WO2024256537A1 - Procédé et système de détection de séquence d'acides nucléiques - Google Patents

Procédé et système de détection de séquence d'acides nucléiques Download PDF

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WO2024256537A1
WO2024256537A1 PCT/EP2024/066358 EP2024066358W WO2024256537A1 WO 2024256537 A1 WO2024256537 A1 WO 2024256537A1 EP 2024066358 W EP2024066358 W EP 2024066358W WO 2024256537 A1 WO2024256537 A1 WO 2024256537A1
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protein
seq
chat
saved
sequence
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Aiko Jurre STEENS
VAN DER John OOST
Raymond Hubert Josèphe STAALS
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Wageningen Universiteit
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

Definitions

  • Class I systems utilize multisubunit Cas complexes, whereas the class II systems use only a single Cas protein to mediate its activity. Different types are generally characterized based on the presence of signature genes (Wright et al., 2016. Cell 164: 29-44).
  • CRISPR-Cas nucleic acid detection diagnostics offer a solution for setting up a decentralized screening platform. CRISPR-Cas based diagnostics have been claimed to be faster than PCR, and cheaper to perform on site (see Sheridan C. (2020) Nature Biotechnology 38: 382 - 384).
  • class 2 systems have primarily been applied for diagnostics, as these systems are simpler to reconstitute.
  • CRISPR defence can be described as a process consisting of three stages: adaptation, expression, and interference (Rath et al., 2015. Biochimie 117: 119— 128; Makarova et al., 2011. Nat Rev Microbiol 9: 467—477).
  • adaptation stage genetic fragments are acquired from foreign invading entities and stored in the CRISPR memory (Jackson et al., 2017. Science 356: eaal5056).
  • This memory comprises the foreign DNA sequences, called spacers, which are separated by repetitive DNA sequences (repeats).
  • CRISPR locus stage II
  • crRNAs CRISPR RNAs
  • the cell expresses Cas proteins, the effectors of the CRISPR/Cas system, which form ribonucleoprotein (RNP) complexes by incorporating the crRNAs.
  • RNP ribonucleoprotein
  • SHERLOCK is in principle similar to DETECTR, but uses a Cas13 nuclease from Leptotrichia wadei. This Cas13 specifically recognizes and cleaves RNA and not DNA. RNA fragments, Cas13 protein crRNA and fluorescent RNA probes are mixed together, and where target RNA is present in the sample, Cas13 recognizes them via crRNA and collateral activity cleaves the fluorescent RNA probes, releasing the fluorophore from the quencher and allowing a signal to be read. Intensity of signal can show the quantity of RNA target in the sample. In vitro transcription of the sample can adapt the test to recognise DNA targets and isothermal amplification by RPA can be used to enrich target molecules and increase sensitivity.
  • Type III CRISPR-Cas systems are adaptive immune systems in bacteria and archaea. These systems use CRISPR-derived RNA (crRNA) guides to target complementary nucleic acids of invading viruses and plasmids. Interestingly, type III systems have many unique features, including a rapidly expanding network of signal transduction pathways to trigger dormancy and cell death (see van Beljouw, S. P. B., et al., (2022) Nat. Rev. Microbiol.21: 21 – 23; Steens, J. A., et al. (2022) Biochem. Soc. Trans. 50: 1353–1364 (2022); and Steens, J. A., et al., (2022) Mol.
  • crRNA CRISPR-derived RNA
  • a typical type III operon encodes multiple Cas proteins that form a type III effector complex together with the mature crRNA. These complexes will bind complementary target RNA sequences, which initiate at an exposed seed region at the 3’ end of the crRNA guide. Seed binding initiates complete base pairing between the target RNA and the crRNA, resulting in the activation of Cas10, the characteristic multidomain subunit of the type III complex (see Steens, J. A. et al. (2021) Nat. Commun.12: 1–12.
  • the HD domain of activated Cas10 degrades ssDNA substrates in a non-sequence specific manner, whereas its Palm domain acts as a cyclase to convert ATP into signalling molecules called cyclic oligoadenylates (cOA) of 3-6 AMP moieties
  • cOA cyclic oligoadenylates
  • CARF and SAVED proteins have been characterized, and despite their different activities, they all are geared towards killing the host, stopping the spread of the invading nucleic acid (e.g., phage progeny, plasmid propagation, etc.) in a process known as abortive infection.
  • invading nucleic acid e.g., phage progeny, plasmid propagation, etc.
  • type III systems indicated that proteases also play a role in type III immunity, as exemplified by the TPR-CHAT (Csx29) protease which associates with the type III-E complex, and a cOA-activated Lon-like protease (CalpL) in a type III-B system (see van Beljouw, S. P. B. et al.
  • the cOA amplifies the cell’s response by activating a CARF-domain nuclease Csx1 which results in more general RNA degradation.
  • Rouillon et al. suggest that a better understanding of type III CRISPR systems could be used to improve the yield of products, like yoghurt, that depend on healthy bacteria. Also suggested is that further research in this area could also lead to new antibiotics that over-activate type III CRISPR to destroy bacterial cells. [0009]
  • Niewoehner O et al., (2017) Nature 548: 543 – 548 describes CRISPR-associated protein Csm6 in prokaryotes and its function as a standalone RNase that degrades invader RNA transcripts.
  • Csx30 is described as being an endogenous protein substrate that is site-specifically cleaved by the RNA-activated Craspase.
  • This protease activity is switched off with a target RNA cleavage by gRAMP and is not activated by RNA targets containing a matching protospacer flanking sequences.
  • the Craspase is proposed as a target RNA-activated protease with self-regulating activity.
  • WO2020/256553 A1 discloses a Type III CRISPR/Cas-related ribonucleic acid detection system and its use in diagnostic applications.
  • the detection system comprises (a) an effector complex comprising a Type III CRISPR-associated effector protein (Cas) and at least one CRISPR RNA (crRNA) that binds to a target nucleic acid molecule, and (b) means for directly or indirectly determining a level of cyclic oligoadenylate (cOA).
  • the detection system is a Type III Cas, a Type IIIB Cas or a Type IIIB Cmr.
  • the Type III Cas is preferably from a thermophilic organism such as Thermus thermophilus.
  • the level of cOA is detected by using an inorganic pyrophosphatase which releases pyrophosphate (PPi) which is then measured by a colorimetric-, fluorometric-, fluorescent- or bioluminescent-based assay.
  • the pyrophosphatase can be chosen to be from a thermophilic organism such as Thermus thermophilus and this conveniently allows for an isothermal reaction when used with a preferred Type III CRISPR/Cas system.
  • Another way of detecting the level of cOA is to use a cOA-dependent, non-specific effector endoribonuclease such as Csx1, and a detectable substrate for said endoribonuclease.
  • CARF CRISPR-associated Rossmann fold
  • SAVED effector domains
  • CARF-Lon fusion protein wherein the Lon domain is active in a family of housekeeping proteases. Further, the authors speculate that a CARF-Lon protein might be activated by cOA towards an aggressive host killing activity.
  • Another non-nuclease protein associated type III immunity described by Steens J A et al., (2022) is a Lon-SAVED effector which contains a C-terminal SAVED sensing domain, consisting of two CARF-like domains, fused to a N-terminal Lon protease domain.
  • a cOA4 messenger molecule binds to the protein and induces an allosteric change that activates it to cleave a specific target protein, CRISPR-T.
  • CRISPR-T a specific target protein
  • the 32 kDa CRISPR-T protein is cleaved by activated CRISPR-Lon into two fragments ( ⁇ 23 and ⁇ 10 kDa).
  • the ⁇ 23 kDa fragment bears structural similarity to MazF, which is known to be a toxin known that cleaves specific rRNA, mRNA and tRNA molecules, leading to abortive infection [0015] Makarova K. S.
  • CARF CRISPR-associated Rossmann Fold
  • a clustered regularly interspaced short palindromic repeats (CRISPR) based nucleic acid sequence detection system comprising: (a) a type III CRISPR-associated effector protein complex which when comprised with a targeting RNA substantially complementary to a desired target polynucleotide sequence recognises that target sequence in a nucleic acid molecule and cleaves the nucleic acid molecule and produces a cyclic oligoadenylate (coA x ) messenger; (b) a cyclic oligoadenylate (coA x )-dependent protease; and (c) a protein or polypeptide susceptible to cleavage by the protease of (b) and from which a detectable signal is generated whether directly or indirectly by cleavage of said protein or polypeptide by the protease of (b).
  • CRISPR clustered regularly interspaced short palindromic repeats
  • a detection system of the invention further comprises the targeting RNA which is substantially complementary to the desired target polynucleotide.
  • the coA x -dependent protease may be a second messenger oligonucleotide or dinucleotide synthetase-associated and fused to various effector domains-caspase HetF associated with Tetratricopeptide repeat (TPR) (SAVED-CHAT) protein; or a CHAT-SAVED protein.
  • TPR Tetratricopeptide repeat
  • SAVED-CHAT protein may have an amino acid sequence of SEQ ID NO: 14, or a sequence of at least 70% identity therewith, or a functional fragment thereof.
  • the SAVED-CHAT protein may, independently or additionally to the aforementioned reference sequence or variants or fragments thereof, may comprise one or more of the following amino acid (peptide) motifs in any combination, wherein X is any amino acid: GX(V/A)X(I/L)PXXX(H/Y)LGXXL [SEQ ID NO: 16] (P/N)XAXXXDXXXXL(R/E)XXXPX(I/V)(V/L)HXX(S/G)HG [SEQ ID NO: 17] DXX(A/S)XXF(S/A)XX(F/L)YXXLXXXXX(I/V)XAF [SEQ ID NO: 18] [0021]
  • the SAVED-CHAT protein may comprise an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or a
  • the CHAT-SAVED protein may have an amino acid sequence of SEQ ID NO: 15, or a sequence of at least 70% identity therewith, or a functional fragment thereof.
  • the CHAT-SAVED protein may, independently or additionally to the aforementioned reference sequence or variants or fragments thereof, may comprise one or more of the following amino acid (peptide) motifs in any combination: LRLVVLXACX [SEQ ID NO: 19] IPAVIASQMPLS [SEQ ID NO: 20] XPLPLAXXXGX[SEQ ID NO: 21] IHLFLAXPXSXAX [SEQ ID NO: 22] wherein X is any amino acid, wherein underlined amino acids are not substituted, and wherein specified amino acids may independently be substituted conservatively with another amino acid, as herein defined.
  • a "functional fragment” or “active fragment” refers to a protein or polypeptide fragment that substantially retains the native protein function or activity.
  • a respective range of possible coAx messengers is available for tailoring to a cyclic oligoadenylate (coAx)-dependent protease. Accordingly, x in coAx may be selected from 2, 3, 4, 5 or 6; preferably wherein the coAx is coA3.
  • the protein or polypeptide susceptible to cleavage by the protease of (b) preferably comprises an amino acid sequence susceptible to cleavage at a site therein by the coA x -dependent protease, so as to generate at least two peptide portions. At least one of these peptide portions may comprise a detectable marker. In this way, the detection of the marker and thereby the peptide portion signals the activity of the coAx-dependent protease, which in turn signals the recognition and cleavage activity of the RNA-guided type III CRISPR-associated effector protein complex on a target nucleic acid in a sample.
  • fluorescent markers which may be fluorescent molecules or moieties.
  • the protein or polypeptide Prior to cleavage the protein or polypeptide may also have a quencher molecule or moiety attached so that in the absence of cleavage there is no fluorescence, or the wavelength of fluorescence is different from the wavelength of fluorescence without the quencher.
  • the quencher may be attached to a different cleavage portion of the peptide than the fluorescent marker.
  • the fluorescent marker may be attached to the N- or C-terminus of the protein or polypeptide, and the quencher may be attached to the respective C- or N-terminus of the protein or polypeptide. More details of fluorescent labels and quenchers is provided hereinafter.
  • the protein or polypeptide (c) may be a PCaspase protein comprising an amino acid sequence of SEQ ID NO: 7 or a sequence of at least 70% identity therewith, or polypeptide fragment thereof, and wherein protease activity of PCaspase protein is activated by the action of the coAx-dependent protease.
  • the detection system preferably further comprises (d) a protein or polypeptide substrate for the PCaspase protein or polypeptide to act on. When this substrate (d) is cleaved it provides the or a detectable signal.
  • the proteins or polypeptides of (c) and/or (d) may serve to provide for the detection of the protease of (b).
  • the detection can be direct and/or indirect, or both direct and indirect.
  • the protein or polypeptide substrate (d) is preferably cleaved into at least two peptide portions, wherein at least one peptide portion comprises a detectable label.
  • the detectable label may be a fluorescent moiety; optionally wherein the protein substrate also comprises a quencher moiety which on cleavage is not comprised in the cleavage portion comprising the fluorescent moiety.
  • the quencher may be attached to a different cleavage portion of the peptide than the fluorescent marker.
  • the fluorescent marker may be attached to the N- or C-terminus of the protein or polypeptide, and the quencher may be attached to the respective C- or N-terminus of the protein or polypeptide. More details of fluorescent labels and quenchers is provided hereinafter.
  • the protein or polypeptide substrate (d) may be of any amino acid sequence and suitable structure to be able to function as a reporter protein.
  • the susceptibility of the substrate (d) to protease activity may be provided by an portion of the amino acid sequence that is not folded or structured in a particular way, and which comprises a lysine (K) and/or arginine (R) amino acid.
  • a useful substrate (d) is casein.
  • the type III CRISPR-associated effector comprises Cas10 with an amino acid sequence of SEQ ID NO: 9 or a sequence of at least 70% identity therewith.
  • the Cas10 is the large subunit of the Cmr complex of proteins and comprised within the effector complex along with other Cas proteins (Cmr 1, Cmr3, Cmr 4, Cmr 5 and Cmr 6).
  • the type III CRISPR complex is the type III-B system from Haliangium ochraceum DSM 14365, although other suitable type III CRISPR complexes may be used from other bacteria.
  • Cmr1, Cmr2 (Cas10), Cmr3, Cmr4, Cmr5 and Cmr6 with amino acid sequences as set forth in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively.
  • a CRISPR complex comprised of any one or more of these proteins, preferably all six of these proteins. Variants of each protein are possible of at least 70% identity with any of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively.
  • any of the six individual proteins may be a variant or not, and may be a variant of any degree of sequence identity from 70% to 99% with the relevant reference sequence. Therefore a wide variety of possible type III-B CRISPR complex variants are possible based on the system from Haliangium ochraceum DSM 14365.
  • the detection systems herein are defined both with and without a sample of nucleic acid.
  • the samples of nucleic acid may be of any kind, whether RNA and/or DNA containing.
  • the target nucleic acid molecule in a sample is an RNA.
  • the invention also provides a method of detecting a target polynucleotide sequence in a sample of nucleic acid, comprising combining in a reaction mixture: (a) a type III CRISPR-associated protein (Cas); (b) a targeting RNA substantially complementary to a desired target polynucleotide sequence, wherein (b) in combination with (a) forms an effector complex; (c) a cyclic oligoadenylate (coAx)-dependent protease; and (d) a protein or polypeptide susceptible to cleavage by the protease of (c) and from which a detectable signal is generated whether directly or indirectly by cleavage of said protein or polypeptide by the protease of (c).
  • a reaction mixture comprising combining in a reaction mixture: (a) a type III CRISPR-associated protein (Cas); (b) a targeting RNA substantially complementary to a desired target polynucleotide sequence, wherein (b) in
  • the coA x -dependent protease is preferably a second messenger oligonucleotide or dinucleotide synthetase-associated and fused to various effector domains-caspase HetF associated with Tetratricopeptide repeat (TPR) (SAVED-CHAT) protein comprising an amino acid sequence of any of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or a sequence of at least 70% identity with any of SEQ ID Nos 1 – 6, or any active fragment thereof.
  • TPR Tetratricopeptide repeat
  • the coAx may be selected from a coAx wherein x is 2, 3, 4, 5 or 6; preferably wherein the coAx is coA3.
  • the protein or polypeptide of (d) comprises an amino acid sequence susceptible to cleavage at a site therein by the coAx-dependent protease so as to generate at least two peptide portions. The cleavage of the protein or polypeptide (d) provides a signal based on the reduction of the amount of detectable full length protein or polypeptide and/or the appearance of one or more fragments of the protein or polypeptide.
  • Detection may be of the protein or polypeptide or fragments thereof directly based on size and/or via a label which can be measured qualitatively and/or quantitatively.
  • at least one portion of a cleaved peptide comprises a detectable marker.
  • Particularly useful markers include fluorescent molecules or moieties which are, ideally, prior to cleavage, covalently linked to the protein or polypeptide (d), and remain covalently linked to the peptide(s) after protease cleavage.
  • Förster resonance energy transfer FRET is a well-known technique involving two fluorescent chromophores for studying protein structure and interactions.
  • the technique is well employed in all aspects of the present invention for detecting protease cleavage activity of the protein or polypeptide (d).
  • Many fluorescent molecule pair combinations including dye-quencher combinations, will be well known to a person of skill in the art and adapted for use in the present invention for detecting cleavage activity, whether by appearance and/or disappearance of fluorescence of suitably tagged proteins or polypeptides (d).
  • a preferred protein or polypeptide (d) for use in methods or other aspects of the invention is a PCaspase protein comprising an amino acid sequence of SEQ ID NO: 7 or a sequence of at least 70% identity therewith, or polypeptide fragment thereof, and wherein protease activity of PCaspase protein is activated by the action of the coAx-dependent protease.
  • SEQ ID NO: 7 is the peptidase C14 caspase catalytic subunit p20 from Haliangium ochraceum (strain DSM 14365 / JCM 11303 / SMP-2).
  • Functional variants of this PCaspase protein are included within the scope of the invention, as may be defined in terms of percentage identity with SEQ ID NO: 7. Functional variants include fragments of the full length sequence or full length variants thereof. Variations in sequence may comprise conservative and/or non- conservative amino acid substitutions.
  • a protein or polypeptide substrate (e) is provided for the PCaspase protein or polypeptide. When this protein or polypeptide substrate (e) is cleaved then a detectable signal for this cleavage activity of the PCaspase protein is generated.
  • the protein or polypeptide substrate (e) is cleaved into at least two peptide portions, and wherein at least one peptide portion comprises a detectable label.
  • the detectable label may be a fluorescent molecule or moiety ideally covalently attached to the protein or polypeptide (e).
  • the amino acid sequence of the protease cleavage site in the protein or polypeptide (e) may be replicated and within a suitable motif may be engineered into other possible protein or polypeptide substrates.
  • the protein substrate (e) comprises an amino acid sequence portion, optionally an unstructured portion, comprising a lysine (K) or arginine (R) amino acid.
  • a particularly useful protein substrate is a casein.
  • the type III CRISPR-associated protein (Cas) is Cas10 (SEQ ID NO: 9 or sequence of at least 70% identity therewith).
  • the Cas10 is the large subunit of the Cmr complex of proteins and comprised within the effector complex along with other Cas proteins.
  • the type III CRISPR complex is the type III-B system from Haliangium ochraceum DSM 14365, although other suitable type III CRISPR Cas proteins and complexes with targeting RNA may be used from other bacteria.
  • a suitable CRISPR complex may be comprised of any one or more of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, or sequences of at least 70% identity therewith. Any of the six individual proteins making up the complex may be a variant or not, and may be a variant of any degree of sequence identity from 70% to 99% with the relevant reference sequence.
  • the essential reagents minus the sample are assembled and optionally combined, following which there may be the step of combining the reagents with a sample (g) comprising nucleic acid; preferably comprising RNA.
  • a sample comprising nucleic acid; preferably comprising RNA.
  • the method of the invention may be carried out whereby the reagents (reaction components) can be combined at different times and in different combinations.
  • the type III CRISPR-associated protein (Cas) may be combined with the targeting RNA first to form an effector complex, prior to combining together with the other components of the reaction mixture.
  • reaction components are readily ascertained and understood by a person of skill in the art for optimisation of the individual reactions in the method/system, taking into consideration the format, e.g. liquid phase, or solid phase, or combination of solid and liquid phase reactions and the methods/instrumentation used for detection of the marker or markers.
  • the sequential order of combining of reaction components is: (g), then (a)/(b), then (c), then (d), then (e).
  • the invention includes a polynucleotide comprising a nucleotide sequence encoding one or more of: i. a type III CRISPR complex comprising proteins with amino acid sequences of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or sequences of at least 70% identity therewith; ii.
  • a SAVED-CHAT protein with an amino acid sequence of SEQ ID NO: 14 or a sequence of at least 70% identity therewith, or a functional fragment thereof; iii. a SAVED-CHAT protein with an amino acid sequence comprising one or more of the following peptide motifs: GX(V/A)X(I/L)PXXX(H/Y)LGXXL [SEQ ID NO: 16]; (P/N)XAXXXDXXXXL(R/E)XXXPX(I/V)(V/L)HXX(S/G)HG [SEQ ID NO: 17]; or DXX(A/S)XXF(S/A)XX(F/L)YXXLXXXXX(I/V)XXAF [SEQ ID NO: 18], wherein X is any amino acid; iv.
  • a CHAT-SAVED protein with an amino acid sequence of SEQ ID NO: 15, or a sequence of at least 70% identity therewith, or a functional fragment thereof; v. a CHAT-SAVED protein with an amino acid sequence comprising one or more of the following peptide motifs: LRLVVLXACX [SEQ ID NO: 19]; IPAVIASQMPLS [SEQ ID NO: 20]; XPLPLAXXXGX [SEQ ID NO: 21]; or IHLFLAXPXSXAX [SEQ ID NO: 22], wherein X is any amino acid, wherein underlined amino acids are not substituted, and wherein specified amino acids may independently conservatively substituted; vi.
  • a PCaspase with an amino acid sequence of SEQ ID NO: 7 or a sequence of at least 70% identity therewith
  • a PCk with an amino acid sequence of SEQ ID NO: 42, or a sequence of at least 70% identity therewith
  • a PC- ⁇ with an amino acid sequence of SEQ ID NO: 43, or a sequence of at least 70% identity therewith.
  • a polynucleotide as aforementioned may comprise a combination of nucleotide sequences selected from: (i) and (ii), optionally further comprising (vi) and/or (vii) and/or (viii); (i) and (iii); optionally further comprising (vi) and/or (vii) and/or (viii); (i) and (iv) optionally further comprising (vi) and/or (vii) and/or (viii); (i) and (v) optionally further comprising (vi) and/or (vii) and/or (viii).
  • a polynucleotide as aforementioned may further comprise a nucleotide sequence encoding a guide RNA with a sequence specific for a desired target nucleic acid sequence.
  • the invention also includes a plasmid comprising any of the aforementioned polynucleotides which lack a gRNA encoding sequence; optionally wherein the plasmid is a native conjugative plasmid, or an expression plasmid.
  • the invention further includes a viral vector comprising any of the aforementioned polynucleotides or plasmids which lack a gRNA encoding sequence; optionally wherein the viral vector is a bacteriophage.
  • the invention also provides a composition comprising any of the aforementioned polynucleotides, plasmids, viral vectors, or nanoparticle compositions which lack a gRNA encoding sequence.
  • the invention further provides a plasmid comprising an aforementioned polynucleotide which additionally comprises a gRNA encoding sequence; optionally wherein the plasmid is a native conjugative plasmid, or an expression plasmid.
  • the invention also provides a viral vector comprising an aforementioned polynucleotide which additionally comprises a gRNA encoding sequence, or a plasmid comprising such a polynucleotide; optionally wherein the viral vector is a bacteriophage.
  • the invention further provides a composition comprising an aforementioned polynucleotide which additionally comprises a gRNA encoding sequence, or a plasmid comprising such a polynucleotide, and a nanoparticle.
  • the invention also provides a composition comprising an aforementioned polynucleotide which additionally comprises a gRNA encoding sequence, a plasmid, viral vector or nanoparticle composition comprising such a polynucleotide.
  • the invention also provides a kit comprising (a) a plasmid as aforementioned which does not comprise a gRNA encoding sequence, a viral vector as aforementioned which does not comprise a gRNA encoding sequence, a nanoparticle composition as aforementioned which does not comprise a gRNA encoding sequence; and (b) a separate polynucleotide, plasmid, vector or nanoparticle composition comprising a nucleotide sequence encoding a guide RNA with a sequence specific for a desired target nucleic acid sequence.
  • the invention further includes a composition comprising: (a) a type III CRISPR-associated protein (Cas); (b) a targeting RNA substantially complementary to a desired target polynucleotide sequence, wherein the combination of (a) and (b) provides an effector complex; and (c) a cyclic oligoadenylate (coAx)-dependent protease.
  • Such compositions of the invention may, as already noted, be in solid and/or liquid phase, depending on the particular format of the detection assay used, and also depending on the particular marker used to detect the various proteolytic cleavage events.
  • composition of the invention may further comprise (d) a protein or polypeptide susceptible to cleavage by the protease of (c) and from which a detectable signal is generated whether directly or indirectly by cleavage of said protein or polypeptide by the protease of (c).
  • the coAx-dependent protease (c) may be a second messenger oligonucleotide or dinucleotide synthetase-associated and fused to various effector domains-caspase HetF associated with TPR (SAVED-CHAT) protein comprising an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6, or a sequence of at least 70% identity with any of SEQ ID Nos 1 – 6, or any active fragment thereof.
  • TPR SAVED- CHAT
  • Functional variants of this TPR (SAVED- CHAT) protein are included within the scope of the invention, as may be defined in terms of percentage identity with SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
  • Functional variants include fragments of the full length sequence or full length variants thereof. Variations in sequence may comprise conservative and/or non-conservative amino acid substitutions.
  • the protein or polypeptide (d) may be a PCaspase protein comprising an amino acid sequence of SEQ ID NO: 7 or a sequence of at least 70% identity therewith, or polypeptide fragment thereof, and wherein protease activity of PCaspase protein is activated by the action of the coA x -dependent protease.
  • Functional variants of this PCaspase protein optionally from other species of microorganism, are included within the scope of the invention, as may be defined in terms of percentage identity with SEQ ID NO: 7.
  • Functional variants include fragments of the full length sequence or full length variants thereof. Variations in sequence may comprise conservative and/or non-conservative amino acid substitutions.
  • Composition as herein described may further comprise (e) a protein or polypeptide substrate for the PCaspase protein or polypeptide, which when cleaved provides the detectable signal.
  • the protein or polypeptide substrate (e) is cleaved into at least two peptide portions, and wherein at least one peptide portion comprises a detectable label.
  • a useful detectable label is a fluorescent molecule or moiety and, ideally, prior to cleavage, these are covalently linked to the protein or polypeptide (d), and may remain covalently linked to the peptide(s) after protease cleavage.
  • FRET Förster resonance energy transfer
  • the substrate (e) preferably comprises a portion of amino acid sequence, optionally an unstructured portion, comprising a lysine (K) or arginine (R) amino acid.
  • a useful protein substrate is casein.
  • the type III CRISPR-associated protein (Cas) is preferably Cas10 (SEQ ID NO: 9 or sequence of at least 70% identity therewith); more preferably the type III CRISPR complex is the type III-B system from Haliangium ochraceum DSM 14365. The sequences of the six Cmr proteins and their variants are elsewhere described herein.
  • compositions of the invention may further comprise (f) a sample comprising nucleic acid; preferably wherein the nucleic acid is RNA.
  • the invention also includes a device for detecting a polynucleotide molecule comprising a defined nucleic acid sequence in a sample, comprising a container, wherein the container comprises (i) a type III CRISPR-associated effector protein (Cas), (ii) a targeting RNA substantially complementary to the defined RNA sequence, wherein the combination of (i) and (ii) provides an effector complex; (iii) a cyclic oligoadenylate (coA x )- dependent protease; and (iv) a protein or polypeptide susceptible to cleavage by the protease of (v) and from which a detectable signal moiety is generated whether directly or indirectly by cleavage of said protein or polypeptide by the protease of (iii).
  • the container may itself form the device of the invention, or the container may be comprised in a device which further includes mechanical, electrical and/or software elements needed in combination to provide a fully operational detection device.
  • the device is preferably presented and available without a sample such that it is in readiness to receive any given sample. Therefore the invention also includes a detection device as described herein further comprising a sample being interrogated for a defined nucleic acid sequence therein.
  • a detection device as described herein further comprising a sample being interrogated for a defined nucleic acid sequence therein.
  • the targeting RNA substantially complementary to the defined RNA sequence is therefore selected to discriminate positively in respect of the defined nucleic acid sequence in the sample. This provides in all aspects of the invention the degree selectivity and specificity needed for detecting the sequence of interest in a sample.
  • the invention includes any device comprising a detection system as herein described and such devices may preferably comprise multiple arrayed ribonucleic add detection systems, each of the arrayed ribonucleic acid detection systems preferably being directed to specifically detect different respective target nucleic add molecules.
  • a device for detecting a polynucleotide molecule comprising a defined sequence in a sample comprising a lateral flow substrate comprising (a) a sample receiving portion, (b) a flow through portion, and (c) a capture portion, wherein the flow through portion comprises (i) a type III CRISPR-associated protein (Cas), (ii) a targeting RNA substantially complementary to the defined RNA sequence, wherein the combination of (i) and (ii) provides an effector complex; (iii) a cyclic oligoadenylate (coA x )-dependent protease; and (iv) a protein or polypeptide susceptible to cleavage by the protease of (iii) and from which a detectable signal is generated whether directly or indirectly by cleavage of said protein or polypeptide by the protease of (iii); and wherein the capture portion retains the detectable signal moiety.
  • the flow through portion comprises (i) a type III C
  • devices in accordance with the invention may comprise any of the necessary other components necessary for a working device, whether standalone or for coupling to other equipment for generating a test result.
  • the invention also provides a kit of parts for the purposes of detecting of a polynucleotide molecule comprising a defined nucleic acid sequence in a sample, the kit comprising at least one container, (i) a type III CRISPR-associated protein (Cas), (ii) a cyclic oligoadenylate (coAx)-dependent protease; and (iii) a protein or polypeptide susceptible to cleavage by the protease of (ii) and from which a detectable signal moiety is generated whether directly or indirectly by cleavage of said protein or polypeptide by the protease of (ii).
  • a type III CRISPR-associated protein Cas
  • a cyclic oligoadenylate (coAx)-dependent protease a protein or polypeptide susceptible to cleavage by the protease of (ii) and from which a detectable signal moiety is generated whether directly or indirectly by cleavage of said protein or
  • the kit may include written material in physical or virtual form providing instructions for operation and use of the kit.
  • the kit may be for use with other devices and assay equipment. Therefore the at least one container may be a reaction vessel within which the detection of labelled substances also takes place.
  • the at least one container may contain one or all of the aforementioned components, but where less than all of the components are comprised in a single container then the kit provides a plurality of containers. Some containers may comprise a single component, other containers may comprise combinations of components.
  • a single empty container may be provided as a reaction vessel into which a sample is introduced, whether before, after or simultaneously with one or more of the other reaction components.
  • kits in accordance with the invention as defined above may further comprise (iv) a targeting RNA substantially complementary to the defined RNA sequence which is being detected for in the sample.
  • kits can be presented to the user for a predetermined sample sequence and therefore ready to use, as compared to a kit which is ready to configure by the user with a selected targeting RNA in order to detect a chosen sequence in a sample.
  • a kit of the invention may have a separate container for each of components (i), (ii), (iii) and (iv).
  • the Haliangium species of bacteria from which a preferred type III Cas protein is derived are from the marine environment, and so temperatures for operation of the method and system of the invention mainly correspond to those ambient native temperatures. If a type III Cas protein is used from a thermophilic organism then the usable temperatures are consequently higher within the range of tolerance of the other components of the system, the preferred elements being the SAVED-CHAT protein and optionally the PCaspase protein. Consequently, the methods and systems of the invention may be operated at temperatures in the range from about 4 °C to about 38 °C.
  • Narrower ranges of temperature such as from about 10 °C to about 36 °C, from about 15 °C to about 36 °C, from about 20 °C to about 36 °C and from about 25 °C to about 36 °C may provide a suitable reaction temperature to be used.
  • the term “about” is used because it is clear to a person of skill in the art that temperatures may not be so precise during the reactions of the assay carried out in accordance with the invention that they cannot vary within + 0.5 °C or + 0.6 °C and yet achieve reproducible and accurate results.
  • the type III Cas protein and resulting effector complex described herein may be modified to lack cleavage activity but still generate coAx and therefore drive the activation of the coAx activated protease.
  • a skilled person will be able to identify the necessary cleavage-dead mutations through a process of design, expression and testing of various mutated versions of the type III Cas effector complex and constituent proteins.
  • base pairing affinity and “complementarity” may be used interchangeably and refer to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types.
  • a percent identity i.e. complementarity in relation to a reference sequence, in the various descriptions of the invention, represents the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% identity).
  • Perfectly complementary means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence, and this is a preferred condition for antisense oligonucleotide binding to the targeting RNA which corresponds to 100% identity for a length of targeting RNA molecule which is the same length as the antisense oligonucleotide.
  • substantially complementary refers to a degree of identity that is at least 90%, 95%, 97%, 98%, 99%, or 100% between the portion of the antisense oligonucleotide and the equivalent length of targeting RNA molecule. This may also correspond to nucleic acids that hybridize under stringent conditions.
  • stringent conditions for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions surrounding the nucleic acids, temperature, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used.
  • the Tm is the temperature at which more than 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand.
  • Hybridization 5x SSC at 65 °C for 16 hours; wash twice: 2x SSC at room temperature (RT) for 15 minutes each; wash twice: 0.5x SSC at 65 °C for 20 minutes each.
  • High Stringency (allows sequences that share at least 80%> identity to hybridize) Hybridization: 5x - 6x SSC at 65 °C - 70 °C for 16 - 20 hours; wash twice: 2x SSC at RT for 5 - 20 minutes each; wash twice: lx SSC at 55 °C - 70 °C for 30 minutes each.
  • the degree of identity may be any of: at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 97%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%
  • the targeting RNA molecule is designed to have complementarity, where hybridization between a target sequence and the RNA targeting molecule promotes the formation of a RNA-targeting complex.
  • Targeting RNA molecules in accordance with the invention may include mature crRNA, guide RNA (gRNA) or single guide RNA (sgRNA) and these terms can be used interchangeably.
  • gRNA guide RNA
  • sgRNA single guide RNA
  • a targeting RNA has a sufficient complementarity with the target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR enzyme or Cascade complex to the target sequence.
  • the degree of complementarity between a targeting RNA and its corresponding target sequence may be more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more, with optimal algorithmic alignment.
  • optimal alignment may be determined using, for example, any of the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • CRISPR Clustering Regularly Interspaced Short Palindromic Repeats
  • the genomic region includes one or more CRISPR-associated effector protein (Cas)-encoding genes that are located in the vicinity of the CRISPR loci.
  • CRISPR crRNA refers to a CRISPR-derived RNA molecule comprising a spacer sequence and 5 and 3 repeat-derived termini.
  • Said CRISPR crRNA preferably has a length of at least 30 nucleotides, more preferred at least 34 nucleotides, more preferred at least 40 nucleotides, more preferred at least 46 nucleotides.
  • Said CRISPR crRNA preferably is less than 1000 nucleotides, preferably less than 200 nucleotides, preferably less than 100 nucleotides.
  • RNA molecule may include ribonucleic acid nucleotide analogues such as inosine, uridine, xanthine, hypoxanthine, 2,6-diaminopurine, and 6,8- diaminopurine-based ribonucleotides and desoxyribonucleotides.
  • CRISPR-associated effector protein refers to a protein that is associated with CRISPR crRNA.
  • CRISPR/Cas systems are presently grouped into two classes. Class I systems utilize multisubunit Cas complexes, whereas Class II systems use only a single Cas protein to mediate its activity.
  • effector complex refers to a CRISPR-Cas ribonucleoprotein complex that has nuclease activity and may cleave and inactivate an invading nucleic acid sequence that comprises complementary sequences to the spacer sequence in the CRISPR crRNA.
  • cyclic oligoadenylate refers to a ring structure comprising 2, 3, 4, 5 or 6 molecules of Adenosine Mono Phosphate (AMP).
  • AMP Adenosine Mono Phosphate
  • Type III Cas refers to a RNA-targeting, multiple subunit CRISPR-associated complex that comprises at least a Cas10 protein.
  • a type IIIA Cas as is used herein, which falls under within the scope of the aforementioned “type III Cas”, is an RNA-targeting Type 3 CRISPR/Cas complex that has unspecific DNase activity upon binding to a target RNA molecule.
  • Type IIIA Cas include, for example, Type IIIA Csm complexes from Staphylococcus thermophilus, Thermus thermophilus and Staphylococcus epidermis.
  • Type IIIB Cas refers to a RNA-targeting Type 3 CRISPR-Cas complex that lacks unspecific DNase activity. Said Type IIIB Cas complex is composed of six to seven proteins.
  • Type IIIB Cas include, for example, Type IIIB Cmr complexes from Pyrococcus furiosus, Thermus thermophilus and Sulfolobus solfataricus, as well as the preferred type III-B Cas from Haliangium ochraceum DSM 14365.
  • biosensor or “biological sensor”, as is used herein, refers to a sensing device comprising a CRISPR-based ribonucleic acid system according to the invention.
  • the signal is preferably converted by means of a suitable transducer into a measurable electrical parameter such as a current or voltage.
  • a general structure of Type III CRISPR/Cas complexes is that they comprise multiple subunits of Cas7 and Cas11 (Staals et al., 2013. Mol. Cell 52: 135-145; Staals et al., 2014. Mol.
  • Cas7 provides RNase activity upon recognition of target RNA by the pre-loaded RNA guide. It has been shown that target recognition promotes the production of cyclic oligoadenylates (cOA) by the Cas10 Palm domain (Kazlauskiene et al., (2017) Science 357(6351): 605-609).
  • cOA cyclic oligoadenylates
  • Preferred fluorescent labels in accordance with any aspect of the invention may be selected from any of Atto425 (ATTO-TEC GmbH, Siegen, Germany), Atto 647N (ATTO- TEC GmbH, Siegen, Germany), YakimaYellow (Epoch Biosciences Inc, Bothell, WA, USA), Cal610 (BioSearch Technologies, Petaluma, CA, USA), Cal635 (BioSearch Technologies, Petalumam, CA, USA), FAM (Thermo Fisher Scientific Inc., Waltham, MA USA), TET (Thermo Fisher Scientific Inc., Waltham, MA USA), HEX ((Thermo Fisher Scientific Inc., Waltham, MA USA), cyanine dyes such as Cy5, Cy5.5, Cy3, Cy3.5, Cy7 (Thermo Fisher Scientific Inc., Waltham, MA USA), Alexa dyes (Thermo Fisher Scientific Inc., Waltham, MA USA), Tamra (Thermo Fisher Scientific Inc., Waltham, MA USA), ROX (Thermo Fisher Scientific Inc., Walt., Walt
  • Said substrate is preferably labelled at the 5’ end with a detectable label, preferably a fluorescent label.
  • Quenchers useful in the invention including, for example, tetramethylrhodamine TAMRA.
  • Preferred quenchers include Black Hole Quencher®- 1 (BHQ1) and BHQ2 (Biosearch Technologies, Petaluma, CA, USA).
  • BHQ1 dark quencher has strong absorption from 480 nm to 580 nm, which provides quenching of fluorophores that fluoresce in this range, such as FAM, TET, CAL Fluor® Gold 540, JOE, HEX, CAL Fluor Orange 560, and Quasar® 570 dyes.
  • the BHQ2 dark quencher has strong absorption from 599 nm to 670 nm, which provides quenching of fluorophores that fluoresce in this range, such as Quasar® 570, TAMRA, CAL Fluor® Red 590, CAL Fluor Red 610, ROX, CAL Fluor Red 635, Pulsar® 650, Quasar 670 and Quasar 705 dyes.
  • BHQ1 and BHQ2 may quench fluorescence by both FRET and static quenching mechanisms.
  • Ribonucleic acid detection systems according to the invention involve an in vitro assembly of a CRISPR Type III ribonucleoprotein complex, preferably a Type IIIB complex.
  • Said proteins preferably are expressed and purified from a suitable expression system.
  • Commonly used expression systems for heterologous protein production include E. coli, Bacillus spp., baculovirus, yeast, fungi, most preferably filamentous fungi or yeasts such as Saccharomyces cerevisiae and Pichia pastoris, eukaryotic cells such as Chinese Hamster Ovary cells (CHO), human embryonic kidney (HEK) cells and PER.C6® cells (Thermo Fisher Scientific, MA, USA) and plants.
  • the efficiency of expression of recombinant proteins in heterologous systems depends on many factors, both on the transcriptional level and the translational level.
  • Cas proteins preferably are produced using prokaryotic cells, preferably E. coli.
  • Said Cas proteins are preferably produced by expression cloning of the proteins to a prokaryotic cell of interest, preferably E. coli.
  • Said expression construct, preferably DNA is preferably produced by recombinant technologies, including the use of polymerases, restriction enzymes, and ligases, as is known to a skilled person.
  • said expression construct is provided by artificial gene synthesis, for example by synthesis of partially or completely overlapping oligonucleotides, or by a combination of organic chemistry and recombinant technologies, as is known to the skilled person.
  • Cas proteins may be isolated from a thermophilic organism by expression of a tagged Cas protein in said thermophilic organism, and isolation of ribonucleoprotein complex comprising said Cas proteins on the basis of the tag. Said isolated ribonucleoprotein complexes can be isolated using the tagged Cas protein.
  • Said expression construct is preferably codon-optimised to enhance expression of the Cas proteins in a prokaryotic cell of interest, preferably E. coli. Further optimization preferably includes removal of cryptic splice sites, removal of cryptic polyA tails and/or removal of sequences that lead to unfavourable folding of the mRNA.
  • the expression construct preferably encodes a protein export signal for secretion of the Cas proteins out of the cell into the periplasm of prokaryotes, allowing efficient purification of the Cas proteins.
  • Methods for purification of Cas proteins are known in the art and are generally based on chromatography such as affinity chromatography and ion exchange chromatography, to remove contaminants. In addition to contaminants, it may also be necessary to remove undesirable derivatives of the product itself such as degradation products and aggregates. Suitable purification process steps are provided in Berthold and Walter (1994) Biologicals 22: 135 - 150.
  • recombinant Cas proteins may be tagged with one or more specific tags by genetic engineering to allow the protein attach to a column specific to the tag and therefore be isolated from impurities.
  • the purified protein is then exchanged from the affinity column with a decoupling reagent.
  • Conventional tags for proteins such as histidine tag, are used with an affinity column that specifically captures the tag (e.g., a Ni-IDA column for the histidine tag) to isolate the protein from other impurities.
  • the protein is then exchanged from the column using a decoupling reagent according to the specific tag (e.g., imidazole for histidine tag).
  • Suitable further tags include c-myc domain EQKLISEEDL (SEQ ID NO: 24), hemagglutinin tag YPYDVPDYA (SEQ ID NO: 25), maltose-binding protein, glutathione-S- transferase, FLAG tag peptide, biotin acceptor peptide, streptavidin-binding peptide and calmodulin-binding peptide, as presented in Chatterjee (2006) Curr. Opin. Biotech.17: 353 - 358. Methods for employing these tags are known in the art and may be used for purifying Cas proteins. [00105] Methods for expression proteins in E.
  • Cas proteins are expressed in E. coli from a codon- optimized expression construct. Said construct is placed in a bicistronic expression plasmid containing a Strep-tag and amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln- (Gly/Ser) at the N-terminus, which amino acid sequence is recognized by a Tobacco Etch Virus (TEV) protease.
  • TSV Tobacco Etch Virus
  • the protein of interest is eluted using Buffer B (100 mM Tris-HCl, 150 mM NaCl & 2.5 mM D-desthiobiotin).
  • Buffer B 100 mM Tris-HCl, 150 mM NaCl & 2.5 mM D-desthiobiotin.
  • the protein is cleaved from the affinity tag by addition of TEV protease and left to incubate overnight at 4 °C.
  • the protein of interest is separated from the mixture by a HisTrap and StrepTrap affinity chromatography step, from which the flow through is collected. If required, an additional size exclusion chromatography is added to achieve higher purity.
  • Cas proteins of use in the invention comprise Cas10 and may comprise Csm or Cmr proteins, at least Cmr 1 and Cmr 4, preferably Cmr 1-6, or at least Csm 2, preferably Csm 2 - 5, more preferably Csm 1 - 6.
  • mutants of said proteins including insertion mutants, deletion mutants, chimeric proteins and amino acid substituted proteins which retain the respective functions required for the system and method described herein may also be used according to the invention.
  • catalytically dead mutant Cmr and/or Csm complexes are preferably created and used in a detection system of the invention.
  • catalytically dead refers to the target RNA-digesting activity of the CRISPR-based ribonucleic acid system according to the invention. These mutants are referred to as dCmr and dCsm.
  • the mutations are introduced in the Cmr4 and Csm3 subunits that are responsible for target binding and cleaving and are selected for abolishment of target cleavage, while maintaining target binding.
  • cleavage-dead mutations include a E227A and E228 double mutant of Cmr4, and Cmr4 D86A (Ramia et al., (2014) Cell Reports 9: 1610 - 1617; Zhu and Ye, (2015) Nucleic Acids Res 43: 1257 - 1267).
  • a Csm3 D32A is a good candidate for a cleavage-dead Type IIIA Csm mutant (Samai et al., (2015) Cell 161: 1164-1174).
  • Jia et al. (2019) Mol Cell 73: 264 – 277 and Park et al.
  • the methods of the invention for detecting specific RNA sequences may be used in human healthcare, veterinary diagnostics, detection of plant pathogens, detection of water contaminants and the detection of food and feed contaminants.
  • the methods of the invention for detecting specific RNA sequences may be used for detecting beneficial organisms. In general, the methods of the invention can be used for detection of bacterial, fungal, archaeal, protest, protozoal, eukaryotic, viral and viroidal pathogens.
  • nucleic acid material is preferably isolated from a biological fluid, preferably from cerebrospinal fluid, saliva, nasopharyngeal secretion, oropharyngeal secretion, sweat, urine stool, or blood.
  • a biological fluid preferably from cerebrospinal fluid, saliva, nasopharyngeal secretion, oropharyngeal secretion, sweat, urine stool, or blood.
  • blood includes blood plasma, which is prepared by removing red and white blood cells, for example by centrifugation, and blood serum, which is prepared by formation of a blood clot, and removal of the clot using, for example, a centrifuge.
  • a preferred biological fluid is blood.
  • nucleic acid material may be purified from a sample using, for instance, a combination of physical and chemical methods.
  • Commercially available systems for nucleic acid isolation are preferably used, such as the NucliSENS® easyMAG® or NucliSENS® miniMAG® nucleic acid extraction system (bioMerieux, Marcy l'Etoile, France), or a MagNA Pure 96 System (Roche Diagnostics, Almere, The Netherlands).
  • RNA may be isolated from a sample by any technique known in the art, including but not limited to suitable commercial RNA isolation kits include Trizol (Invitrogen; Carlsbad, California), RNAqueous® (Applied Biosystems/Ambion, Austin, Tx), Qiazol® (Qiagen, Hilden, Germany), Agilent Total RNA Isolation Kits (Agilent; Santa Clara, California), RNA-Bee® (Tel-Test. Friendswood, Texas), the RNeasy mini kit (Qiagen, Venlo, The Netherlands), and MaxwellTM 16 Total RNA Purification Kit (Promega; Madison, Wisconsin).
  • RNA preferably mRNA
  • the isolated RNA is preferably reverse transcribed with the aid of a RNA-dependent DNA polymerase into single or double stranded cDNA.
  • diagnostics such as detection of urinary tract infection, respiratory tract infection, (for example SARS-CoV-2 and respiratory syncytial virus (RSV)), blood infection (sepsis), expression of antibiotic resistance markers such as methicillin-resistant Staphylococcus aureus markers and extended spectrum beta - lactamases markers, gastrointestinal infection, skin infection, odontogenic infection, vaginal infection such as Candidiasis, Trichomonas vaginalis, and Gardnerella, male reproductive system infection, tropical infectious disease such as malaria, trypanosoma, dengue fever, Zika fever, chickungunya fever, detection of sexually transmissible diseases caused by Chlamydia spp., gonorrhea,
  • the Type III CRISPR/Cas methods and systems according to the invention may further be used for veterinary diagnostics including detection of cattle infectious diseases such as mastitis, bluetongue, foot and mouth disease, Salmonella spp., Klebsiella spp., Campylobacter spp., pig infectious diseases such as respiratory diseases, dermatitis, diarrhea, and infections by porcine parvovirus; sheep and goat infectious diseases such as Clostridial diseases, soremouth, pneumonia and infections with Rift Valley Disease Virus; poultry infectious diseases such as infectious bronchitis, Salmonella spp.; feline infectious diseases such as infections with feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV), respiratory infections; canine infectious diseases such as rabies, and infections with Bordetella, Leptospira, and Borellia.
  • cattle infectious diseases such as mastitis, bluetongue, foot and mouth disease, Salmonella spp., Klebsiella spp.,
  • the Type III CRISPR/Cas detection method or system according to the invention may further be used for detection of plant pathogens such as detection of certain fungi such as Ascomycetes species and Basidiomycetes species, certain fungi-like organisms such as oomycetes and phytomyxea, certain bacteria such as Burkholderia, proteobacteria, and Pseudomonas species; viruses, viroids and virus- like organisms such as tobacco mosaic virus, cauliflower mosaic virus; nematodes such as Meloidogyne chitwoodii and M. fallax; and protozoa and algae such as Phytomonas and Cephaleuro.
  • the Type III CRISPR/Cas detection system or methods according to the invention may further be used for detection of water contaminants, including detection of bacterial contamination such as Vibrio cholerae, E. coli, Shigella spp., Legionella spp., Salmonella spp.; viral contamination such as hepatitis A, hepatitis E, poliovirus; algae contamination such as presence of Desmodesmus spp; and parasitic contamination such as presence of Dracunculiasis spp.
  • the Type III CRISPR/Cas detection systems or methods according to the invention may further be used for detection of food and feed contaminants including bacterial contamination such as presence of Clostridium botulinum, E.
  • Type III CRISPR/Cas detection system may further be used for detection of any organism, as all organisms generate RNA during infection.
  • Said organisms include bacteria such as Bacillus species, Clostridium species, Enterobacter species, Escherichia species, Enterococcus species, Klebsiella species, Listeria species, Legionella species, Salmonella species, Staphylococcus species, Streptococcus species, and combinations thereof; viruses including DNA viruses such as hepatitis B virus, adenovirus, human papilloma virus; RNA viruses such as Influenza virus, Hepatitis A/C/D/E, polio virus, tobacco mosaic virus, Coronavirus, and HIV; viroids; Archaea; fungi such as Aspergillus species, Ascomycetes species, Candida species; protozoa; and parasites such as Trypanosoma species.
  • bacteria such as Bacillus species, Clostridium species, Enterobacter species, Escherichia species, Enterococcus species, Klebsiella species, Listeria species, Legionella species, Salmonella species, Staphylococcus species, Str
  • Type III CRISPR/Cas detection systems or methods according to the invention for detection of RNA will find explicit benefit in a diagnostic setting. Two such diagnostic settings are explained herein below. However, a person skilled in the art will without undue effort be able to apply the invention to a range of possible further diagnostic settings that will benefit from the Type III CRISPR/Cas detection system according to the invention for detection of RNA. [00122] If necessary, for example to increase detection levels, the methods of the invention for detecting specific RNA sequences can be preceded by amplification of target sequences.
  • Amplification may be performed by any suitable amplification system including, for example, ligase chain reaction (LCR), isothermal ribonucleic acid amplification systems such as nucleic acid sequence-based amplification (NASBA) and cleavage-based signal amplification of RNA, transcription mediated amplification, strand displacement amplification and, polymerase chain reaction (PCR).
  • LCR ligase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • PCR polymerase chain reaction
  • RNA preferably is reverse transcribed prior to, or during the amplification reaction.
  • a preferred amplification reaction is a single tube, isothermal reaction such as NASBA, loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), recombinase polymerase amplification (RPA) reaction, and nicking enzyme amplification reaction (NEAR).
  • a preferred single tube, isothermal reaction is a RPA) reaction (TwistDx Ltd., Cambridge, UK).
  • Said single tube, isothermal reaction such as RPA preferably is integrated with the CRISPR-based ribonucleic acid detection system of the invention as a “one pot” reaction system.
  • An advantage of such one pot, or single tube, system is a reduced risk for contamination of the samples, or cross-contamination of different samples.
  • Methods for the isolation of nucleic acid material include, but are not limited to organic extraction, chelex extraction, solid phase extraction, magnetic beads, and/or anion exchange.
  • sample preparation steps are also required to solve readout interference by other factors including, but not limited to, proteases in patient samples, salt concentrations, and pH.
  • Specific qPCR primers as described in van der Zee et al. (2016) PLOS ONE 11: e0150755) may be used for detection of UTI-causing pathogens.
  • the CRISPR/Cas-based ribonucleic acid detection method and system according to the invention may be manifest as a device.
  • Said device preferably comprises one or more detection systems that target one or more specific RNA sequences.
  • Said device may comprise openings such as inlet and outlet ports, for the introduction and extraction of fluids into and from the device. Said openings may be connected to valves, tubes, channels, chambers, syringes and/or pumps.
  • the devices may be connected to fluid flow actuators that allow directional movement of fluids within the microfluidic device.
  • Example actuators include, but are not limited to, syringe pumps, mechanically actuated recirculating pumps, electroosmotic pumps, bulbs, bellows, diaphragms, or bubbles intended to force movement of fluids.
  • the devices are connected to controllers with programmable valves that work together to move fluids through the device. Additionally, a temperature control mechanism may be provided for incubation of the reaction mixture at a desired temperature.
  • Said CRISPR/Cas-based ribonucleic acid detection system preferably is present in a biosensor, preferably by using a disposable cartridge.
  • a preferred biosensor provides methods and means for detecting interaction of the CRISPR/Cas-based ribonucleic acid detection system with a target nucleic acid.
  • a preferred biosensor comprises a reusable hand-held reader capable of simple push-button operation for automated analysis of samples, and cost-effective disposable cartridges, preferably disposable microfluidic sensor cartridges, that have been functionalized to provide optimal detection and/or quantification of multiple clinically relevant agents such as pathogens.
  • a possible biosensor is a lateral flow test device, for example based on the accumulation of quantifiable substances such as magnetic particles.
  • a device or biosensor in accordance with the invention is a Point of Care (POC) testing device, which is a transportable, portable, and handheld instrument or test kit that allows to collect a sample and obtain the results in a very short period of time at or near the location of the patient so that the treatment plan can be adjusted as necessary.
  • POC Point of Care
  • Said device preferably comprises means that allow a rapid, low- cost, and reliable determination of the presence or absence of a target nucleic acid and, preferably also, a quantification of said target nucleic acid.
  • a POC comprising a CRISPR/Cas-based ribonucleic acid detection system preferably is directed to detecting a limited number of target nucleic acid molecules, including 5 or less target nucleic acid molecules, 4 or less target nucleic acid molecules, 3 or less target nucleic acid molecules, such as 2 target nucleic acid molecules and 1 target nucleic acid molecule.
  • the crRNA ribonucleoprotein complexes are preferably present at discrete positions ribonucleic acid detection system, allowing to determine a level of cOA for each of the individual crRNA ribonucleoprotein complexes.
  • An array comprising a CRISPR/Cas-based ribonucleic acid detection system of the invention may target at least 5 different target nucleic acid molecules, preferably at least different 10 target nucleic acid molecules, preferably at least different 20 target nucleic acid molecules, preferably at least different 50 target nucleic acid molecules, preferably at least different 100 target nucleic acid molecules; or any number of target nucleic acid molecules from 2 to as many as 12,000 target nucleic acid molecules.
  • said different target nucleic acid molecules may all be directed to different organisms such that each of the crRNA molecules is derived from, and is used to detect, a different target organism such as different bacteria, viruses, fungi, protozoa, and/or parasites.
  • Said different crRNA molecules may also be chosen such that a subset of the different crRNA molecules are directed to the same organisms. Said subset may comprise 2 different crRNA molecules, 3 different crRNA molecules, 4 different crRNA molecules, 5 different crRNA molecules.
  • the number of different crRNA molecules that are directed to the same organism preferably is limited to a maximum of 10.
  • a specific crRNA molecule may be present in multiple copies in a CRISPR/Cas-based ribonucleic acid detection system according to the invention, for example in multiple wells of a microtiter plate such as a 48 well plate, a 96 well plate, a 192 well plate, a 384 well plate or a 768 well plate. Detection of a target organism by multiple copies of a crRNA molecule provides confirmation that the identification of the target organism is correct.
  • Figure 1A is a schematic diagram showing the operon for the type III-B CRISPR system from Haliangium ochraceum DSM 14365, together with associated proteins.
  • Figure 1B is a schematic diagram showing the arrangement of components in the type III-B protein complex.
  • Figure 1C is a schematic diagram of the interaction between the SAVED-CHAT protein when activated by coA 3 and the PCaspase protein.
  • Figure 2a is schematic representation of the H.
  • FIG. 1 is a genomic neighborhoods diagram for SAVED-CHAT and PCaspase.
  • Figure 2c shows the domain architecture of SAVED-CHAT.
  • Figure 2d shows SAVED domain phylogenetic midpoint-rooted tree.
  • Figure 2e shows cyclic oligonucleotide-based antiphage signaling system (CBASS) type operons containing Haliangium SAVED clade domains with associated proteins containing cyclase, protein kinase, Sigma-70 and transposase domains.
  • Figure 3a is a schematic representation of the second H. ochraceum type III CRISPR-Cas operon and its associated genes: SAVED-CHAT, PCc- ⁇ , PCi, PCk, and PCaspase (locus tags Hoch_5578-5588).
  • Figure 3b are CHAT domain general and individual focal clade phylogenetic trees.
  • Figure 3c is a phylogenetic tree of caspase domain focal clade.
  • Figures 3d and 3e are phylogenetic trees of PCk and PCc- ⁇ , respectively. All trees are midpoint-rooted, scale bars indicate substitutions per position in the alignment.
  • Figure 4a is an amino acid sequence alignment of certain CHAT domain- containing proteins known in the art. The shading corresponds to the category of amino acid (i.e. polar, basic, etc.,) in accordance with the Clustal coding scheme.
  • WP_015211748.1/1-592 is the SAVED domain-containing protein from Oscillatoria nigro-viridis; WP_046712262.1/1-650 [SEQ ID NO: 3] is the SAVED domain- containing protein from Myxococcus fulvus; WP_095983898.1/1-651 [SEQ ID NO: 4] is the SAVED domain containing-protein from Cystobacter fuscus; WP_082175316.1 [SEQ ID NO: 5] is the SAVED domain-containing protein from Archangium gephyra; WP_012830652.1 [SEQ ID NO: 6] is the CHAT domain-containing protein from Haliangium ochraceum; WP_012826486.1 [SEQ ID NO: 1] is the CHAT domain- containing protein from Haliangium ochraceum.
  • Figure 2 is in grey-scale yet in original colour version follows the Clustal default colour coding as follows: Category Colour Residue at position Hydrophobic BLUE A,I,L,M,F,W,V C Positive RED K,R charge Negative MAGENTA E charge D Polar GREEN N Q S,T Cysteines PINK C Glycines ORANGE G Prolines YELLOW P Aromatic CYAN H,Y Unconserved WHITE any / gap [00145]
  • Figure 4b is an alignment of SAVED-CHAT protein sequences known in the art. The SAVED and CHAT domains are indicated. The residues involved in cOA binding are shown as solid triangles and protease activity shown as open triangles.
  • Figure 4c is an alignment of CHAT-SAVED protein sequences known in the art.
  • Figure 5 is evolutionary tree built using a neighbourhood-joining algorithm from the sequence-alignment presented in Figure 4a.
  • Figure 6a is a photograph of an SDS-PAGE gel showing the results of a SAVED- CHAT cleavage assay showing its dependency on coA3 for cleaving PCaspase.
  • Figure 6b is a photograph of an SDS-PAGE gel showing the results of cleavage activity of SAVED-CHAT on PCc- ⁇ and PCi.
  • Figure 6c is a photograph of an SDS-PAGE gel showing the results of cleavage activity of activated PCaspase on PCc- ⁇ and PCi.
  • Figure 6d is a photograph of a native SDS-PAGE gel showing oligomerization of SAVED-CHAT upon coA 3 addition.
  • Figure 7 is a photograph of an SDS-PAGE gel wherein comparison with various controls shows that only through coA 3 activation of SAVED-CHAT, PCaspase is cleaved. Plus and minus above each well indicate presence and absence of coA 3 respectively.
  • FIG. 8 is a photograph of an SDS-PAGE gel showing the co-factor requirements of PCaspase cleavage by SAVED-CHAT.
  • Figure 9 is a photograph of an SDS-PAGE gel showing how PCi inhibits PCc- ⁇ cleavage by PCaspase. An increasing concentration of PCi reduces the ability PCaspase to cleave PCc- ⁇ , cleavage of PCi is also incomplete. Molar ratios of PCi:PCaspase range from 1:20 – 2:1.
  • Figure 10 is a photograph of an SDS-PAGE gel showing various control that cleaved PCaspase, by cA3 activated SAVED-CHAT, leads to the cleavage of a reporter protein (casein). A catalytically dead version of caspase-like (dPCaspase) does not cleave casein.
  • Figure 11 is a photograph of an SDS-PAGE gel showing co-factor requirements PCaspase cleavage of casein. SDS-PAGE protein cleavage assays with SAVED-CHAT, PCaspase and casein, demonstrating that the activity of PCaspase does not require co- factors.
  • Figure 12 is a photograph of an SDS-PAGE gel showing how PCi inhibits casein cleavage by PCaspase. An increasing concentration of PCi reduces the ability PCaspase to cleave casein and complete cleavage of PCi. Molar ratios of PCi:PCaspase range from 1:20 – 2:1.
  • Figure 13b is a real-time fluorescence assay plot of increasing molar ratio (PCi:PCaspase) of PCi, which reduces the cleavage activity of a FAM-peptide substrate by activated PCaspase.
  • Figure 13c is a real-time fluorescence assay plot for increasing cOA 3 concentrations.
  • the sensitivity of the FAM-peptide visualization method for cOA 3 is 15.6 nM.
  • Figure 14 is a plasmid map of expression vector pJS-BCD.
  • Figure 15a is a 2D class averages of SAVED-CHAT bound to cOA 3 , showing side and top views. In the side view, the filament curvature is evident, with the CHAT domain at the periphery of the arch. In the top view, singlet filaments form a partial inter-filament doublet, with two singlets running in opposite polarities forming cross-fiber contacts spanning ⁇ 3 monomers.
  • Figures 15b and 15c are 3.1 ⁇ -resolution cryo-EM reconstruction and model of the cOA3-bound SAVED-CHAT filament.
  • FIG. 15d is a close-up view of the CHAT-CHAT singlet intra-filament interface, consisting of a four-helix bundle.
  • Figure 15e is a close-up view of the CHAT-CHAT doublet inter-filament interface.
  • Figure 15f shows cOA3 binding site, at the interface between adjacent SAVED domains.
  • Figure 15g shows the CHAT domain active site. The residues H375 and C422 that comprise catalytic dyad (central dark portion) are 3.8 ⁇ apart and located within the substrate-binding channel beneath two unstructured gating loops.
  • Figure 16a shows flexibility of SAVED-CHAT monomer.
  • Four AlphaFold2 models were aligned based on the SAVED domain. Solid spheres correspond to the same residue in all four models, highlighting the flexibility.
  • Figure 16b shows alignment of a single SAVED-CHAT proteolytic active site with TPR-CHAT (Csx29) bound to type III-E CRISPR effector in complex with activating non- self target RNA and substrate peptide Csx30.
  • FIG. 17 is a diagram of a model of the type III-B CRISPR-Cas system and its associated genes in H. ochraceum. Binding of coA 3 to the SAVED domain of SAVED- CHAT induces oligomerization that activates its CHAT domain. Activated SAVED-CHAT cleaves PCaspase, which subsequently mediates further downstream events by cleaving PCi (inhibitor of PCaspase), PCc- ⁇ (a transcriptional response) and potentially also PCk (likely phosphorylation / toxicity) and/or other (host) proteins. Broken lines indicate hypothesized events.
  • PCi inhibitor of PCaspase
  • PCc- ⁇ a transcriptional response
  • PCk likely phosphorylation / toxicity
  • Figure 18 shows how PCaspase activation reduces the transformation efficiency of a target plasmid.
  • A is a schematic overview of the experimental setup in E. coli BL21- AI.
  • B is data of transformation efficiencies (relative to the non-target control) of target or non-target plasmid in E. coli co-expressing H. ochraceum type III CRISPR-Cas complex and different combinations of effector proteins.
  • C is data of transformation efficiencies (relative to the non-target control) when SAVED-CHAT and PCaspase are co-expressed with PCi and PC- ⁇ . Error bars represent the standard deviation of the mean.
  • Figure 19 shows linearized plasmid maps and transformation efficiencies with repressed protein expression for the plasmid challenge assay.
  • (A) is a map of the H.
  • ochraceum type III CRISPR-Cas expression plasmid pHochTypeIII with cmr1-cmr6 from the SAVED-CHAT genomic neighbourhood, csb2 from a co-occurring type I-G system, and the associated CRISPR array with a single spacer sequence targeting a protospacer on pTarget.
  • B are maps of the effector expression plasmids, showing the different combinations of effectors used in the study. pEcNucC and pEmpty were used as positive and negative controls respectively.
  • C are maps of the pTarget and pNon-target, having identical backbones except for a protospacer on pTarget.
  • (D) is data showing transformation efficiencies (relative to the non-target) of pTarget or pNon-target in E. coli with repressed expression of H. ochraceum type III CRISPR-Cas complex and different combinations of SAVED-CHAT and PCaspase.
  • Figure 20 shows expression of the post-cleaved PCaspase fragments in E. coli. Transformation efficiencies of plasmids constitutively expressing PCaspase (“WT”), or the N- (aa 1-153) and/or C-terminal (aa 154-666) PCaspase fragment(s).
  • WT PCaspase
  • the inventors have discovered a novel class of allosterically-activated proteases associated with type III CRISPR-Cas systems. Various aspects of the invention are as hereinbefore defined.
  • cAx-dependent protease is a SAVED-CHAT or CHAT-SAVED protein
  • it may be as hereinbefore defined with reference to any of the reference sequences SEQ ID NO: 14 or SEQ ID NO: 15 or any variant sequence of at least 70% identity therewith, including functional fragments thereof.
  • a variant sequence may have any percentage identity from 70% to 100%.
  • the SAVED-CHAT protein may comprise one or more of the following amino acid (peptide) motifs, wherein X is any amino acid: GX(V/A)X(I/L)PXXX(H/Y)LGXXL [SEQ ID NO: 16] (P/N)XAXXXDXXXXL(R/E)XXXPX(I/V)(V/L)HXX(S/G)HG [SEQ ID NO: 17] DXX(A/S)XXF(S/A)XX(F/L)YXXLXXXXX(I/V)XXAF [SEQ ID NO: 18] [00177] Additionally, or alternatively to (a) the amino acid structure of the SAVED-CHAT protein and/or (b) the one or more amino acid (peptide) motifs, as hereinbefore defined, the SAVED-CHAT protein may comprise one or more of the following amino acid (peptide) motifs, wherein X is any amino acid:
  • the SAVED-CHAT may comprise one or more of the following amino acid (peptide) motifs, wherein X is any amino acid and wherein the underlined amino acid is not subject to substitution or change, but any of the other specified amino acids may be substituted conservatively as elsewhere herein defined: LRLVVLXACX [SEQ ID NO: 19] IPAVIASQMPLS [SEQ ID NO: 20] XPLPLAXXXGX [SEQ ID NO: 21] IHLFLAXPXSXAX [SEQ ID NO: 22] [00179] Further possible SAVED-CHAT proteins are described in Makarova K.
  • CHAT-SAVED proteins are set forth in Table 2 below are described in EMBL-EBI Interpro Classification of protein Families: 52cb831ac#table as set forth in Table 1 below.
  • proteins listed in Table 1 are variants of these proteins having at least 70% identity to the amino acid sequences thereof, or functional fragments of these proteins or sequence variants.
  • proteins listed in Table 2 are variants of these sequences of at least 70% identity therewith, or functional fragments thereof.
  • B2 SAVED A0A3A8RJB2_9DELT protein 5 AB032C SAVED domain- D7X99 A0A3A8R CHAT- containing _1865 Corallococcus sp.
  • L35 SAVED A0A3A8RL35_9DELT protein 0 AB032C CHAT domain- D7Y15 A0A3A8T CHAT- containing _4039 Corallococcus sp.
  • W57 SAVED T protein 5 AB030 SAVED domain- EDM0 A0A3M9Y CHAT- A0A3M9YX02_9CYA containing 5_336 Leptolyngbya sp.
  • X02 SAVED N protein 85 IPPAS B-1204 SAVED domain- EOO71 A0A4Q5Z CHAT- containing _1269 Myxococcaceae
  • the mutations may be conservative or non-conservative amino acid substitutions.
  • “Conservative amino acid substitutions” refer to the interchangeability of residues having similar side chains, and thus typically involves substitution of an amino acid in a polypeptide with amino acids within the same or similar defined class of amino acids.
  • an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid, e.g., alanine, valine, leucine, and isoleucine; an amino acid with a hydroxyl side chain may be substituted with another amino acid with a hydroxyl side chain, e.g., serine and threonine; amino acids having aromatic side chains may be substituted with another amino acid having an aromatic side chain, e.g., phenylalanine, tyrosine, tryptophan, and histidine; an amino acid with a basic side chain may be substituted with another amino acid with a basic side chain, e.g., lysine and arginine; an amino acid with an acidic side chain may be substituted with another amino acid with an acidic side chain, e.g., aspartic acid or glutamic acid; and a hydrophobic or hydrophilic amino acid may be substituted with another hydrophobic or hydrophilic amino acid, respectively
  • Non-conservative substitution refers to substitution of an amino acid in a polypeptide with an amino acid with significantly differing side chain properties.
  • Non- conservative substitutions may use amino acids between, rather than within, the defined groups and may affect (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain.
  • an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.
  • “Deletion” refers to modification of a polypeptide by removal of one or more amino acids in comparison to a wild-type or control polypeptide.
  • Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, or 3 or more amino acids of the polypeptide while retaining enzymatic activity. Deletions can comprise a continuous segment or can be discontinuous. [00186] The inventors have discovered a type III-B system in Haliangium ochraceum DSM 14365 that is flanked by a second messenger oligonucleotide or dinucleotide synthetase- associated and fused to various effector domains (SAVED) -Caspase HetF associated with TPR (CHAT) protein and a Caspase-like gene (see Figures 1A and 2a).
  • the type III CRISPR-Cas system from Haliangium ochraceum DSM 14365 comprises a multi-subunit type III-B protein complex and this, together with suitably complementary guide/crRNA, targets RNA molecules for cleavage (see Figure 1B).
  • Base pairing of the target RNA triggers the target RNA cleavage, but also activates the production of cyclic oligoadenylate (cOA) second messenger molecules, made from ATP.
  • cOA cyclic oligoadenylate
  • the inventors identify a set of genes that resides close to an operon encoding a type III-B CRISPR-Cas crRNA-guided protein complex (Cmr1-6) in the Haliangium ochraceum DSM 14365 genome. These are shown in Figures 1A and 2a.
  • the inventors observed a SAVED-CHAT gene, which encodes a fusion protein of a SAVED sensory domain and a CHAT domain (related to cysteine proteases that include the caspases, known to be involved in controlled cell death in eukaryotes). Further downstream, the inventors observe a gene encoding a caspase-like protein, which is named PCaspase (Prokaryotic Caspase).
  • PCc- ⁇ Prokaryotic Caspase-controlled sigma factor
  • PCi Prokaryotic Caspase inhibitor, due to predicted structural homology with the CI-2 family of serine protease inhibitors
  • PCk Prokaryotic Caspase-controlled kinase
  • ochraceum SAVED domains formed a monophyletic clade ( Figure.3d) and were likely acquired once and duplicated, leading to the two SAVED copies to which CHAT domains of different origin were added subsequently.
  • This scenario is further supported by phylogenies of PCaspase, PCc- ⁇ , and PCk (see Figures 2b - 2e), which all support monophyly of the two respective copies.
  • the SAVED domains are most closely related to those of other Myxococcota and may be part of a conserved system in these bacteria.
  • CBASS cyclic oligonucleotide-based antiphage signaling system
  • SAVED-CHAT & PCaspase cleavage activity [00191] All type III-associated proteins were purified from H. ochraceum (except for PCk, which could not be cloned either individually or in combination with PCc- ⁇ and PCi, likely due to toxicity) and conducted in vitro cleavage assays with SAVED-CHAT incubated with any of the three other proteins. [00192] Protein cleavage assays were also performed with SAVED-CHAT and PCaspase in combination with either PCc- ⁇ or PCi alone or with PCc- ⁇ and PCi together.
  • SAVED-CHAT complex formation on native PAGE 1 ⁇ M SAVED- CHAT was incubated with or without 1 ⁇ M coA 3 for 1h at 35 °C in a buffer containing 125 mM NaCl, 10 mM Tris-HCl (pH 8.0), and 1 mM DTT. Afterwards, the reaction was run on a native 4-20% polyacrylamide gel, stained with Coomassie blue, and visualized (Biorad Gel Doc XR).
  • Figure 6a, 7 and 8 show that SAVED-CHAT specifically cleaves PCaspase, in a coA 3 -dependent and co-factor independent manner, whereas no cleavage was observed for PCc- ⁇ and PCi (Fig.6b).
  • SAVED-CHAT cleaved PCaspase into at least three defined fragments in addition to a myriad of products.
  • Figure 7 shows how a catalytically dead version of SAVED-CHAT (dSAVED-CHAT, H375A, C422A) abrogated this activity.
  • Figure 6c shows cleavage of PCaspase by coA3-induced SAVED-CHAT resulted in the subsequent cleavage of PCc- ⁇ and PCi into defined cleavage products.
  • Example 2 Activated PCaspase protein has general protease activity
  • Biologically unrelated casein protein was used as a substrate for activated PCaspase in an in vitro cleavage assay.
  • Activated PCaspase leads to the degradation of casein in a co-factor independent manner, while SAVED-CHAT by itself does not (see Figures 10a and 11).
  • a catalytic mutant of PCaspase (dPCaspase, H78A, C145A) abrogates this activity completely.
  • a PCi titration experiment demonstrates that PCi inhibits PCaspase activity on casein, demonstrating this inhibitory effect is independent from PCc- ⁇ ( Figure 12).
  • Example 3 Prototype fluorescent in vitro diagnostic assay
  • a small fluorophore-quencher peptide was provided as a substrate, which is specifically cleaved by activated PCaspase ( Figure 13a).
  • Figure 13a Real-time PCaspase activity assay.
  • SAVED-CHAT/PCaspase activity assays were conducted in vitro in activity buffer (125 mM NaCl, 10 mM Tris, 1 mM DTT, pH 8.0) to which different combinations of component were added: SAVED-CHAT (0.5 ⁇ M), PCaspase (0.5 ⁇ M), coA 3 (15.6 nM to 1 ⁇ M), and/or FAM-peptide substrate (5 ⁇ M) (Eurogentec AS-60579-01). Assays were incubated for one hour at 37 °C with a FAM channel measurement at 1 min intervals in a Thermo Scientific Quantstudio 1 RT-qPCR instrument running Quantstudio Design & Analysis software (v1.5.2).
  • Example 4 Structural basis for SAVED-CHAT activation
  • 10 ⁇ M SAVED-CHAT was mixed with 125 ⁇ M coA3.2.5 ⁇ l of complex was immediately applied to C-flat grids (1.2/1.3, 300 mesh) which had been plasma-cleaned for 30 seconds in a Solarus 950 plasma cleaner (Gatan) with a 4:1 ratio of O2/H2.
  • Grids were blotted with Vitrobot Mark IV (Thermo Fisher) for 6 seconds, blot force 0 at 4oC & 100% humidity, and plunge-frozen in liquid ethane.
  • Data were collected on a FEI Glacios cryo- TEM equipped with a Falcon 4 detector. Data was collected in SerialEM, with a pixel size of 0.94 ⁇ , a defocus range of -1.5 - -2.5 ⁇ m, and a total exposure time of 15s resulting in a total accumulated dose of 40 e/ ⁇ 2 which was split into 60 EER fractions.
  • Motion correction, CTF estimation and particle picking was performed on-the-fly using cryoSPARC Live v4.0.0-privatebeta.
  • SAVED-CHAT oligomerization results in long, curved filaments with the SAVED domain on the inside and the CHAT domains at the periphery, with a curvature of ⁇ 10o between each monomer ( Figure 15a).
  • SAVED-CHAT monomers assemble via head-to-tail oligomerization, with a single coA3 bound at the interface between two SAVED domains, forming ‘singlet’ filaments.
  • the CHAT-CHAT inter- filament doublet interface is mediated by an unusual ⁇ - ⁇ stacking interaction between two R349 residues (that is, the same residue from different monomers), reinforced by additional electrostatic contacts (Figure 15e).
  • the coA3 is buried within the intra-filament interface between two SAVED domains, and participates in a plethora of hydrogen bonds, electrostatic and stacking interactions (Figure 15f). This network of contacts suggests that coA 3 acts as a molecular glue to bridge SAVED domain intra-filament interactions, which subsequently provides a platform for CHAT domain rigidification, doublet formation and ultimately substrate capture.
  • cOA3 ochraceum and its effector components, where detection of target RNA results in the generation of coA 3 (not shown in Figure 17).
  • the synthesized cOA3 binds to the sensory domain of SAVED-CHAT. Acting as a molecular glue, cOA3 participates in a multitude of interactions that initially stabilize SAVED:SAVED dimerization ( Figures 15a - g). The subsequent multimerization of the unusual antiparallel SAVED-CHAT doublet filament results in the activation of the CHAT domains, a unique mechanism for CARF and SAVED effector proteins ( Figures 15a - g).
  • the activated CHAT domains cleave and activate PCaspase, which in turn becomes an active protease, cleaving at least PCc- ⁇ and PCi into defined products ( Figure 6a - d).
  • PCaspase might have a broader substrate repertoire, as demonstrated by the cleavage of the casein and FAM-peptide substrates ( Figures 13b and c).
  • Example 5 SAVED-CHAT and PCaspase activation leads to a strong defense mechanism phenotype Plasmid interference assay [00214] To assess SAVED-CHAT/PCaspase-mediated in vivo response, electrocompetent E. coli BL21-AI (Invitrogen) carrying pHochTypeIII and one of the pEffectors (see Figures 18A, 19A and 19B) were prepared for target/non-target plasmid transformation. [00215] For pHochTypeIII, H.
  • orchaceum type III-B cmr1-6, csb2, and a minimal CRISPR array carrying one spacer sequence were placed individually under the control of T7 promoters.
  • the various effector genes (and mutants or fragments thereof) were cloned into one operon and placed under the control of a constitutively expressing lacUV5 promoter.
  • a non-coding RNA was placed under the control of a trc promoter and lacO. This non-coding RNA carries the protospacer targeted by the spacer of the crRNA guide encoded by the CRISPR array on pHochTypeIII.
  • the transformations were carried out in biological triplicates, using 100 ng of target or non-target plasmid, by electroporation (BTX electroporation system). After transformation, all cells were recovered in 1 mL of LB at 37 °C for 1h. Ten-fold dilutions were plated on LB agar medium containing 0.2% arabinose, 34 ⁇ g/mL chloramphenicol, 50 ⁇ g/mL carbenicillin, 50 ⁇ g/mL kanamycin, and either 1 mM IPTG or 0.2% glucose to induce or repress target RNA transcription, respectively. Finally, the plates were incubated overnight at 30 °C, and transformation efficiencies were quantified.
  • the SAVED-CHAT and PCaspase genes were expressed in different combinations from the pEffector plasmid (see Figure 19B).
  • NucC a cA3-responsive nuclease that causes abortive infection
  • Escherichia. coli MS115-1 and an empty vector were used as positive and negative controls, respectively (see Figure 19B).
  • Target and non-target plasmids encoded an isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) inducible protospacer that was complementary and non complementary to the spacer, respectively (see Figure 19C).
  • IPTG isopropyl- ⁇ -D-thiogalactopyranoside

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Abstract

Un système de type lll-B de Haliangium ochraceum contient deux protéases de type caspase, SAVED-CHAT et PCaspase (caspase procaryote). L'oligomérisation de SAVED-CHAT induite par le tri-adénosine monophosphate cyclique (AMP) active l'activité protéolytique des domaines CHAT, qui clivent et activent spécifiquement la PCaspase. Ensuite, la PCaspase activée clive une multitude de protéines, ce qui conduit à un fort phénotype d'interférence in vivo dans Escherichia coli. Ceci fournit un procédé basé sur CRISPR-Cas de détection d'un ARN cible, ce par quoi une signalisation de détection est déclenchée par l'intermédiaire d'une cascade d'activités protéolytiques associées à la caspase. L'invention concerne également des polynucléotides, des plasmides, des vecteurs et des complexes nucléoprotéiques comprenant des acides nucléiques codant pour les composants du système.
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