protospacer Adjacent Motif
  • the protospacer may be immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence in the target sequence selected from 5'-AAC-3' and 5'-ATG-3'.
  • PAM Protospacer Adjacent Motif
  • the protospacer may be immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence in the target sequence which is 5'-AAC-3'.
  • the protospacer may be immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence having any of the features described elsewhere herein.
  • PAM Protospacer Adjacent Motif
  • the spacer sequence may be from (about) 28 to 32 nucleotides in length.
  • the spacer sequence may be about 32 nucleotides in length.
  • the spacer sequence may be 32 nucleotides in length.
  • the spacer sequence may be 32 nucleotides in length.
  • the spacer sequence may have any of the features described elsewhere herein (e.g. in the First Aspect of Detailed Description, in Concept 10 and in Concept 30).
  • the protospacer may have any of the features described elsewhere herein (e.g. in the First Aspect of Detailed Description, in Concept 10 and in Concept 30).
  • the spacer may be identical to the complement of the protospacer in the target sequence across its entire length.
  • the spacer may be (about) 80% (for example (about) 90%) identical to the complement of the protospacer in the target sequence across its entire length.
  • the spacer may be identical to the complement of the protospacer in the target sequence across the first 1 to 28 nucleotides which are immediately 5' of the PAM sequence in the target sequence, and (about) 80% (e.g. (about) 90%) identical across the rest of the nucleotides in the protospacer.
  • the I-TevI cleavage site nucleotide sequence may be as described elsewhere herein. In one embodiment, the I-TevI cleavage site nucleotide sequence is 5' of the protospacer in the target sequence.
  • the I-TevI cleavage site nucleotide sequence may be is between (about) 3 and 7 nucleotides in length, for example between (about) 4 and 6 nucleotides in length.
  • the I-TevI cleavage site nucleotide sequence may be about 5 nucleotides in length.
  • the I-TevI cleavage site nucleotide sequence may comprise the motif 5'-CNNNG-3'.
  • the I-TevI cleavage site nucleotide sequence may be selected from 5'-CCACG-3', 5'-CACCG-3', 5'-CATAG-3', 5'-CTAAG-3', 5'-CTGAG- 3', 5'-CTTAG-3', 5'-CCTCG-3', 5'-CAGCG-3', 5'-CACTG-3', 5'-CGACG-3', 5'-CGATG-3', 5'-CTCCG-3', 5'-CTACG-3', 5'-CTGTG-3', 5'-CTGCG-3', 5'-CCGTG-3', 5'-CCTAG-3', 5'-CCATG-3', 5'-CAAAG-3', 5'- CAGAG-3', 5'-CATTG-3' and 5'-CATCG-3'.
  • the I-TevI cleavage site nucleotide sequence may be selected from 5'-CCACG-3', 5'-CACCG-3', 5'-CATAG-3', 5'-CTAAG-3', 5'-CTGAG-3', 5'-CTTAG-3', 5'- CCTCG-3' and 5'-CAGCG-3', or is selected from 5'-CCACG-3', 5'-CACCG-3', 5'-CATAG-3', 5'-CTAAG- 3' and 5'-CTGAG-3'.
  • the I-TevI cleavage site nucleotide sequence may be selected from 5'-CCACG- 3', 5'-CACCG-3' and 5'-CATAG-3'.
  • the I-TevI cleavage site nucleotide sequence may be selected from 5'-CCACG-3' and 5'-CACCG-3'.
  • the I-TevI cleavage site nucleotide sequence may have the nucleotide sequence of SEQ ID No:42.
  • the I-TevI spacer nucleotide sequence may be as described elsewhere herein. In one embodiment, the I-TevI spacer nucleotide sequence is 5' of the protospacer in the target sequence.
  • the I-TevI spacer nucleotide sequence may be between (about) 10 and 35 nucleotides in length, such as from 15 to 35, or from 20 to 35 in length.
  • the I-TevI spacer nucleotide sequence may be between (about) 29 and 33 nucleotides in length.
  • the I-TevI spacer nucleotide sequence may be between (about) 30 and 32 nucleotides in length.
  • the I-TevI spacer nucleotide sequence may be any other nucleotides in length.
  • the I-TevI spacer nucleotide sequence may at least (about) 80% (e.g. (about) 90%) identical to the nucleotide sequence of SEQ ID No:43.
  • the I-TevI spacer nucleotide sequence may be 100% identical to the nucleotide sequence of SEQ ID No:43.
  • sequences a) to d) are each immediately adjacent to each other.
  • the I-TevI cleavage site nucleotide sequence is as described elsewhere herein (e.g. as described in Concept 43).
  • the I-TevI spacer nucleotide sequence is as described elsewhere herein (e.g. as described in Concept 44).
  • the PAM sequence is as described elsewhere herein (e.g. as described in Concept 39).
  • the protospacer sequence is as described elsewhere herein (e.g. as in the First Aspect of Detailed Description, or in Concept 10, or Concept 30).
  • Concept 46A A cell which comprises one or more vector(s), fusion protein(s) or fusion protein complexes described in any Configuration, Concept, aspect, example, embodiment, option or other feature as described elsewhere herein.
  • a eukaryotic cell which comprises one or more vector(s), fusion protein(s) or fusion protein complexes described in any Configuration, Concept, aspect, example, embodiment, option or other feature as described elsewhere herein.
  • a prokaryotic cell comprising one or more vector(s), fusion protein(s) or fusion protein complexes described in any Configuration, Concept, aspect, example, embodiment, option or other feature as described elsewhere herein.
  • a prokaryotic cell comprising one or more vector(s), fusion protein(s) or fusion protein complexes described in any Configuration, Concept, aspect, example, embodiment, option or other feature as described elsewhere herein, wherein the cell is not a bacterial cell (for example an E coii, Pseudomonas or Klebsiella cell) that comprises endogenous nucleotide sequences that encode the polypeptides of a) to e) as recited in Concept 1A.
  • bacterial cell for example an E coii, Pseudomonas or Klebsiella cell
  • the cell is a vertebrate, mammal or human cell.
  • composition comprising one or more vector(s), fusion protein(s) or fusion protein complexes described in any Configuration, Concept, aspect, example, embodiment, option or other feature as described elsewhere herein.
  • compositions described herein may be as described elsewhere herein.
  • a composition described herein may be an in vitro composition.
  • a composition described herein may be comprised by a medical container.
  • compositions comprising one or more vector(s), fusion protein(s) or fusion protein complexes described in any Configuration, Concept, aspect, example, embodiment, option or other feature as described elsewhere herein and further comprising a diluent, excipient or carrier.
  • the pharmaceutical compositions are for use in any of the methods of medical treatment described herein.
  • vector(s), fusion protein(s) or fusion protein complexes described in any Configuration, Concept, aspect, example, embodiment, option or other feature as described elsewhere herein for use in therapy There is also provided one or more vector(s), fusion protein(s) or fusion protein complexes described in any Configuration, Concept, aspect, example, embodiment, option or other feature as described elsewhere herein for use in therapy.
  • the pharmaceutical compositions may be comprised within a medical device (such as an ampoule, a syringe, or an inhaler).
  • the pharmaceutical compositions may be formulated in a tincture, a capsule or a slow-release formulation.
  • the pharmaceutical compositions may be an oral tablet, comprised within a blister pack.
  • the pharmaceutical composition comprising one or more vector(s), fusion protein(s) or fusion protein complexes as described herein is formulated for oral or rectal administration. In one embodiment, the pharmaceutical composition is formulated for oral administration. In one embodiment, the pharmaceutical composition is formulated as a capsule or coated tablet.
  • the formulation comprising the one or more vector(s), fusion protein(s) or fusion protein complexes described herein may be freeze dried prior to encapsulation.
  • the pharmaceutical composition comprising one or more vector(s), fusion protein(s) or fusion protein complexes as described herein is a lyophilised formulation.
  • the pharmaceutical composition comprising one or more vector(s), fusion protein(s) or fusion protein complexes as described herein is an encapsulated formulation to be released in the lower gut of a subject.
  • a composition (optionally an in vitro composition or wherein the composition is comprised by a medical container) comprising a crRNA or nucleic acid encoding the crRNA; and one, more or all of a) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO: 1; b) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:2; c) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:3; d) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:4; and e) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO: 5 wherein
  • the crRNA comprises a spacer that is cognate to a first protospacer, wherein the protospacer is f) not found in E. coir, g) is a eukaryotic cell protospacer; or h) an animal (optionally mammal or human), plant or fungus cell protospacer; or
  • B a protospacer of an E. coii is devoid of endogenous nucleotide sequences that encode the polypeptides recited in parts a) to e) above.
  • the composition comprises (a)-(e). In an example, the composition comprises (a)-(d). In an example, the composition comprises (a)-(c). In an example, the composition comprises (a)-(b). In an example, the composition comprises (d)-(e). In an example, the composition comprises (c)-(e). In an example, the composition comprises (b)-(e).
  • the composition comprises (a). In an example, the composition comprises (b). In an example, the composition comprises (c). In an example, the composition comprises (d). In an example, the composition comprises (e).
  • the crRNA is operable in a cell to guide the polypeptide(s), protein(s), fusion protein(s) and complexes described herein to a target sequence comprised by a nucleic acid in the cell.
  • a complex that comprises one, more or all of polypeptides (a)-(e) and the crRNA, wherein the crRNA is capable of guiding the complex to a protospacer comprised by a target DNA.
  • the complex comprises (a)-(e).
  • a composition (optionally an in vitro composition or wherein the composition is comprised by a medical container) comprising a crRNA or nucleic acid encoding the crRNA; and nucleic acid encoding one, more or all of a) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO: 1; b) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:2; c) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:3; d) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:4; and e) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO: 5 wherein
  • the crRNA comprises a spacer that is cognate to a first protospacer, wherein the protospacer is f) not found in E. coir, g) is a eukaryotic cell protospacer; or h) an animal (optionally mammal or human), plant or fungus cell protospacer; or
  • the composition comprises a vector that encodes at least 2, 3 or 4, of said polypeptides.
  • (a)-(e) are encoded by the same vector.
  • the composition comprises a vector encoding all of (a)-(e).
  • composition nucleic acid encodes (a)-(e). In an example, the composition nucleic acid encodes (a)-(d). In an example, the composition nucleic acid encodes (a)-(c). In an example, the composition nucleic acid encodes (a)-(b). In an example, the composition nucleic acid encodes (d)-(e). In an example, the composition nucleic acid encodes (c)-(e). In an example, the composition nucleic acid encodes (b)-(e).
  • the crRNA is operable in a cell to guide the polypeptide(s) to a target sequence comprised by a nucleic acid in the cell.
  • a complex that comprises one, more or all of polypeptides (a)-(e) and the crRNA, wherein the crRNA is capable of guiding the complex to a protospacer comprised by a target DNA.
  • the complex comprises (a)-(e).
  • Any method herein may be an in vitro method.
  • the method is carried out on a cell comprised by a subject, such as a human, animal (e.g. a mammal, e.g. a rodent, mouse or rat), plant, insect or fungus (e.g. yeast).
  • a subject such as a human, animal (e.g. a mammal, e.g. a rodent, mouse or rat), plant, insect or fungus (e.g. yeast).
  • modifying e.g. modifying a target sequence of a DNA, polynucleotide or cell
  • modifying may be wherein the modifying cuts, edits, blocks, marks or labels the target sequence.
  • the cRNA of the composition (or nucleic acids encoding the crRNA whereby the cRNA are expressed in the cell) and the nucleic acid of the composition encoding the polypeptide(s) or protein(s), whereby the polypeptide(s) or protein(s) are expressed in the cell; (Ill) wherein the crRNA form a complex with the polypeptide(s) or protein(s) to guide the complex to the target sequence.
  • the modifying may be downregulation of a gene comprising the target sequence.
  • the modifying may be downregulation of a gene adjacent to (e.g. within 2kb of) the target sequence.
  • the modifying may be cutting the target sequence (e.g. when Py comprises a nuclease or a nickase).
  • the modifying may be introduction of one or more mutations in the target sequence (e.g. when Py comprises a nuclease, nickase, prime editor or base editor).
  • the modifying may be performing base editing in the target sequence (e.g. when Py is a base editor).
  • the modifying may be performing prime editing in the target sequence (e.g. when Py is a prime editor).
  • the modifying may be methylating one or more nucleotides in the target sequence (e.g. when Py is a methyltransferase).
  • the modifying may be methylasing one or more nucleotides in the target sequence (e.g. when Py is a methylase).
  • the modifying may be reverse transcribing the target sequence, or reverse transcribing an RNA in the cell to produce a DNA that is inserted at the target sequence (e.g. when Py is a reverse transcriptase).
  • the modifying may be de-activating transcription of a gene in the cell, such as a gene comprising or adjacent to the target sequence (e.g. when Py is a transcription deactivator).
  • the modifying may be de-activating translation of a gene in the cell, such as a gene comprising or adjacent to the target sequence (e.g. when Py is a translation deactivator).
  • the cRNA of the composition (or nucleic acids encoding the crRNA whereby the cRNA are expressed in the cell) and the nucleic acid of the composition encoding the polypeptide(s), whereby the polypeptide(s) are expressed in the cell; c) wherein the crRNA form a complex with the polypeptide(s) to guide the complex to the target sequence.
  • the complex modifies (e.g. cuts or edits) a coding target sequence coding or a non-coding target sequence, optionally wherein (i) a coding strand but not non-coding strand of DNA is modified or (ii) a non-coding strand but not coding strand of DNA is modified.
  • the modifying herein may usefully modify (i) a coding strand but not non-coding strand of DNA or (ii) a non-coding strand but not coding strand of DNA.
  • Concept 48C The method of Concept 48B, wherein the complex is guided to the target sequence and modifies the target sequence, optionally wherein the complex comprises a component selected from a) a nuclease which cuts the target sequence; b) a deaminase (such as a cytosine or adenine deaminase) which carries out deamination of the target sequence; c) a base editor which base edits the target sequence; d) a prime editor which prime edits the target sequence; e) a reverse transcriptase which reverse transcribes the target sequence or which reverse transcribes an RNA in the cell to produce a DNA that is inserted at the target sequence; f) a methyltransferase that methylates DNA in the cell; g) a methylase that methylases DNA in the cell; h) an acetylase that acetylases DNA in the cell; i) an acetyltransferase that subjects DNA
  • the complex further comprises a base editor (e.g. as described elsewhere herein).
  • the complex further comprises a prime editor (e.g. as described elsewhere herein).
  • the complex further comprises a nuclease (e.g. as described elsewhere herein).
  • Concept 49A The method of Concept 48C, wherein the cell is a eukaryotic, animal, plant, insect or fungus cell.
  • an animal herein is a non-human mammal or vertebrate.
  • an animal cell herein is a non-human mammal or vertebrate cell.
  • the cell is a plant cell, bacteria cell, fungal cell, mammalian cell, insect cell, or archaeon cell.
  • a cell herein may be ex vivo or in vivo.
  • a cell herein is a pluripotent cell, such as a pluripotent stem cell.
  • a cell herein is stem cell, such as an embryonic stem cell.
  • a cell herein is a totipotent cell.
  • the cell is a human cell.
  • the cell is not a human cell and not comprised by a human or human embryo.
  • any method herein is not any of the following:
  • any method herein is not a method of treatment on the human or animal body.
  • any method herein is not a surgical method.
  • inhibition is at least (about) 20, 30, 40, 50, 60, 70, 80, 90 or 95% compared to replication in an identical cell that has not been exposed to the vector(s) or composition. In an example, inhibition is 100%.
  • the target sequence is within (about) 2 kb upstream (5Q or downstream (3Q of a gene of interest.
  • the target sequence may be upstream (5') of the gene of interest.
  • the target sequence may be downstream (3Q of the gene of interest.
  • the target sequence may be within (about) 1.75kb, within (about) 1.5kb, within (about) 1.25kb, within (about) lkb, within (about) 0.75kb, within (about) 0.5kb, or within (about) 0.25kb upstream (5Q or downstream (3Q of a gene of interest.
  • the target sequence may be within a gene of interest, for example may be within the promoter, exon(s) and/or intron(s) of the gene of interest. In one embodiment, the target sequence is within the promoter of the gene of interest.
  • the CasS3 protein can have a wide range of influence, with binding effects being shown within approximately 2kb either side (i.e. 5' and 3') of the protospacer sequence in the target sequence.
  • the CasS system is used for CRISPRi (i.e. in any method described herein of upregulation or downregulation of a gene of interest)
  • the modifying is a substitution of one or more nucleotides in the target sequence.
  • the modifying is one or more C to T substitutions in the nucleotides of the target sequence when Py is a CBE.
  • the CBE is a PmCDAl cytidine deaminase, for example as described elsewhere herein. Concept 55.
  • a method of treating or preventing a disease or condition that is mediated by target cells in a subject comprising carrying out a method as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example as described in any one of Concepts 48 to 54) to modify the target cells, wherein said contacting comprises administering the vector(s), protein(s) (including fusion protein(s) or fusion protein complexes) or composition to the subject and wherein the modification treats or prevents the disease or condition.
  • Target cells may be, for example, pathogenic bacterial cells. They may be antibiotic resistance bacterial cells and the modification re-sensitises the bacterial cell to the antibiotic.
  • Concept 56 The method of Concept 55, wherein the subject is a human, animal or plant.
  • SNP single nucleotide polymorphisms
  • progeria syndrome and mandibuloacral dysplasia are caused by a missense SNP C.1580G>T SNP in the LMNA gene encoding the lamin A/C protein, and an SNP in the F5 gene causes Factor V Leiden thrombophilia. Correction of these SNPs by targets substitutions could be used to ameliorate diseases.
  • the modification upregulates or downregulates expression of a gene in the cells. In one embodiment, the modification downregulates expression of a gene in the cells. In one embodiment, the modification inhibits expression of a gene in the cells. In one embodiment, the modification blocks expression of a gene in the cells.
  • the modification is insertion of a new gene or pathway of genes (e.g. an operon), which may be used to produce substances which are beneficial to the cells.
  • the new genes may produce beneficial metabolites, or therapeutics.
  • the modification is insertion of a new gene or pathway of genes (e.g. an operon), which may be used to remove substances which are detrimental to the cells.
  • the new genes may metabolise harmful toxins.
  • Concept 58B The method of Concept 56 or 57, wherein the modification adds a new nucleotide sequence to the genomes of the cells for expression of a protein encoded by the new sequence.
  • the protein is a heterologous protein.
  • the protein is a therapeutic protein, e.g. an antibody, antibody fragment (such as an antibody single variable domain), TCR (T-cell receptor) binding site, TCR variable domain, hormone, incretin, growth factor, anti-cancer agent, neurotransmitter or enzyme.
  • the modification modulates the expression of a nucleotide sequence comprised by a plasmid. In one embodiment, the modification modulates the expression of a nucleotide sequence which is not comprised by a plasmid. For example, the expression is upregulated. For example, the expression is down regulated.
  • Concept 59 Vector(s), a protein(s) (inclusion fusion protein(s) and fusion protein complexes), cell(s) or composition as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example as described in any one of Concepts 1-58) for use in a method of treating or preventing a disease or condition in a human or animal, wherein the method as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example as described in any one of Concepts 55 to 58).
  • Any vector herein may be a) a plasmid vector (optionally a conjugative plasmid) b) a transposon vector (optionally a conjugative transposon); c) a virus vector (optionally a phage, AAV or lentivirus vector); d) a phagemid (optionally a packaged phagemid); or e) a nanoparticle (optionally a lipid nanoparticle).
  • a method of introducing a targeted edit in a target polynucleotide comprising contacting the target polynucleotide with a fusion protein or fusion protein complex as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein, wherein the protein or fusion protein complex is in combination with a crRNA which comprises a spacer that is cognate to a first protospacer in a target sequence (for example as described in the First Aspect of Detailed Description, Concept 10 or in Concept 30 or in any one of Concepts 38 to 45 when dependent on Concept 30) wherein the first protospacer in the target sequence is comprised by the polynucleotide, wherein the crRNA hybridizes to the protospacer to guide the protein (e.g. the ribonucleoprotein complex) whereby the protein (e.g. the ribonucleoprotein complex) edits the polynucleotide.
  • a crRNA which comprises a spacer that is cognate to a first protospacer in a
  • the method may be carried out in a parental cell that comprises the polynucleotide.
  • a progeny cell or organism that is derived from the edited parental cell, wherein the progeny cell or organism retains the edit in its genome.
  • the method may comprise one or more steps of culturing the edited cell to produce said progeny cell or a plurality of such progeny cells.
  • the PAM may be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39).
  • a container comprising a plurality of proteins operable for use with a crRNA to form a ribonucleoprotein complex for protospacer targeting in a polynucleotide, the protein(s)s comprising any of the protein(s) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example as described in Concept 13), wherein the complex is operable with any of the protospacer adjacent motifs (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39), and the container is not a cell, and the proteins are mixed with an in vitro buffer.
  • PAMs protospacer adjacent motifs
  • Concept 62B A container comprising a plurality of proteins operable for use with a crRNA to form a ribonucleoprotein complex for protospacer targeting in a polynucleotide, the proteins comprising any of the fusion protein(s) or fusion protein complexes described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example as described in any one of Concepts 14 to 29), wherein the complex is operable with any of the protospacer adjacent motifs (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39), and the container is not a cell, and the proteins are mixed with an in vitro buffer.
  • Concept 62C A container comprising a plurality of proteins operable for use with a crRNA to form a ribonucleoprotein complex for protospacer targeting in a polynucleotide, the proteins comprising any of the fusion protein(s) or fusion
  • a container comprising a protein or plurality of proteins operable for use with a crRNA to form a ribonucleoprotein complex for protospacer targeting in a polynucleotide, the protein(s) comprising one, more or all polypeptides selected from a) a polypeptide that comprises an amino acid that is at least (about)80% identical to SEQ ID NO:1; b) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:2; c) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:3; d) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:4; and e) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO: 5 wherein the complex is operable with a protospacer adjacent motif (PAM) having the sequence 5'-AAG-3', the container is
  • the PAM may be any one of the PAMs described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39)
  • a protein or plurality of proteins operable for use with a crRNA to form a ribonucleoprotein complex for protospacer targeting in a polynucleotide comprising one, more or all polypeptides selected from a) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO: 1; b) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:2; c) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:3; d) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO:4; and e) a polypeptide that comprises an amino acid that is at least (about) 80% identical to SEQ ID NO: 5 wherein the complex is operable with a protospacer adjacent motif (PAM) having the sequence 5'-AAG-3'.
  • the PAM may be any protospacer adjacent motif
  • PAMs protospacer adjacent motifs
  • the protospacer adjacent motif has the sequence 5'-AAG-3'.
  • a method of targeting a polynucleotide comprising a) contacting the polynucleotide with the protein(s), (including fusion protein(s) and fusion protein complexes) as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein, wherein the protein(s) is/are in combination with a crRNA which comprises a spacer that is cognate to a first protospacer in a target sequence; b) allowing the formation of a ribonucleoprotein complex comprising the protein(s) and crRNA, wherein the complex is guided to a target sequence comprised by the polynucleotide to modify the polynucleotide or replication thereof.
  • the targeting may be in a cell.
  • the targeting may be in vitro.
  • Concept 65 The method of Concept 64, wherein the polynucleotide is comprised by a chromosome or an episome.
  • the polynucleotide is comprised by a chromosome. In one embodiment, the polynucleotide is comprised by a plasmid. In one embodiment, the polynucleotide is not comprised by a plasmid.
  • Concept 66B The method of Concept 65, wherein the method edits the polynucleotide.
  • the editing may insert a base or nucleic acid sequence in the polynucleotide.
  • the editing may delete a base or nucleic acid sequence in the polynucleotide.
  • the editing may substitute a base or nucleic acid sequence in the polynucleotide.
  • a kit comprising a) One or more protein(s) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example as described in Concept 13); and b) A crRNA, (or one or more nucleic acids encoding a crRNA), wherein the crRNA is cognate to any of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example a PAM having one of the sequences recited in Concept 39); wherein said polypeptide(s) are operable for use with the crRNA to form a ribonucleoprotein complex for protospacer targeting in a polynucleotide.
  • a kit comprising a) One or more fusion protein(s) or fusion protein complexes described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example as described in any one of Concepts 14 to 29), or one, or more nucleic acids encoding such polypeptide(s); and b) A crRNA, or one or more nucleic acids encoding a crRNA), wherein the crRNA is cognate to any of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example a PAM having one of the sequences recited in Concept 39); wherein said polypeptide(s) are operable for use with the crRNA to form a ribonucleoprotein complex for protospacer targeting in a polynucleotide.
  • a kit comprising a) One or more vector(s) as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example as described in any one of Concepts 1 to 12, or any one of Concepts 31 to 45), or one, or more nucleic acids encoding such polypeptide(s); and b) A crRNA, or one or more nucleic acids encoding a crRNA), wherein the crRNA is cognate to any of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example a PAM having one of the sequences recited in Concept 39); wherein said polypeptide(s) are operable for use with the crRNA to form a ribonucleoprotein complex for protospacer targeting in a polynucleotide.
  • a kit comprising a) One or more polypeptides as recited in Concept 7, or one, or more nucleic acids encoding such polypeptide(s); and b) A crRNA, (or one or more nucleic acids encoding a crRNA, wherein the crRNA is cognate to PAM having the sequence 5'-AAG-3'; wherein said polypeptide(s) are operable for use with the crRNA or guide RNA to form a ribonucleoprotein complex for protospacer targeting in a polynucleotide.
  • the complex further comprises one or more Cascade Cas proteins, e.g. one, more or all of a Type I CasA-E proteins.
  • the complex may further comprise a Cas3 and optionally one or more cognate Type I Cascade proteins.
  • the complex may further comprise a Cas3, 9, 10, 12 or 13.
  • function of a Type-S system or components can be provided together with function on one or more proteins of a different Type of CRISPR/Cas system.
  • the complex may comprise a helicase fused to a nuclease.
  • the nuclease may be, for example, a Cas3, 9, 10, 12 or 13.
  • the nuclease is a Cas3.
  • the nuclease is a Cas9.
  • the complex further comprises a MutH protein.
  • the complex is an isolated complex.
  • the complex may be in vitro.
  • the complex may be comprised by a medical container, such as an IV bag, medical vial or medical injection device.
  • Concept 68A A ribonucleoprotein complex comprising a) One or more protein(s) as described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example as described in Concept 13); and b) A crRNA which is cognate to any of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39).
  • a ribonucleoprotein complex comprising a) One or more fusion protein(s) or fusion protein complexes described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example as described in any one of Concepts 14 to 29); and b) A crRNA which is cognate to any of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39).
  • Concept 68D The complex of Concept 68C, wherein the complex is devoid of a) a Cas nuclease; b) a DNA nuclease or RNA nuclease; or c) a nuclease.
  • a method of transcriptional control or replication of a target DNA comprising contacting the target DNA with any of the complexes described herein (for example with the complex of Concept 68), wherein the complex is devoid of a DNA nuclease and the complex binds to the target DNA, thereby controlling the transcription or replication of the target DNA.
  • Concept 70A A method of controlling replication of a target RNA, comprising contacting the target RNA or a DNA encoding the RNA with any of the complexes described herein (for example with the complex of Concept 68), wherein the complex binds to the target RNA or DNA, thereby controlling the transcription of the target RNA.
  • Concept 70B A method of controlling transcription of a target RNA, comprising contacting the target RNA or a DNA encoding the RNA with any of the complexes described herein (for example with the complex of Concep 68), wherein the complex binds to the target RNA or DNA, thereby controlling the transcription of the target RNA.
  • the DNA is comprised by a chromosome. In one embodiment, the DNA is comprised by a plasmid. In one embodiment, the DNA is not comprised by a plasmid.
  • Concept 72 A method of editing a target DNA, comprising contacting the target DNA with any of the complexes described herein (for example with the complex of Concept 68), wherein the complex binds to the target DNA, thereby editing the target DNA.
  • Concept 73A A method of editing a target DNA, comprising contacting the target DNA with any of the complexes described herein (for example with the complex of Concept 68), wherein the complex binds to the target DNA, thereby editing the target DNA.
  • a method for cleaving a double stranded DNA comprising contacting said dsDNA with any of the complexes described herein (for example with the complex of Concept 68), wherein the complex comprises a fusion protein in which Py comprises a nuclease, wherein the dsDNA comprises a protospacer sequence flanked at its 5' by any one of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39), whereby the nuclease cleaves the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer.
  • a method for cleaving a double stranded DNA comprising contacting said dsDNA with the complex of Concept 68C or 68D, wherein the complex comprises a nuclease, wherein the dsDNA comprises a protospacer sequence flanked at its 5' by a PAM having the sequence 5'-AAG-3' or a PAM that is identical except for one base change, whereby the nuclease cleaves the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer.
  • a method for cleaving a single strand in a double stranded DNA comprising contacting said dsDNA with any of the complexes described herein (for example with the complex of Concept 68), wherein the complex comprises a fusion protein in which Py comprises a nickase, wherein the dsDNA comprises a protospacer sequence flanked at its 5' by any one of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39), whereby the nickase cleaves a single strand of the dsDNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer.
  • dsDNA double stranded DNA
  • a method for cleaving a single stranded DNA comprising contacting said DNA with the complex of Concept 68C or 68D, wherein the complex comprises a nickase, wherein the DNA comprises a protospacer sequence flanked at its 5' by a PAM having the sequence 5'- AAG-3' or a PAM that is identical except for one base change, whereby the nickase cleaves a single strand of the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer.
  • the nickase may be any nuclease described herein.
  • the nickase may be a DNA nickase.
  • the nickase may be selected from Nt.BstNBI, Nb.BsrDI, Nb.BtsI, Nt.AIwI, Nb.BbvCI, Nt.BbvCI, and Nb.BsmI (available from New England BioLabs).
  • the nickase a synthetic Cas nickase (e.g. nCas9 or Cas9D10A).
  • Concept 75A A method of marking or identifying a region of a DNA, comprising contacting said DNA with any of the complexes described herein (for example with the complex of Concept 68), wherein the DNA comprises a protospacer sequence flanked at its 5' by any one of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39), whereby the complex binds to the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer, and optionally wherein the complex comprising a fusion protein in which Py comprises a detectable label.
  • the complexes described herein for example with the complex of Concept 68
  • the DNA comprises a protospacer sequence flanked at its 5' by any one of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39)
  • the complex bind
  • a method of modifying transcription of a region of a DNA comprising contacting said DNA with any of the complexes described herein (for example with the complex of Concept 68), wherein the DNA comprises a protospacer sequence flanked at its 5' by any one of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39), whereby the complex binds to the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer, whereby the complex up- or down-regulates transcription of the region of DNA or an adjacent gene.
  • PAMs protospacer sequence flanked at its 5' by any one of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39)
  • a method of modifying transcription of a region of a DNA comprising contacting said DNA with the complex of Concept 68C or 68D, wherein the DNA comprises a protospacer sequence flanked at its 5' by a PAM having the sequence 5'-AAG-3' or a PAM that is identical except for one base change, whereby the complex binds to the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer, whereby the complex up- or down-regulates transcription of the region of DNA or an adjacent gene.
  • the complex down-regulates transcription. Down-regulation may be at least about 50% compared to transcription without the complex present. In an embodiment, the downregulation is at least about 60%, 70%, 80% or 90%. In another embodiment, the downregulation is at least about 95%, 96%, 97%, 98% or 99%. In another embodiment, the downregulation is 100%, i.e. transcription of the region of DNA is blocked completely.
  • downregulation may be desirable when a gene is overexpressed and is causative of a negative phenotype, but complete removal of the protein would be detrimental. Downregulation of a target gene can be useful in making and studying knockout phenotypes where generation of a complete knockout would be lethal to the cell or organism.
  • blocking transcription of an essential gene in a pathogenic bacteria can be used to kill the pathogenic bacterium whilst leaving other, beneficial bacteria unaffected. Equally, pathogenic bacteria can be killed by blocking the transcription of the origin of replication. Blocking non-essential genes in a cell or organism is useful for studying knock-outs and phenotype behaviour(s).
  • a method of modifying a target dsDNA of a cell without introducing dsDNA breaks comprising producing in the cell with any of the complexes described herein (for example with the complex of Concept 68), wherein the complex targets a target dsDNA comprised by the cell, the target dsDNA comprising a protospacer sequence flanked at its 5' by any one of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39), whereby the complex binds to the dsDNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer, whereby the dsDNA is modified without introducing dsDNA breaks.
  • any of the complexes described herein for example with the complex of Concept 68
  • the complex targets a target dsDNA comprised by the cell, the target dsDNA comprising a protospacer sequence flanked at its 5' by any one of the
  • a method of modifying a target dsDNA of a cell without introducing dsDNA breaks comprising producing in the cell with the complex of Concept 68C or 68D, wherein the complex targets a target DNA comprised by the cell, the target DNA comprising a protospacer sequence flanked at its 5' by a PAM having the sequence 5'-AAG-3' or a PAM that is identical except for one base change, whereby the complex binds to the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer, whereby the DNA is modified without introducing breaks in the DNA.
  • the modifying may be the introduction of one or more substitutions when the complex is a complex of Concept 68B and Py is a base editor or a prime editor (such as any of the base editors or prime editors described elsewhere herein).
  • the modifying may be the introduction of one or more insertions and/or deletions (INDELS) when the complex is a complex of Concept 68B and Py is a nuclease or nickase and the cell is a cell which is able to repair the dsDNA break or the ssDNA break i//a the NHEJ mechanism.
  • INDELS insertions and/or deletions
  • a method of inhibiting the growth or proliferation of a cell without introducing lethal dsDNA breaks comprising producing in the cell any of the complexes described herein (for example a complex of Concept 68), wherein the complex targets a target DNA comprised by the cell, the target DNA comprising a protospacer sequence flanked at its 5' by any one of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, whereby the complex binds to the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer, whereby replication of the DNA is inhibited without introducing lethal dsDNA breaks in the DNA, thereby inhibiting the growth or proliferation of a cell.
  • the complexes described herein for example a complex of Concept 68
  • the complex targets a target DNA comprised by the cell, the target DNA comprising a protospacer sequence flanked at its 5' by any one of the (PAMs) described in any Configuration, Concept, aspect, example, embodiment, whereby the
  • a method of inhibiting the growth or proliferation of a cell without introducing lethal dsDNA breaks comprising producing in the cell the complex of Concept 68C or 68D, wherein the complex targets a target DNA comprised by the cell, the target DNA comprising a protospacer sequence flanked at its 5' by a PAM having the sequence 5'-AAG-3' or a PAM that is identical except for one base change, whereby the complex binds to the DNA in the region defined by complementary binding of a spacer sequence of the crRNA to the protospacer, whereby replication of the DNA is inhibited without introducing breaks in the DNA, thereby inhibiting the growth or proliferation of a cell.
  • the growth or proliferation of a cell may be inhibited by the introduction of one or more insertions and/or deletions (INDELS) when the complex is a complex of Concept 68B and Py is a nickase which introduces single stranded breaks in DNA and the cell is a cell which is able to repair the dsDNA break or the ssDNA break via the NHEJ mechanism.
  • the growth or proliferation of a cell may be inhibited by downregulating or blocking the transcription of an essential gene in the cell when the complex is a complex of Concept 68A. Essential genes are well-known to those skilled in the art.
  • the growth or proliferation of a cell may be inhibited by downregulating or blocking the transcription of a chromosomal origin of replication in the cell when the complex is a complex of Concept 68A.
  • Concept 80 A method of treating or preventing a disease or condition in a human, animal, plant or fungus subject, the method comprising carrying out the method of any one of Concepts 69 to 79, wherein cells of the subject comprise said DNA or RNA and the cells mediate the disease or condition.
  • a coding strand but not non-coding strand of DNA is modified. In another embodiment, a non-coding strand but not coding strand of DNA is modified.
  • Concept 81B The method of any one of Concepts 69 to 80, wherein the complex modifies (e.g. cuts, edits, blocks, marks or labels) a coding target sequence coding or a non-coding target sequence, optionally wherein (i) a coding strand but not non-coding strand of DNA is modified or (ii) a non-coding strand but not coding strand of DNA is modified.
  • the complex modifies (e.g. cuts, edits, blocks, marks or labels) a coding target sequence coding or a non-coding target sequence, optionally wherein (i) a coding strand but not non-coding strand of DNA is modified or (ii) a non-coding strand but not coding strand of DNA is modified.
  • the modifying may be blocking.
  • the modifying may be downregulating transcription of a coding target sequence.
  • the modifying may be cutting a coding target sequence.
  • the modifying may be editing a coding target sequence. The base editor may introduce one or more substitutions in the coding target sequence.
  • nucleic acid vectors or one or more nucleic acids comprising at least one nucleotide sequence selected from SEQ ID NOs:7-ll, wherein a said nucleotide sequence is operably connected to a heterologous promoter, synthetic promoter, eukaryotic promoter or non- bacterial promoter.
  • nucleic acid vectors or one or more nucleic acids comprising at least one nucleotide sequence selected from SEQ ID NOs:7-ll, wherein a said nucleotide sequence is comprised by a cell that is a eukaryotic cell, a non-bacterial cell, or a cell that is not a bacterial cell (e.g. an E coli, Pseudomonas or Klebsiella cell) that comprises endogenous nucleotide sequence comprising SEQ ID NOs:7-ll.
  • nucleic acid vectors or one or more nucleic acids comprising at least one nucleotide sequence selected from SEQ ID NOs:7-ll, wherein a said nucleotide sequence is operably connected to a heterologous promoter, synthetic promoter, eukaryotic promoter or non- bacterial promoter and a said nucleotide sequence is comprised by a cell that is a eukaryotic cell, a non-bacterial cell, or a cell that is not a bacterial cell (e.g. an E coll, Pseudomonas or Klebsiella cell) that comprises endogenous nucleotide sequence comprising SEQ ID NOs:7-ll.
  • a eukaryotic cell e.g. an E coll, Pseudomonas or Klebsiella cell
  • nucleic acid(s) of Concept 85 wherein the nucleic acid is comprised by a vector or the nucleic acids are comprised by one or more vectors.
  • Concept 87 The nucleic acid(s) of Concept 85 or 86, wherein the DNA is comprised by a chromosome.
  • a ribonucleoprotein CRISPR/Cas complex comprising a plurality of Cas proteins and a crRNA, wherein the RNA is capable of guiding the complex to a protospacer comprised by a target DNA, wherein the 5'end of the protospacer is flanked by any one of theprotospacer adjacent motifs (PAMs) described in any Configuration, Concept, aspect, example, embodiment, option or other feature herein (for example having one of the sequences recited in Concept 39)', wherein a) the complex is devoid of a DNA nuclease and is capable of modifying DNA without introducing a double stranded DNA break; b) the complex is devoid of a Cas3,and a CaslO; and c) the complex does not comprise all of the Cas proteins of a Type I, II, III, IV, V or VI CRISPR/Cas complex.
  • PAMs protospacer adjacent motifs
  • Concept 90 The nucleic acid(s) or complex of any one of Concepts 85 to 89, wherein the complex is capable of modifying (i) a coding strand but not non-coding strand of DNA or (ii) a non-coding strand but not coding strand of DNA.
  • Concept 91 The nucleic acid(s) or complex of any one of Concepts 85 to 90, wherein the complex modifies (e.g. cuts, edits, blocks, marks or labels) a coding target sequence or a non-coding target sequence of the DNA.
  • the complex modifies (e.g. cuts, edits, blocks, marks or labels) a coding target sequence or a non-coding target sequence of the DNA.
  • the plurality of Cas proteins comprises Cas-Sl.1, S2.1, S3.1, S4.1 and S5.1.
  • Concept 93 The nucleic acid(s) or complex of any of Concepts 85 to 92, wherein the complex further comprises an effector protein domain for modifying DNA, optionally wherein the effector domain comprises nuclease activity, nickase activity, recombinase activity, reverse transcriptase, helicase, deaminase activity, methyltransferase activity, methylase activity, acetylase activity, acetyltransferase activity, transcriptional activation activity, or transcriptional repression activity.
  • the effector domain comprises nuclease activity, nickase activity, recombinase activity, reverse transcriptase, helicase, deaminase activity, methyltransferase activity, methylase activity, acetylase activity, acetyltransferase activity, transcriptional activation activity, or transcriptional repression activity.
  • the complex comprises a component selected from a) a nuclease which cuts the target site; b) a deaminase (such as a cytosine or adenine deaminase) which carries out deamination of the target site; c) a base editor which base edits the target site; d) a prime editor which prime edits the target site; e) a reverse transcriptase which reverse transcribes the target site or which reverse transcribes an RNA in the cell to produce a DNA that is inserted at the target site; f) a methyltransferase that methylates DNA in the cell; g) a methylase that methylases DNA in the cell; h) an acetylase that acetylases DNA in the cell; i) an acetyltransferase that subjects DNA in the cell to acetyltransferase activity; j) a transcription activator that activates transcription
  • Concept 94 A cell comprising the nucleic acid(s) or complex of any one of Concepts 85 to 93, wherein the DNA is comprised by a chromosome of the cell.
  • Concept 95 A cell comprising the nucleic acid(s) or complex of any one of Concepts85 to 93, wherein the DNA is comprised by a plasmid in the cell.
  • a method of modifying a DNA comprising a) contacting the DNA with the nucleic acid(s) defined in any one of Concepts 85 to 87 and 89 to 93; b) allowing the formation of a ribonucleoprotein complex comprising the Cas protein(s) and crRNA, wherein the complex is guided to a target site comprised by the DNA to modify the DNA.
  • a method of inhibiting the replication of a plasmid comprising DNA comprising a) contacting the DNA with the nucleic acid(s) defined in any one of Concepts 85 to 87 and 89 to 93; b) allowing the formation of a ribonucleoprotein complex comprising the Cas protein(s) and crRNA, wherein the complex is guided to a target site comprised by the DNA to inhibit replication of the plasmid.
  • Concept 99 A method of inhibiting the transcription of a nucleotide sequence comprised by a DNA, the method comprising a) contacting the DNA with the nucleic acid(s) defined in any one of Concepts 85 to 87 and 89 to 93; b) allowing the formation of a ribonucleoprotein complex comprising the Cas protein(s) and crRNA, wherein the complex is guided to a target site comprised by the nucleotide sequence to inhibit transcription thereof.
  • Concept 100 A method of inhibiting the transcription of a nucleotide sequence comprised by a DNA, the method comprising a) contacting the DNA with the nucleic acid(s) defined in any one of Concepts 85 to 87 and 89 to 93; b) allowing the formation of a ribonucleoprotein complex comprising the Cas protein(s) and crRNA, wherein the complex is guided to a target site comprised by the nucleotide sequence to inhibit transcription thereof.
  • Concept 100 A method of inhibiting the transcription of
  • a method of inhibiting the growth or proliferation of a cell comprising DNA, the method comprising a) contacting the DNA with the nucleic acid(s) defined in any one of Concepts 85 to 87 and 89 to 93; b) allowing the formation of a ribonucleoprotein complex comprising the Cas protein(s) and crRNA, wherein the complex is guided to a target site comprised by the DNA to inhibit growth or proliferation of the cell.
  • Concept 101 A method of treating or preventing a disease or condition that is mediated by target cells in a human or animal subject, the method comprising carrying out the method of any one of Concepts 97 to 100 to modify the target cells, wherein said contacting comprises administering the nucleic acid(s) to the subject and wherein the modification treats or prevents the disease or condition.
  • Concept 102 The method of Concept 101, wherein the target cells are cells of the subject that comprise a nucleic acid defect and the modification corrects the defect.
  • Concept 103 The method of Concept 101 or 102, wherein the modification a) adds a new functionality to the cells, optionally the modification upregulates or downregulates expression of a gene in the cells or adds a new nucleotide sequence to the genomes of the cells for expression of a protein encoded by the sequence; or b) modulates the expression of a nucleotide sequence comprised by a chromosome or episome (optionally a plasmid) of the cell; or c) inhibits growth or proliferation of the cells.
  • the modification a) adds a new functionality to the cells, optionally the modification upregulates or downregulates expression of a gene in the cells or adds a new nucleotide sequence to the genomes of the cells for expression of a protein encoded by the sequence; or b) modulates the expression of a nucleotide sequence comprised by a chromosome or episome (optionally a plasmid) of the cell; or c) inhibit
  • the cell may be a bacterial cell of any genus or species in Table 3.
  • the method may modify a cell of any genus or species in Table 3.
  • the DNA or polynucleotide may be comprised by a cell of any genus or species in Table 3.
  • the method may kill or inhibit the growth or proliferation of a cell of any genus or species in Table 3.
  • the cell may be a cell of a genus or species that is different from one of the genera or species respectively in Table 3, for example, the cell is not an E. coli cell, or the cell is not a Klebsiella cell.
  • the cell may be an archaea, such as a methanogen.
  • the method may kill or inhibit the growth or proliferation of the archaea.
  • the method may edit the genome of the bacterial or archaeal cell.
  • Any prokaryotic cell (e.g. bacterial or archaeal cell) or fungal cell herein may be comprised by a microbiome, such as a microbiome of a human, animal, plant or environment (such as soil or a waterway).
  • the microbiome is comprised by the gastrointestinal tract (GI tract) of a human or animal.
  • the microbiome is comprised by the urinary system of a human or animal, e.g. comprised by a bladder, kidney or urethra.
  • the microbiome is comprised by the blood of a human or animal.
  • the microbiome is comprised by the eye or nose or ear of a human or animal.
  • the microbiome is comprised by the hair of a human or animal.
  • the microbiome is comprised by the skin of a human or animal.
  • the microbiome is comprised by a leaf, root, stem or seed of a plant.
  • a disease or condition herein may be selected from any of the following:
  • a heart or cardiovascular disease or • A cancer condition, e.g. heart attack, strokdOor • An autoimmune disease or condition, e.g. atrial fibrillation; SLE;
  • a kidney disease or condition e.g. e.g. rheumatoid arthritis, psoriasis, chronic kidney disease (CKD); eczema, asthma, ulcerative colitis, colitis,
  • CKD chronic kidney disease
  • pancreas disease or condition 45 Crohn's disease or IBD;
  • a lung disease or condition e.g. cystic • Autism; fibrosis or COPD; • ADHD;
  • a genital disease or condition e.g. a condition; vaginal, labial, penile or scrotal disease or • Miscarriage; condition; • A blood clotting condition;
  • a sexually-transmissible disease ssor • Bronchitis; condition, e.g. gonorrhea, HIV infection, • Dry or wet AMD; syphilis or Chlamydia infection;
  • Neovascularisation e.g. of a tumour or in
  • a nasal disease or condition • Fibrosis, e.g. liver or lung fibrosis; • A fungal disease or condition, e.g. An allergy, eg, a nut, grass, pollen, dust thrush; mite, cat or dog fur or dander allergy;
  • Fibrosis e.g. liver or lung fibrosis
  • a fungal disease or condition e.g. An allergy, eg, a nut, grass, pollen, dust thrush; mite, cat or dog fur or dander allergy;
  • a metabolic disease or condition e.g. Malaria, typhoid fever, tuberculosis or obesity, anorexia, diabetes, Type I or cholera; Type II diabetes. 25 Depression;
  • Ulcer(s) e.g. gastric ulceration or skin Mental retardation; ulceration; Microcephaly;
  • a bone disorder and age of the subject
  • the CasS complexes described herein are fused to or complexed with a nuclease which is able to cut either single- or double-stranded DNA
  • the resulting nucleases can be used to target a specific protospacer within the same cell at a target site that results in death or a reduction in growth of the cells.
  • the CasS-nuclease complexes or fusions described herein are harboured within a cell, which also comprises other modifications, such as cells which are designed to produce a molecule of interest, e.g. cells which contain one or more exogenous genes for the conversion of one metabolite into another, or contain one or more exogenous genes which reduce the amounts of a disadvantageous molecule in the local environment.
  • the exogenous gene(s) may be comprised by one or more operons.
  • the exogenous gene(s) may be under the control of one or more promoters, which may be constitutive or inducible, in particular constitutive promoters.
  • the one or more exogenous genes comprised by one or more operons may be engineered into the chromosome of the cell, which may have advantages of stable transmission and reduced ability to transfer to nearby cells.
  • the one or more exogenous genes comprised by one or more operons may be engineered into a plasmid which is introduced into the cell (e.g. via a conjugative plasmid or transduction particle such as an engineered phage).
  • the CasS-nuclease complexes or fusions are under the control of a promoter which allows for transcription only under certain exogenous conditions (e.g. an inducible promoter)
  • the CasS-nuclease complexes or fusions can be activated to kill (or reduce growth of) the cell at a desired point in time, if the one or more exogenous genes comprised by one or more operons are comprised by the chromosome of the cell and the target sequence is also comprised by the chromosome of the cell.
  • a plasmid may be destroyed or the expression of the one or more exogenous genes comprised by one or more operons may be prevented (or reduced) when the one or more operons is comprised by a plasmid in the cell and the target sequence is also comprised by the plasmid.
  • the target sequence which is recognised by the CasS-nuclease complexes or fusions is longer than the PAM and protospacer of the CasS itself (because it further includes the nuclease cleavage site nucleotide sequence and optionally a nuclease spacer nucleotide sequence), which may be advantageous to enable selective targeting and reduce off-target cutting.
  • the protospacer may be located anywhere in the chromosome of the cell which harbours the CasS-nuclease complexes or fusions, and if the nuclease introduces a double-stranded or single-stranded DNA break, the cell will be killed.
  • the CasS-nuclease complexes or fusions target a gene which is essential for growth of the cell, and the DNA modification prevents expression of the essential gene, resulting in either cell death or a severe reduction in growth or proliferation of the cell.
  • a gene may be present in either the chromosome of the cell which harbours the CasS- nuclease complexes or fusions, or in a plasmid in the cell.
  • the CasS-nuclease complexes or fusions target one or more exogenous genes (e.g. comprised by one or more operons), and the DNA modification results in a prevention or reduction of expression of the one or more exogenous genes.
  • exogenous genes may be present in either the chromosome of the cell which harbours the CasS-nuclease complexes or fusions, or in a plasmid in the cell.
  • protein moiety means one or more proteins which are assembled together, either covalently (e.g. via direct fusion of amino acids or other covalent bonds such as disulfide bonds) or non-covalently (e.g. in a protein complex, where individual protein subunits are associated by a combination of non-covalent forces, such as hydrophobic, ionic and hydrogen bonds, or van der Waals forces).
  • non-covalent forces such as hydrophobic, ionic and hydrogen bonds, or van der Waals forces.
  • a method of modifying a target nucleic acid sequence in a prokaryotic cell which cell comprises one or more nucleic acid sequence(s) encoding a first protein moiety and a crRNA, wherein the method comprises expressing the first protein moiety and the crRNA in said cell to form a ribonucleoprotein complex, wherein the crRNA guides the first protein moiety to the target sequence; and wherein the crRNA comprises a spacer sequence which hybridises to a protospacer comprised by the target sequence; and wherein the complex modifies the target sequence in the cell and results in a reduction in growth or death of the cell, or wherein the complex modifies the target sequence and prevents or reduces expression of one or more exogenous genes comprised by the cell; and wherein the first protein moiety comprises: a) a polypeptide (Px) comprising an amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID Nos: 1-5; and b) a heterologous polypeptide (Py) which
  • the polypeptide (Px) may be any of the CasS proteins or combinations of proteins described elsewhere herein.
  • the nuclease, nickase, crRNA, target nucleic acid sequence may be any of those described elsewhere herein.
  • the protein moiety may be a protein complex.
  • the protein moiety may be a fusion of different protein subunits.
  • the protein moiety may comprise one of a CasSl, a CasS2, a CasS3, a CasS4 or a CasS5 protein as described elsewhere herein (including % homologies).
  • the protein moiety may comprise two proteins selected from a CasSl, a CasS2, a CasS3, a CasS4 and a CasS5 protein as described elsewhere herein (including % homologies).
  • the protein moiety may comprise three proteins selected from a CasSl, a CasS2, a CasS3, a CasS4 and a CasS5 protein as described elsewhere herein (including % homologies).
  • the protein moiety may comprise four proteins selected from a CasSl, a CasS2, a CasS3, a CasS4 and a CasS5 protein as described elsewhere herein (including % homologies).
  • the protein moiety may comprise all of a CasSl, a CasS2, a CasS3, a CasS4 and a CasS5 protein as described elsewhere herein (including % homologies).
  • the protein moiety is a fusion protein of the formula A-B-C as described elsewhere herein.
  • the protein moiety comprises proteins as described in the concepts herein.
  • Py is a nuclease.
  • the fusion may be as described in concept 14, 14A, 14B or 14C. Any of the additional features described for those concepts may be applied here.
  • Arrangement 3 The method according to arrangement 2, wherein Px is fused to Py wa a linker, such as a polypeptide linker.
  • the linkers may be any of the linkers described herein.
  • Arrangement 4 The method of any one of arrangements 1 to 3, wherein the first protein moiety is in the form of a first protein moiety complex which comprises at least two further polypeptides, wherein the two further polypeptides each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No: 2, SEQ ID No:4 and SEQ ID No:5, and wherein the first protein moiety complex comprises amino acid sequences that are at least 90% identical to each of the sequences of SEQ ID No:2, SEQ ID No:4 and SEQ ID No:5, and wherein the method comprises expressing the further polypeptides from one or more (e.g. one) vector(s), or from one or more (e.g. one) nucleic acid sequence(s) in the cell.
  • the method comprises expressing the further polypeptides from one or more (e.g. one) vector(s), or from one or more (e.g. one) nucleic acid sequence(s) in the cell.
  • Arrangement 5 The method of arrangement 4, wherein the first protein moiety complex comprises at least three further polypeptides, and wherein the first protein moiety complex comprises amino acid sequences that are at least 90% identical to each of the sequences of SEQ ID No:2, SEQ ID No:3, SEQ ID No:4 and SEQ ID No:5.
  • Arrangement 6 The method of arrangement 4, wherein the first protein moiety complex comprises at least three further polypeptides, and wherein the first protein moiety complex comprises amino acid sequences that are at least 90% identical to each of the sequences of SEQ ID No:l, SEQ ID No:2, SEQ ID No:4 and SEQ ID No:5.
  • Arrangement 7 The method of arrangement 4, wherein the first protein moiety complex comprises at least four further polypeptides, and wherein the first protein moiety complex comprises amino acid sequences that are at least 90% identical to each of the sequences of SEQ ID No:l, SEQ ID No:2, SEQ ID No:3, SEQ ID No:4 and SEQ ID No:5.
  • Arrangement 8 The method of any one of arrangements 1 to 3, wherein Px comprises an amino acid sequence that is selected from an amino acid sequence that is at least 90% identical to the sequence selected from the sequences of SEQ ID Nos:l, 3, and 4; wherein the first protein moiety is in the form of a fusion protein complex which comprises at least two further polypeptides, wherein the two further polypeptides each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:2 and SEQ ID No:5, and
  • polypeptide which is at least 90% identical to a sequence selected from SEQ ID Nos: 1, 3, and 4 and is not based on the amino acid sequence of the polypeptide of Px;
  • polypeptide which is at least 90% identical to a sequence selected from SEQ ID Nos: 1, 3, and 4 and is not based on the amino acid sequence of the polypeptide of Px, and, if present, is not based on the amino acid sequence of the polypeptide recited in part (i), and wherein the method comprises expressing the further polypeptides from one or more (e.g. one) nucleic acid sequence(s) in the cell.
  • Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: 1 and the first protein moiety is in the form of a fusion protein complex which comprises two further polypeptides which each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:2 and SEQ ID No:5.
  • Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: 3 and the first protein moiety is in the form of a fusion protein complex which comprises two further polypeptides which each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:2 and SEQ ID No:5.
  • Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:4 and the first protein moiety is in the form of a fusion protein complex which comprises two further polypeptides which each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:2 and SEQ ID No:5.
  • Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: l
  • the first protein moiety is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:3, SEQ ID No:2 and SEQ ID No:5.
  • Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: l
  • the first protein moiety is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:4, SEQ ID No: 2 and SEQ ID No: 5.
  • Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: 3
  • the first protein moiety is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:l, SEQ ID No: 2 and SEQ ID No: 5.
  • Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: 3
  • the first protein moiety is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:4, SEQ ID No: 2 and SEQ ID No: 5.
  • Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:4, and the first protein moiety is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:l, SEQ ID No: 2 and SEQ ID No: 5.
  • Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:4, and the first protein moiety is in the form of a fusion protein complex which comprises three further polypeptides, wherein the three further polypeptides each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:3, SEQ ID No:2 and SEQ ID No:5.
  • Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: l
  • the first protein moiety is in the form of a fusion protein complex which comprises four further polypeptides, wherein the four further polypeptides each have an amino acid sequence that is at least 90% identical to a sequence selected from the sequences of SEQ ID No:2, SEQ ID No: 5, SEQ ID No:3 and SEQ ID No:4.
  • the fusion protein is expressed from a first nucleic acid in the cell, and the two or more (e.g. three or four) further polypeptides are expressed from a second nucleic acid within the cell. In another embodiment, the fusion protein and the two or more (e.g. three or four) further polypeptides are expressed from a single nucleic acid within the cell.
  • Arrangement 9 The method of any one of arrangements 4 to 8, wherein all of the further polypeptides are expressed from one nucleic acid sequence comprised by the cell.
  • Arrangement 10 The method of any preceding arrangement, wherein at least one nucleic acid (for example each nucleic acid sequence) is comprised by a vector, optionally a plasmid vector.
  • Arrangement 11 The method of any one of arrangements 2 to 10, wherein Py is fused to the C- terminus of Px.
  • Py may alternatively be fused to the N-terminus of Px.
  • nuclease cleavage site nucleotide sequence may be an
  • the I-TevI cleavage site nucleotide sequence may be as described elsewhere herein, such as in concept 43 (and any of the additional features described for that concept.)
  • the I-TevI cleavage site nucleotide sequence is 5'-CNNNG-3', such as wherein the I-TevI cleavage site nucleotide sequence has the nucleotide sequence of SEQ ID No:42.
  • the nuclease spacer nucleotide sequence is present. In another embodiment, the nuclease spacer nucleotide sequence is absent.
  • the nuclease spacer nucleotide sequence is an I-TevI spacer nucleotide sequence.
  • the I-TevI spacer nucleotide sequence may be as described elsewhere herein, such as in Concept 44, or any of the additional features described for that concept.
  • the PAM sequence may be as described elsewhere herein, such as in Concept 39, or any of the additional features described for that concept.
  • the protospacer sequence may be as described elsewhere herein, such as in the First Aspect of Detailed Description, or in Concept 10, or Concept 30 or any of the additional features described for that concept.
  • nucleic acid sequence comprised by the cell' means that the nucleic acid is integrated into the genome of the cell, in particular into the chromosome of the cell.
  • the first protein moiety complex comprises a fusion protein complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a first vector comprised by the cell, and the crRNA is encoded by a second vector comprised by the cell.
  • the first protein moiety complex comprises a fusion protein complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a first nucleic acid comprised by the cell (in particular, a first nucleic acid in the chromosome of the cell), and the crRNA is encoded by a first vector comprised by the cell.
  • the first protein moiety complex comprises a fusion protein complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a first nucleic acid comprised by the cell (in particular, a first nucleic acid in the chromosome of the cell), and the crRNA is encoded by a second nucleic acid comprised by the cell (in particular, a second nucleic acid in the chromosome of the cell).
  • the first protein moiety complex comprises a fusion protein complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a first vector comprised by the cell, and the crRNA is encoded by a first nucleic acid comprised by the cell (in particular, a first nucleic acid in the chromosome of the cell).
  • the first protein moiety complex comprises a fusion protein, which is encoded by a first vector comprised by the cell, complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a second vector comprised by the cell, and the crRNA is encoded by a third vector comprised by the cell.
  • the first protein moiety complex comprises a fusion protein, which is encoded by a first nucleic acid comprised by the cell (in particular, a first nucleic acid in the chromosome of the cell), complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a first vector comprised by the cell, and the crRNA is encoded by a second vector comprised by the cell.
  • the first protein moiety complex comprises a fusion protein, which is encoded by a first vector comprised by the cell, complexed with at least two further (or three further or four further) polypeptides (e.g.
  • the first protein moiety complex comprises a fusion protein, which is encoded by a first vector comprised by the cell, complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a second vector comprised by the cell, and the crRNA is encoded by a nucleic acid comprised by the cell (in particular, a nucleic acid in the chromosome of the cell).
  • the first protein moiety complex comprises a fusion protein, which is encoded by a first nucleic acid comprised by the cell (in particular, a first nucleic acid in the chromosome of the cell), complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a second nucleic acid comprised by the cell (in particular, a second nucleic acid in the chromosome of the cell), and the crRNA is encoded by a vector comprised by the cell.
  • the first protein moiety complex comprises a fusion protein, which is encoded by a first nucleic acid comprised by the cell (in particular, a first nucleic acid in the chromosome of the cell), complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a vector comprised by the cell, and the crRNA is encoded by a second nucleic acid comprised by the cell (in particular, a second nucleic acid in the chromosome of the cell).
  • the first protein moiety complex comprises a fusion protein, which is encoded by a vector comprised by the cell, complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a first nucleic acid comprised by the cell (in particular, a first nucleic acid in the chromosome of the cell), and the crRNA is encoded by a second nucleic acid comprised by the cell (in particular, a second nucleic acid in the chromosome of the cell).
  • the first protein moiety complex comprises a fusion protein, which is encoded by a first nucleic acid comprised by the cell (in particular, a first nucleic acid in the chromosome of the cell), complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a second nucleic acid comprised by the cell (in particular, a second nucleic acid in the chromosome of the cell), and the crRNA is encoded by a third nucleic acid comprised by the cell (in particular, a third nucleic acid in the chromosome of the cell).
  • a fusion protein which is encoded by a first nucleic acid comprised by the cell (in particular, a first nucleic acid in the chromosome of the cell), complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein) which are encoded by a second nucleic acid comprised by
  • the first protein moiety complex comprises a fusion protein is complexed with at least two further (or three further or four further) polypeptides (e.g. as described anywhere elsewhere herein), and each of the fusion protein, the at least two further (or three further or four further) polypeptides, and the crRNA are encoded by a single nucleic acid comprised by the cell (in particular, a single nucleic acid in the chromosome of the cell).
  • Arrangement 14 The method of arrangement 13, wherein the first protein moiety is in the form of a first protein moiety complex which comprises at least two further polypeptides, wherein the first protein moiety is encoded by a single first nucleic acid sequence (e.g. comprised by a first vector), and wherein the at least two further polypeptides are encoded by a second nucleic acid sequence (e.g. comprised by a second vector), optionally wherein all of the further polypeptides are encoded by a second nucleic acid sequence (e.g. comprised by a second vector).
  • the first and/or second nucleic acid sequences may be nucleic acid sequences comprised by the cell (i.e. integrated into the genome or, in particular, into the chromosome of the cell).
  • Arrangement 15 The method of arrangement 13, wherein the first protein moiety is in the form of a first protein moiety complex which comprises at least two further polypeptides, wherein the first protein moiety complex is encoded by: a) a first nucleic acid sequence encoding the first protein moiety which comprises Py fused to Px, and in which Px comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No:l, SEQ ID No: 3 or SEQ ID No:4; and b) a second nucleic acid sequence encoding the at least two further polypeptides encoding amino acid sequences that are at least 90% identical to the sequences of SEQ ID Nos:l, 2, 3, 4 and/or 5, optionally wherein the first protein moiety complex comprises an amino acid sequence at least 90% identical to the each of the sequences of SEQ ID Nos:l, 2, 3, 4 and 5.
  • the first and/or second nucleic acid sequences may be nucleic acid sequences comprised by the cell (i.e. integrated into the genome or, in particular, into the chromosome of the cell).
  • Arrangement 16 The method of any one of arrangements 13 to 15, wherein the crRNA is encoded by the first vector or nucleic acid sequence, or is encoded by the second vector or nucleic acid sequence.
  • the first and/or second nucleic acid sequences may be nucleic acid sequences comprised by the cell (e.g. integrated into the genome or, in particular, into the chromosome of the cell).
  • Arrangement 17 The method of any preceding arrangement, wherein at least one of the nucleic acid sequences encoding the first protein moiety and/or the at least two further polypeptides is operably connected to a promoter that is i. heterologous to the at least one nucleic acid sequence(s) encoding the first protein moiety and/or the at least two further polypeptides; ii. a eukaryotic cell promoter; iii. an animal promoter (optionally a mammalian or human promoter); iv. a plant promoter; v. a fungus promoter (optionally a yeast promoter); vi. an insect promoter; vii. a virus promoter (optionally a virus, AAV or lentivirus promoter); or viii. a synthetic promoter.
  • a promoter that is i. heterologous to the at least one nucleic acid sequence(s) encoding the first protein moiety and/or the at least two further polypeptides;
  • the promoters may be any of the promoters described elsewhere herein.
  • Arrangement 18 The method of any one of arrangements 11 to 15, wherein at least one of the nucleic acid sequences encoding the first protein moiety and/or the at least two further polypeptides is operably connected to an inducible promoter.
  • Arrangement 19 The method of arrangement 18, wherein the inducible promoter is selected from an aTc (anhydrotetracycline)-inducible Tet promoter, an IPTG or lactose-inducible Lac promoter, a benzoic acid-inducible XylS/Pm promoter, an arabinose-inducible Ara promoter, a rhamnose- inducible Rha promoter, a bile acid-inducible BetA promoter, temperature controlled promoters and porphyrin-inducible promoters, and synthetic derivatives thereof.
  • aTc anhydrotetracycline
  • Tet promoter an IPTG or lactose-inducible Lac promoter
  • benzoic acid-inducible XylS/Pm promoter an arabinose-inducible Ara promoter
  • a rhamnose- inducible Rha promoter a bile acid-inducible BetA promoter
  • aTc (anhydrotetracycline)-inducible Tet promoters are described, for example, in Rodriguez-Garcia et al., "Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyced' , Nucleic Acids Research, 33(9), 1 May 2005, Page e87, https://doi.org/10.1093/nar/gni086 and in Rincon & Farny, "Unlocking the strength of inducible promoters in Gram-negative bacteria", Microb. Biotechnol., 16(5), 2023, 961-976, doi: 10.1111/1751-7915.14219, both of which are incorporated herein by reference in their entirety.
  • IPTG or lactose-inducible Lac promoters are described, for example, in Rincon & Farny, supra.
  • Benzoic acid-inducible XylS/Pm promoters are described, for example, in Gawin eta/., " The Xy/S/Pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering' , Microb. Biotechnol., 10(4), 2017, 702-718, doi: 10.1111/1751-7915.12701, which is incorporated herein by reference in its entirety.
  • Arabinose-inducible Ara promoters are described, for example, in Guzman et al, " Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter, J. Bacteriol., 177(14), 1995, 4121-30, doi: 10.1128/jb.l77.14.4121-4130.1995, which is incorporated herein by reference in its entirety.
  • Rhamnose-inducible Rha promoters are described, for example, in Wegerer et al, "Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations", BMC Biotechnology, volume s, Article number: 2, 2008, doi: https://doi.org/10.1186/1472-6750-8-2; Kelly et al, "4 Rhamnose-Inducible System for Precise and Temporal Control of Gene Expression in Cyanobacterid' , ACS Synth.
  • Bile acid-inducible BetA promoters are described for example, in Ruiz et al, "Controlled Gene Expression in Bifidobacteria by Use of a Biie-Responsive Element', Appl. Environ. Microbiol., 78(2), 2012, 581-585, doi: 10.1128/AEM.06611-11, which is incorporated herein by reference in its entirety.
  • Temperature controlled promoters are described, for example, in Villaverde et al, "Fine regulation of cI857-controiied gene expression in continuous culture of recombinant Escherichia coil by temperaturd', Appl. Environ. Microbiol., 59(10), 1993, 3485-3487, doi: 10.1128/aem.59.10.3485-3487.1993, which is incorporated herein by reference in its entirety.
  • Porphyrin-inducible promoters are described for example in W02020/252370A1 (Novome Biotechnologies, Inc.), which is incorporated herein by reference in its entirety.
  • inducible promoters that may be used include chondroitin sulfate-inducible promoters, and arabinogalactan-inducible promoters, which are described for example, in Mimee etal, supra.
  • Arrangement 20 The method of arrangement 18 or arrangement 19, wherein the method further comprises the step of exposing (or subjecting) the cell to conditions to induce expression of the inducible promoter.
  • Arrangement 21 The method of any preceding arrangement, wherein the crRNA comprises two repeat sequences.
  • the crRNA may be as described elsewhere herein.
  • Arrangement 22 The method of any preceding arrangement, wherein the crRNA comprises a repeat sequence comprising the nucleotide sequence of SEQ ID No:6 or SEQ ID No:70.
  • Arrangement 23A The method of any preceding arrangement, wherein Py is a nuclease.
  • Arrangement 23B The method of any preceding arrangement, wherein Py is an I-TevI nuclease.
  • Arrangement 23C The method of any preceding arrangement, wherein Py is an I-TevI nuclease which is fused to the N-terminus of Px.
  • Arrangement 23D The method of any preceding arrangement, wherein Py is an I-TevI nuclease which is fused to the N-terminus of Px, which comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID No: l.
  • the amino acid sequence may be at least (about) 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to SEQ ID No:l.
  • the identity is at least (about) 80%.
  • the identity is at least (about) 90 or 95%.
  • the identity is at least (about) 96%.
  • the identity is at least (about) 97%.
  • the identity is at least about 98%.
  • the identity is at least about 99%.
  • the I-TevI nuclease may be as described elsewhere herein (e.g. as in Concept 14).
  • Arrangement 24 The method of arrangement 23, wherein the I-TevI nuclease is devoid of a complete DNA binding domain.
  • the I-TevI nuclease does not comprise a complete DNA binding domain.
  • the I-TevI nuclease which is devoid of a complete DNA binding domain may be as described elsewhere herein, such as in Concept 22, or any of the additional features described for that concept.
  • Arrangement 25 The method of arrangement 23 or arrangement 24, wherein the I-TevI nuclease comprises a N-terminal catalytic domain.
  • the expressed I-TevI may comprise amino acids 1 to 92 of SEQ ID No:45.
  • the expressed I-TevI may comprise the amino acid sequence of SEQ ID No:45.
  • Arrangement 26A The method of any one of arrangements 23 to 25, wherein the I-TevI nuclease is a bacteriophage I-TevI nuclease.
  • Arrangement 26B The method of any one of arrangements 23 to 25, wherein the I-TevI nuclease is a T4 bacteriophage I-TevI.
  • the I-TevI nuclease may be a T4 bacteriophage I-TevI.
  • the I-TevI nuclease may be from a T4 bacteriophage.
  • the I-TevI nuclease may be encoded by the intron of bacteriophage T4.
  • the I- TevI nuclease may be encoded by a nucleic acid sequence of SEQ ID No:44.
  • Arrangement 27A The method of any one of arrangements 23 to 26, wherein the expressed I- TevI nuclease comprises amino acids 1 to 206 of a naturally occurring I-TevI nuclease.
  • Arrangement 27B The method of any one of arrangements 23 to 26, wherein the expressed I- TevI nuclease comprises the amino acid sequence of SEQ ID No:45.
  • Arrangement 28 The method of any one of arrangements 23 to 27, wherein the target sequence comprises, in 5' to 3' orientation: a) an I-TevI cleavage site nucleotide sequence; b) an I-TevI spacer nucleotide sequence; c) a PAM sequence; and d) a protospacer sequence (optionally wherein the protospacer sequence is about 32 base pairs in length (e.g. is 32 base pairs in length), and optionally wherein sequences a) to d) are each immediately adjacent to each other.
  • the target sequence comprises, in 5' to 3' orientation: a) an I-TevI cleavage site nucleotide sequence; b) an I-TevI spacer nucleotide sequence; c) a PAM sequence; and d) a protospacer sequence (optionally wherein the protospacer sequence is about 32 base pairs in length (e.g. is 32 base pairs in length), and optionally wherein sequences a) to
  • I-TevI cleavage site nucleotide sequence may be as described elsewhere herein, such as in Concept 43, or any of the additional features described for that concept.
  • the I-TevI spacer nucleotide sequence may be as described elsewhere herein, such as in Concept 44, or any of the additional features described for that concept.
  • the PAM sequence may be as described elsewhere herein, such as in Concept 39, or any of the additional features described for that concept.
  • the protospacer sequence may be as described elsewhere herein, such as in the First Aspect of Detailed Description, or in Concept 10, or Concept 30, or any of the additional features described for those concepts.
  • Arrangement 30A The method of any preceding arrangement, wherein the protospacer is immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence in the target sequence selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG-3', 5'-AAT-3', 5'-ACA-3', 5'-ACT- 3', 5'-ATC-3', 5'-ATA-3', 5'-GAG-3', 5'-TAG-3', 5'-ACC-3', 5'-AGG-3', 5'-ATT-3', 5'-GAC-3' and 5'- GTG-3'.
  • PAM Protospacer Adjacent Motif
  • Arrangement 30C The method of any preceding arrangement, wherein the protospacer is immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence in the target sequence selected from 5'-AAC-3', 5'-ATG-3', 5'-AAA-3', 5'-AAG-3', 5'-ACG-3', 5'-AAT-3' and 5'-ACA-3'.
  • PAM Protospacer Adjacent Motif
  • Arrangement 30D The method of any preceding arrangement, wherein the protospacer is immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence in the target sequence is selected from 5'-AAC-3' and 5'-ATG-3'.
  • PAM Protospacer Adjacent Motif
  • Arrangement 30E The method of any preceding arrangement, wherein the protospacer is immediately adjacent to a Protospacer Adjacent Motif (PAM) sequence in the target sequence is 5'-AAC-3'.
  • PAM Protospacer Adjacent Motif
  • the PAM is according to Arrangement 30E.
  • Arrangement 31A The method of any preceding arrangement, wherein the crRNA spacer sequence is from 25 to 39 nucleotides in length.
  • Arrangement 31B The method of any preceding arrangement, wherein the crRNA spacer sequence is from 28 to 32 nucleotides in length.
  • Arrangement 31C The method of any preceding arrangement, wherein the crRNA spacer sequence is about 32 nucleotides in length.
  • the crRNA spacer sequence may be as described elsewhere herein, such as in Concept 40, or any of the additional features described for that concept.
  • Arrangement 32 The method of any ones of arrangements 31A-D, wherein base pairs 1 to 28 of the spacer are identical to the complement of nucleotides 1 to 28 of the protospacer sequence which is immediately 5' of the PAM sequence in the target sequence.
  • the protospacer may be as described elsewhere herein, such as in the First Aspect of Detailed Description, or in Concept 10, or Concept 30, or any of the additional features described for those concepts.
  • Arrangement 33 The method of arrangement 31A-D or arrangement 32, wherein the spacer is identical to the complement of the protospacer in the target sequence.
  • the spacer may be as described elsewhere herein, such as in Concept 42, or any of the additional features described for that concept.
  • Arrangement 34 The method of any preceding arrangement, wherein Py is an I-TevI nuclease, and the I-TevI nuclease recognises an I-TevI cleavage site nucleotide sequence which is 5' or 3' (e.g. 5') of the protospacer in the target sequence.
  • the I-TevI cleavage site nucleotide sequence is 5' of the protospacer.
  • I-TevI cleavage site nucleotide sequence may be as described elsewhere herein, such as in Concept 43 or 44, or any of the additional features described for those concepts.
  • Arrangement 35A The method of arrangement 34, wherein the I-TevI cleavage site nucleotide sequence is between about 3 and 7 nucleotides in length.
  • Arrangement 35B The method of arrangement 34, wherein the I-TevI cleavage site nucleotide sequence is about 5 nucleotides in length.
  • Arrangement 35C The method of arrangement 34, wherein the I-TevI cleavage site nucleotide sequence is 5'-CNNNG-3'.
  • Arrangement 36A The method of any one of arrangements 35A-C, wherein the I-TevI cleavage site nucleotide sequence is selected from 5'-CCACG-3', 5'-CACCG-3', 5'-CATAG-3', 5'-CTAAG-3', 5'- CTGAG-3', 5'-CTTAG-3', 5'-CCTCG-3', 5'-CAGCG-3', 5'-CACTG-3', 5'-CGACG-3', 5'-CGATG-3', 5'- CTCCG-3', 5'-CTACG-3', 5'-CTGTG-3', 5'-CTGCG-3', 5'-CCGTG-3', 5'-CCTAG-3', 5'-CCATG-3', 5'- CAAAG-3', 5'-CAGAG-3', 5'-CATTG-3' and 5'-CATCG-3'.
  • Arrangement 36B The method of any one of arrangements 35A-C, wherein the I-TevI cleavage site nucleotide sequence is selected from 5'-CCACG-3', 5'-CACCG-3', 5'-CATAG-3', 5'-CTAAG-3', 5'- CTGAG-3', 5'-CTTAG-3', 5'-CCTCG-3' and 5'-CAGCG-3'.
  • Arrangement 36C The method of any one of arrangements 35A-C, wherein the I-TevI cleavage site nucleotide sequence is selected from 5'-CCACG-3', 5'-CACCG-3', 5'-CATAG-3', 5'-CTAAG-3' and 5'-CTGAG-3'.
  • Arrangement 36D The method of any one of arrangements 35A-C, wherein the I-TevI cleavage site nucleotide sequence is selected from 5'-CCACG-3', 5'-CACCG-3' and 5'-CATAG-3'.
  • Arrangement 36E The method of any one of arrangements 35A-C, wherein the I-TevI cleavage site nucleotide sequence is selected from 5'-CCACG-3' and 5'-CACCG-3'
  • Arrangement 36F The method of any one of arrangements 35A-C, wherein the I-TevI cleavage site nucleotide sequence has the nucleotide sequence of SEQ ID No:42.
  • the I-TevI cleavage site nucleotide sequence has the nucleotide sequence of SEQ ID No:42.
  • Arrangement 37 The method of any preceding arrangement, wherein Py is an I-TevI nuclease, and the I-TevI nuclease recognises an I-TevI spacer nucleotide sequence which is 5' or 3' (e.g. 5') of the protospacer in the target sequence.
  • the I-TevI spacer nucleotide sequence is 5' of the protospacer.
  • I-TevI cleavage site nucleotide sequence may be as described elsewhere herein, such as in Concept 44, or any of the additional features described for that concept.
  • Arrangement 38A The method of arrangement 37, wherein the I-TevI spacer nucleotide sequence is between about 10 and 35 nucleotides in length.
  • Arrangement 38B The method of arrangement 37, wherein the I-TevI spacer nucleotide sequence is between about 29 and 33 nucleotides in length.
  • Arrangement 38C The method of arrangement 37, wherein the I-TevI spacer nucleotide sequence is about 31 nucleotides in length.
  • Arrangement 38D The method of arrangement 37, wherein the I-TevI spacer nucleotide sequence is at least about 90% identical to the nucleotide sequence of SEQ ID No:43.
  • Arrangement 38E The method of arrangement 37, wherein the I-TevI spacer nucleotide sequence has the nucleotide sequence of SEQ ID No:43.
  • I-TevI spacer nucleotide sequence has the nucleotide sequence of SEQ ID No:43.
  • Arrangement 39 The method of any preceding arrangement, wherein the cell comprises one or more exogenous genes comprised by one or more operons (e.g. by one operon).
  • Modified bacteria are proving useful to provide therapeutic effects in patients, such as in humans.
  • a bacteria can be engineered to produce drugs which provide a therapeutic effect in a microbiome (e.g. in any microbiome described herein), or to contain metabolic pathways which convert one detrimental molecule or metabolite into one which either has a neutral effect in the local environment, or indeed even a beneficial effect in said environment.
  • a microbiome e.g. in any microbiome described herein
  • metabolic pathways which convert one detrimental molecule or metabolite into one which either has a neutral effect in the local environment, or indeed even a beneficial effect in said environment.
  • CasS-nuclease complexes or fusions as described herein can provide a biocontainment strategy for such engineered bacteria.
  • any exogenous genes can be reduced or prevented by targeted modification of the operon, (i) the growth or destruction of the cell in its entirety can be achieved by modification of a gene essential to growth or maintenance of the cell, or (iii) the cell can be destroyed (i.e. killed) by cutting the chromosome of the cell.
  • the cell comprises a single operon under the control of a single promoter, which operon comprises one or more exogenous genes.
  • the cell comprises two operons, each under the control of a separate promoter, each operon comprising one or more exogenous genes.
  • One operon may comprise one or more exogenous genes encoding an importer of a molecule of interest.
  • One operon may comprise one or more exogenous genes encoding an exporter of a molecule of interest.
  • One operon may comprise one or more exogenous genes encoding for expression of a molecule of interest.
  • One operon may comprise one or more exogenous genes encoding for conversion of a first molecule of interest into a second molecule of interest.
  • the target sequence may be comprised by the operon, e.g. by a promoter or a coding or non-coding sequence of the operon.
  • the target sequence is immediately adjacent to an operon, e.g. immediately upstream of a promoter comprised by the operon.
  • Metabolic pathways, exogenous genes, exporters and importers may be those described in W02016/141108, W02016/201380, WO2017/074566, WO2017/123418, WO2017/136792, W02019/014391, WO2019/232415, W02020/097424, W02020/232063, W02020/257610, W02020/257707, WO2021/146394, WO2021/146397, WO2021/163421, WO2021/173808, WO2021/188618, WO2021/188819, WO2021/242897, WO2022/072636, WO2022/146718,
  • WO2022/150779, W02022/204406, WO2022/221273 and WO2023/225667 (all owned by Synlogic Operating Company, Inc); US10,993,930B2, US10,300,043B2, US11, 878,00262, WO2020/123483, WO2022/169909 (all owned by Novome Biotechnologies, Inc), each of which is incorporated herein by reference in its entirety.
  • the patient is an animal.
  • the patient may be a livestock or companion pet animal (e.g. a cow, pig, goat, sheep, horse, dog, cat or rabbit).
  • the patient may be an insect (an insect at any stage of its lifecycle, e.g. egg, larva or pupa).
  • Uses of engineered bacteria for animal husbandry, veterinary and food purposes are described in WO2020/229372 (Folium Food Science Limited), which is incorporated herein by reference in its entirety.
  • the engineered bacteria may have applications in modification of environmental microbiota (e.g. a waterway microbiota, a water microbiota, a soil microbiota).
  • the environmental microbiota may be a marine or waterway (e.g. lake, canal, river or reservoir) environmental microbiota, an example, the water environment is drinking water intended for human consumption or sewage water.
  • the soil environmental microbiota is a microbiota comprised by soil of farming land or soil at a mining site (e.g. a mineral or metal mining site).
  • the environmental microbiota may be an air environmental microbiota.
  • the environmental microbiota may be an agricultural environmental microbiota.
  • the environmental microbiota is an oil or petroleum recovery environmental microbiota, e.g. an oil or petroleum field or well.
  • the environmental microbiota may be an environmental microbiota in or on a foodstuff or beverage for human or non-human animal consumption.
  • the engineered bacteria can be used to convert a harmful or detrimental environmental microbiota agent, such as a soil or waterway component, into a less toxic or safe environmental microbiota agent or component, for example.
  • AcidithiobaciHus bacteria produce sulphuric acid, which frequently damages sewer pipes.
  • Ferrobacillus ferrooxidans directly oxidises iron to iron oxides and iron hydroxides.
  • Other bacteria produce various acids, both organic and mineral, or ammonia.
  • aerobic bacteria like Thiobacillus thiooxidans, Thiobacillus thioparus, and Thiobacillus concretivorus, all three widely present in the environment, are the common corrosion-causing factors resulting in biogenic sulphide corrosion.
  • the engineered bacteria described herein may breakdown these harmful products (e.g. iron hydroxide, iron oxides, sulfuric acid) or may alter the bacteria such that they no longer produce those harmful products.
  • the engineered bacteria may be for use in an industry or the environment is an industrial environment, wherein the industry is an industry of a field selected from the group consisting of the medical and healthcare; pharmaceutical; human food; animal food; plant fertilizers; beverage; dairy; meat processing; agriculture; livestock farming; poultry farming; fish and shellfish farming; veterinary; oil; gas; petrochemical; water treatment; sewage treatment; packaging; electronics and computer; personal healthcare and toiletries; cosmetics; dental; non-medical dental; ophthalmic; non-medical ophthalmic; mineral mining and processing; metals mining and processing; quarrying; aviation; automotive; rail; shipping; space; environmental; soil treatment; pulp and paper; clothing manufacture; dyes; printing; adhesives; air treatment; solvents; biodefence; vitamin supplements; cold storage; fibre retting and production; biotechnology; chemical; industrial cleaning products; domestic cleaning products; soaps and detergents; consumer products; forestry; fishing; leisure; recycling; plastics; hide, leather and suede; waste management; funeral and undertaking; fuel; building; energy; steel
  • Arrangement 40 The method of arrangement 39, wherein the one or more operons are under the control of a constitutive promoter.
  • Constitutive promoters may be as described elsewhere herein, such as the constitutive promoter of the bo/A gene (SEQ ID No: 14) (pboiA).
  • Arrangement 41 The method of arrangement 39 or arrangement 40, wherein the one or more operons produce a first molecule of interest in the cell.
  • the first molecule of interest may be a therapeutic protein, e.g. an antibody, antibody fragment (such as an antibody single variable domain), TCR (T-cell receptor) binding site, TCR variable domain, hormone, incretin, growth factor, anti-cancer agent, neurotransmitter or enzyme.
  • a therapeutic protein e.g. an antibody, antibody fragment (such as an antibody single variable domain), TCR (T-cell receptor) binding site, TCR variable domain, hormone, incretin, growth factor, anti-cancer agent, neurotransmitter or enzyme.
  • Arrangement 42 The method of arrangement 41, wherein the molecule of interest is selected from a therapeutic molecule and a beneficial metabolite.
  • the therapeutic molecule may be a therapeutic protein, e.g. as described above.
  • Arrangement 43 The method of arrangement 39 or arrangement 40, wherein the one or more operons comprise one or more exogenous genes for the conversion of a first molecule of interest (e.g. a metabolite) within the cell to a second molecule of interest (e.g. a beneficial molecule).
  • a first molecule of interest e.g. a metabolite
  • a second molecule of interest e.g. a beneficial molecule
  • the one or more operons may metabolise a harmful toxin into a second molecule which may either be neutral to the cell (e.g. able to be secreted without any detrimental effect) or may be beneficial to the cell or to the local environment to the cell (e.g. may cause beneficial effects in the gut microbiome).
  • the conversion is of a harmful metabolite (e.g. tryptophan) to one which is beneficial to the gut (e.g. indole-3-acetic acid, IAA).
  • Arrangement 44 The method of any one of arrangements 39 to 43, wherein the one or more exogenous genes encodes an exporter and/or importer of a further molecule of interest.
  • Arrangement 45 The method of any preceding arrangement, wherein the target sequence is comprised by the chromosome of the cell.
  • This embodiment is useful when the bacterial cell has been engineered to include exogenous gene operons which are designed to produce a molecule of interest (e.g. cells which contain one or more exogenous genes for the conversion of one metabolite into another, or contain one or more exogenous genes which reduce the amounts of a disadvantageous molecule in the local environment) and the operon(s) have been engineered into the chromosome for stable transmission between generations.
  • exogenous gene operons which are designed to produce a molecule of interest (e.g. cells which contain one or more exogenous genes for the conversion of one metabolite into another, or contain one or more exogenous genes which reduce the amounts of a disadvantageous molecule in the local environment) and the operon(s) have been engineered into the chromosome for stable transmission between generations.
  • the expression of the CasS-nuclease complexes or fusions may cut the target sequence, and result in death of the cell, stopping the expression of the one or more exogenous genes.
  • the target sequence is comprised by the chromosome of the cell, and the modification cuts the DNA and results in the death of the cell.
  • the expression of the CasS-nuclease complexes or fusions may modify the target sequence which is comprised by a gene essential to growth or maintenance of the cell, or where the target sequence is comprised by the operon, and results in a reduction of growth of the cell, reducing the expression of the one or more exogenous genes in the operon.
  • the expression of the CasS-nuclease complexes or fusions may modify the target sequence which is comprised by the one or more exogenous genes in the operon, and results in a reduction of expression of the one or more exogenous genes.
  • the target sequence is comprised by the chromosome of the cell, and the modification results in the death of the cell.
  • the modification may be double-stranded cutting of the chromosome.
  • the target sequence is comprised by the chromosome of the cell, and the modification results in a reduction in growth of the cell.
  • the modification may be singlestranded cutting of the chromosome.
  • the target sequence is comprised by the chromosome of the cell, and the modification prevents or reduces expression of one or more exogenous genes comprised by the cell.
  • the modification may be single-stranded cutting of the chromosome.
  • Arrangement 46 The method of any one of arrangements 1 to 44, wherein the target sequence is comprised by a plasmid within the cell.
  • This alternative embodiment is useful when the bacterial cell has been engineered to include a plasmid or vector which comprises exogenous gene operons which are designed to produce a molecule of interest (e.g. cells which contain one or more exogenous genes for the conversion of one metabolite into another, or contain one or more exogenous genes which reduce the amounts of a disadvantageous molecule in the local environment).
  • a plasmid or vector which comprises exogenous gene operons which are designed to produce a molecule of interest (e.g. cells which contain one or more exogenous genes for the conversion of one metabolite into another, or contain one or more exogenous genes which reduce the amounts of a disadvantageous molecule in the local environment).
  • the expression of the CasS-nuclease complexes or fusions may cut the target sequence in the plasmid, and result in destruction of the plasmid, also stopping the expression of the exogenous operon.
  • the target sequence is comprised by a plasmid within the cell, and the modification prevents or reduces expression of one or more exogenous genes comprised by the cell.
  • the modification may be double-stranded cutting of the plasmid which prevents expression of one or more exogenous genes by destroying the plasmid.
  • the modification may be single-stranded cutting of the plasmid which reduces expression of one or more exogenous genes.
  • the target sequence may be comprised within an antibiotic resistance gene comprised by a plasmid within the cell, and the method further comprises administration of the antibiotic to which the resistance gene conferred protection, thereby resulting in death of the cell which comprised the antibiotic resistant-plasmid.
  • the one or more exogenous genes may be comprised by the chromosome of the cell or by a second plasmid within the cell.
  • Arrangement 47 The method of arrangement 46, wherein the modification prevents or reduces expression of one or more exogenous genes comprised by the cell.
  • Arrangement 48A The method of any one of arrangements 39 to 47, wherein the target sequence is comprised by the one or more operons.
  • Arrangement 48B The method of any one of arrangements 39 to 47, wherein the target sequence is immediately adjacent to one operons (e.g. is immediately upstream of the promoter of one operon).
  • Arrangement 49 The method of arrangement 48A, wherein the target sequence is comprised by a promoter within the one or more operons.
  • Arrangement 50A The method of any preceding arrangement, wherein the modification of the target nucleic acid is cutting of the nucleic acid.
  • Arrangement 50B The method of any preceding arrangement, wherein the modification of the target nucleic acid is double-stranded cutting of the nucleic acid.
  • the cutting is of single stranded DNA or RNA, wherein Py comprises a nickase or RNA nuclease.
  • Py comprises a nickase or RNA nuclease.
  • the nickase, RNA nuclease and single stranded DNA or RNA cutting may be as described elsewhere herein, such as in the Detailed Description, in Concept 8B, Concept 73B, Concept 74A, 74B or 74C, or Concept 79B or any of the additional features described for those concepts.
  • the modification is cutting of a double-stranded DNA
  • Py comprises a nuclease, such as an I-TevI nuclease.
  • the nuclease and double-stranded DNA cutting may as described elsewhere herein, such as in the Detailed Description, in Concept 8B, Concept 73A or 73C or any of the additional features described for those concepts.
  • Mechanisms for selectively killing bacteria may as described elsewhere herein, such as in the Detailed Description, in Concept 76B or any of the additional features described for those concepts.
  • Arrangement 52 The method of arrangement 51, wherein the method is carried out on a population of cells, and wherein the method results in death of at least (about) 70% of the cells in the population which comprise the target sequence.
  • the method results in death of at least (about) 80%, (about) 85%, (about) 90%, (about) 92%, (about) 93%, (about) 94%, (about) 95%, (about) 96%, (about) 97%, (about) 98%, (about) 99% of the cells in the population which comprise the target sequence.
  • it is not always possible to kill the entire population of bacteria, due to events like escape mutants forming. However, it is desired that most of the population is killed.
  • Arrangement 53 The method of any one of arrangements 1 to 50, wherein the modification of the target nucleic acid modulates (e.g. up- or down-reg ulates) expression of a gene comprised by the cell, and wherein the growth of the cell is reduced.
  • the modification of the target nucleic acid modulates (e.g. up- or down-reg ulates) expression of a gene comprised by the cell, and wherein the growth of the cell is reduced.
  • Mechanisms and features relating to regulating expression of genes in bacteria(e.g. to reduce growth) may as described elsewhere herein, such as in the Detailed Description, in Concept 48B, Concept 53, Concept 58A, Concept 58C, Concept 76A, Concept 76B, Concept 82 or Concept 103 or any of the additional features described for those concepts.
  • Arrangement 54 The method of arrangement 53, wherein the method results in death of the cell, optionally wherein the method is carried out on a population of cells, and the method results in death of at least (about) 70% (for example at least (about) 80%, (about) 85%, (about) 90%, (about) 92%, (about) 93%, (about) 94%, (about) 95%, (about) 96%, (about) 97%, (about) 98%, (about) 99%) of the cells in the population which comprise the target sequence.
  • 70% for example at least (about) 80%, (about) 85%, (about) 90%, (about) 92%, (about) 93%, (about) 94%, (about) 95%, (about) 96%, (about) 97%, (about) 98%, (about) 99%) of the cells in the population which comprise the target sequence.
  • the method results in death of at least (about) 80%, (about) 85%, (about) 90%, (about) 92%, (about) 93%, (about) 94%, (about) 95%, (about) 96%, (about) 97%, (about) 98%, (about) 99% of the cells in the population which comprise the target sequence. If the expression of an essential gene or a gene essential to maintenance of the cell is sufficiently reduced, then the cell may die, as opposed to no longer reproducing, and being in an inert, stable state (stationary phase). As is known to those skilled in the art, it is not always possible to kill the entire population of bacteria in this embodiment, due to e.g. some of the cells remaining in the stationary phase. However, it is desired that most of the population is killed.
  • Arrangement 55 The method of arrangement 53, wherein the method is carried out on a population of cells, and the method results in a reduction in growth of at least (about) 70% of the cells in the population which comprise the target sequence.
  • the method results in a reduction in growth of at least (about) 80%, (about) 85%, (about) 90%, (about) 92%, (about) 93%, (about) 94%, (about) 95%, (about) 96%, (about) 97%, (about) 98%, (about) 99% of the cells in the population which comprise the target sequence.
  • the cells are growing (e.g. in the exponential phase)
  • the speed of growth is reduced.
  • the growth of the population is arrested, such that the cells enter the stationary phase.
  • a modified prokaryotic cell comprising a target sequence which comprises, in 5' to 3' orientation: a) a nuclease cleavage site nucleotide sequence; b) optionally a nuclease spacer nucleotide sequence; c) a PAM sequence; and d) a protospacer sequence.
  • one or more of a) to d) are non-native (i.e. exogenous) sequence(s) to the cell. In another embodiment, all of a) to d) are non-native (i.e. exogenous) sequences to cell. In another embodiment, a) and/or b) are non-native (i.e. exogenous) sequences to cell.
  • the nuclease cleavage site nucleotide sequence may be an I-TevI cleavage site nucleotide sequence.
  • the I-TevI cleavage site nucleotide sequence may be as described elsewhere herein, such as in concept 43 (and any of the additional features described for that concept.)
  • the I-TevI cleavage site nucleotide sequence is 5'-CNNNG-3', such as wherein the I-TevI cleavage site nucleotide sequence has the nucleotide sequence of SEQ ID No:42.
  • nuclease spacer nucleotide sequence is present. In another embodiment, the nuclease spacer nucleotide sequence is absent.
  • the nuclease spacer nucleotide sequence is an I-TevI spacer nucleotide sequence.
  • the I-TevI spacer nucleotide sequence may be as described elsewhere herein, such as in Concept 44, or any of the additional features described for that concept.
  • the PAM sequence may be as described elsewhere herein, such as in Concept 39, or any of the additional features described for that concept.
  • the protospacer sequence may be as described elsewhere herein, such as in the First Aspect of Detailed Description, or in Concept 10, or Concept 30 or any of the additional features described for that concept.
  • a protospacer sequence (optionally wherein the protospacer sequence is about 32 base pairs in length (e.g. is 32 base pairs in length) or is as recited in arrangement 29, optionally wherein sequences a) to d) are each immediately adjacent to each other.
  • one or more of a) to d) are non-native (i.e. exogenous) sequence(s) to the cell. In another embodiment, all of a) to d) are non-native (i.e. exogenous) sequences to cell. In another embodiment, a) and/or b) are non-native (i.e. exogenous) sequences to cell.
  • I-TevI cleavage site nucleotide sequence may be as described elsewhere herein, such as in Concept 43, or any of the additional features described for that concept.
  • the I-TevI spacer nucleotide sequence may be as described elsewhere herein, such as in Concept 44, or any of the additional features described for that concept.
  • the PAM sequence may be as described elsewhere herein, such as in Concept 39, or any of the additional features described for that concept.
  • the protospacer sequence may be as described elsewhere herein, such as in the First Aspect of Detailed Description, or in Concept 10, or Concept 30, or any of the additional features described for those concepts.
  • Arrangement 58 The modified cell of arrangement 56 or arrangement 57, wherein the cell further comprises one or more vector(s), or one or more nucleic acid sequence(s) encoding (i) a first protein moiety or first protein moiety complex, and (ii) a crRNA as recited in any one of arrangements 1 to 19 or 21 to 27 or 31 to 33.
  • Arrangement 59 The modified cell of any one of arrangements 56 to 57, wherein the cell comprises one or more exogenous genes comprised by one or more operons (e.g. by one operon).
  • Arrangement 60 The modified cell of arrangement 59, wherein the one or more operons are under the control of a constitutive promoter.
  • Arrangement 61 The modified cell of arrangement 58 or arrangement 59, wherein the one or more operons produce a first molecule of interest in the cell.
  • Arrangement 62 The modified cell of arrangement 61, wherein the molecule of interest is selected from a therapeutic molecule and a beneficial metabolite.
  • Arrangement 63 The modified cell of arrangement 59 or arrangement 60, wherein the one or more operons comprise one or more exogenous genes for the conversion of a first molecule of interest (e.g. a metabolite) within the cell to a second molecule of interest (e.g. a beneficial molecule).
  • a first molecule of interest e.g. a metabolite
  • a second molecule of interest e.g. a beneficial molecule
  • Arrangement 64 The modified cell of any one of arrangements 59 to 63, wherein the one or more exogenous genes encodes an exporter and/or importer of a further molecule of interest.
  • Arrangement 65 A composition (optionally an in vitro composition or wherein the composition is comprised by a medical container) comprising the cell recited in any one of arrangements 56 to 64.
  • composition may be as described elsewhere herein, such as in the Detailed Description, in Concept 47A or any of the additional features described for that concept.
  • Arrangement 66 A method of modifying a prokaryotic cell to comprise a target sequence wherein the target sequence comprises, in 5' to 3' orientation: a) a nuclease cleavage site nucleotide sequence; b) optionally a nuclease spacer nucleotide sequence; c) a PAM sequence; and d) a protospacer sequence, and optionally wherein sequences a) to d) are each immediately adjacent to each other, said method comprising introducing one or more modifications into the cell to produce the target sequence; and introducing into the cell one or more vector(s) or one or more nucleic acid sequence(s) encoding (i) a first protein moiety or first protein moiety complex, and (ii) a crRNA as recited in any one of arrangements 1 to 19 or 21 to 27 or 31 to 33.
  • one or more of a) to d) are non-native (i.e. exogenous) sequence(s) to the cell. In another embodiment, all of a) to d) are non-native (i.e. exogenous) sequences to cell. In another embodiment, a) and/or b) are non-native (i.e. exogenous) sequences to cell.
  • the nuclease cleavage site nucleotide sequence may be an I-TevI cleavage site nucleotide sequence.
  • the I-TevI cleavage site nucleotide sequence may be as described elsewhere herein, such as in concept 43 (and any of the additional features described for that concept.)
  • the I-TevI cleavage site nucleotide sequence is 5'-CNNNG-3', such as wherein the I-TevI cleavage site nucleotide sequence has the nucleotide sequence of SEQ ID No:42.
  • the nuclease spacer nucleotide sequence is present. In another embodiment, the nuclease spacer nucleotide sequence is absent.
  • the nuclease spacer nucleotide sequence is an I-TevI spacer nucleotide sequence.
  • the I-TevI spacer nucleotide sequence may be as described elsewhere herein, such as in Concept 44, or any of the additional features described for that concept.
  • the PAM sequence may be as described elsewhere herein, such as in Concept 39, or any of the additional features described for that concept.
  • the protospacer sequence may be as described elsewhere herein, such as in the First Aspect of Detailed Description, or in Concept 10, or Concept 30 or any of the additional features described for that concept.
  • Arrangement 67 A method of modifying a prokaryotic cell to comprise a target sequence wherein the target sequence comprises, in 5' to 3' orientation:
  • a protospacer sequence (optionally wherein the protospacer sequence is about 32 base pairs in length (e.g. is 32 base pairs in length) or is as recited in arrangement 29, and optionally wherein sequences a) to d) are each immediately adjacent to each other, said method comprising introducing one or more modifications into the cell to produce the target sequence; and introducing into the cell one or more vector(s) or one or more nucleic acid sequence(s) encoding (i) a first protein moiety or first protein moiety complex, and (ii) a crRNA as recited in any one of arrangements 1 to 19 or 21 to 27 or 31 to 33.
  • one or more of a) to d) are non-native (i.e. exogenous) sequence(s) to the cell. In another embodiment, all of a) to d) are non-native (i.e. exogenous) sequences to cell. In another embodiment, a) and/or b) are non-native (i.e. exogenous) sequences to cell.
  • I-TevI cleavage site nucleotide sequence may be as described elsewhere herein, such as in Concept 43, or any of the additional features described for that concept.
  • the I-TevI spacer nucleotide sequence may be as described elsewhere herein, such as in Concept 44, or any of the additional features described for that concept.

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Abstract

La technologie décrite dans la présente invention concerne de nouvelles fusions de nucléase et de nickase de nouveaux systèmes CRISPR/Cas et leur utilisation en tant que système autodestructeur ("kill switch") à des fins de bioconfinement dans des bactéries modifiées et/ou ingéniérisées.
PCT/EP2024/074264 2023-08-31 2024-08-30 Nouveau type de système crispr/cas Pending WO2025046062A1 (fr)

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