Synthetic CAS proteins

Abstract

The present invention relates to a novel RNA-guided Cas nuclease having the sequence shown in SEQ. ID. No.1 and designated α-synCs. It shares low sequence homology with known Cas nucleases and has the following advantageous properties: (i) ability to target for nucleolytic cleavage all of dsDNA, ssDNA and ssRNA (ii) able to non-sequence-specifically trans-cleave all of dsDNA, ssDNA and ssRNA regardless of which target it has been activated by (ii) appears to have no PAM specificity (iii) ability to use a wide range of crRNAs belonging to a plurality of Cas nucleases for targeted cleavage and (iv) ability to process pre-crRNA transcripts.

Description

[0001] SYNTHETIC CAS PROTEINS

[0002] Field of the Invention

[0003] The present invention relates to a novel RNA-guided Cas nuclease not found in nature which is able to target for nucleolytic cleavage all of dsDNA, ssDNA and ssRNA, is able to non- sequence-specifically trans-cleave all of dsDNA, ssDNA and ssRNA regardless of which target it has been activated by, has relaxed PAM requirement, is able to use a wide range of crRNAs belonging to a plurality of Cas nucleases for targeted cleavage, is able to process pre- crRNA transcripts, and shares low sequence homology with known Cas effector nucleases. The inventors have termed this nuclease a-synCas. The present invention further relates to novel variants of a-synCas including nucleases, nucleic acids capable of expressing a polypeptide as now disclosed, products comprising such a polypeptide, and methods of using a-synCas and variants thereof to detect nucleic acids and to modify or regulate a target nucleic acid sequence.

[0004] Background to the Invention

[0005] Introduction. Native CRISPR-Cas systems provide immunity to prokaryotes and archaea in response to invading nucleic acids - DNA or RNA - from infectious genetic elements. Cas effector nucleases guided by RNA molecules (gRNAs) recognize specific regions of the foreign genome and cleave it for inactivation. Since the first Cas effector nuclease, Cas9, was repurposed as a gene-editing tool in 2012 (Jinek et al.), such CRISPR systems have revolutionised the field of genome engineering and nucleic acid-based diagnostics.

[0006] CRISPR RNA (crRNA). Target recognition by Cas effector nucleases is accomplished by a structural non-coding crRNA that complexes with the Cas effector nuclease and through basepairing complementarity locates the Cas-crRNA complex to its target nucleic acid sequence site for nucleolytic cleavage. To enable such Cas nuclease function, the crRNA may or may not need to complex with another RNA such as a trans-activating CRISPR RNA (tracrRNA) or a short-complementarity untranslated RNA (scoutRNA; see Harrington et a / .2020). The dual RNA or single RNA that binds to and guides the Cas effector nuclease to enable targeted nucleic acid cleavage is called the guide RNA (gRNA). Hence, the terms ‘crRNA’ and ‘gRNA’ may in some instances be used interchangeably, e.g. Cas12a nucleases require for targeted cleavage a single RNA oligonucleotide commonly referred to as the crRNA and acting as the gRNA. In contrast, a Type II Cas nuclease such as a Cas9 requires a crRNA and a tracrRNA to provide a guide RNA for targeted cleavage, either in the form of a dual RNA or linked to provide a single oligonucleotide (commonly termed a sgRNA). Guide RNA is known to contain an element, for example a hairpin loop-type structure, which enables its binding to the Cas effector nuclease. This non-targeting region of the gRNA is broadly called the scaffold region and is specific both in sequence and therefore structure to the CRISPR system and species to which it belongs or from which it derives.

[0007] PAM requirements. In addition to crRNA recognition, initiation of Cas-crRNA target binding may require a motif to be present within the target nucleic acid substrate, termed the protospacer adjacent motif (PAM) for DNA or protospacer flanking site (PFS) for RNA. This is important for distinguishing self from non-self in native anti-viral defence systems. However, it is less helpful for some desired applications arising from the repurposing of Class 2 CRISPR- Cas systems as a therapeutic or diagnostic tool.

[0008] CRISPR-Cas diversity & classification. The number and diversity of known CRISPR-Cas systems has been constantly expanding since the first demonstration of in vitro DNA-editing using Cas9 in 2012 (Jinek et al.). The classification of these enzymes is evolving accordingly. Today CRISPR systems are classified into two classes, Class 1 or Class 2, and various types (Type l-VI). Class 1 contains Types I, III and IV and Class 2 contains Types II, V and VI as reviewed by Makarova et al. (2020). Each type may have subtypes. The distinguishing feature of Class 2 systems is that Cas effector nucleases of these systems consist of a single, multidomain protein as exemplified by such enzymes as Cas9 (Type II), Cas12a and Cas14a (Type V), and Cas13a (Type VI).

[0009] Domain architectures of Type II, V, and VI. Cas effector nucleases are characterised as Type II by containing two nuclease domains, both an HNH-like and RuvC-like endonuclease domain, and associated catalytic residues required for double-strand DNA endonuclease cleavage resulting predominantly in blunt ends. Each domain is responsible for the cleavage of one strand of the target DNA. By contrast, Type V Cas effector nucleases (Cas12 nucleases) are currently known to only contain a RuvC-like endonuclease domain that cleaves both strands in a staggered manner. RNA-targeting Type VI effector nucleases (Cas13 nucleases) are unrelated to Type II and Type V systems, seem to be characterised by containing two HEPN domains, and in their native environment apparently target either invading RNA genomes or the RNA transcripts of invading DNA genomes. Type VI systems are the first and so far only family of CRISPR-Cas systems to exclusively cleave RNA using HEPN domains. Pre-crRNA processing. In their native setting, crRNAs are derived from a long pre-crRNA transcript which requires processing into mature crRNAs. A distinguishing feature of seemingly all Type V and VI Cas effector nucleases is their ability to process their own pre- crRNA into crRNAs.

[0010] Trans-cleavage. Another distinguishing feature of seemingly all Type V and VI Cas effectors is their ability to trigger non-specific, indiscriminate, collateral cleavage (trans-cleavage) of certain types of non-target nucleic acids. Such trans-cleavage abilities are believed to be triggered upon target recognition by the Cas-crRNA complex.

[0011] Diagnostics. The ability of Type V and VI family Cas effector nucleases to unleash indiscriminate nuclease activity upon target recognition lends such nucleases to diagnostic applications. Broadly the most common forms of CRISPR-based diagnostics rely on the collateral cleavage activity of Type V and VI Cas effector nucleases. In such diagnostic applications, Type V or VI Cas enzymes are provided with crRNAs that are complementary to a target to be detected. Upon provision of the test sample and target recognition by the Cas- crRNA complex, the Cas enzyme indiscriminately cleaves a reporter nucleic acid in its surrounding that it is capable of cleaving regardless of sequence. This indiscriminate cleavage, commonly referred to as collateral cleavage, allows for amplification of the signal of target nucleic acid detection, since one detected molecule results in several trans-cleaved substrates. When such nucleic acid trans-substrates are labelled with both a fluorophore and a quencher for example, their cleavage releases the fluorophore and thus creates fluorescence. This enables a fluorescent readout once target nucleic acids have been detected. Various other methods are in common usage for detection of collateral cleavage activity by Cas nucleases, including, for example, detection of reporter cleavage by lateral flow assay. Variations on application of such collateral cleavage for nucleic acid target detection exist depending on the type of Cas enzyme used, the sample preparation method, and the assay readout; a good review is provided by Kaminski, M.M. et al., 2021 , CRISPR-based diagnostics, Nature Biomedical Engineering, 5(7), pp.643-656.

[0012] Cas properties in diagnostics. The diverse properties of various Cas effector nucleases predispose them to distinct diagnostic applications. Some of the most important features determining the utility of Cas effector nucleases in diagnostic applications are the types of nucleic acids they can cis-target and the types of nucleic acids they are able to indiscriminately trans-cleave. Cis-targets and trans-substrates may each be one or more of single-stranded (ss)DNA, double-stranded (ds)DNA or single-stranded (ss)RNA. Briefly reviewing the Cas enzymes of greatest relevance to the present disclosure, Cas12a has been shown to cis-target only ssDNA and dsDNA and to trans-cleave only ssDNA (Chen et al., 2018); these same activities are shared by Type V Cas effector nucleases Cas12b, Cas12c, Cas12d, Cas12h, Cas12i and Cas12j (Cas<t>) (Tong et al., 2021). Cas13a (formerly C2c2) has been shown to cis-target and trans-cleave ssRNA (Abudayyeh et aL, 2016; East-Seletsky et aL, 2016). Cas12a2 has been shown to cis-target only ssRNA and to trans-cleave ssDNA, dsDNA and ssRNA (Bravo et aL, 2023; Dmytrenko et aL, 2023). A further important feature of Cas effector nucleases for diagnostic, therapeutic and other applications is the sequence of the PAM or PFS required for target recognition and cleavage. Less restrictive sequence requirements enable broader and more customisable targeting. All the aforementioned Cas effector nucleases have defined PAM or PFS sequence requirements: Cas12a: TTTN (Zetsche et aL, 2015), Cas12j (Cas$): TTN (Wang et a!., 2023), Cas13a: H (A / C / U); Abudayyeh et aL, 2016); Cas12a2: GAAAG (Dmytrenko et al. 2023). Note that some RNA-targeting Cas13 orthologues do not require a PFS (Huynh et al., 2023).

[0013] Broadening PAMs. Attempts have been made to engineer Cas variants with broadened PAM recognition abilities. Techniques such as structure-guided mutagenesis and directed evolution through random mutagenesis of the PAM interaction domain (PID) of SpCas9 followed by subsequent selection are reviewed by Collias & Beisel (2020). Walton et al. (2020), by application of structure-guided mutagenesis, achieved the SpRY nuclease variant which recognises a consensus NR (R= A or G) PAM and with less preferred recognition a RY (Y=C or T) PAM. The most relaxed PAM requirements reported to date are for an ancestral Cas9 (anCas9), obtained through phylogenetic reconstruction techniques, which appears to require no PAM at all (see PCT publication WO2022 / 248607).

[0014] Cas problems. The applications of Cas nucleases face several problems. As a therapeutic tool, Cas enzymes suffer due to limitations such as the generation of unwanted mutations at similar (off-target) loci, production of multiple alleles leading to genetic mosaicism, low efficiency and possible induction in the host of an immune response. One study found that blood samples from human donors showed a high percentage with antibodies to SpyCas9 and Staphylcoccus aureus Cas9 (Charlesworth et aL, 2019). Furthermore, the requirement of a PAM or PFS can limit the utility of Cas effector nucleases across all applications including therapeutics and diagnostics. The utility of Cas nucleases is further restricted across all their applications by the types of nucleic acids they are able to cleave in cis or in trans, in particular trans-substrate requirements are crucial for diagnostic applications. Such restrictions may necessitate the research and development of multiple Cas nucleases to detect various nucleic acid substrates as desired. There has remained a need for truly multi-purpose Cas nucleases to enable ‘all-in-one’ diagnostic solutions Thus, despite the diversity of known naturally-occurring Class II CRISPR-Cas systems, still further diversity is desired to amongst other things achieve diverse nucleic acid targeting desires, especially in the therapeutics and diagnostics fields.

[0015] Summary of the Invention

[0016] The inventors in this instance have now designed and expressed in E. coli at high level a nonnative Cas enzyme which extends the (cis-)targeting ability observed with Cas12a2 from just ssRNA to all of ssRNA, ssDNA and dsDNA with exhibition of the same collateral (trans-)cleavage activities against all of ssRNA, ssDNA and dsDNA. In other words, said nonnative Cas enzyme is able to cis-target all of ssDNA, dsDNA and ssRNA and upon recognition of each of these targets is able to then trans-cleave all of ssDNA, dsDNA and ssRNA. This enzyme is designated alpha-synCas (a-synCas) and consists of the amino acid sequence as set out in SEQ ID NO. 1.

[0017] Alpha-SynCas has additionally been found to have (a) a relaxed PAM requirement, being able to efficiently linearize plasmid DNA with non-T-rich, non-A-rich, non-G-rich and non-C-rich PAM sequences of 3 nucleotides, e.g. CCC or TTT; indeed appearing PAM-less and demonstrating virtually no PAM preferences in an unbiased PAM detection assay, (b) ability to use a crRNA as a single oligonucleotide gRNA selectable from crRNAs suitable for employment by a plurality of Cas enzymes of Type II and Type V, for example selected from crRNAs suitable for a Cas12a from a plurality of species, e.g. crRNAs suitable for any of the Cas 12a of Francisella novicida (FnCas12a), Lachnospiraceae bacterium (LbCas12a) or Acidaminococcus sp. (AsCas12a), or a crRNA suitable for a Cas9, e.g. SpCas9 (i.e. a Type II crRNA without any tracrRNA), or a crRNA suitable for a phage Cas<t> (Type V) and (c) an ability to process its own crRNA from a pre-crRNA transcript, e.g. process a crRNA from a FnCas12a pre-crRNA transcript in vitro under conditions whereby the same pre-crRNA transcript will be processed to provide a crRNA by FnCas12a.

[0018] It will be understood from (b) above that an important property of a-SynCas is promiscuity towards gRNA scaffold recognition consistent with an ability to use for targeted cis-nucleic acid cleavage a variety of crRNAs suitable for a Type V CRISPR-Cas system and even merely a crRNA of a Type II CRISPR-cas system without any tracrRNA interaction as exemplified and noted above. The noted functional properties of a-synCas, including its versatile cis- and trans-cleavage abilities, relaxed PAM requirement, broad-ranging pre-crRNA processing abilities and versatile gRNAs compatibilities, make this nuclease and variants that retain its properties a highly useful addition to the toolbox of available Cas effector nucleases for a number of reasons.

[0019] Firstly, a- synCas is especially useful for nucleic-acid based diagnostics owing primarily to its cis-cleavage abilities, which provide versatility in detecting all of dsDNA, ssDNA and ssRNA nucleic acids, and owing also to its versatile trans-cleavage abilities which allow for all of dsDNA, ssDNA and ssRNA to be used as nucleic acid reporters (e.g. fluorescently labelled ones) in such diagnostic assays. Accordingly, a-synCas can be used instead of Cas12 enzymes such as DNA-targeting Cas12a, Cas12f (formerly Cas14) and Cas12j (formerly Cas<t>) and RNA-targeting Cas12a, instead of RNA-targeting Cas 13 (formerly C2c2) enzymes, or instead of any other Cas nuclease, thus providing a single-enzyme solution for targeting all of dsDNA, ssDNA and ssRNA. Alpha-synCas is of especial interest for multiplex screening of nucleic acid sequences in part due to its pre-crRNA processing abilities, being able to process a single pre-crRNA transcript into one or more crRNAs for subsequent complexing and RNA- guided nucleolytic cleavage. Alpha-synCas has opened the door to what the inventors term the MONTANA approach (Multimodal One-enzyme Targeting of Nucleic Acids).

[0020] Secondly, a-synCas can be used for modifying and regulating target nucleic acid sequences with relaxed PAM requirement compared to known Cas effector nucleases such as Cas12 enzymes or SpCas9. This is useful for broadly all its applications including diagnostics and therapeutics. Relaxed PAM requirements broaden the targeting space or targeting range achievable with Cas effector nucleases.

[0021] Thirdly, low sequence homology between a-synCas and SpCas9, as well as other known Cas effectors, makes it a useful alternative therapeutic tool which avoids the problem of treating subjects with pre-existing antibodies against SpCas9 or S. pyogenes more broadly; a significant proportion of humans (-60%) are believed to have immunity against S. pyogenes meaning the majority of individuals will have an immune response to SpCas9.

[0022] Alpha-synCas has low sequence identity to native Cas12 enzymes (e.g., 52% to FnCas12a, 26% identity to Sulfuricurvum sp. PC08-66 Cas12a [SuCas12a2]) but comparison with such enzymes can provide direction to attaining, by application of routine techniques, useful variants in addition to variants with one or more conservative substitutions, which retain the same functional activities including targeting and collateral cleavage activities. In particular, variant amino acid positions have been identified compared to the known native FnCas12a as discussed below which are considered essential for retaining its described functional activities including its targeting and collateral cleavage activities.

[0023] Thus in one aspect there is now provided a Cas nuclease comprising or consisting of

[0024] (a) the nuclease having the amino acid sequence as set out in SEQ ID NO 1 and designated a-synCas; or

[0025] (b) a Cas nuclease variant of the amino acid sequence of SEQ ID NO: 1 which retains

[0026] (i) the ability when complexed with a guide RNA (gRNA) to target and cleave any of a dsDNA, ssDNA and ssRNA and, when activated by such targeting, the capability to non-specifically trans-cleave any of dsDNA, ssDNA and ssRNA or retains (i) and / or

[0027] (ii) a relaxed PAM requirement comparable to a-synCas; and which preferably also retains one or both of the following functional properties:

[0028] (iii) ability to use a crRNA as a single oligonucleotide gRNA selectable from crRNAs suitable for employment by a plurality of Cas enzymes of Type II and Type V as discussed above; and

[0029] (iv) ability to process its own crRNA from a pre-crRNA transcript.

[0030] As a preferable embodiment of the invention, a Cas nuclease of the invention comprises or consists of

[0031] (a) the nuclease having the amino acid sequence as set out in SEQ ID NO 1 and designated a-synCas; or

[0032] (b) a variant of the amino acid sequence of SEQ ID NO: 1 which retains

[0033] (i) the ability when complexed with a gRNA to target and cleave any of a dsDNA, ssDNA and ssRNA and, when activated by such targeting, the capability to non- specifically trans-cleave any of dsDNA, ssDNA and ssRNA and which preferably also retains one or more of the following functional properties:

[0034] (ii) a relaxed PAM requirement comparable to a-synCas;

[0035] (iii) ability to use a crRNA as a single oligonucleotide gRNA selectable from crRNAs suitable for employment by a plurality of Cas enzymes of Type II and Type V, for example selected from crRNAs suitable for a Cas12a from a plurality of species, e.g. crRNAs suitable for any of FnCas12a, LbCas12a and AsCas12a, or a crRNA suitable for a Cas9, e,g. SpCas9, or a crRNA suitable for a phage Cas<|) and

[0036] (iv) ability to process its own crRNA from a pre-crRNA transcript, e.g. process a crRNA from a FnCas12a pre-crRNA transcript in vitro under conditions whereby the same pre- crRNA transcript will be processed to provide a crRNA by FnCas12a. A variant of a-synCas may be especially preferred that retains all of the functional properties (i)-(iv) as noted above.

[0037] Useful modifications to Cas nucleases are well-known in the art such as the addition of a nuclear localisation signal to the N- or C-terminus, or the mutation of certain key catalytic amino acid residues which modify their behaviour. Accordingly, a Cas nuclease according to the invention as discussed above may be fused at the N- or C-terminus to an additional amino acid sequence, e.g. a nuclear localization signal. The invention also provides variants of a Cas nuclease as discussed above derivable by catalytic site mutagenesis to vary its cis-targeting and / or collateral trans-cleavage ability, for example such a variant which is either (a) a nickase which exhibits nickase activity upon targeting to a dsDNA or (b) is a dead Cas (dCas) without nuclease activity, possibly said nickase or dCas being linked with a non-Cas effector protein such as a DNA modification domain (i.e. non-Cas effector protein) for genetic modification or regulation.

[0038] It is envisaged that novel functional domains of a-synCas or variants thereof as now disclosed can be taken in isolation and optionally combined with functional domains of other proteins such as Cas nucleases to achieve customisable assembly of various functionalities thus resulting in chimeric polypeptides with user-defined capabilities. For example, the cis- and trans-cleavage abilities of a-synCas might be combined with domains from a smaller Cas nuclease such as Cas12j (also known as Cas<t>; see Tong et al. 2021) to yield a small Cas effector with the same cis- and trans-cleavage abilities of a-synCas.

[0039] In a further aspect, the invention therefore provides a novel functional domain corresponding to or derivable from a parent Cas nuclease or variant as discussed above, said parent Cas nuclease or variant retaining all the properties (i) -(iv) as noted above or being a nickase or dCas variant thereof obtained by catalytic site mutagenesis, e.g. a-synCas or a nickase or dCas modification thereof. For example, such a functional domain may provide the cleavage abilities of a- synCas or the relaxed PAM requirement of a-synCas. The invention extends to any chimeric cas polypeptide incorporating such a domain, e.g. a chimeric cas nuclease.

[0040] The invention additionally extends to nucleic acids capable of expressing a polypeptide of the invention.

[0041] In another aspect, there is provided a combination product comprising or consisting of

[0042] (i) a Cas nuclease, variant or chimeric Cas polypeptide according to the invention, or a polynucleotide capable of expressing the same and (ii) a crRNA for targeting said Cas nuclease, variant or chimeric Cas polypeptide to a target sequence, or a polynucleotide capable of expressing the same, for example where (i) and (ii) are expression vectors or where (i) and (ii) provide a ribonucleoprotein complex wherein said Cas nuclease, variant or chimeric Cas polypeptide is complexed with a crRNA.

[0043] Such a combination product may be packaged and / or formulated to provide a pharmaceutical composition. This may include packaging into virus particles, virus-like particles or lipid particles for example, or any other commonly used packaging means applied in the art to Cas nucleases for therapeutic purposes.

[0044] It will be appreciated that the invention extends to use of Cas nucleases of the invention for in vitro detection of one or more target nucleic acids in a variety of samples where the collateral cleavage ability of the Cas nuclease is relied upon for detection. As indicated above, Cas nucleases of the invention are especially of interest for multiplex screening for a plurality of target nucleic acid sequences where the Cas nuclease can be provided with a single pre- crRNA transcript for provision of all required crRNAs for targeting.

[0045] Moreover, it will be appreciated that the invention extends the options for modifying or regulating target nucleic acid sequences as further discussed below and now claimed.

[0046] Detailed Description of the Invention

[0047] The following terms are well understood in the art and are used herein in conventional manner but for completeness are now commented upon:

[0048] “Cas effector nuclease” and “Cas nuclease” are used interchangeably to refer to the CRISPR-associated (Cas) enzymes that are nucleases associated with the cellular adaptive immune system widely referred to simply as CRISPR or nucleases which function in the same manner by targeting using a guide RNA, e.g. Cas nucleases include a-synCas, Cas9 and Cas12a.

[0049] “Nucleic acid” collectively refers to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA).

[0050] “Target”, “cis-target” and “cis-substrate” are used interchangeably to refer to the nucleic acid substrate of a Cas nuclease that is complementary, at least partially, to the targeting region of its crRNA. “Cis-recognition” refers to the ability of a Cas effector nuclease to recognise its cistarget.

[0051] “Trans-target” and “trans-substrate” are used interchangeably to refer to nucleic acids that do not necessarily share any complementarity with a crRNA but are nevertheless subject to cleavage, regardless of sequence, by a Cas nuclease through its indiscriminate cleavage or trans-cleavage abilities once activated by cis-substrate recognition.

[0052] “Cis-targeting”, “cis-cleavage” and “cis-activity” are used interchangeably to refer to the cleavage of a cis-target nucleic acid by a Cas nuclease.

[0053] “Trans-targeting”, “trans-cleavage”, “trans-activity”, “indiscriminate targeting”, “indiscriminate cleavage, “indiscriminate activity”, “collateral targeting”, “collateral cleavage”, “collateral activity”, “non-specific targeting”, “non-specific cleavage” and “non-specific targeting” or combinations thereof such as “indiscriminate collateral trans-cleavage” are used interchangeably to refer to the non-sequence-specific cleavage by a Cas effector nuclease of non-target nucleic acid substrates, an activity which is believed in the art to be enabled by cis-target recognition by a Cas-gRNA complex (or Cas-crRNA complex).

[0054] “Guide RNA” (“gRNA”) refers to the one or more RNA molecules which complex with a Cas nuclease and guide it to its target site to enable cis substrate cleavage as determined by base-pairing between the targeting region of the gRNA (historically called the spacer sequence) and the target nucleic acid. In other words, the sequence of the targeting region of a gRNA matches that of the target nucleic acid site optionally with a number of mismatches. The constituent components of a gRNA vary across CRISPR family types and subtypes (as reviewed by Makarova et al 2020), but all gRNAs comprise at least a CRISPR RNA (“crRNA”) which contains said targeting region; gRNAs of certain Cas nucleases further provide a crRNA in a dual RNA form comprising a transactivating CRISPR RNA (“tracrRNA”) or a short-complementarity untranslated RNA (“scoutRNA”). The binding of a gRNA to its cognate Cas nuclease is determined by the “repeat region” of the crRNA, referring to the region of the crRNA that is not the targeting region (i.e. , a crRNA consists of a targeting region and a repeat region) and which when complexed with a tracrRNA or scoutRNA is involved in said complexing interaction through base pairing complementarity with said tracrRNA or scoutRNA. Said repeat region forms a specific tertiary structure, optionally in combination with a tracrRNA or scoutRNA, required for complex formation with its cognate Cas nuclease. “Target strand” (“TS”) refers to the strand of a nucleic acid molecule that is bound by the gRNA, or more specifically the crRNA component of a gRNA as defined herein.

[0055] “Non-target strand” (“NTS”) refers to the strand of a double-stranded nucleic acid molecule that is not the target strand, and is therefore complementary to the target strand, in other words it is not bound by the gRNA, in other words its sequence matches the sequence of the targeting-region of the gRNA.

[0056] “Nickase” refers to a nuclease capable of cleaving only one strand of DNA, in other words only one strand of a double-stranded DNA.

[0057] Functional features

[0058] As hereinbefore noted, provided as an aspect of the invention is a Cas nuclease, designated alpha-synCas (a-synCas), comprising or consisting of the amino acid sequence of SEQ ID NO 1 , which is considered an important addition to the CRISPR- cas system toolbox. It enables Multimodal One-enzyme Targeting of Nucleic Acids or the MONTANA approach.

[0059] By way of interesting and advantageous properties of a-synCas, it is now reported that: a-synCas is not known in nature and only has 52% identity to FnCas12a and 26% identity to SuCas12a2. Nevertheless, it can functionally employ an FnCas12a crRNA as shown in Figure 3a, b.

[0060] - As previously highlighted above, it has broad cis-target and trans-substrate nucleic acid compatibilities; it is capable of RNA-guided sequence-specific target (cis-target) nucleic acid targeting and cleavage (cis-targeting or cis-cleavage) of all of dsDNA, ssDNA and ssRNA as well as the ability upon cis-target recognition to indiscriminately cleave (trans-target or trans-cleave) all of dsDNA, ssDNA and ssRNA nucleic acid in its surrounding regardless of their sequence (trans-substrates). Results in Figure 1 demonstrate its ability to cleave all of dsDNA, ssDNA and ssRNA while Figure 2 demonstrates that upon exposure to each of these cis-targets it has the ability to trans- cleave all of dsDNA, ssDNA and ssRNA.

[0061] - As demonstrated using an in vitro unbiased PAM preference detection assay visualised as a PAM wheel and WebLogo in Figure 4, a-synCas does not show specificity of PAM recognition. Figure 4 shows that a-synCas is capable of cleaving target nucleic acids regardless of the 3 or 7 nucleotide sequence that was directly upstream of the PAM. Additional in vitro cleavage experiments testing specific PAMs provided in plasmid DNA (as shown by Figure 3c) have demonstrated the successful cutting of target nucleic acids with TTT (a non-G-rich and non-C-rich PAM), CCC (a non-A-rich and non-T-rich PAM), TTG, TCA and TGG PAMs. Thus based on the conditions tested and described in the examples, which are broadly in line with the characterisation of other Cas nucleases in the literature, a-synCas can be designated as being PAM-less. It will be recognised that variants of aSynCas which retain a comparable relaxed PAM requirement may be identified by application of equivalent PAM preference detection assays with up to 7 nucleotide variant sequences representing possible PAM provision. Comparable relaxed PAM requirement may be equated with no evident PAM specificity by such testing.

[0062] - As also previously highlighted above, a-synCas shows high flexibility in gRNA requirements. As shown by Figure 3a and Figure 3b, a-synCas can utilise for guide RNA purpose crRNAs corresponding to those of any of a wide variety of existing CRISPR family types and species, including at least the crRNAs corresponding to those of FnCas12a,, LbCas12a, AsCas12a, Cas<t> and SpCas9. Such broad-ranging versatility in gRNA requirement, spanning crRNAs of know Type II and Type V Cas nucleases and a breadth of species including both bacteria and archaea, is highly advantageous.

[0063] It has been shown to be capable of processing into a crRNA the pre-crRNA of FnCas12a as demonstrated by Figure 3d. Given its above described ability to function as a nuclease using the crRNAs corresponding to those of all of FnCas12a, , LbCas12a, AsCas12a, Cas<t>a ns SpCas9, it is envisaged that a-synCas has more general ability to process its own crRNA from a pre-crRNA transcript corresponding to a wider range of CRISPR family types, subtypes and species.

[0064] Alpha-synCas shares similarities with Cas12 enzymes such as Sulfuricurvum sp. PC08-66 Cas12a2 (Bravo et al., 2023; Dmytrenko et al., 2023) but with a number of advantageous differences specifically with regard to its above described ability to cis-target all of dsDNA, ssDNA and ssRNA whilst retaining the ability to trans-cleave all of the same nucleic acid substrates, its PAM-less nature and its flexible crRNA (i.e. gRNA) requirements.

[0065] While comparisons in this disclosure are made between a-synCas and Cas12 enzymes and a-synCas and SpCas9 for illustrative purposes, it should be noted that a-synCas shares properties with Cas nucleases from multiple CRISPR families. More particularly, the ability of a-synCas to process a crRNA from a pre-crRNA transcript is shared with Type V and VI Cas nucleases, its ability to target all of dsDNA, ssDNA and ssRNA as well as its relaxed PAM requirement are shared to some extent with an ancestral version of Cas9 termed ancestral Cas (anCas) (Alonso-Lerma et al., 2023), while its flexible gRNA requirements and its combined cis- and trans-cleavage abilities are seen as unique; asynCas is deemed to provide a unique collection of advantageous properties compared to any CRISPR family type reported to date.

[0066] Alpha-synCas has been shown to be functional in both eukaryotic and bacterial cells. As shown in Figure 6, a-synCas is capable of driving indel formation (gene editing) at targeted loci DNMT1 , AAVS1 , EMX when expressed from a humanised gene in human HEK293T cells together with provision of a suitable crRNA (i.e. gRNA). Indeed, these studies support high editing efficacy being achievable with of a-synCas in eukaryotic cells; a-synCas was observed to drive editing in HEK293T cells under conditions where FnCas12a proved ineffective. In bacterial cells functional cleavage has been demonstrated by expressing a-synCas in such cells together with a crRNA programmed to target a transformation plasmid whereby successful targeting by a-synCas can be assessed by antibiotic selection. An over two orders of magnitude decrease in successfully selected transformants demonstrated the functionality of a-synCas in bacteria when induced by ITPG as shown in Figure 5.

[0067] The ability of certain Cas nucleases to indiscriminately trans-cleavage nucleic acids is believed in the art to be enabled by RNA-guided cis-target nucleic acid recognition by the Cas effector nuclease. Thus binding of an a-synCas-crRNA complex to its cis-target nucleic acid substrate is believed to enable indiscriminate trans-cleavage activity (collateral cleavage activity) by the Cas nuclease. Activation of a Cas nuclease for such trans-cleavage may refer to the exposure of a Cas-gRNA complex to a cis-target nucleic acid substrate, or to the recognition of a cis- target by a Cas-gRNA complex, or to the binding of a Cas-gRNA complex to a cis-target nucleic acid substrate whether or not such binding is accompanied by cleavage of the cis- target substrate.

[0068] As mentioned above and further expanded on below in the exemplification with reference to Figure 4, a-synCas may be designated as PAM-less, i.e. exhibiting no PAM specificity requirement for effective cis-targeting in a dsDNA plasmid. Furthermore, it has been shown to have virtually no preference for any potential PAM sequence as also demonstrated in Figure 4; PAM preferences for a-synCas would be expected directly upstream of a target site.

[0069] As also previously noted, a-synCas is able to utilise gRNAs corresponding to crRNAs of a plurality of existing CRISPR family types, including by way of illustration herein at least the crRNAs corresponding to those of FnCas12a, LbCas12a, AsCas12a, Cas<t> and SpCas9 (see Zetsche et al 2015, Examples, Figure 3a, b). Such broad and non-specific crRNA use is not a property which has been reported previously for any known Cas nuclease. Furthermore, the ability of a-SynCas to process into a crRNA the pre-crRNA transcript corresponding to FnCas12a as shown in Figure 3d can be envisaged as applicable to other pre-crRNA transcripts capable of providing crRNAs compatible with a-synCas, especially for example a Type V Cas enzyme pre-crRNA transcript, e.g. any Cas12a pre-crRNA transcript.

[0070] It will be appreciated that the teaching herein of the functional properties of a-synCas enables the attainment of novel a-synCas variants by recognised routine development techniques which share one or more desired characteristics as noted herein compared with a-synCas. More particularly of interest are such variants which share with a-synCas:

[0071] (a) the ability when complexed with a guide RNA (e.g. crRNA, crRNA:tracrRNA complex, or sgRNA) to target and cleave any of a dsDNA, ssDNA and ssRNA and, when exposed to such targets or when activated by such targeting, the capability to non- specifically trans-cleave any of dsDNA, ssDNA and ssRNA or (a) and / or

[0072] (b) a relaxed PAM requirement comparable to a-synCas; possibly together with

[0073] (c) ability to use a gRNA selectable from crRNAs suitable for employment by a plurality of Cas enzymes of Type II and Type V for target recognition; and / or

[0074] (d) ability to process its own crRNA from a pre-crRNA transcript.

[0075] Preferred may be an a-synCas variant with the above-noted versatile cis-targeting and transtargeting capabilities (characteristic (a)), possibly in combination with characteristic (b), possibly alternatively or additionally in combination with one or both of characteristics (c) and (d). Preferred may also be selection of an a-synCas variant with the above-noted relaxed PAM requirement comparable to a-synCas (characteristic (b)), possibly in combination with at least one or both of characteristics (c) and (d), optionally supplemented with the cleavage abilities of a-synCas (characteristic (a)). However, as hereinbefore indicated a variant of a-synCas may be especially preferred that retains all of its functional characteristics as noted above.

[0076] Either a-synCas or an a-synCas variant nuclease as discussed above may be converted to a variant which is either a nickase or a dead Cas (dCas) with no nuclease activity by catalytic site mutagenesis. Optionally such a nickase or dCas may be linked, e.g. fused, with a variety of non-Cas effector proteins such as a non-Cas effector protein for genetic modification or regulation, e.g. a base editor, a reverse transcriptase for prime editing, a transcriptional or epigenetic regulator, a transposase, an integrase, a retron or a non-Cas nuclease domain such as a Fok1 nuclease domain. Such linkage may be achieved in same manner as well- known for linkage of other Cas nickases or dCas to non-Cas effector proteins. Such linkage may be via linkage to a guide RNA, such as linkage of a base editor via an RNA aptamer extension of a guide and an RNA binding domain as taught in W02017 / 011721 (Rutgers University). See also Collantes et al. (2021) Development and Characterisation of a Modular CRISPR and RNA Aptamer Mediated Base Editing System. a-synCas variants of the invention may also comprise an N- or C-terminal linkage to a nonnuclease effector so as to provide a chimeric fusion protein, for example an N- terminal or C- terminal sequence may comprise a nuclear localisation signal (NLS, e.g., SV40 NLS, nucleoplasmin NLS, or any other NLS known in the art), ora cell-penetrating peptide sequence (e.g., TAT peptide). Such a fusion protein may provide a non-Cas effector protein for genetic modification or regulation as indicated above, e.g. a deaminase for base editing ,a reverse transcriptase, e.g. for prime editing, a transcriptional activation domain (e.g., herpes simplex virus VP16 or VP64 and others), a transcriptional repressor domain (e.g., KRAB, ERD, SID, and others), a silencing domain (e.g., HP1), a domain that can recruit an RNA such as a long non-coding RNA, a histone modification domain (e.g., histone acetyltransferase, histone deacetylase, histone methyltransferase, histone demethylase and more), or any other domain known in the art to be useful in a fusion protein construct comprising a Cas nuclease or a nickase or dCas derived therefrom.

[0077] It will be appreciated that variants of a-synCas which have one or more amino acid changes by way of substitution, deletion or insertion, e.g. one or more conservative substitutions, may be employed similarly as a Cas9 or Cas12a endonuclease in terms of for example targeting and / or cleaving a target nucleic acid, or achieving trans-cleavage through cis-target recognition. Of especial interest and forming part of the invention are such variants that retain the ability to cis-cleave and trans-cleave all of dsDNA, ssDNA and ssRNA, and variants which have relaxed PAM requirement comparable to a-synCas.

[0078] From the above, it will be evident that a-synCas and variants thereof as discussed herein are seen as highly useful novel additions to the toolbox of Cas proteins.

[0079] Derivation of a-synCas nuclease variants

[0080] Nuclease variants of a-synCas which form part of the invention may have varying degrees of sequence identity to SEQ ID NO. 1 subject to retaining its desired distinguishing characteristic or characteristics. Variants may for example have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity (i.e. similarity) to SEQ ID NO. 1 as ascertainable using pairwise sequence alignment algorithms well known in the art such as the Needleman- Wunsch, Myers and Miller, or Smith-Waterman algorithms for example. Of particular interest are deemed a-synCas variants which exhibit at least 70% sequence identity to SEQ ID NO 1 or higher, or only have one or more conservative substitutions compared to SEQ ID NO 1 , and retain at least the cleavage abilities of a-synCas as discussed above or at least those cleavage abilities and / or the relaxed PAM requirement, preferably together with one or both of the properties specified as (iii) and (iv) or (c) and (d) above.

[0081] Experimental procedures described in the examples allow such functionalities to be tested and determined and thereby it is envisaged that such variants with at least 70% identity to SEQ ID NO. 1 can be ascertained by application of no more than routine development.

[0082] As indicated above, a-synCas variants of the invention may have one or more conservative substitutions compared to SEQ ID NO: 1 which can be expected to result in retention of the same functional characteristics. Conservative substitutions involve replacing one amino acid with a different amino acid with similar biochemical properties. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0083] By aligning the amino acid sequence of a-synCas provided in SEQ ID NO: 1 to the sequence of wild-type FnCas12a (Francisella novicida 11112 Cas12a) (SEQ ID NO: 35) and use of published crystal structure information elucidating the interactions of this known Cas12a with a crRNA and target, the inventors have been able to identify changes in known key catalytic residues of Cas12a which are present in SEQ ID NO 1. These changes as set out in Table 1 below are considered structurally implicated in accounting for the distinguished functional properties of a-synCas, thus providing a structural roadmap towards attaining a-synCas variants which have all its described functional properties.

[0084] Table 1 . Mutations in key catalytic residues identified in a-synCas with respect to FnCas12a. NTS: nontarget strand. TS: target strand.

[0085] By way of shorthand, the above-noted amino acid changes in a-synCas compared to wild-type FnCas12a are hereinafter referred to as: R800K, R948H, V862T, K871 R, A850K, F873Y, N700S, S702del, P703del, R692Y, Q704del, Q125K, Y410W, K397T, K341A, T1063S, A676N, K131del, R800K, V862T, K871 R, K956L, R948H, A850K, K895N, K1287S, I586L, N607D (where del refers to deletion or missing, and substitutions in SEQ ID NO 1 need to additionally take account of alignment).

[0086] Accordingly, desirable Cas nuclease variants of a- synCas are viewed as variants which in comparison to FnCas12a retain all the amino acid substitutions and deletions exhibited by the sequence of SEQ ID NO 1 when compared to the same Cas12a sequence as noted above, or the same substitutions or deletions except for one or more conservative substitutions. Such variants are envisaged as capable of retaining all the functional properties of a-synCas noted herein which distinguish that enzyme from known Cas12 enzymes and also Type VI Cas enzymes such as Cas13a and Type II Cas enzymes such as Cas9. It will be appreciated that armed with the above information on amino acid changes in a- synCas, the same changes may be transferred into any Cas12 enzyme including Francisella tularensis subsp. Novicida U112 Cas12a (FnCas12a), Lachnospiraceae bacterium MC2017 Cpf1 (Lb3Cas12a), Butyrivibrio proteoclasticus Cas 12a (BpCas12a), Peregrinibacteria bacterium Cas 12a (PeCas12a), Parcubacteria bacterium Cas 12a (PbCas12a), Smithella sp. SC_K08D17 Cas12a (SsCas12a), Acida mi nococcus sp. BV3L6 Cas 12a (AsCas12a), Lachnospiraceae bacterium MA2020 Cas 12a (Lb2Cas12a), Candidatus Methanoplasma termitum Cas12a (CMtCas12a), Eubacterium eligens Cas12a (EeCas12a), Moraxella bovoculi 237 Cas 12a (MbCas12a), Leptospira inadai Cas 12a (LiCas12a), Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a), Porphyromonas crevioricanis Cas12a (PcCas12a), Prevotella disiens Cas12a (PdCas12a), Porphyromonas macacae Cas12a (PmCas12a), any Cas12a2 enzyme including Sulfuricurvum sp. PC08-66 Cas12a2, any Cas12f enzyme (formerly Cas14) including Cas12f from an uncultured archaeon as reported by Harrington et al. (2018), and any Cas12j enzyme (formerly Cas<t>). In this way it is envisaged that functional equivalents of a-synCas may be attained with application of routine techniques for enzyme sequence mutation and testing for required functional characteristics, e.g. starting from any Cas12a. Testing may be readily carried out as exemplified.

[0087] It will be appreciated that a-synCas and all variants thereof of the present invention will have certain functional protein domains. For the full-length a-synCas protein, these domains are provided in the table below. It is envisaged that these functional domains of a-synCas will be isolatable and combinable with functional domains from other Cas nuclease effectors resulting in chimeric Cas nucleases. For example, a domain identified in a-synCas may be replaceable with a corresponding domain in another Cas nuclease, thereby introducing the functional properties of a-synCas associated with that domain into another Cas nuclease. Testing of such chimeric fusion Cas nucleases has been for example reported for 560 synthetic chimeras by Liu et al (2019).

[0088] Table 2. Functional protein domains present in a-synCas, shown with comparison to FnCas12a

[0089] Variants of a-synCas forming part of the invention therefore include a functional domain corresponding to a domain of a parent Cas nuclease or variant of the invention (a parent with all the functional characteristics of a- synCas noted herein or a nickase or dCas variant thereof) for example a functional domain providing the cleavage abilities specified above, or a functional domain providing the relaxed PAM requirement of a-synCas. Included as part of the invention therefore is also a chimeric Cas polypeptide incorporating a domain as described above, for example a chimeric Cas nuclease.

[0090] Nucleic acids and ribonucleoproteins (RNPs)

[0091] Another aspect of the invention provides nucleic acids for expression of a-synCas, a variant thereof, or polypeptide derived therefrom as discussed herein, e.g. expression vectors or gene constructs for expression of such proteins. In such a construct, the coding sequence for a- synCas or a variant, functional domain or chimeric Cas polypeptide of the invention may be preferably codon-optimised for expression in the chosen host cells, e,g. bacterial cells, human cells (either ex vivo human cells or cells of human cell line) or plant cells. For example, a codon-optimised coding sequence for expression of a-synCas in E. coli cells has been found to enable good expression for isolation of the nuclease from such cells as detailed in the exemplification below. A humanised coding sequence for a-synCas has also enabled successful gene targeting using this novel Cas enzyme to be observed in human cells in vitro. Promoters for such expression may be selected from promoters well known in the field of CRISPR-Cas system application.

[0092] An expression vector capable of expressing a polypeptide of the invention may be employed together with a gRNA for targeting, or together with a second polynucleotide capable of expressing such a gRNA (e.g., crRNA, crRNA:tracrRNA complex, or sgRNA) or, or may itself be capable of expressing the desired gRNA. Thus, one or more vectors providing a-synCas or a variant thereof as herein taught, and a suitable guide RNA, may be provided for transfection into cells optionally together with other nucleic acids, e.g. to provide sequences for homology-directed repair applications. Such combinations of components may be provided in the form of a pharmaceutical composition with a pharmaceutically acceptable excipient. Alternatively, a-synCas and variants as herein taught may be provided together with gRNA as a ribonucleoprotein (RNP) complex for transfection into cells, e.g. by electroporation into isolated cells. Such proteins may be provided in the form of a pharmaceutical composition with a pharmaceutically acceptable excipient. The purification of a-synCas and variants along with RNP formation is described in the examples and is well known in the art.

[0093] Accordingly now provided is a combination product (hereinafter the combination product of the invention) comprising or consisting of

[0094] (i) a Cas nuclease, variant or chimeric Cas polypeptide of the invention, or a polynucleotide capable of expressing the same and

[0095] (ii) a crRNA for targeting said Cas nuclease, variant or chimeric Cas polypeptide to a target sequence, or a polynucleotide capable of expressing the same, for example where (i) and (ii) are expression vectors or (i) and (ii) provide a ribonucleoprotein complex wherein said Cas nuclease, variant or chimeric Cas polypeptide is complexed with a crRNA.

[0096] Such a combination product may be supplemented with an one or more additional protein and / or nucleic acid components depending on the intended application , e.g. as indicated above a nucleic acid to provide a nucleic acid sequence for insertion into a target nucleic acid sequence for homology-directed repair or for serving as a DNA repair template for guiding the outcome of the modified target sequence.

[0097] Such a combination product may be packaged and / or formulated to provide a pharmaceutical composition by various means. As hereinbefore indicated, known means for therapeutic delivery of nucleic acids may be employed including packaging into virus particles, virus-like particles or lipid particles, e.g. a composition employing an adeno-associated virus (AAV) or lipid nanoparticle (LNP) for expression construct delivery.

[0098] Nucleic acid detection assays

[0099] Alpha-synCas and those functional variants described herein that retain at least the ability to functionally recognise or target a nucleic acid target and the ability to functionally trans-cleave at least one type of nucleic acid trans-target are useful for nucleic acid-based diagnostic applications. Preferably Cas nuclease variants of the present invention which can target all of dsDNA, ssDNA, ssRNA and which can in addition trans-target all of dsDNA, ssDNA and ssRNA provide the most versatility in such applications. In aggregate, such diagnostic application methods rely upon binding of the Cas effector nuclease such as a-synCas or its functional variant to its RNA-guided target, which induces its indiscriminate trans-cleavage ability leading to non-specific cleavage of labelled nucleic acid reporters which provide the assay readout. Such methods forming part of the invention therefore include contacting a sample known or suspected to include a target nucleic acid of interest, whether that is dsDNA, ssDNA or ssRNA, with (a) a-synCas or a functional nuclease variant thereof, (b) a guide RNA functionally compatible with such a nuclease and (c) a labelled reporter nucleic acid that is commonly ssDNA or ssRNA. When the nuclease binds its gRNA- complementary target the non-specific nuclease activity leads to cleavage of the reporter nucleic acid thus producing a signal. Methods for measuring the signal from the labelled detector nucleic acid are known in the art and can include e.g. detecting one or more of a gold nanoparticle, a fluorophore, fluorescence polarization, colloid phase transition / dispersion, electrochemical signals, and semiconductor-based signals. The labelled reporter nucleic acid may produce an amount of detectable signal prior to being cleaved, with the amount of detectable signal being reduced once cleaved. Alternatively, the signal emanating from the labelled reporter nucleic acid may increase once cleaved. The labelled reporter nucleic acid may comprise a quencher / fluor pair. As hereinbefore, a general review of such diagnostic applications is provided by Kaminski et al. (2021).

[0100] Methods of nucleic acid-based diagnostic applications relying on non-specific collateral cleavage activity by a Cas enzyme once activated by target recognition are well-known.

[0101] Thus now provided are methods for detecting in a sample one or more target nucleic acids wherein a-synCas or a nuclease variant thereof of the invention is employed (capable of nonspecific collateral cleavage of at least one type of nucleic acid - dsDNA, ssRNA, dsDNA, or a combination thereof), the Cas nuclease being provided together with one or more gRNAs each capable of targeting the Cas nuclease to target sequence(s) and successful targeting with activation of the Cas nuclease being determined by detecting collateral cleavage of a reporter nucleic acid. The reporter will not hybridise with any gRNA employed. The full cis-targeting activities exhibited by a-synCas may be utilised with a gRNA being employed which provides a targeting region complementary to an RNA or DNA encoding an RNA whereby all of a dsDNA, ssDNA and ssRNA complementary to said targeting region are targeted by the Cas nuclease and generally either ssDNA or ssRNA is employed as a reporter nucleic acid, e.g. a ssDNA or ssRNA labelled with a fluorescent label and quencher whereby cleavage of the reporter is detectable by measurement of fluorescence signal.

[0102] In a particular embodiment, the one or more target nucleic acids are from bacterial pathogens. The term “bacterial pathogen” refers to a type of bacterium that can cause disease in humans, animals, or plants. These pathogens invade host tissues, evade immune defences, and produce toxins or other harmful effects that lead to illness.

[0103] In a particular embodiment, the bacterial pathogen is selected from the group consisting of: Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Salmonella, Mycobacterium tuberculosis, Vibrio cholerae, Acinetobacter baumannii, Klebsiella pneumoniae, Helicobacter pylori and a combination thereof.

[0104] In a more particular embodiment, the bacterial pathogen is selected from the group consisting of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and a combination thereof.

[0105] In another particular embodiment, the one or more target nucleic acids are 16S RNA or 16S DNA.

[0106] In another aspect, the present invention relates to an in vitro method for determining the presence of an infection by a bacterial pathogen in a subject, wherein the method comprises detecting in a sample from the subject one or more target nucleic acids from the bacterial pathogen using the methods for detecting in a sample one or more target nucleic acids described above.

[0107] In these methods, a Cas nuclease or a nuclease variant thereof of the invention is employed, which is capable of non-specific collateral cleavage activity, said Cas nuclease being provided together with one or more gRNAs each capable of targeting the Cas nuclease to target sequence(s) and said targeting with activation of the Cas nuclease being determined by detecting collateral cleavage of a reporter nucleic acid. Preferably, said gRNA provides a targeting region complementary to an RNA or DNA encoding said RNA whereby all of a dsDNA, ssDNA and ssRNA complementary to said targeting region are targeted by a Cas nuclease according to the invention and either a ssDNA or ssRNA is employed as a reporter nucleic acid, e.g. a ssDNA or ssRNA labelled with a fluorescent label and quencher whereby cleavage of the reporter nucleic acid by said Cas nuclease results in detectable fluorescence.

[0108] The sample may be any sample containing a nucleic acid sequence of interest for detection. In a particular embodiment, the sample is a biological sample. A biological sample can be isolated from any suitable biological tissue or fluid; nevertheless, said sample is preferably a biofluid from the subject under study. Said biological sample or biofluid could be a blood, serum, plasma, urine or saliva sample. It may be an environmental sample, e.g. a sewage-derived sample, water sample or soil- derived sample. It may also be a preparation derived from such samples such as a nucleic acid preparation.

[0109] In such detection application, a-synCas’ ability to be able to detect all of dsDNA, ssDNA and ssRNA targets, and trans-cleave all of dsDNA, ssDNA and ssRNA reporter nucleic acid transtargets may be seen as advantageous. For example, a-synCas or a variant thereof retaining the same cleavage abilities will be able to detect the genomes or genomic fragments of ssDNA or ssRNA viruses directly without the need for reverse transcription, or the reverse transcribed ssDNA or dsDNA products of ssRNA viral genomes (i.e. cDNA).

[0110] Such methods relying on provision of a pre-crRNA from which a-synCas or a functional variant can process any required crRNA lend themselves to multiplex detection applications. Thus multiplex screening may be achieved for a plurality of target nucleic acid sequences where each crRNA of a required plurality of crRNAs is processed from a single pre-designed pre- crRNA transcript by a Cas nuclease of the invention whereby it targets the different nucleic acid targets of interest. This may simplify assay design and cost.

[0111] In another aspect, the present invention relates to the use of the combination product of the invention for determining the presence of an infection by a bacterial pathogen.

[0112] Use in modifying and regulating nucleic acids

[0113] It will be recognised that a-synCas and variants thereof as now disclosed may be employed in the whole panoply of genetic modification techniques envisaged for known Cas effector nucleases such as Cas9 or Cas12a. As hereinbefore indicated, these extend to use of nickases and dcas of the invention in linkage with a non-Cas effector protein for gene modification or regulation, e,g. use in linkage with a base editor where linkage may be by nuclease fusion or via an RNA extension of a guide and an RNA binding domain as taught in W02017 / 011721. Suitably the Cas effector variant linked with a non-nuclease effector may be any a-synCas obtained in accordance with the invention or any variant thereof with nuclease activity which has been modified by for example conventional catalytic site mutagenesis to have nickase only activity or no nuclease activity, e.g. a dCas which has no cleavage ability but nevertheless retains the relaxed PAM specificity and broad gRNA compatibilities observed with a-synCas. Thus, in a further aspect, the invention provides a method for modifying or regulating a target nucleic acid sequence, e.g. a target DNA sequence, the method comprising contacting the target sequence with (i) a Cas protein as taught herein and (ii) a guide RNA (e.g. a single crRNA), wherein said guide RNA targets the Cas protein to the target sequence, and wherein (a) said contacting is in vitro with an isolated target nucleic acid or in a cell, for example an ex vivo cell, with the proviso that the method is not a method of modifying the germ line identity of a human being; or (b) the method is not a method of medical treatment practised on the human or animal body. The target sequence may optionally be further contacted with an exogeneous nucleic acid sequence for insertion into the target nucleic acid sequence or for serving as a DNA repair template for guiding the outcome of target sequence modification.

[0114] For such a method, a combination of the invention providing a Cas protein, e.g. a- synCas or a variant Cas nuclease and a suitable guide RNA, may be introduced into any of a range of host cells commonly of interest in relation to genomic modification. Such methods extend for example to genetic modification of human or animal cells ex vivo. They may also find use in genetic modification of plants, e.g. by modifying target sequences possibly but not exclusively in protoplasts, and genetic modification of microbial cells.

[0115] A method of the invention for modifying or regulating a target nucleic acid sequence in plant cells, e.g. by use of a- synCas to enable gene modification by cleavage and homologous recombination or converted to a base editor, may further comprise obtaining a modified plant therefrom and seeds thereof. For example, such a method may find use in introducing or improving pest resistance, improving drought tolerance, lengthening fruit storage durability or beneficially changing nutritional content.

[0116] It will be appreciated that the invention also extends to a combination of the invention as discussed above for use in a method of therapeutic treatment by modifying or regulating a target nucleic acid sequence, e.g. a DNA sequence. Again, optionally, the combination may provide in addition to a Cas polypeptide and suitable guide RNA, a nucleic acid for insertion into the target nucleic acid sequence or for serving as a template for guiding the outcome of target sequence modification.

[0117] As indicated above, the low sequence identity of, a-synCas to known Cas effectors, for example to SpyCas9, is considered advantageous in relation to contemplating such use. Such use may embrace for example Cas action in pathogenic bacteria or for manipulation of the gut microbiome or skin microbiome. The invention is further illustrated below by the following non-limiting examples and with reference to the accompanying figures as now specified.

[0118] Brief Description of the Figures

[0119] Figure 1 : Cis-activity of a-synCas endonuclease on DNA, ssDNA and ssRNA substrates.

[0120] (a) In vitro cleavage assay for FnCas12a and a-synCas on a 4,007 bp plasmid substrate (Zero- Blunt TOPO) at different reaction times. Supercoiled plasmids became linearised upon cleavage. Quantification of total cleavage at different reaction times with exponentially fitted trendlines. Values reported as mean ± standard deviation (SD), where n (sample size) = 3.

[0121] (b) In vitro cleavage assay for FnCas12a and a-synCas on a ssDNA M13 phage substrate (M13MP18 substrate) at different reaction times. Quantification of total cleavage at different reaction times with exponentially fitted trendlines. Values reported as mean ± SD, where n = 3. (c) In vitro cleavage assay for a-synCas on a ssRNA substrate at different reaction times. Quantification of total cleavage at different reaction times with exponentially fitted trendline. Values reported as mean ± SD, where n = 3.

[0122] Figure 2: Substrate target recognition by a-synCas triggers collateral degradation of dsDNA, ssDNA and ssRNA in vitro. Collateral cleavage (trans-cleavage) of non-target nucleic acid substrates ((a) dsDNA, (b) ssDNA and (c) ssRNA) by the a-synCas-crRNA complex with either dsDNA, ssDNA or ssRNA target substrates (activators) and time-course analysis of collateral cleavage of non-target dsDNA, ssDNA or ssRNA. Values reported as mean ± SD, where n = 3.

[0123] Figure 3: Promiscuous activities of a-synCas. In vitro cleavage assay of a supercoiled dsDNA substrate using a-synCas complexed with crRNAs from (a) different CRISPR system types and (b) Cas12a from different species: (i) FnCas12a: Francisella novicida U112 Cas12a, (ii) SpCas9: Streptococcus pyogenes Cas9, (iii) LbCas12a: Lachnospiraceae bacterium ND2006 Cas12a and (iv) AsCas12a: Acidaminococcus sp. BV3L6. (c) In vitro cleavage assay using a- synCas provided with a variety of 3nt PAMs (TTG, TTT, CCC, TCA and TCGO. Supercoiled plasmids become linearised upon cleavage, (d) In vitro processing of FnCas12a pre-crRNA transcript with purified FnCas12a and a-synCas. Bands corresponding to 65 nt and 49 nt reveal successfully processed crRNAs as shown by Zetsche et al. (2015). (f,g) Quantification of maximum fluorescence signal generated after incubating a-synCas-crRNA with various activators (x-axis) together with a custom (f) trans-ssRNA-FQ or (g) trans-ssDNA-FQ reporter. The FnCas12a control used a dsDNA activator. Error bars represent the mean ± SD, where n = 3. Figure 4: PAM recognition profile of a-synCas. PAM wheels (Krona plots) and WebLogo to enable the assessment of the 3 and 7 nucleotide (nt) PAM preferences of a-synCas.

[0124] Figure 5: Demonstration of editing activity of a-synCas in E. coli by plasmid interference assay relying on antibiotic selection as described in the exemplification. The graph shows determination of plasmid targeting by a-synCas under non-induction (- IPTG) and induction (+ IPTG) conditions as measured by colony formation units (CFU), where the target plasmid provides chloramphenicol resistance.

[0125] Figure 6: Demonstration of editing activity of a-synCas in eukaryotic cells, (a) Gels of surveyor assays and (b) corresponding quantification of indel efficiency achieved by a-synCas and FnCas12a at DNMT1 , AAVS1 and EMX1 target sites respectively in HEK293T cells. The full length target sites as well as the two smaller cleaved products are labelled by size.

[0126] Figure 7: Demonstration of a-synCas exhibits broad PAM recognition activity in human cells, (a) On-target activities of a-synCas and enAsCas12a across sites encoding canonical and non-canonical PAM sequences, quantified using TIDE (Brinkman, E. K., et al., 2014, Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res 42, e168). Error bars represent the mean ± SEM, where n = 3. (b) Assessment of the gene editing activities of a-synCas and enAsCas12a on-target sites harbouring canonical and non-canonical PAM sequences (n = 13). NS (non-significant differences), P > 0.05; *, P < 0.05 (two-tailed Welch’s t-tests). NS difference for those target sites with PAMs recognized by enAsCas12 (i.e., 5-TTTC-3' , 5-TTTG-3' , 5-TCCT-3' , 5-TCCC-3' , 5-TTAC-3) indicated as empty symbols.

[0127] Figure 8: Demonstration of a-synCas as molecular diagnostic tool for PAM-free dsDNA target recognition, (a) Schematic of in vitro collateral cleavage assay for dsDNA recognition by a- synCas. The a-synCas-gRNA complex recognizes and cleavage the dsDNA target, which triggers non-specific collateral cleavage on ssDNA. (b) Sensitivity of a-synCas and LbCas12a using a dsDNA with canonical PAM (5-TTTC-3) as target substrate and ssDNA beacon as collateral substrate. Velocities were obtained by linear regression analysis of the linear regions of the progress curves, (c) Velocities for dsDNA target recognition and posterior collateral cleavage of ssDNA beacon by a-synCas and LbCas12a at RT. (d) Velocities for dsDNA target recognition and posterior collateral cleavage of ssDNA beacon by a-synCas and LbCas12a at 37°C. Error bars represent the mean ± SD, where n = 3. NS: not significant, P > 0.05; ***P < 0.001 (two-tailed Welch’s t-tests). (e) 16S dsDNA from four different pathogens including both conserved and variable region used for dsDNA target detection with non-canonical PAM sequence (5-GTCG-3). (f) Specific detection of pathogens based on 16S rDNA with a-synCas and each specific crRNA using universal primers for specific amplification of variable region (light grey, V1) using conserved regions of 16S rDNA (dark grey). Ab: Acinetobacter baumannir, Ec: Escherichia coir, Kp: Klebsiella pneumoniae’, Sa: Staphylococcus aureus. Error bars represent the mean ± SD, where n = 3.

[0128] Figure 9: Demonstration of in vitro PAM-flexible activity of a-synCas. (a) In vitro activity of a- synCas and FnCas12a against supercoiled DNA as a function of PAM sequence variation, (b) In vitro activity of a-synCas against FAM-labelled dsDNA substrates with different PAMs (-: negative control with a-synCas + non-target crRNA; +: sample with a-synCas + target crRNA).

[0129] Figure 10: Demonstration of in vivo activity of a-synCas as RNP. On-target activities of a- synCas on (a) HeLa and (b) Hs27 fibroblast cells across sites encoding canonical and non- canonical PAM sequences, quantified using TIDE (Brinkman, E. K., et al., 2014). Error bars represent the mean ± SEM, where n = 2.

[0130] Figure 11 : Progress curve for dsDNA target recognition and posterior collateral cleavage of ssDNA beacon by a-synCas and LbCas12a at 37 °C and room temperature (RT). Error bars represent the mean ± SD, where n = 3.

[0131] Figure 12: On target recognition of variable regions of 16S rDNA from four different pathogens by a-synCas and LbCas12a. Progress curve for dsDNA target recognition and posterior collateral cleavage of ssDNA beacon by a-synCas and LbCas12a at 37 °C. Non target control sample was prepared using nuclease-free water instead of the nucleic acid target activator. Error bars represent the mean ± SD, where n = 3.

[0132] Figure 13: Demonstration of specificity of a-synCas. Mismatch profile of a-synCas was measured using mismatched dsDNAs that differed from target site by 1 or 2 nt as target substrates and ssDNA beacon as collateral substrate. Velocities were obtained by linear regression analysis of the linear regions of the progress curves. Error bars represent the mean ± SD, where n = 3. ***P < 0.001 (two-tailed Welch’s t-tests).

[0133] Examples In these examples, there is reference to the ability of a-synCas to target substrates in cis or in trans, in other words its ability for cis-cleavage and trans-cleavage, respectively. Cis-cleavage (or cis-targeting or cis-activity) refers to targeting of a substrate which shares sequence complementarity with the crRNA, and which is therefore the intended target of a-synCas as predetermined by the crRNA. Trans-cleavage (or trans-targeting or trans-activity), also referred to as collateral cleavage or activity, indiscriminate cleavage or activity, or non-specific cleavage or activity, refers to the non-specific cleavage of any nucleic acid substrate regardless of its sequence identity; trans-cleavage is believed to be activated upon cis-target recognition by a CRISPR nuclease (here a-synCas).

[0134] Example 1 : a-synCas can cis-cleave target dsDNA, ssDNA and ssRNA

[0135] To determine the targeting activity of a-synCas (SEQ ID NO: 1), the inventors recombinantly expressed and purified a-synCas, then tested its enzymatic activities in vitro (Figure 1). To determine the nucleic acid target preference of a-synCas, dsDNA, ssDNA or ssRNA substrates (dsDNA PAM sequence: TGG; ssDNA and ssRNA no PAM was considered) containing a region of complementarity to the crRNA (i.e. target site) were incubated with the a-synCas-crRNA complex for various periods of time (crRNAs for dsDNA, ssDNA and ssRNA are in SEQ ID NO: 2, 3 and 4, respectively). See Figures 1a-c.

[0136] Surprisingly, and in contrast to other wild-type or engineered CRISPR-Cas effectors, which to the best of the inventors’ knowledge only exert cis-cleavage activity over either one or two of these types of substrates, a-synCas was activated by and cis-cleaved all three types of crRNA- complementary target substrates: dsDNA, ssRNA and ssDNA. This demonstrates the advantageously versatile cis-cleavage activity of a-synCas. In the case of dsDNA, a-synCas cleaved almost 100% of the plasmid dsDNA resulting in linearised dsDNA products, revealing its double-stranded break (DSB) activity. In the case of single-stranded (ss) substrates, namely ssDNA and ssRNA, the activity of the a-synCas enzyme produced their degradation as efficiently as that of dsDNA.

[0137] These results demonstrate that a-synCas can successfully target and cis-cleave dsDNA, ssDNA and ssRNA target substrates.

[0138] Example 2: a-synCas can trans-cleave non-target dsDNA, ssDNA and ssRNA

[0139] In this Example, the inventors examined whether the specific substrate targeting activity (in cis) of a-synCas could induce indiscriminate nuclease activity, in other words whether it had the ability to trans-cleave non-crRNA-complementary targets (Figure 2). Trans-cleavage activity is defined as the ability of Cas effector nucleases to trigger non-sequence-specific collateral cleavage upon target recognition (cis-recognition). Trans-substrates therefore refer to those substrates which bear no sequence complementarity to the crRNA, or less sequence complementarity than would be required for a-synCas to successfully cleave it with a given crRNA (in other words, trans-substrates may have partial crRNA-complementarity but less than required for successful cis-cleavage). Trans-cleavage activity appears in CRISPR Class II type V and type VI systems, such as the Cas12 and Cas13 families, respectively. This activity is the basis for the application of such enzymes in the detection of nucleic acids.

[0140] Surprisingly, a-synCas exhibits highly promiscuous trans-cleavage activities as demonstrated by various combinations of cis-targets (activators) and trans-substrates (Figure 2). In in vitro cleavage assays, for cis-target activators the inventors employed short linear nucleic acid fragments for dsDNA, ssDNA, and ssRNA (SEQ ID NO: 6-8, respectively) (each one targeted using the crRNA in SEQ ID NO: 9), and for trans-substrates either pUC18 plasmid for dsDNA, M13 phage for ssDNA or a linear nucleic acid fragment for ssRNA (SEQ ID NO: 10). The inventors found that a-synCas robustly and indiscriminately degraded dsDNA (Figure 2a), ssDNA (Figure 2b) and ssRNA (Figure 2c) trans-substrates after cis-targeting dsDNA, ssDNA and ssRNA substrates complementary to the crRNA guide. In other words, a-synCas demonstrated the ability to use either dsDNA, ssDNA or ssRNA as activators (i.e. , a-synCas has the ability to target these nucleic acid substrates) to then successfully trans-cleave dsDNA, ssDNA and ssRNA trans-substrates. By contrast, other Cas effectors of Type V and Type VI indiscriminately degrade only ssDNA after either dsDNA or ssDNA targeting (e.g. Cas12a), ssRNA after RNA targeting (e.g. Cas13) or dsDNA, ssDNA and ssRNA after RNA targeting (e.g. Cas12a2). Therefore, such promiscuity in both cis-recognition and trans-activity has not yet been previously reported for any Cas effector nuclease.

[0141] In summary, these results show that in addition to a-synCas being able to target and cis-cleave dsDNA, ssDNA and ssRNA substrates, targeting of each of these substrates allows a-synCas to trans-cleave dsDNA, ssDNA and ssRNA trans-substrates.

[0142] Example 3: Guide RNA scaffold promiscuity of a-synCas

[0143] To determine the promiscuity of a-synCas towards guide RNA scaffold recognition, in vitro cleavage assays were performed targeting plasmids by incubating a-synCas with crRNAs either from different CRISPR system types (Figure 3a) or from different species within a given CRISPR system type (Figure 3b). When complexed with crRNAs from different CRISPR system types, namely Cas9 (Type II, SEQ ID NO: 11), Cas12a or bacteriophage Cas<t> (both Type V; Cas<t> crRNA sequence in SEQ ID NO: 12), or when complexed with Cas12a crRNAs (i.e. gRNAs) derived from different species (Francisella novicida U112, SEQ ID NO: 13 for Figure 3a, SEQ ID NO: 14 for Figure 3b; Lachnospiraceae bacterium ND2006, SEQ ID NO: 15; Acidaminococcus sp. BV3L6, SEQ ID NO: 16), a-synCas was able to linearise target plasmid DNA. Alpha-synCas is therefore functionally active in its ability to cis-cleave target nucleic acids when complexed with at least any of these crRNAs.

[0144] In summary, a-synCas has promiscuous gRNA scaffold requirement; it is able to use as a guide crRNAs suitable for a Cas12a from a plurality of species, crRNAs suitable for other Type V Cas enzymes (e.g. a phage Cas<t>) or a crRNA of a Type II Cas as illustrated by use of a crRNA for SpCas9 without any tracrRNA component.

[0145] Example 4: Relaxed PAM specificity of a-synCas

[0146] To preliminarily gauge the PAM requirements of a-synCas, the inventors performed in vitro cleavage assays using the same target sequence but with different PAMs cloned into plasmid Zero-Blunt TOPO (the target sequence is in SEQ ID NO: 17, crRNA sequence in SEQ ID NO: 18). Alpha-synCas exhibited little to no preference for any PAM tested and was able to linearise plasmid DNA even with non-T rich, non-A-rich (e.g. CCC), non-G-rich and non-C-rich (e.g. TTT) PAM sequences (Figure 3c).

[0147] Further testing of PAM requirement with libraries of 3 or 7 random nucleotides as further discussed below was consistent with a-synCas not showing specificity for any PAM to enable activation.

[0148] To determine accurately and without bias any PAM preference of a-synCas, the inventors designed a DNA library with 7 random nucleotides (NNNNNNN) and a crRNA to target the sequence downstream of these random nucleotides (SEQ ID NO: 20; the crRNA sequence used to target this library is in SEQ ID NO: 19). The 7 random nucleotides served as a pool of all potential PAMs. This PAM library (SEQ ID NO: 20) was cloned into plasmid pUC18 and an 855 bp sequence was amplified from the resulting pUC18 plasmid library to produce a linear DNA fragment library containing the 7 random nucleotides upstream of the target sequence. This library was then subject to in vitro cleavage using a-synCas and the aforementioned crRNA. Successful cleavage produced two fragments, one 597 bp fragment and a smaller 258 bp fragment containing the PAM sequence. The inventors separated and extracted the uncleaved fragment from an agarose gel and analysed it by Ion Torrent Next Generation Sequencing (NGS). Through this analysis, depletion among the 7 random nucleotides is able to reveal any PAM preferences.

[0149] From the sequencing data, the inventors analysed the frequency of depletion of each 3 or 7 nucleotide PAM compared with those reads obtained from a negative control PCR fragment which was not treated with a-synCas, allowing a fold-change in frequency to be calculated for each 3 or 7 nucleotide PAM in the library. Then, the inventors calculated the total proportion against the overall frequency of each PAM in the library. The calculated frequencies were plotted on a PAM wheel and WebLogo to visualize the PAM preferences of a-synCas. Results showed that a-synCas does not show specificity towards recognizing any 3 nt or 7 nt PAM (Figure 4).

[0150] With reference to Figure 4, at most extremely weak PAM preference was shown, but significantly the PAM requirement for a-synCas target cleavage may be given as NNN, i.e, it may be categorized as PAM-less.

[0151] Example 5: crRNA processing activity of a-synCas

[0152] The inventors additionally tested the ability of a-synCas to process precursor RNA transcripts from which crRNAs are derived, referred to as pre-crRNA. By using a single customized pre- crRNA transcript (SEQ ID NO: 21 , obtained from Zetsche et al., 2017), the inventors showed a-synCas was as good as FnCas12a in processing FnCas12a crRNA (Figure 3d). This ability is highly valuable for the application of a-synCas in multiplexed genome editing, enabling the use of a customised CRISPR array for multiple targeting (which may be referred to as multiplex targeting or simply multiplexing).

[0153] Example 6: Diagnostic application of a-synCas

[0154] The collateral activity of CRISPR single effector nucleases has previously been repurposed for molecular diagnostics. To confirm that a-synCas could be repurposed as a biotechnological tool for nucleic acid detection, the inventors demonstrated the ability of a-synCas to detect both RNA and ssDNA in an in vitro assay system. The a-synCas-crRNA complex was incubated with a crRNA-complementary dsDNA, ssDNA or ssRNA target (the same as employed as the activators for Figure 2, SEQ ID NO: 6-8, respectively; crRNA in SEQ ID NO: 9), and these complexes were then in turn incubated with either a non-crRNA-complementary ssRNA (Figure 3f) or ssDNA (Figure 3g). These non-target ssRNA and ssDNA substrates were labelled with a fluorophore that emits fluorescence once the nucleic acid substrates to which they are attached have been cleaved, as this cleavage spatially separates the fluorophore from a quencher located on the same molecule. Consequently, fluorescence is only detected if the a-synCas-crRNA complex exerts indiscriminate trans-cleavage activity. The inventors were able to detect trans-cleavage of both ssDNA and ssRNA trans-substrates via fluorescence regardless of whether the cis-target substrate was dsDNA, ssDNA or ssRNA at 37 °C; a-synCas even outperformed the positive control FnCas12a (Figure 3f,g).

[0155] In summary, these results show that a-synCas can be repurposed as a nucleic acid diagnostic tool to detect any of the substrates that it can cis-target, those being dsDNA, ssDNA and ssRNA.

[0156] Example 7: Demonstration of a-synCas activity in bacterial cells

[0157] To determine the activity of a-synCas in bacteria, a plasmid interference assay was used. E. coli cells containing (i) a plasmid for IPTG- inducible expression of both a-synCas and a crRNA (crRNA sequence in SEQ ID NO: 22) plus also providing kanamycin resistance and (ii) transformed with a target plasmid capable of providing additionally chloramphenicol resistance plus sequence for targeting by the crRNA were cultured with or without IPTG and in the presence of both chloramphenicol and kanamycin. IPTG induction of a-synCas and crRNA expression led to a-synCas-crRNA-mediated destruction of the target plasmid as determined by measurement of cell forming units (CFU / mL). Under induction conditions (+IPTG), there was an approximately 1000-fold reduction in CFU / mL in comparison with no induction (-IPTG, non-induced a-synCas-crRNA) conditions, demonstrating successful targeting of dsDNA plasmid in a bacterial cell (Figure 5).

[0158] Example 8: Demonstration of a-synCas activity in eukaryotic cells

[0159] To determine activity of a-synCas in a eukaryotic system, HEK293T cells were transfected with an expression plasmid carrying the humanized a-synCas gene (SEQ ID NO: 23). Three crRNAs (SEQ ID NO: 24-26) were designed to target the DNMT1 , EMX1 and AAVS1 loci with T-rich PAMs (TTTC, TTTG, TTTG, respectively). The expression plasmid for expression of a- synCas was co-transfected with a DNA fragment for expressing the crRNA and, after 48 hours of transfection, the genomic DNA from HEK293T was extracted and a fragment of each of the three targeted loci was PCR amplified. As a positive control, FnCas12a (with crRNA sequences 24, 25 and 26 for DNMT1 , EMX1 and AAVS1 , respectively) was used. A T7E1 endonuclease assay was performed using the fragments to confirm genome editing.

[0160] Alpha-synCas mediated robust genome editing in the human cell line at all three loci, showing robust indel (15-35%) formation in human cells compared with the lack of activity of FnCas12a (see Figure 6).

[0161] Example 9: Sequence homology to known Cas nucleases

[0162] The inventors aligned the protein sequences of a-synCas (SEQ ID NO: 1) to those of FnCas12a (SEQ ID NO: 35), SpCas9 (SEQ ID NO: 36), Sulfuricurvum sp. PC08-66 Cas12a2 (SuCas12a2, SEQ ID NO: 37), Leptotrichia shahii Cas13 (LsCas13, SEQ ID NO: 38), Oscillibacter sp. Cas12f1 (OsCas12f1 , SEQ ID NO: 39) using BLAST available on the NCBI website. Sequence comparisons to SpCas9, LsCas13 and OsCas12f1 resulted in “No significant similarity found”. Sequence comparison results to FnCas12a and SuCas12a2 are provided in the table below.

[0163] Table 3. Sequence alignment of a-synCas with the following Cas enzymes.

[0164] Example 10: a-synCas exhibits robust PAM-flexible editing in human cells

[0165] Given the PAM flexibility of the ancestral version of SpCas9 (Alonso Lerma et al., 2023) the inventors explored the PAM requirements for a-synCas. The inventors applied an in vitro cleavage assay in which purified a-synCas loaded with an in v / tro-transcribed crRNA complex was incubated with a linear DNA target flanked seven random nucleotides. The PAM- containing cut DNA fragment was then gel purified and submitted for amplicon sequencing. The resulting normalized read counts revealed a modest preference for C / T at the -2 and -3 positions. However, all sequences were identified in the gel-extracted DNA suggesting that the resurrected nuclease has PAM biases but no obvious requirements. This PAM-flexible activity was verified in vitro using both the non-T-rich PAM sequences 5 -ATTG-3', 5 -ATTT-3', 5 -ACCC-3', 5 -ATCA-3' and 5 -ATGG-3' on a supercoiled plasmid, as well as 5 -TAAA-3', 5'- TCCC-3', and 5 -TGGG-3' on a FAM-labelled dsDNA substrate (Figure 9). Given the parallels to the PAM flexibility of the ancestral form of SpCas9 (Alonso Lerma et al., 2023), flexible PAM recognition could be a general feature of resurrected Cas nucleases. After demonstrating the ability of a-synCas to target non-canonical sites in vitro, the inventors evaluated its targeting flexibility in human cells in comparison to the engineered Cas12a variant enAsCas12a (Kleinstiver B. P. et al., 2019, Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat Biotechnol 37, 276-282). enAsCas12a nuclease was selected because it has been associated with the broadest PAM recognition of Cas12a nucleases to-date, with recognition of canonical numerous non-canonical PAMs (Kleinstiver et al., 2019). To provide a more comprehensive analysis, the inventors targeted 13 sites in four human genes (DNMT1 , CFTR, FANC and RLINX) and analysed the frequency of indels by Sanger-based indel detection methodology, which provides precise results for indels with a frequency above 1-2% (Brinkman, E. K. et al., 2014). These experiments yielded robust and editing activities (ranges 30-37%) at two target sites with a canonical PAM (5-TTTC-3, 5-TTTG-3) as well as two target sites with T / C-rich PAM (5-TCCT-3, 5-TCCC-3) PAMs (ranges 22-29%), but also at sites with PAMs not recognized efficiently by enAsCas12a such as 5-TCGG-3 (40%), 5-TATT-3 (38%), 5-AGTT-3 (17%) or 5-TGTT-3 (17%) (Figure 7a). At four sites with a 5-TTAC-3, 5-TCGC-3, 5-CGGT-3 or 5-TAAC-3 PAM, both nucleases yielded poor indel frequencies (< 5%), either due to limited PAM recognition or other factors such as poor sgRNA performance or site accessibility. Across the other sites, a-synCas efficiently edited those targeted by enAsCas12a and, more importantly, demonstrated robust genome editing at sites not recognized by enAsCas12a (Figure 7b). These data show that a-synCas enables robust editing of sites with non-canonical PAMs, with a tested PAM preference towards 5-NRYN-3 and 5-NYRN-3 in eukaryotic cells, extending beyond those recognized by enAsCas12a. Additionally, a-synCas could drive indel formation when transfected as a ribonucleoprotein (RNP) complex in two other cell lines (HeLa and Hs27 fibroblast cells) (Figure 10). Across the four target sites in four genes (DNMT1 , SOD1 , FANCF, CFTR) representing both canonical and non-canonical PAMs, a-synCas showed robust editing between 10-60%. Finally, off-target analysis of five on-target sites revealed that the indel frequencies at the predicted / potential off-target sites (Bae, S. et al., 2014, Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475) were below 1 %, much lower than those at corresponding on-target sites (Table 4). Thus, a-synCas exhibits PAM- flexible editing in human cells that outperforms enAnCas12a in a head-to-head comparison. ble 4. Off-targets analysis

[0166]

[0167]

[0168]

[0169] Example 11 : a-synCas accepts RNA, ssDNA and dsDNA targets and collateral substrates as well as altered crRNAs

[0170] Traditional CRISPR diagnostics systems based on collateral cleavage by Cas12 nucleases are restricted by the presence of PAM sequence before the target sequence, compromising the universal recognition of any target. Based on PAM-flexible recognition of a-synCas, the inventors envisioned that combining the collateral cleavage of a-synCas next to its PAM- flexible recognition, they could overcome this main limitation of this technology. To test this hypothesis, the inventors first compared the collateral cleavage of a-synCas with those exposed by wild-type LbCas12a (Chen, J. S. et al., 2018), a widely used Cas12a nuclease for molecular diagnostics (Kaminski, M. M., et al., 2021 , CRISPR-based diagnostics. Nat Biomed Eng 5, 643-656), after the recognition of a dsDNA target with a T-rich PAM (Figure 8a). The activity exhibited by a-synCas (0.0045 All / s) was lower to that of LbCas12a (0.0051 All / s) under equivalent conditions (Figure 8 b, d) at 37°C. Reducing the temperature from 37°C to 25°C (room temperature, RT, Figure 11) significantly decreases the activity of both nucleases (Figure 8b). However, at RT, a-synCas retains its ability to recognize target substrates at low concentrations (2 pM, Figure 8c) compared to LbCas12a which shows a higher detection limit (10 nM, Figure 8c). This is in line with previously reported results, where higher temperatures yielded optimal cleavage activity for Ca12a nucleases (Huyke, D. A. et al. 2022, Enzyme Kinetics and Detector Sensitivity Determine Limits of Detection of Amplification-Free CRISPR- Cas12 and CRISPR-Cas13 Diagnostics. Anal Chem 94, 9826-9834, and Nalefski, E. A. et al. 2021 , Kinetic analysis of Cas12a and Cas13a RNA-Guided nucleases for development of improved CRISPR-Based diagnostics. iScience 24, 102996). Thus, a-synCas exhibits catalytic properties comparable to LbCas12a, a leading nuclease for CRISPR-based diagnostics while also offering PAM flexibility and measurable activity at room temperature well suited for point-of-care molecular diagnostics.

[0171] Given the ability of a-synCas to recognize non-canonical PAMs with similar activities to that of LbCas12a, the inventors applied a-synCas in application requiring dsDNA recognition and PAM flexibility: detection of a short (21-bp) variable region of a single amplicon of 16S rDNA (Figure 8e). By applying this approach, it is possible to amplify the target region using universal primers that anneal to the conserved region of 16S rDNA and thus avoid difficulties associated with multiplexed pre-amplification. To evaluate this approach, the inventors designed four different crRNAs that hybridizes to four variable regions (from hypervariable region V1) of bacterial 16S rDNA with a non-canonical PAM (GTCG). Importantly, this region does not possess a T-rich stretch and thus cannot be recognized by traditional Cas12a-based diagnostics. Specifically, the inventors selected four bacterial pathogens, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus, relevant pathogens associated with sepsis in newborns (Srinivasan, R. et al. 2015, Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens. PLoS One 10, e0117617). Then, the inventors evaluated the collateral cleavage activity for each 16S dsDNA fragment and each crRNA. They found that a-synCas triggered fluorescence release only when there was a match between the dsDNA 16S fragment and the crRNA (Figure 8f). As expected, LbCas12a was not able to recognize any of these DNA targets (Figure 12). The inventors noticed crRNA from K. pneumoniae also triggered fluorescence release when paired with the other three pathogens, likely due to gRNA sequence similarity with seed region as previously reported (results not published). In fact, the mismatch profile analysis of a-synCas showed that the protein did not tolerate single or double mismatches at positions 1-20, only triggering a reduced fluorescence signal to single mismatches at positions 7-20 nt or double mismatches at the end of the PAM distal region (Figure 13). However, this signal was significantly reduced compared to that displayed by a-synCas with a target dsDNA substrate. These results demonstrate the specificity and flexibility of a-synCas for molecular diagnostics.

[0172] Methods

[0173] Protein purification a-synCas gene was synthesized and codon optimized for E. coli cell expression (SEQ ID NO: 34). Alpha-synCas was cloned in pET-28A(+) expression vector and transformed in E. coli BL21 (DE3) (Life Technologies) for protein expression. Cells were incubated in LB medium at 30 °C at 160 rpm until OD600 reached 0.6, IPTG (400pM) was added for protein induction overnight at 18 °C. Cells were pelleted by centrifugation at 5000 g. Pellets were resuspended in extraction buffer (Tris 50 mM, NaCI 500 mM, pH 8, Imidazole 10 mM) supplemented with EDTA Free protease inhibitor (Thermo). Then, the pellet was sonicated for 3 cycles for 10 min at 30% amplitude. Cell debris was separated by ultracentrifugation at 33,000 G for 1 h. For purification, the supernatants were mixed with Ni-NTA agarose beads (Thermo) and incubated for 1 hour. Then, the beads were washed with 50 x column volumes with 40 mM imidazole. After that, imidazole concentration was reduced to 10 mM. an aliquot of 3C protein was added. After 1 hour of incubation, protein was eluted in elution buffer (Tris 50 mM, NaCI 500 mM, pH 8, Imidazole 10 mM). Protein was further purified by size exclusion chromatography using a Superdex 200 HR column (GE Healthcare) and eluted in 50 mM Tris pH 7.5, 150 mM NaCI, 2 mM MgCh. For protein purification verification sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used with 8% gels. The protein concentration was calculated by measuring the absorbance at 280 nm in Nanodrop 2000C. FnCas12a was purified by the same protocol.

[0174] In vitro targeted cleavage

[0175] To make a-synCas-gRNA ribonucleoproteins (RNPs) in all assays, a-synCas was incubated for 10 min at 37 °C with gRNA (e.g. crRNA) at a 1 :3 ratio in 1 x NEB 3.1 buffer (50 mM Tris- HCI pH 7.9, 100 mM NaCI, 10 mM MgCI2, 100 pg ml-1 BSA). For analysis of targeted cleavage on supercoiled DNA, 30 pL reactions of 170 nM of a-synCas-gRNA and 100 nM of dsDNA target (Figures 1a and 3a: Zero-Blunt TOPO plasmid, Figure 3b: pUC18 plasmid, Figure 3c: Zero-Blunt TOPO plasmid with different fragments cloned into it containing the same target seguence as shown in SEQ ID NO: 17 but different PAM seguences) in 1 x NEB 3.1 buffer (50 mM Tris-HCI pH 7.9, 100 mM NaCI, 10 mM MgCI2, 100 pg ml-1 BSA) were incubated at 37 °C for varying incubation times. In the case of ssDNA substrate, 30 uL reactions of 80 nM of a-synCas-gRNA and 30 nM of ssDNA target (M13 phage DNA) in 1 x NEB 3.1 buffer were incubated at 37 °C for varying incubation times. Reactions were stopped by adding 6X loading dye (NEB) with EDTA and running 1-2% agarose gel. Gels were dyed with SYBR gold (ThermoFisher) and imaged with ChemiDoc XRS + System (Bio-Rad). Cleavage was guantified by Imaged. Finally, for ssRNA targeted cleavage, 30 pL reactions of 250 nM of a-synCas-gRNA and 120 nM of ssRNA target (SEQ ID NO: 5) in 1 x NEB 3.1 buffer were incubated at 37 °C for varying incubation times. Reactions were stopped by adding 2X loading dye (NEB) with EDTA and running 10% TBE polyacrylamide gels. Gels were dyed with SYBR gold (ThermoFisher) and imaged with ChemiDoc XRS + System (Bio-Rad). Cleavage was guantified by Imaged.

[0176] In vitro non-targeted (trans) cleavage

[0177] For analysis of targeted cleavage, 30 pL reactions of 70 nM of a-synCas-crRNA, 70 nM of cistarget substrate activators (dsDNA: SEQ ID NO: 6, ssDNA: SEQ ID NO: 7, ssRNA: SEQ ID NO: 8, all of which were targeted using the crRNA in SEQ ID NO:9) and 120 nM of collateral trans-substrate (dsDNA: pUC18 plasmid, ssDNA: M13 phage, ssRNA: SEQ ID NO: 10) in 1x NEB3.1 were incubated at 37 °C for varying incubation times. Reactions were stopped by adding 2X loading dye (NEB) with EDTA or 6X loading dye (NEB) with EDTA and then run on 10% TBE polyacrylamide gels (for ssRNA) or 1-2% agarose gel (for dsDNA and ssDNA). Gels were dyed with SYBR gold (ThermoFisher) and imaged with ChemiDoc XRS + System (BioRad). Cleavage was guantified by Imaged. A control without cis- activator was employed to show cleavage which can be accounted for by the assay conditions.

[0178] Pre-crRNA processing Pre-crRNA arrays were synthesized using the HiScribe T7 High Yield RNA Synthesis Kit (NEB). T7 transcription was performed for 16 h and then RNA was purified using the Monarch RNA Cleanup Kit (NEB). In vitro cleavage was performed in 30pL reactions with purified recombinant a-synCas (330 mM) and in v / fro-transcribed pre-crRNA arrays (100 nM) were incubated at 37 °C 1 x NEB 3.1 buffer for 1 hour. Reactions were stopped by adding 2X loading dye (NEB) with EDTA and running 10% TBE polyacrylamide gels. Gels were dyed with SYBR gold (ThermoFisher) and imaged with ChemiDoc XRS + System (Bio-Rad).

[0179] PAM determination assay

[0180] PAM determination assay was performed by incubating 170 nM of a-synCas-crRNA (targeting 23 nt downstream the 7 random nucleotides) and 100 nM of PCR fragment (855 bp) from the DNA library in 1 x NEB 3.1 buffer (50 mM Tris-HCI pH 7.9, 100 mM NaCI, 10 mM MgCI2, 100 pg ml-1 BSA) for 1 hour at 37°C. Reaction was stopped added 6X loading dye (NEB) with EDTA and run 2% agarose gel. Gels were dyed with SYBR gold (ThermoFisher) and imaged with ChemiDoc XRS + System (Bio-Rad). The uncleaved 855 bp fragment was purified from the agarose gel with GeneJet Gel Extraction kit (ThermoFisher). The fragment was sequenced by Ion Torrent and the obtained reads were mapped to the reference sequence (SEQ ID NO: 27). The reads that aligned to the reference with 0 mismatches were selected and the frequency was calculated for each PAM. For each PAM sequence, enrichment (i.e. foldchange) was calculated as follows:

[0181] Total control reads Experiment PAM reads enrichment = - - - — x - - - - —

[0182] Total experiment reads Control PAM reads

[0183] For web-logo, the average of the Iog2(enrichment) was calculated per each nucleotide at each position.

[0184] Nucleic acid detection with ssRNA and ssDNA reporter probes.

[0185] For nucleic acid detection with fluorescent reporter probes, 70 nM of a-synCas-crRNA (crRNA SEQ ID NO: 9) complex was combined with RNase or DNase Alert (250 nM, IDT) and 70 nM of target substrate (dsDNA: SEQ ID NO: 6, ssDNA: SEQ ID NO: 7, or ssRNA: SEQ ID NO: 8) in 100 pL reactions in a 96-well plate (Corning). A background control was prepared with nuclease-free water instead of target substrate. For the DNase Alert reaction, a positive control was prepared using FnCas12a-crRNA instead of a-synCas-crRNA and a dsDNA target substrate (SEQ ID NO: 6) as the target substrate The reactions were incubated at 37 °C and monitored for reporter fluorescence (RNase Alert: excitation 490 / emission 520, DNAse Alert: excitation 530 / emission 560) using the multi-mode microplate reader (BioTek Instruments).

[0186] Nucleic acid detection by a-synCas with ssRNA and ssDNA reporter probes. For nucleic acid detection using fluorescent reporter probes, a 70 nM concentration of the a- synCas -crRNA complex was combined with RNase or DNase Alert (250 nM, IDT) and a 70 nM concentration of the target substrate (dsDNA, ssDNA, or ssRNA) for 2 hours and 30 minutes at 37°C, respectively. The trans-cleavage assay was performed in 100 pL reactions and the resulting end-point fluorescence was measured using a multi-mode microplate reader (BioTek Instruments), with excitation / emission wavelengths of 490 / 520 nm for RNase Alert and 500 / 560 nm for DNase Alert. A negative control was prepared using nuclease-free water instead of the nucleic acid target.

[0187] For a-synCas comparison with LbCas12a, the wild-type LbCas12a was purified using pMBP- LbCas12a expression plasmid [pMBP-LbCas12a was a gift from Jennifer Doudna (Addgene plasmid # 113431 ; RRID:Addgene_113431)]. Protein purification was conducted as previously described and the protein stocks were diluted into storage buffer (10 mM Tris-HCI, 300 mM NaCI, 1 mM DTT, 0.1 mM EDTA, 500 pg / ml BSA, 50% Glycerol, pH 7.5) at 1 pM. For in vitro collateral cleavage assay, purified proteins (a-synCas and LbCas12a, 100 nM), gRNA (100 nM), target dsDNA (10, 2, 0.2, 0.02, 0.002 nM, respectively) and single-strand DNA reporter (DNase alert, 1000 nM, IDT) were added into 1 x NEB r2.1 (Supplementary Table 11). The trans-cleavage assay was performed in 5 pL reactions by measuring the fluorescence on a multi-mode microplate reader (BioTek Instruments), using a 96-well V bottom plate (Thermo) with excitation / emission wavelengths 500 / 560 nm. Time-courses were run for 2 h with an interval of 1 min between reads. Observed rates were obtained by regression analysis of the linear regions of the progress curves. A negative control was prepared using nuclease-free water instead of the nucleic acid target.

[0188] For 16S rDNA detection of multiple pathogens related with sepsis, purified proteins (a-synCas and LbCas12a, 100 nM), gRNA (100 nM), target dsDNA fragments (10 nm) and single-strand DNA reporter (DNase alert, 1000 nM, IDT) were added into 1 x NEB r2.1

[0189] Bacterial cell culture and transfection

[0190] Editing in bacteria was performed in E. coli BL21 containing a a-synCas nuclease and crRNA- expressing plasmid (pET_28a(+)) enabling IPTG induction of cas nuclease and crRNA expression and also providing kanamycin resistance. Bacterial culture was grown overnight and used to inoculate fresh ZymoBroth with 50 pg / mL kanamycin to an O.D. of 0.4-0.6 and made competent using a Mix & Go kit (Zymo). Immediately after, the competent E. coli pool was transformed with 500 ng of the target plasmid (a modified version of pBAD33 plasmid containing a subcloned LacZ gene and providing chloramphenicol resistance) and incubated on ice for 15 min. The crRNA target for a-synCas was located in the LacZ gene. The cells were recovered for 2 h at 37 °C with shaking in 500 pL of LB without antibiotics. Next, the cultures were sequentially diluted, and the dilutions were plated on LB agar plates containing 50 pg / mL kanamycin and 25 pg / mL chloramphenicol in the absence or presence of IPTG (0.3 mM). After colonies appeared, CFU / mL was calculated.

[0191] HEK293T cell culture and transfection

[0192] Human HEK293T cells (ATCC) were maintained in DMEM (Gibco) supplemented with 10% heat-inactivated FBS (Gibco) in an incubator at 37°C and 5% CO2. HEK293T cells were seeded onto 24-well plates (Thermo) 24 hrs before transfection. Cells were transfected using jetPrime (Polyplus) at 70-80% confluency following the manufacturer’s recommended protocol. For each well of a 24-well plate, a total of 500 ng plasmid DNA (humanized version of a-synCas, SEQ ID NO: 23, cloned into pCDNA 3.1 ; or FnCas12a expression plasmid Addgene 69976) and 500 ng of PCR amplicons comprising a U6 promoter driving expression of the crRNA for targeting each gene (DNMT1 , EMX1 , AAVS1 , crRNA sequences in SEQ ID NO: 24, 25, 26, respectively) were used.

[0193] Surveyor nuclease assay for genome modification in HEK293T cells

[0194] HEK293T cells were transfected with DNA as described above. Cells were incubated at 37°C for 48 hrs after transfection before genomic DNA extraction. Genomic DNA was extracted using the GENELUTE™ mammalian genomic DNA Miniprep kit (Merck) following the manufacturer’s protocol. Editing activity of a-synCas was evaluated using EnGen Mutation Detection Kit (NEB). Briefly, the genomic region flanking the target site for each gene was PCR amplified using Q5® Hot Start High Fidelity (PCR primers for DNMT1 : SEQ ID NO: 28, 29; EMX1 : SEQ ID NO: 30, 31 ; AAVS1 : SEQ ID NO: 32, 33) and PCR products were subjected to a denaturation and re-annealing process to enable heteroduplex formation. After reannealing, products were treated with T7 endonuclease I, and analysed on 2% agarose gels. Gels were dyed with SYBR gold (ThermoFisher) and imaged with ChemiDoc XRS + System (Bio-Rad). Cleavage was quantified by Imaged. Quantification was based on relative band intensities. Indel percentage were determined using the following formula, % Indels = 100 x (1 - where a is the integrated intensity of the undigested PCR product band, and b and c are the integrated intensities of each cleavage product band.

[0195] References

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[0207] Gasiunas, G., Young, J.K., Karvelis, T., Kazlauskas, D., Urbaitis, T., Jasnauskaite, M., Grusyte, M.M., Paulraj, S., Wang, P.H., Hou, Z. and Dooley, S.K., 2020. A catalogue of biochemically diverse CRISPR-Cas9 orthologs. Nature Communications, 11(1), p.5512.

[0208] Harrington, L.B., Burstein, D., Chen, J.S., Paez-Espino, D., Ma, E., Witte, I.P., Cofsky, J.C., Kyrpides, N.C., Banfield, J.F. and Doudna, J.A., 2018. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science, 362(6416), pp.839-842.

[0209] Harrington, L. B., Ma, E. Chen, J.S., Witte, I.P., Gertz, D., Paez-Espino, D., Al-Shayeb, B., Kyrpides, N. C., Burstein, D., Banfiled, J. F. and Doudna, J. A., 2020. A ScoutRNA Is Required for Some Type V CRISPR-Cas Systems. Molecular Cell, 79, 416-424.

[0210] Huyke, D. A. et al. Enzyme Kinetics and Detector Sensitivity Determine Limits of Detection of Amplification-Free CRISPR-Cas12 and CRISPR-Cas13 Diagnostics. Anal Chem 94, 9826- 9834, doi:10.1021 / acs.analchem.2c01670 (2022).

[0211] Huynh, N., Depner, N., Larson, R. and King-Jones, K., 2020. A versatile toolkit for CRISPR- Cas13-based RNA manipulation in Drosophila. Genome Biology, 21 (1), pp.1-29.

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[0214] Kaminski, M.M., Abudayyeh, O.O. , Gootenberg, J.S., Zhang, F. and Collins, J. J., 2021. CRISPR-based diagnostics. Nature Biomedical Engineering, 5(7), pp.643-656.

[0215] Liu, R.M., Liang, L.L., Freed, E., Chang, H., Oh, E., Liu, Z.Y., Garst, A., Eckert, C.A. and Gill, R.T., 2019. Synthetic chimeric nucleases function for efficient genome editing. Nature Communications, 10(1), p.5524.

[0216] Makarova, K.S., Wolf, Y.I., Iranzo, J., Shmakov, S.A., Alkhnbashi, O.S., Brouns, S.J., Charpentier, E., Cheng, D., Haft, D.H., Horvath, P. and Moineau, S., 2020. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), pp.67-83.

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[0227] SEQUENCES

[0228] SEQ ID NO: 1

[0229] >a-synCas

[0230] MKMFSNFTNQ YPLSKTLRFE LKPVGKTLEH IEKKGLLEQD EKRAEDYKKV KKIIDEYHKD FIEEALNNVK LNGEGLEEYY ELYFKKNKDD KDKKKKEFEK IQDNLRKQIV EAFKNHEKYK NLFKKELIKE DLPNWLKNSE DTGEEDKETV EKFKNFTTYF TGFHENRKNM YSDEEKSTAI AYRLIHENLP KFLDNMKVFE KIKEKHPEAE QLEKTLKDLG EEEVLGGNNV EDIFSLDYFN HTLTQSGIDI YNTIIGGKTE EDGNKKIQGL NEYINLYRQK NNEKNRKLPK LKPLYKQILS DRESLSFIPE AFENDEELLE AIEEFYENLN FSNNNEATNV LEKLKELLSN LADYDLNKIY IRNDTSLTDI SQKIFGDWSV IKDALNAHYD QTYPKKKKKK SKEKLEEKRE KWLKKQKYFS IAELQEALDS YCKESDESKE QKENSIADYF KTLAQTKNET DKKTDLIENI KSKYQYPNDK KLAQDKEFKD VEKIKAFLDS IMNLQHFVKP LHLVKGGSAG AEMEKDEAFY SEFEALYEEL SQVIPLYNKV RNYLTQKPYS TEKIKLNFEN STLLDGWDVN KETDNTSVLL RKDGLYYLGI MNKKHNKVFE NIPESNENDK CYEKMDYKLL PGANKMLPKV FFSNKNIDYF NPSAEILEIY ENGTHKKSGD NFNLDDCHKL IDFFKESINK HEDWKKFGFK FSPTSSYEDI

[0231] SGFYREVEQQ GYKISFKNIS ESYIDELVDE GKLYLFQIYN KDFSPYSKGK PNLHTLYWKA LFDEENLKDV VYKLNGEAEV FYRKASINET IVHKANEPIK NKNPLNPKKQ STFEYDIIKD RRYTVDKFQF HVPITMNFKA EGNSNINDEV NEFLKGNAPD VNIIGIDRGE RHLLYLTLID QKGKIVEQDS LNTITNEHNETDYHALLDDK EKERDKARKS WGTIENIKEL KEGYLSQWH KIAKLMVEHN AIVVMEDLNF GFKRGRFKVE KQVYQKFEKM LIDKLNYLVD KDKEPNEPGG LLNAYQLTNK FESFQKMGKQ SGFLFYVPAW NTSKIDPTTG FVNLFHPRYE NVEKAKEFFN KFDSIRYNSE KDYFEFAFDY NNFTEKAEGT KWTVCTYGER IKTYRNADKN NQWDSKEVNV TEEFKNLFDE YNIDYKNGND LKEAILSQDD ADFFKSLLHL LRLTLQMRNS ITGTEIDYII SPVANENGEF FDSRKADESL PKDADANGAY HIARKGLWVL EQIKQTDDLK KVNLAISNKE WLEFVQERKN SEQ ID NO: 2

[0232] >crRNA targeting dsDNA, Fig. 1a

[0233] AATTTCT ACT GTTGT AGATtcctaaggcgttaccccaat

[0234] SEQ ID NO: 3

[0235] > crRNA targeting ssDNA, Fig. 1a

[0236] AATTTCTACTGTTGTAGATAACGAACCACCAGCAGAAGA

[0237] SEQ ID NO: 4

[0238] >crRNA targeting ssRNA, Fig. 1a

[0239] AATTTCT ACT GTT GT AGATtcctaaggcgttaccccaat

[0240] SEQ ID NO: 5

[0241] >ssRNA target, Fig. 1c

[0242] GGGCCAGUGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAAAUAUGGGCUGUCCG

[0243] AUCGUAUAACAGGAUUCCGCAAUGGGGUUACCGCUUAAGCAUUAGGGGAGCUCGAU

[0244] UACUUGGUAGAACAGCAAUCUACUCGACCUGCAGGCAUGCAAGCUUGGCGUAAUCAU

[0245] GUGUUUAUCCGCUCACAAUUCCACACAACAUACGAGCCGGAAGCAUAAAGCGUAAUC

[0246] AUGGUCAUAGCUGUUUCCUG

[0247] SEQ ID NO: 6

[0248] >dsDNA target, Fig. 2, Fig. 3f,g ggatccTAATACGACTCACTATAGGGACAGGCTAGCATATTGTCCTAAGGCGTTACCCCAA

[0249] TGGcgaattcgtaatcCCCTCGAG

[0250] SEQ ID NO: 7

[0251] >ssDNA target, Fig. 2, Fig. 3f,g

[0252] CTCGAGGGgattacgaattcgCCATTGGGGTAACGCCTTAGGACAATATGCTAGCCTGTCCCT

[0253] ATAGTGAGTCGTATTAggatcc

[0254] SEQ ID NO: 8

[0255] >ssRNA target, Fig. 2, Fig. 3f,g

[0256] CTCGAGGGgattacgaattcgCCATTGGGGTAACGCCTTAGGACAATATGCTAGCCTGTC

[0257] GG

[0258] SEQ ID NO: 9 >crRNA targeting dsDNA, ssDNA, and ssRNA, Fig. 2

[0259] AATTTCT ACT GTTGT AGATtcctaaggcgttaccccaat

[0260] SEQ ID NO: 10

[0261] >ssRNA trans-substrate, Fig. 2b

[0262] GGCCAGUGAAUUCGAGCUCGGUACCCGGGGAUCCUCUAGAAAUAUGGAUUACUUGG

[0263] UAGAACAGCAAUCUACUCGACCUGCAGGCAUGCAAGCUUGGCGUAAUCAUGGUCAUA

[0264] GCUGUUUCCUGUGUUUAUCCGCUCACAAUUCCACACAACAUACGAGCCGGAAGCAUA

[0265] AAG

[0266] SEQ ID NO: 11

[0267] >SpCas9 crRNA, Fig. 3a gtcctaaggcgttaccccaagttttagagcta

[0268] SEQ ID NO: 12

[0269] >Cas<t> crRNA, Fig. 3a

[0270] CAACGATTGCCCCTCACGAGGGGACtcctaaggcgttaccccaat

[0271] SEQ ID NO: 13

[0272] >FnCas12a crRNA, Fig. 3a

[0273] AATTTCT ACT GTT GT AGATtcctaaggcgttaccccaat

[0274] SEQ ID NO: 14

[0275] >FnCas12a crRNA, Fig. 3b

[0276] AATTTCTACTGTTGTAGATCCAGTCACGACGTTGTAAAACGA

[0277] SEQ ID NO: 15

[0278] >LbCas12a crRNA, Fig. 3b

[0279] AATTTCTACTAAGTGTAGATCCAGTCACGACGTTGTAAAACGA

[0280] SEQ ID NO: 16

[0281] >AsCas12a crRNA, Fig. 3b

[0282] TAATTTCTACTCTTGTAGATCCAGTCACGACGTTGTAAAACGA

[0283] SEQ ID NO: 17

[0284] >target sequence, Fig. 3c g ag gg gtaccg ag ctcg aattcg SEQ ID NO: 18

[0285] >crRNA sequence, Fig. 3c

[0286] AATTTCTACTGTTGTAGATgaggggtaccgagctcgaattcg

[0287] SEQ ID NO: 19

[0288] > crRNA sequence used for PAM wheel, Fig. 4

[0289] AATTT CT ACT GTTGT AG AT ggtaccgagctcgaattcgtaat

[0290] SEQ ID NO: 20

[0291] >PAM library

[0292] N N N N N N N ggtaccgagctcgaattcgtaat

[0293] SEQ ID NO: 21

[0294] > Pre-crRNA transcript sequence, Fig. 3d

[0295] GGGGGTCTTTTTTTGCTGATTTAGGCAAAAACGGGTCTAAGAACTTTAAATAATTTCTAC

[0296] TGTTGTAGATGAGAAGTCATTTAATAAGGCCACTGTTAAAAGTCTAAGAACTTTAAATAA

[0297] TTTCTACTGTTGTAGATGCTACTATTCCTGTGCCTTCAGATAATTCAGTCTAAGAACTTTA

[0298] AATAATTTCTACTGTTGTAGATGTCTAGAGCCTTTTGTATTAGTAGCCGGTCTAAGAACT

[0299] TTAAATAATTTCTACTGTTGTAGATTAGCGATTTATGAAGGTCATTTTTTTGTCTAGCTTT

[0300] AATGCGGTAGTTTATCACAGTTAAATTGCTAACG

[0301] SEQ ID NO: 22

[0302] >crRNA sequence used in bacteria, Fig. 5

[0303] AATTTCTACTGTTGTAGATCCAGTCACGACGTTGTAAAACGA

[0304] SEQ ID NO: 23

[0305] >Humanised a-synCas gene, Fig. 6

[0306] ATGAAGATGTTCAGCAACTTTACCAACCAGTACCCTCTGTCTAAAACCTTGCGGTTCGA

[0307] GCTGAAGCCCGTGGGCAAGACCCTGGAACACATCGAGAAGAAGGGCCTGCTGGAACA

[0308] AGATGAAAAACGGGCCGAGGACTACAAAAAGGTCAAGAAGATCATCGACGAGTACCAC

[0309] AAGGACTTCATCGAGGAGGCTCTGAACAATGTGAAGCTTAATGGCGAGGGCCTGGAAG

[0310] AATACTACGAGCTGTACTTCAAGAAAAACAAGGATGATAAGGATAAGAAGAAGAAAGAG

[0311] TTCGAGAAAATCCAGGACAACCTGAGGAAGCAGATCGTCGAAGCCTTCAAGAATCACG

[0312] AGAAGTACAAGAACCTGTTTAAAAAGGAACTGATCAAGGAAGATCTGCCTAACTGGCTG

[0313] AAAAACTCCGAGGATACAGGCGAGGAGGATAAGGAAACCGTTGAGAAGTTCAAAAATTT

[0314] CACAACATACTTCACAGGCTTCCACGAGAACCGGAAGAATATGTACAGCGACGAGGAA AAGAGCACAGCCATCGCCTATCGGCTGATCCACGAGAACCTGCCCAAGTTCCTGGACA

[0315] ATATGAAGGTCTTTGAGAAGATCAAGGAAAAGCATCCTGAGGCAGAGCAGCTGGAGAA

[0316] GACGCTGAAGGATCTGGGCGAAGAAGAGGTGCTGGGCGGTAACAATGTGGAAGACAT

[0317] CTTCTCTCTGGACTACTTCAACCACACCCTGACACAGAGCGGCATCGACATCTACAACA

[0318] CCATCATTGGAGGAAAGACCGAGGAAGACGGCAATAAGAAAATTCAGGGCCTGAATGA

[0319] GTATATCAACCTGTACAGACAGAAAAACAACGAGAAGAATAGAAAGCTGCCCAAGCTGA

[0320] AGCCCCTGTACAAGCAGATCCTGTCTGATAGAGAGAGCCTGAGCTTCATCCCTGAAGC

[0321] CTTCGAAAACGATGAGGAACTGCTGGAGGCCATCGAGGAGTTCTACGAGAACCTGAAT

[0322] TTCAGCAACAACAATGAGGCCACAAACGTGCTCGAGAAGCTCAAGGAGCTGCTCTCCA

[0323] ATCTGGCCGACTATGACCTGAACAAAATCTACATCAGAAACGACACCAGCCTGACCGAC

[0324] ATCAGCCAAAAGATCTTCGGCGATTGGAGCGTGATCAAAGACGCCCTGAACGCCCACT

[0325] ACGACCAGACCTACCCTAAAAAGAAAAAAAAGAAGTCTAAGGAGAAACTGGAAGAGAAA

[0326] AGAGAGAAGTGGCTGAAGAAACAAAAGTACTTCTCCATCGCCGAACTGCAGGAGGCCC

[0327] TCGACAGCTACTGTAAAGAGTCTGACGAGTCCAAAGAGCAGAAAGAAAACAGCATTGC

[0328] CGACTACTTCAAGACACTGGCTCAGACCAAGAACGAAACCGACAAAAAGACCGACCTG

[0329] ATCGAAAACATCAAAAGCAAGTACCAGTACCCCAACGACAAAAAGCTTGCTCAGGATAA

[0330] GGAGTTTAAAGACGTGGAAAAGATCAAGGCTTTCCTGGACAGCATCATGAACCTTCAAC

[0331] ACTTCGTGAAGCCCCTGCACCTGGTGAAGGGAGGCAGCGCCGGAGCCGAGATGGAAA

[0332] AGGACGAGGCCTTCTACTCGGAGTTCGAGGCCCTGTACGAAGAACTGAGCCAGGTGAT

[0333] CCCACTGTACAACAAAGTGCGGAACTACCTGACCCAGAAGCCTTACAGCACAGAGAAG

[0334] ATCAAGCTGAATTTCGAGAACTCTACCCTGCTGGATGGCTGGGACGTGAACAAGGAGA

[0335] CAGACAACACCTCGGTGCTGCTGAGAAAGGATGGCCTGTACTACCTGGGAATCATGAA

[0336] CAAGAAGCACAACAAGGTGTTTGAGAACATCCCTGAAAGCAACGAGAACGACAAGTGC

[0337] TACGAGAAGATGGACTACAAGCTTCTGCCAGGCGCCAACAAGATGCTGCCTAAGGTGT

[0338] TCTTCAGCAACAAGAATATCGACTACTTCAACCCCAGCGCCGAGATTCTGGAGATCTAT

[0339] GAGAATGGCACCCACAAAAAGAGCGGCGACAACTTCAACCTGGACGACTGCCACAAAC

[0340] TGATCGATTTCTTCAAGGAGAGCATCAATAAGCATGAGGATTGGAAGAAATTCGGCTTT

[0341] AAGTTTTCTCCTACATCTAGCTACGAAGACATCAGCGGCTTCTACCGAGAGGTGGAGCA

[0342] GCAGGGCTACAAGATCTCTTTCAAGAACATCAGCGAAAGCTACATCGACGAACTGGTG

[0343] GACGAGGGCAAACTCTACCTGTTCCAGATCTACAATAAGGACTTTTCCCCCTACAGCAA

[0344] GGGCAAGCCTAACTTGCATACATTATATTGGAAGGCCCTGTTCGACGAAGAGAATCTGA

[0345] AGGACGTGGTGTACAAGCTGAACGGAGAGGCCGAAGTCTTTTACCGCAAGGCCAGCAT

[0346] CAACGAGACTATCGTGCACAAGGCGAATGAACCTATCAAGAACAAGAACCCCCTGAAT

[0347] CCTAAGAAACAGAGCACCTTCGAGTACGACATCATCAAGGACCGGCGGTACACCGTTG

[0348] ACAAGTTCCAGTTCCACGTGCCCATCACCATGAACTTCAAGGCAGAGGGCAACAGCAA

[0349] CATCAACGACGAGGTGAATGAGTTCCTGAAGGGCAACGCCCCTGACGTGAATATCATC

[0350] GGCATCGATAGAGGCGAAAGACACCTGCTGTACCTGACCCTGATCGACCAGAAGGGCA AGATCGTGGAGCAGGACTCCCTGAACACCATCACCAACGAGCACAACGAGACTGATTA

[0351] CCACGCCCTGCTGGACGACAAGGAGAAGGAGAGAGACAAGGCTAGAAAGAGCTGGGG

[0352] CACAATCGAAAATATCAAGGAACTGAAGGAAGGATACCTAAGTCAGGTCGTGCACAAGA

[0353] TCGCTAAACTGATGGTGGAACACAACGCCATCGTGGTGATGGAAGACCTGAACTTTGG

[0354] CTTTAAGAGAGGCAGATTCAAAGTGGAAAAGCAGGTGTACCAGAAGTTCGAGAAAATG

[0355] CTGATTGACAAGCTGAATTACCTGGTTGACAAGGATAAAGAGCCTAACGAACCTGGCG

[0356] GACTGCTGAATGCCTACCAACTGACAAACAAGTTCGAGTCTTTCCAGAAGATGGGCAAG

[0357] CAAAGCGGATTCCTGTTCTACGTGCCAGCCTGGAACACCTCCAAGATTGATCCTACCAC

[0358] CGGCTTCGTGAACTTGTTTCACCCTAGATACGAAAACGTGGAAAAAGCCAAGGAGTTCT

[0359] TCAACAAGTTCGACAGCATTAGATACAATAGCGAAAAAGATTATTTCGAGTTCGCCTTTG

[0360] ACTACAACAACTTCACCGAAAAGGCTGAGGGCACCAAGTGGACCGTGTGCACCTACGG

[0361] CGAAAGAATCAAAACCTACCGGAACGCCGATAAGAACAACCAGTGGGATAGCAAGGAG

[0362] GTGAACGTGACCGAGGAGTTTAAAAATCTCTTCGATGAATACAACATCGACTACAAGAA

[0363] CGGCAACGACCTCAAGGAAGCCATCCTGTCCCAGGACGATGCCGACTTCTTCAAGTCC

[0364] CTGCTGCACCTGCTGAGACTGACCCTGCAGATGAGAAATAGCATCACAGGCACAGAGA

[0365] TCGATTATATCATCTCCCCAGTGGCCAACGAGAACGGCGAGTTCTTCGACTCCCGGAA

[0366] GGCTGATGAGAGCCTGCCCAAGGACGCGGATGCCAACGGCGCCTACCATATCGCCAG

[0367] AAAAGGTCTGTGGGTGCTGGAACAGATCAAGCAGACAGATGACCTGAAGAAGGTGAAC

[0368] CTGGCCATCAGCAACAAGGAGTGGCTGGAGTTCGTGCAAGAGCGGAAGAAC

[0369] SEQ ID NO: 24

[0370] > crRNA for DNMT1 , Fig. 6

[0371] AATTT CTACTGTTGT AG AT ctgatggtccatgtctgttactc

[0372] SEQ ID NO: 25

[0373] >crRNA for EMX1 , Fig .6

[0374] AATTT CTACTGTTGT AG ATtcctccggttctggaaccacacc

[0375] SEQ ID NO: 26

[0376] > crRNA for AAVS1 , Fig .6

[0377] AATTTCTACTGTTGTAGATCTTACGATGGAGCCAGAGAGGAT

[0378] SEQ ID NO: 27

[0379] >Reference sequence for mapping, Fig. 4

[0380] CCCCAAN N N N N N Nggtaccgagctcgaattcgtaatcatggtcatagctg

[0381] SEQ ID NO: 28 >Primer DNMT1 FW

[0382] CTGGGACTCAGGCGGGTCAC

[0383] SEQ ID NO: 29

[0384] >Primer DNMT1 RV

[0385] CCTCACACAACAGCTTCATGTCAGC

[0386] SEQ ID NO: 30

[0387] >Primer EMX1 FW

[0388] CCATCCCCTTCTGTGAATGT

[0389] SEQ ID NO: 31

[0390] >Primer EMX1 RV

[0391] GGAGATTGGAGACACGGAGA

[0392] SEQ ID NO: 32

[0393] >Primer AAVS1 FW

[0394] GGGCTGGCTACTGGCCTTAT

[0395] SEQ ID NO: 33

[0396] >Primer AAVS1 RV

[0397] TATCTGTCCCCTCCACCCCA

[0398] SEQ ID NO: 34

[0399] >Bacterial optimised a-synCas gene

[0400] AAGATGTTTAGTAACTTCACCAACCAGTACCCGCTGAGCAAAACTCTGCGTTTTGAGCT

[0401] GAAACCGGTTGGTAAAACCCTGGAGCACATTGAGAAAAAAGGTCTGCTTGAGCAAGAT

[0402] GAAAAACGGGCAGAGGACTACAAAAAAGTCAAAAAAATCATCGACGAGTATCATAAAGA

[0403] CTTTATTGAAGAAGCGCTGAATAACGTAAAACTAAACGGCGAGGGCTTAGAGGAGTATT

[0404] ACGAGCTGTACTTTAAGAAAAACAAAGATGATAAGGACAAGAAAAAGAAGGAGTTCGAA

[0405] AAGATCCAAGACAACTTACGCAAGCAAATCGTTGAAGCGTTCAAAAACCATGAAAAGTA

[0406] TAAGAACTTGTTCAAGAAGGAACTTATCAAAGAAGATTTGCCGAATTGGCTGAAAAACA

[0407] GTGAGGATACTGGTGAGGAGGATAAAGAAACTGTGGAAAAGTTCAAGAACTTTACCACC

[0408] TACTTCACCGGTTTCCACGAAAATCGTAAAAATATGTATAGCGATGAGGAAAAGAGCAC

[0409] CGCAATTGCGTATCGTCTGATTCATGAAAACCTGCCTAAGTTCCTGGACAATATGAAAG

[0410] TGTTTGAGAAGATCAAAGAGAAGCACCCGGAGGCGGAACAACTGGAGAAGACCTTAAA

[0411] GGACTTGGGCGAGGAGGAAGTCTTAGGTGGTAACAACGTTGAAGACATCTTTTCTCTG GATTACTTTAACCACACCTTGACCCAGTCTGGTATTGATATCTATAACACCATTATTGGC

[0412] GGTAAAACCGAAGAGGACGGTAACAAGAAAATCCAGGGTCTGAACGAATATATTAATTT

[0413] GTACCGCCAGAAGAACAACGAAAAGAACCGCAAACTGCCGAAGCTGAAACCACTGTAC

[0414] AAGCAGATCCTGAGCGACCGTGAAAGCTTGAGCTTTATCCCAGAAGCGTTTGAGAACG

[0415] ATGAAGAGTTGCTAGAAGCGATTGAAGAGTTCTATGAGAACCTCAACTTCAGCAACAAC

[0416] AATGAGGCGACCAATGTCTTAGAAAAGCTTAAAGAACTGCTTAGCAACCTGGCAGACTA

[0417] CGACCTGAACAAGATCTATATCCGCAATGACACCAGCCTGACTGACATCTCGCAAAAGA

[0418] TCTTCGGTGATTGGTCGGTGATCAAGGACGCCCTGAATGCCCATTATGATCAGACCTAC

[0419] CCGAAAAAAAAAAAGAAGAAATCTAAGGAAAAGTTGGAGGAAAAAAGAGAAAAGTGGCT

[0420] GAAAAAGCAAAAATACTTCTCTATCGCGGAGCTGCAGGAGGCACTGGACTCATACTGC

[0421] AAAGAGAGCGATGAAAGCAAGGAGCAGAAAGAAAACTCCATTGCGGATTACTTCAAAAC

[0422] GCTGGCTCAAACCAAAAACGAAACGGACAAGAAAACCGATCTGATCGAGAATATTAAGA

[0423] GCAAATACCAGTATCCGAACGATAAGAAATTGGCGCAGGACAAAGAATTCAAGGACGT

[0424] GGAGAAAATCAAGGCGTTTCTGGACTCCATTATGAACTTACAGCATTTTGTTAAACCACT

[0425] GCACTTGGTCAAGGGTGGTAGCGCGGGTGCTGAAATGGAAAAAGATGAGGCTTTTTAC

[0426] TCCGAGTTTGAAGCTCTGTACGAGGAACTGTCGCAAGTCATCCCGCTATACAATAAAGT

[0427] GCGTAATTATCTGACGCAGAAGCCGTATAGCACCGAAAAAATCAAACTGAATTTTGAAA

[0428] ACAGCACGTTGCTGGATGGCTGGGACGTTAACAAAGAAACCGATAATACCAGCGTTCT

[0429] GTTGCGTAAAGATGGTCTCTACTATCTCGGTATTATGAACAAAAAACACAACAAGGTGTT

[0430] TGAAAACATTCCGGAATCTAACGAGAACGACAAGTGCTATGAAAAGATGGATTACAAAC

[0431] TCCTGCCTGGTGCCAATAAAATGCTGCCGAAGGTTTTTTTCTCCAATAAAAACATCGATT

[0432] ACTTTAACCCGAGCGCTGAGATACTGGAGATTTACGAAAATGGTACGCATAAGAAGAGC

[0433] GGTGATAATTTCAATTTAGACGACTGCCATAAACTGATTGATTTTTTTAAAGAATCAATCA

[0434] ATAAGCACGAAGATTGGAAAAAGTTCGGGTTCAAATTCAGCCCCACGTCGAGCTATGAA

[0435] GACATTAGCGGCTTTTATCGTGAAGTAGAACAGCAGGGTTATAAGATCTCATTCAAGAA

[0436] CATCAGCGAGTCGTATATCGACGAGTTGGTGGATGAAGGCAAGTTATACCTGTTCCAAA

[0437] TTTACAATAAGGATTTCTCCCCGTATTCAAAAGGCAAACCGAATTTACACACCTTGTACT

[0438] GGAAAGCGTTGTTCGACGAGGAGAACCTGAAAGATGTGGTCTATAAACTTAATGGCGA

[0439] GGCGGAAGTGTTCTACCGTAAAGCGAGCATCAACGAGACAATTGTGCACAAAGCGAAC

[0440] GAGCCGATTAAAAACAAAAACCCGCTGAATCCGAAAAAGCAAAGCACCTTTGAGTACGA

[0441] CATCATCAAGGACCGCCGTTATACCGTAGATAAATTTCAATTTCACGTTCCGATTACTAT

[0442] GAATTTTAAGGCCGAAGGTAACTCCAACATTAACGACGAAGTGAACGAATTCCTGAAAG

[0443] GTAACGCTCCGGATGTTAATATTATCGGCATCGACCGCGGCGAGCGCCACCTGTTGTA

[0444] CCTGACCTTGATTGACCAGAAGGGCAAGATCGTGGAGCAGGATAGCTTAAATACGATC

[0445] ACCAATGAGCACAATGAAACTGACTACCATGCGTTGCTGGATGACAAAGAAAAAGAGC

[0446] GTGATAAGGCGCGTAAGAGTTGGGGTACCATCGAAAACATCAAAGAACTGAAAGAGGG

[0447] CTATTTGAGCCAAGTTGTGCATAAAATCGCGAAACTGATGGTAGAGCACAATGCTATCG TGGTTATGGAAGACCTGAACTTTGGTTTTAAGCGTGGTCGTTTTAAAGTTGAAAAACAAG

[0448] TTTATCAGAAGTTCGAAAAAATGCTGATTGACAAATTGAACTATCTGGTTGATAAAGACA

[0449] AGGAACCGAACGAGCCGGGTGGGTTGCTAAACGCATATCAGCTGACCAATAAGTTCGA

[0450] AAGCTTTCAGAAGATGGGTAAACAGTCCGGCTTCCTGTTCTACGTGCCGGCATGGAATA

[0451] CGAGCAAGATTGATCCGACCACAGGCTTCGTGAACTTGTTCCATCCGCGTTACGAGAA

[0452] CGTTGAGAAGGCCAAAGAATTCTTTAACAAGTTCGATTCTATTCGTTATAACTCGGAAAA

[0453] GGACTACTTCGAGTTCGCCTTTGACTATAACAACTTCACCGAAAAGGCGGAGGGCACC

[0454] AAATGGACCGTTTGTACCTACGGCGAGCGCATTAAGACGTACCGCAATGCGGACAAAA

[0455] ACAACCAGTGGGATTCTAAAGAGGTGAATGTCACCGAAGAGTTCAAAAACCTGTTTGAC

[0456] GAGTACAACATCGACTACAAAAACGGCAACGACCTGAAGGAGGCCATACTGAGCCAAG

[0457] ATGATGCGGACTTCTTTAAGTCCCTGCTGCACTTGCTGCGTCTGACTCTGCAGATGCGT

[0458] AATTCCATCACCGGCACCGAGATAGACTACATTATTAGCCCGGTTGCAAATGAGAATGG

[0459] CGAGTTCTTCGATAGCCGTAAAGCCGATGAGTCCCTCCCAAAGGACGCTGACGCAAAC

[0460] GGTGCATATCACATTGCTCGTAAAGGCCTGTGGGTTCTGGAGCAAATTAAGCAAACGG

[0461] ATGACCTGAAAAAAGTGAACTTGGCGATTTCCAACAAGGAATGGCTGGAGTTCGTGCA GGAGCGCAAGAAC

[0462] SEQ ID NO: 35

[0463] >FnCas12a

[0464] MSIYQEFVNKYSLSKTLRFELIPQGKTLENIKARGLILDDEKRAKDYKKAKQIIDKYHQFFIEEI

[0465] LSSVCISEDLLQNYSDVYFKLKKSDDDNLQKDFKSAKDTIKKQISEYIKDSEKFKNLFNQNLID

[0466] AKKGQESDLILWLKQSKDNGIELFKANSDITDIDEALEIIKSFKGWTTYFKGFHENRKNVYSS

[0467] NDIPTSIIYRIVDDNLPKFLENKAKYESLKDKAPEAINYEQIKKDLAEELTFDIDYKTSEVNQRV

[0468] FSLDEVFEIANFNNYLNQSGITKFNTIIGGKFVNGENTKRKGINEYINLYSQQINDKTLKKYKM

[0469] SVLFKQILSDTESKSFVIDKLEDDSDVVTTMQSFYEQIAAFKTVEEKSIKETLSLLFDDLKAQK

[0470] LDLSKIYFKNDKSLTDLSQQVFDDYSVIGTAVLEYITQQIAPKNLDNPSKKEQELIAKKTEKAK

[0471] YLSLETIKLALEEFNKHRDIDKQCRFEEILANFAAIPMIFDEIAQNKDNLAQISIKYQNQGKKDL

[0472] LQASAEDDVKAIKDLLDQTNNLLHKLKIFHISQSEDKANILDKDEHFYLVFEECYFELANIVPL

[0473] YNKIRNYITQKPYSDEKFKLNFENSTLANGWDKNKEPDNTAILFIKDDKYYLGVMNKKNNKIF

[0474] DDKAIKENKGEGYKKIVYKLLPGANKMLPKVFFSAKSIKFYNPSEDILRIRNHSTHTKNGSPQ

[0475] KGYEKFEFNIEDCRKFIDFYKQSISKHPEWKDFGFRFSDTQRYNSIDEFYREVENQGYKLTF

[0476] ENISESYIDSVVNQGKLYLFQIYNKDFSAYSKGRPNLHTLYWKALFDERNLQDVVYKLNGEA

[0477] ELFYRKQSIPKKITHPAKEAIANKNKDNPKKESVFEYDLIKDKRFTEDKFFFHCPITINFKSSG

[0478] ANKFNDEINLLLKEKANDVHILSIDRGERHLAYYTLVDGKGNIIKQDTFNIIGNDRMKTNYHDK

[0479] LAAIEKDRDSARKDWKKINNIKEMKEGYLSQVVHEIAKLVIEYNAIVVFEDLNFGFKRGRFKV

[0480] EKQVYQKLEKMLIEKLNYLVFKDNEFDKTGGVLRAYQLTAPFETFKKMGKQTGIIYYVPAGF

[0481] TSKICPVTGFVNQLYPKYESVSKSQEFFSKFDKICYNLDKGYFEFSFDYKNFGDKAAKGKW TIASFGSRLINFRNSDKNHNWDTREVYPTKELEKLLKDYSIEYGHGECIKAAICGESDKKFFA

[0482] KLTSVLNTILQMRNSKTGTELDYLISPVADVNGNFFDSRQAPKNMPQDADANGAYHIGLKG

[0483] LMLLGRIKNNQEGKKLNLVIKNEEYFEFVQNRNN

[0484] SEQ ID NO: 36

[0485] >SpCas9

[0486] MDKKYSIGLDIGTNSVGWAVITDDYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGEIAEATR

[0487] LKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDE

[0488] VAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLV

[0489] QTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNF

[0490] KSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRLNSEITKAP

[0491] LSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPI

[0492] LEKMDGTEELLAKLNRKDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEK

[0493] ILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASAQSFIERMTNFDKNLPN

[0494] EKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKE

[0495] DYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREM

[0496] IEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNF

[0497] MQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHK

[0498] PENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQ

[0499] NGRDMYVDQELDINRLSDYDVDHIVPQSFIKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKK

[0500] MKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMN

[0501] TKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPK

[0502] LESEFVYGDYKVYDVRKMLAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETN

[0503] GETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWD

[0504] PKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEV

[0505] RKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDN

[0506] EQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTN

[0507] LGAPTAFKYFDTTIDRKRYTSTKEVLDATFIHQSITGLYETRIDLSQLGGD

[0508] SEQ ID NO: 37

[0509] >SuCas12a2

[0510] MLHAFTNQYQLSKTLRFGATLKEDEKKCKSHEELKGFVDISYENMKSSATIAESLNENELVK

[0511] KCERCYSEIVKFHNAWEKIYYRTDQIAVYKDFYRQLSRKARFDAGKQNSQLITLASLCGMY

[0512] QGAKLSRYITNYWKDNITRQKSFLKDFSQQLHQYTRALEKSDKAHTKPNLINFNKTFMVLAN

[0513] LVNEIVIPLSNGAISFPNISKLEDGEESHLIEFALNDYSQLSELIGELKDAIATNGGYTPFAKVT

[0514] LNHYTAEQKPHVFKNDIDAKIRELKLIGLVETLKGKSSEQIEEYFSNLDKFSTYNDRNQSVIV

[0515] RTQCFKYKPIPFLVKHQLAKYISEPNGWDEDAVAKVLDAVGAIRSPAHDYANNQEGFDLNH YPI KVAFDYAWEQLANSLYTTVTFPQEMCEKYLNSI YGCEVSKEPVFKFYADLLYI RKN LAVL EHKNNLPSNQEEFICKINNTFENIVLPYKISQFETYKKDILAWINDGHDHKKYTDAKQQLGFIR GGLKGRIKAEEVSQKDKYGKIKSYYENPYTKLTNEFKQISSTYGKTFAELRDKFKEKNEITKI THFGIIIEDKNRDRYLLASELKHEQINHVSTILNKLDKSSEFITYQVKSLTSKTLIKLIKNHTTKK GAISPYADFHTSKTGFNKNEIEKNWDNYKREQVLVEYVKDCLTDSTMAKNQNWAEFGWNF EKCNSYEDIEHEIDQKSYLLQSDTISKQSIASLVEGGCLLLPIINQDITSKERKDKNQFSKDWN HIFEGSKEFRLHPEFAVSYRTPIEGYPVQKRYGRLQFVCAFNAHIVPQNGEFINLKKQIENFN DEDVQKRNVTEFNKKVNHALSDKEYVVIGIDRGLKQLATLCVLDKRGKILGDFEIYKKEFVRA EKRSESHWEHTQAETRHILDLSNLRVETTIEGKKVLVDQSLTLVKKNRDTPDEEATEENKQK IKLKQLSYIRKLQHKMQTNEQDVLDLINNEPSDEEFKKRIEGLISSFGEGQKYADLPINTMRE MISDLQGVIARGNNQTEKNKIIELDAADNLKQGIVANMIGIVNYIFAKYSYKAYISLEDLSRAY GGAKSGYDGRYLPSTSQDEDVDFKEQQNQMLAGLGTYQFFEMQLLKKLQKIQSDNTVLRF VPAFRSADNYRNILRLEETKYKSKPFGVVHFIDPKFTSKKCPVCSKTNVYRDKDDILVCKEC GFRSDSQLKERENNIHYIHNGDDNGAYHIALKSVENLIQMK

[0516] SEQ ID NO: 38

[0517] >LsCas13

[0518] MGNLFGHKRWYEVRDKKDFKIKRKVKVKRNYDGNKYILNINENNNKEKIDNNKFIRKYINYK KNDNILKEFTRKFHAGNILFKLKGKEGIIRIENNDDFLETEEVVLYIEAYGKSEKLKALGITKKKI IDEAIRQGITKDDKKIEIKRQENEEEIEIDIRDEYTNKTLNDCSIILRIIENDELETKKSIYEIFKNIN MSLYKIIEKIIENETEKVFENRYYEEHLREKLLKDDKIDVILTNFMEIREKIKSNLEILGFVKFYL NVGGDKKKSKNKKMLVEKILNINVDLTVEDIADFVIKELEFWNITKRIEKVKKVNNEFLEKRRN RTYIKSYVLLDKHEKFKIERENKKDKIVKFFVENIKNNSIKEKIEKILAEFKIDELIKKLEKELKKG NCDTEIFGIFKKHYKVNFDSKKFSKKSDEEKELYKIIYRYLKGRIEKILVNEQKVRLKKMEKIEI EKILNESILSEKILKRVKQYTLEHIMYLGKLRHNDIDMTTVNTDDFSRLHAKEELDLELITFFAS TNMELNKIFSRENINNDENIDFFGGDREKNYVLDKKILNSKIKIIRDLDFIDNKNNITNNFIRKFT KIGTNERNRILHAISKERDLQGTQDDYNKVINIIQNLKISDEEVSKALNLDVVFKDKKNIITKIND IKISEENNNDIKYLPSFSKVLPEILNLYRNNPKNEPFDTIETEKIVLNALIYVNKELYKKLILEDDL EENESKNIFLQELKKTLGNIDEIDENIIENYYKNAQISASKGNNKAIKKYQKKVIECYIGYLRKN YEELFDFSDFKMNIQEIKKQIKDINDNKTYERITVKTSDKTIVINDDFEYIISIFALLNSNAVINKI RNRFFATSVWLNTSEYQNIIDILDEIMQLNTLRNECITENWNLNLEEFIQKMKEIEKDFDDFKI QTKKEIFNNYYEDIKNNILTEFKDDINGCDVLEKKLEKIVIFDDETKFEIDKKSNILQDEQRKLS NINKKDLKKKVDQYIKDKDQEIKSKILCRIIFNSDFLKKYKKEIDNLIEDMESENENKFQEIYYP KERKNELYIYKKNLFLNIGNPNFDKIYGLISNDIKMADAKFLFNIDGKNIRKNKISEIDAILKNLN DKLNGYSKEYKEKYIKKLKENDDFFAKNIQNKNYKSFEKDYNRVSEYKKIRDLVEFNYLNKIE

[0519] SYLIDINWKLAIQMARFERDMHYIVNGLRELGIIKLSGYNTGISRAYPKRNGSDGFYTTTAYY KFFDEESYKKFEKICYGFGIDLSENSEINKPENESIRNYISHFYIVRNPFADYSIAEQIDRVSNL LSYSTRYNNSTYASVFEVFKKDVNLDYDELKKKFKLIGNNDILERLMKPKKVSVLELESYNS

[0520] DYIKNLIIELLTKIENTNDTL

[0521] SEQ ID NO: 39

[0522] >OsCas12f1

[0523] MGKGVLAKVMKYELRYLDGCGDFSNMQEQVWALQRQTREILNRSIQIAFQWDCANSEHH

[0524] RKTGEYLDLKTETGYKRLDGHIYNCLKGQYEDMATSNLNATIQKAWKKYNSSKKEILRGSM

[0525] SIPSYKMNQPLTLDKNTVKLSEGERNPIVTLTLFSDKFKRAQGVSNVKFSMPLHDGTQRA

[0526] IFANLMNGTYQLGECQLVYKRPKWFLFVTYKFPPVEHPLDPDKILGVDMGEACALYASTF

[0527] GEHGYLKIDGGEITKYAKKMEARIRSMQKQAAHCGEGRIGHGTKTRVSWYQAKDKVARF

[0528] RDTINHRYSKALIDYALKNQCGTIQMEDLTGIKEDTGFPKFLRHWTYYDLQSKIEAKAAE

[0529] HGIQVVKINPRHTSQRCSRCGHIDKANRTSQADFCCTKCGFSANADFNASQNISIRNIDK

[0530] IIAKAIGANRKQT

[0531] SEQ ID NO: 40

[0532] >A. baumannii, Fig. 8e gcaggcttaa cacatgcaag tcgagcgggg gaaggtagct tgct

[0533] SEQ ID NO: 41

[0534] >E. coli, Fig 8e gcaggcctaa cacatgcaag tcgaacggta acaggaagaa gctt

[0535] SEQ ID NO: 42

[0536] >K. pneumoniae, Fig. 8e gcaggcctaa cacatgcaag tcgagcggta acacagagag cttg

[0537] SEQ ID NO: 43

[0538] >S. aureus, Fig. 8e gcgtgcctaa tacatgcaag tcgagcgaac ggacgagaag cttg

[0539] SEQ ID NO: 44

[0540] >target sequence , Fig. 13 agcgggggaa ggtagcttgc

[0541] SEQ ID NO: 45

[0542] >target sequence, Fig. 13 ggcgggggaa ggtagcttgc

[0543] SEQ ID NO: 46

[0544] >target sequence, Fig. 13 agtgggggaa ggtagcttgc

[0545] SEQ ID NO: 47

[0546] >target sequence, Fig. 13 agcgggagaa ggtagcttgc

[0547] SEQ ID NO: 48

[0548] >target sequence, Fig. 13 agcgggggaa gatagcttgc

[0549] SEQ ID NO: 49

[0550] >target sequence, Fig. 13 agcgggggaa ggtaacttgc

[0551] SEQ ID NO: 50

[0552] >target sequence, Fig. 13 agcgggggaa ggtagcttgt

[0553] SEQ ID NO: 51

[0554] >target sequence, Fig. 13 agtaggggaa ggtagcttgc

[0555] SEQ ID NO: 52

[0556] >target sequence, Fig. 13 agcgggggag agtagcttgc

[0557] SEQ ID NO: 53

[0558] >target sequence, Fig. 13 agcgggggaa ggtagctcac

[0559] SEQ ID NO: 54

[0560] >primer, table 4

[0561] TCCCTCACTCCTGCTCGGTGAA SEQ ID NO: 55

[0562] >primer, table 4

[0563] ACCTTTTGGGCGTGGAGAAGGG

[0564] SEQ ID NO: 56

[0565] >primer, table 4

[0566] CTCCCCCACCCCCTAGGAAAGT

[0567] SEQ ID NO: 57

[0568] >primer, table 4

[0569] TGAAGGTATAGGAGAGGTTTTGGGCT

[0570] SEQ ID NO: 58

[0571] >primer, table 4

[0572] TGCCAGTGGAAGGAGGGAGTGT

[0573] SEQ ID NO: 59

[0574] >primer, table 4

[0575] CCATCCCCTTCTGTGAATGT

[0576] SEQ ID NO: 60

[0577] >primer, table 4

[0578] AGCCACCTCATAAGGAAGGTC

[0579] SEQ ID NO: 61

[0580] >primer, table 4

[0581] GCTGCTGGTATCATGGAAAACATAA

[0582] SEQ ID NO: 62

[0583] >primer, table 4

[0584] CTTTGCTCTCAGCGGGATATAGATG

[0585] SEQ ID NO: 63

[0586] >primer, table 4

[0587] ACCAATGCAGCAGACATCCT SEQ ID NO: 64

[0588] >primer, table 4

[0589] GGGCTGGCTACTGGCCTTAT

[0590] SEQ ID NO: 65

[0591] >primer, table 4

[0592] AGAATTCTCATTGCGACTGTTTTGT

[0593] SEQ ID NO: 66

[0594] >primer, table 4

[0595] ATAGCAGTTTCTCCCATACAACAGT

[0596] SEQ ID NO: 67

[0597] >primer, table 4

[0598] TTGCATAAGAAAAATGCAGCTCTGA

[0599] SEQ ID NO: 68

[0600] >primer, table 4

[0601] CCCAATAAGCTTGCTCTTCTGTTTA

[0602] SEQ ID NO: 69

[0603] >primer, table 4

[0604] GCCCTACATCTGCTCTCCCTCC

[0605] SEQ ID NO: 70

[0606] >primer, table 4

[0607] GCCACCAATTTCTAAACTACAGCTA

[0608] SEQ ID NO: 72

[0609] >primer, table 4

[0610] TATTCACCGCTTTAAATCCTTGGTG

[0611] SEQ ID NO: 73

[0612] >primer, table 4

[0613] ATGGTTCTATGACCATTCCTTAGCA

[0614] SEQ ID NO: 74 >primer, table 4

[0615] GCTGTGTCTGTAAACTGATGGCTAACA

[0616] SEQ ID NO: 75

[0617] >primer, table 4

[0618] TGAGCAGGAAGAATATATAGGCACC

[0619] SEQ ID NO: 76

[0620] >primer, table 4

[0621] TACTACCCTGTTTATCCTGTTGAGC

[0622] SEQ ID NO: 77

[0623] >primer, table 4

[0624] CTTTGTGAGTAAATGGAAAGCCACT

[0625] SEQ ID NO: 78

[0626] >primer, table 4

[0627] AAGTCACTCTGGGGAACACGCC

[0628] SEQ ID NO: 79

[0629] >primer, table 4

[0630] GGGAGGGGGTCAGCATGAAAGG

[0631] SEQ ID NO: 80

[0632] >primer, table 4

[0633] CCCTTTCTGGTGGAGTGTCCCC

[0634] SEQ ID NO: 81

[0635] >primer, table 4

[0636] AGACGACCTTAGATGGAGTGTTGTGT

[0637] SEQ ID NO: 82

[0638] >primer, table 4

[0639] TGCCCAGGAAGTTGCTTCTCCC

[0640] SEQ ID NO: 83

[0641] >primer, table 4 GGAGATTGGAGACACGGAGA

[0642] SEQ ID NO: 84

[0643] >primer, table 4

[0644] ATGCTATTTTATCCTGGGGAGCAG

[0645] SEQ ID NO: 85

[0646] >primer, table 4

[0647] CATGTTGCCTTCCTGTCTATGAATC

[0648] SEQ ID NO: 86

[0649] >primer, table 4

[0650] GTTTCTGTGGGGTTAGGTTTATGTT

[0651] SEQ ID NO: 87

[0652] >primer, table 4

[0653] AAGAAACTCTCACGGCGAG

[0654] SEQ ID NO: 88

[0655] >primer, table 4

[0656] TATCTGTCCCCTCCACCCCA

[0657] SEQ ID NO: 89

[0658] >primer, table 4

[0659] AAACCACTATCTTGCCATCTTCTCT

[0660] SEQ ID NO: 90

[0661] >primer, table 4

[0662] TCTATGGGTCAGCACATGTTTTCTA

[0663] SEQ ID NO: 91

[0664] >primer, table 4

[0665] GACAACAAACTGAGACAGATGCTTT

[0666] SEQ ID NO: 92

[0667] >primer, table 4

[0668] TGAGTTAATTATGCACTTACATTTCTGA SEQ ID NO: 93

[0669] >primer, table 4

[0670] GGGCCGGGAAAGAGTTGCTG

[0671] SEQ ID NO: 94

[0672] >primer, table 4

[0673] ACTTTGAATGAATAACACTGCTGCT

[0674] SEQ ID NO: 96

[0675] >primer, table 4

[0676] GGTTTCTTGGCATCAACATGACTAA

[0677] SEQ ID NO: 97

[0678] >primer, table 4

[0679] TTTTGCTCTCTCTAGGACTTGAACA

[0680] SEQ ID NO: 98

[0681] >primer, table 4

[0682] TTGCATTCTACTCAATTGCATTCTGTGGG

[0683] SEQ ID NO: 99

[0684] >primer, table 4

[0685] CTTCCTCCAGTATTGAAAAGCAGTG

[0686] SEQ ID NO: 100

[0687] >primer, table 4

[0688] TATGAGTTGCATATGGAGGTGAGAG

[0689] SEQ ID NO: 101

[0690] >primer, table 4

[0691] CGTATGTCCATGTTAGCAAGACTTC

[0692] SEQ ID NO: 102

[0693] >target site, table 4

[0694] TTTCCTGATGGTCCATGTCTGTTACTC SEQ ID NO: 103

[0695] >target site, table 4

[0696] TTTCCTGcTGGTCCATGTCTaaTACTC

[0697] SEQ ID NO: 104

[0698] >target site, table 4

[0699] TTTTCTGATGGTCCATacCTGTTACaC

[0700] SEQ ID NO: 105

[0701] >target site, table 4

[0702] TTTTCTtATtGTaCATGTCTGTaACTC

[0703] SEQ ID NO: 106

[0704] >target site, table 4

[0705] TTTCCTGATGGTCCAcacCTGTTACaC

[0706] SEQ ID NO: 107

[0707] >target site, table 4

[0708] TTTGTCCTCCGGTTCTGGAACCACACC

[0709] SEQ ID NO: 108

[0710] >target site, table 4

[0711] TTTCTCCTCaGGTTCTGGAACCAataC

[0712] SEQ ID NO: 109

[0713] >target site, table 4

[0714] TTTCTCCTCCGGcTtTaGAgtCACACC

[0715] SEQ ID NO: 110

[0716] >target site, table 4

[0717] TTTGTtCTtCGGTTCTGaAACCAtACt

[0718] SEQ ID NO: 111

[0719] >target site, table 4

[0720] TTTATggTggGGTTCTGGAACCAaACC SEQ ID NO: 112

[0721] >target site, table 4

[0722] TTTGCTTACGATGGAGCCAGAGAGGAT

[0723] SEQ ID NO: 113

[0724] >target site, table 4

[0725] TTTTCTTttGATGGtGCCAGAGAGGAT

[0726] SEQ ID NO: 114

[0727] >target site, table 4

[0728] TTTCCTTAtGATGaAGCCAGAGAaGcT

[0729] SEQ ID NO: 115

[0730] >target site, table 4

[0731] TTTGCTTttGtTGGAGCCAGAGAGcAg

[0732] SEQ ID NO: 116

[0733] >target site, table 4

[0734] TTTGCTTcCtATGtAaaCAGAGAGGAT

[0735] SEQ ID NO: 117

[0736] >target site, table 4

[0737] TCCGAGCTTCTGGCGGTCTCAAGCACT

[0738] SEQ ID NO: 118

[0739] >target site, table 4

[0740] TTCCAGCTTCTGGtGGTtgCAAGCACT

[0741] SEQ ID NO: 120

[0742] >target site, table 4

[0743] TCCCAGCTTCTGGCaGTCTCAgGCctT

[0744] SEQ ID NO: 121

[0745] >target site, table 4

[0746] TTCCAGCTTCTGcCGGTCaCcAGCAaT SEQ ID NO: 122

[0747] >target site, table 4

[0748] TCCCCAAACTCTCCAGTCTGTTTAAAA

[0749] SEQ ID NO: 123

[0750] >target site, table 4

[0751] TCCCCAAACaCgCCAGTCTGTTTcAgA

[0752] SEQ ID NO: 124

[0753] >target site, table 4

[0754] TTTTCAAACTCaCCAGTaTaTTTAAcA

[0755] SEQ ID NO: 125

[0756] >target site, table 4

[0757] TTTGCAAACaaTtCAGTgTGTTTAAAA

Claims

CLAIMS1 . A Cas nuclease comprising or consisting of(a) the nuclease having the amino acid sequence as set out in SEQ. ID. NO. 1 and designated a-synCas, or(b) a Cas nuclease variant of the amino acid sequence of SEQ. I D. NO. 1 , which retains(i) the ability when complexed with a guide RNA (gRNA) to target and cleave any of a dsDNA, ssDNA and ssRNA and, when activated by such targeting, the capability to non-specifically cleave any of dsDNA, ssDNA and ssRNA , or retains (i) and / or(ii) a relaxed PAM requirement comparable to a-synCas; and which preferably also retains one or both of the following functional properties:(iii) ability to use a crRNA as a single oligonucleotide gRNA selectable from crRNAs suitable for employment by a plurality of Cas enzymes of Type II and Type V, for example selected from crRNAs suitable for a Cas12a from a plurality of species, e.g. crRNAs suitable for any of FnCas12a, LbCas12a and AsCas12a, or a crRNA suitable for a Cas9, e,g. SpCas9, or a crRNA suitable for a phage Cas<|) and(iv) ability to process its own crRNA from a pre-crRNA transcript, e.g. process a crRNA from a FnCas12a pre-crRNA transcript in vitro under conditions whereby the same pre- crRNA transcript will be processed to provide a crRNA by FnCas12a.

2. A Cas nuclease variant as claimed in claim 1 , wherein said variant retains all of the properties specified in (i)-(iv) and in comparison to FnCas12a retains the following amino acid substitutions and deletions as exhibited by the sequence of SEQ. ID. NO. 1 when compared to the sequence of the same Cas12a, or the same substitutions and deletions except for one or more conservative substitutions: R800K, R948H, V862T, K871 R, A850K, F873Y, N700S, S702del, P703del, R692Y, Q704del, Q125K, Y410W, K397T, K341A, T1063S, A676N, K131del, R800K, V862T, K871 R, K956L, R948H, A850K, K895N, K1287S, I586L, N607D.

3. A Cas nuclease variant as claimed in claim 1 , wherein said variant exhibits at least 70% sequence identity to SEQ. ID. NO. 1 , or only has one or more conservative substitutions compared with SEQ. ID. NO. 1 , and retains at least the cleavage abilities specified in claim 1 (i) or at least the cleavage abilities specified in claim (i) and / or the relaxed PAM requirement specified in claim 1 (ii), preferably together with one or both of the properties specified in claim 1 (iii) and (iv).

4. A Cas nuclease as claimed in any one of claims 1 to 3, which is fused at the N- and / or C- terminus to an additional amino acid sequence, e.g. a nuclear localization signal.

5. A variant of a Cas nuclease as claimed in any one of claims 1 to 4 which is derivable from said nuclease by catalytic site mutagenesis to vary its targeting and / or collateral cleavage ability, for example such a variant which is either (a) a nickase which exhibits nickase activity upon targeting to a dsDNA or (b) is a dead Cas (dCas) without nuclease activity, optionally said nickase or dCas being linked with a non-Cas effector protein such as a non-Cas effector protein for genetic modification or regulation.

6. A functional domain corresponding to a domain of a parent Cas nuclease or variant as claimed in any one of claims 1 to 5, said parent Cas nuclease or variant retaining all the properties specified in claim 1 (i)-(iv) or being a nickase or dCas variant, for example a functional domain providing the cleavage abilities as specified in claim 1(i) or the relaxed PAM requirement as specified in claim 1(ii).

7. A chimeric Cas polypeptide incorporating a domain as claimed in claim 6, for example a chimeric Cas nuclease.

8. A nucleic acid capable of expressing a Cas nuclease, variant, domain or chimeric Cas polypeptide according to any one of claims 1 to 7.

9. A combination product comprising or consisting of(i) a Cas nuclease, variant or chimeric Cas polypeptide according to any one of any one of claims 1 to 5 and 7, or a polynucleotide capable of expressing the same and(ii) a crRNA for targeting said Cas nuclease, variant or chimeric Cas polypeptide to a target sequence, or a polynucleotide capable of expressing the same, for example where (i) and (ii) are expression vectors or (i) and (ii) provide a ribonucleoprotein complex wherein said Cas nuclease, variant or chimeric Cas polypeptide is complexed with a crRNA.

10. A combination product as claimed in claim 9 packaged and / or formulated to provide a pharmaceutical composition.

11. A method for detecting in a sample one or more target nucleic acids wherein a Cas nuclease according to any one of claims 1 to 5 and 7 is employed which is capable of nonspecific collateral cleavage activity, said Cas nuclease being provided together with one ormore gRNAs each capable of targeting the Cas nuclease to target sequence(s) and said targeting with activation of the Cas nuclease being determined by detecting collateral cleavage of a reporter nucleic acid, preferably wherein said gRNA provides a targeting region complementary to an RNA or DNA encoding said RNA whereby all of a dsDNA, ssDNA and ssRNA complementary to said targeting region are targeted by a Cas nuclease according to any one of claims 1 to 5 and 7 and either a ssDNA or ssRNA is employed as a reporter nucleic acid, e.g. a ssDNA or ssRNA labelled with a fluorescent label and quencher whereby cleavage of the reporter nucleic acid by said Cas nuclease results in detectable fluorescence.

12. A method as claimed in claim 11 wherein a plurality of crRNAs are employed as single guide RNAs for multiplex screening for a plurality of target nucleic acid sequences, wherein a Cas nuclease according to any one of claims 1 to 5 and 7 is employed which is capable of processing a pre-crRNA transcript to provide its own crRNA and said Cas nuclease is provided with a single pre-crRNA transcript from which it can obtain said plurality of crRNAs and thereby target said plurality of target sequences.

13. A method as claimed in any one of claims 11 or 12, wherein the one or more target nucleic acids are from bacterial pathogens.

14. A method as claimed in claim 13, wherein the one or more target nucleic acids are 16S RNA or 16S DNA.

15. A method as claimed in any one of claims 13 or 14, wherein the bacterial pathogen is selected from the group consisting of Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and a combination thereof.

16. An in vitro method for determining the presence of an infection by a bacterial pathogen in a subject, wherein the method comprises detecting in a sample from the subject one or more target nucleic acids from the bacterial pathogen using the method according to any one of claims 11 to 15.

17. An in vitro method as claimed in claim 16, wherein the sample is a biological sample, preferably blood, serum, plasma, urine or saliva sample.

18. Use of a combination product according to claim 9 for determining the presence of an infection by a bacterial pathogen.

19. A method for modifying or regulating a target nucleic acid sequence, for example a DNA sequence, the method comprising contacting the target sequence with(i) a Cas protein selected from a Cas nuclease, variant and chimeric Cas polypeptide as claimed in any one of claims 1 to 5 and 7 and(ii) a guide RNA, wherein said guide RNA targets the Cas protein to the target sequence, and wherein(a) said contacting is in vitro with an isolated target nucleic acid or in a cell, for example an ex vivo cell, with the proviso that the method is not a method of modifying the germ line identity of a human being; or(b) the method is not a method of medical treatment practised on the human or animal body.

20. A method as claimed in claim 19 wherein a combination according to claim 9 is introduced into host cells, for example ex vivo human or animal cells, microbial cells or plant cells, optionally with additional provision in the host cell of an exogenous nucleic acid sequence for insertion into the target sequence following cleavage or for serving as a DNA repair template for guiding the outcome of target sequence modification.

21. A method as claimed in claim 20 wherein a combination as claimed in claim 9 is introduced into plant cells to modify or regulate a target nucleic acid sequence, said method further comprising obtaining a modified plant from said cells and seeds thereof.

22. A combination as claimed in claim 9 or claim 10 for use in a method of therapeutic treatment by modifying or regulating a target nucleic acid sequence, for example a DNA sequence, optionally wherein said use further comprises provision of a nucleic acid for insertion into the target nucleic acid sequence or for serving as a DNA repair template for guiding the outcome of target sequence modification.

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