Type iii crispr-CAS system and therapeutic uses thereof

Abstract

The present invention is concerned with targeted cell death or arrested cell growth in cells expressing a target RNA by delivering to the cell (i) a guide ribonucleic acid that is complementary to a recognition sequence within the target RNA expressed by the cell, (ii) one or more type III CRISPR-Cas proteins provided at least one protein is capable of producing a signaling molecule and (iii) at least one accessory protein capable of causing cell death or cell growth arrest which accessory protein is activated by the signaling molecule to cause dell death or cell growth arrest, wherein the presence of the target RNA within the cell causes the gRNA and type III CRISPR-Cas proteins to bind to the target RNA thereby producing at least one signaling molecule and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell. In certain examples according to the methods described herein the signaling molecule is a cyclic oligoadenylate and / or S-adenosyl methionine AMP and the accessory protein possesses deoxyribonuclease activity, ribonuclease activity, protease activity, adenosine deaminase activity and nicotinamide adenine dinucleotide (NAD+) nucleosidase activity sufficient to cause targeted cell death or growth arrest of the cell.

Description

[0001] TYPE III CRISPR-CAS SYSTEM AND THERAPEUTIC USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods for targeted cell death or cell growth arrest in a cell expressing a target RNA by delivering to the cell a type III CRISPR-Cas effector complex which is capable of recognising and binding to a target RNA transcript, resulting in activation of at least one accessory protein possessing activity sufficient to cause cell death or arrest cell growth. The methods according to the present invention are particularly well adapted for targeted cell death or cell growth arrest in mammalian cells, including human cells, with significant clinical implications for the treatment of diseases including cancer.

[0004] BACKGROUND OF THE INVENTION

[0005] Bacteria have evolved an extensive repertoire of defence mechanisms such as CRISPR-Cas systems, that provide sequence-specific protection against phages and other mobile genetic elements[l]. CRISPR (clustered regularly interspaced short palindromic repeats) loci contain CRISPR- associated (cas) genes, and CRISPR arrays of repeats and spacers. Arrays produce CRISPR RNAs (crRNA) which assemble with Cas proteins to form an RNA-guided effector complex. There are different CRISPR-Cas types with a range of activities and Cas protein components. There are two classes with six types and many more subtypes, with various DNA / RNA targeting abilities and different Cas proteins involved in the activity of CRISPR systems[2-4]. Class 1 systems involve multi-protein complexes and include type I, III and IV systems. Class 2 systems comprise single, multidomain crRNA-binding proteins and include type II (Cas9), V (Casl2) and VI (Casl3) systems. Despite differences between systems, CRISPR-Cas activity occurs through three stages: 1) the adaptation stage where new spacers are acquired from foreign nucleic acid, 2) the expression stage where Cas proteins and crRNAs are generated and then 3) the interference stage where crRNAs are used to guide the Cas ribonucleoprotein complex to the target sequence for cleavage[5,6]. It is due to these sequence-specific programmability of CRISPR-Cas systems that they have been exploited in a variety of biotechnologies.

[0006] Type III systems involve multi-protein complexes that recognize target RNA and then trigger a series of downstream catalytic events, including but not limited to degradation of DNA, RNA, protein and nicotinamide adenine dinucleotide (NAD)[7]. Type III systems are divided into five subtypes III-A to III-E[8] . Type III pre-crRNA transcripts are cleaved within the repeats by CRISPR-associated nuclease Cas6 and processed at the 3' end by other cellular nucleases[9]. The Cas protein complex forms on the crRNA and uses this as a guide to recognize complementary RNA sequences. The Cas7 family protein forms the backbone of the complex and acts in the catalytic cleavage of target RNA, typically with a 6- nucleotide cleavage periodicity upon base-pairing with the crRNA[10,ll]. Binding to target RNA also activates two catalytic activities in CaslO: 1) the HD domain cleaves single stranded DNA (e.g. in the transcription bubble) and 2) the Palm domain uses ATP to produce intracellular signaling molecules, for example cyclic oligoadenylate (cOA) or S-adenosyl methionine (SAM)-AMP[12-14]. These signaling molecules are allosteric activators of accessory proteins that possess catalytic activity, which provide the bacterium with a second line of defence. The most characterized examples of accessory proteins are ribonucleases (RNases; e.g. Csm6, Csxl) and nucleases (DNases; e.g. Canl, NucC) activated by specific cOA molecules. However, recent characterizations and bioinformatic studies have identified a range of catalytic activities associated with accessory proteins that include proteases, membrane proteins, adenosine deaminases and NADases. Common cOA sensory domains of accessory proteins include CARF (CRISPR-associated Rossmann fold; e.g. Csm6, Csxl) or SAVED (second messenger oligonucleotide or dinucleotide synthetase-associated and fused to various effector domains; e.g. TIR- SAVED), while other mechanisms can also exist (e.g. NucC).

[0007] A variant of the type III- D system (type III-Dv) is of significant interest as it has an unusual series of Cas7 subunit fusions, indicating it may be an evolutionary intermediate between multi-subunit (Class 1) and single effector (Class 2) CRISPR-Cas complexes[3]. The type III-Dv systems contain proteins CaslO, a Cas7-Cas7 fusion, a Cas7-Cas5-Casll fusion, Cas7 with an insertion, and Csxl9, a signature protein of type III-D systems of unknown function. The applicants have determined the structure of the type III-Dv effector complex from Synechocystis sp. PCC6803 and have identified which subunits and amino acids are involved in cleavage of target nucleic acids[ 15, 16] .

[0008] The Applicants recently discovered and characterized the NucC accessory nuclease from Serratia sp. ATCC 39006(17], The Applicants demonstrated Serratia NucC is activated by signal cA3 from the bacterium's type III-A CRISPR-Cas system. Once activated, NucC cleaves DNA in the cell, including genomic DNA, which results in bacterial cell death

[0017] . Despite recent advances in determining the different modes of action by type III systems and their accessory proteins, there is little information concerning clinical and non-clinical applications of type III CRISPR-Cas,. Furthermore, no published study has examined the activity of type III accessory proteins in combination with type III effector complexes in human or eukaryotic cells. The present application seeks to address this limitation by providing the first documented evidence of the application of a type III CRISPR-Cas to cause targeted cell death in mammalian cells by recognizing a specific host RNA sequence and eliciting activation of accessory proteins, including the NucC accessory DNase. This discovery has significant and far-reaching clinical potential, particularly for the in vivo treatment of cancers, autoimmune diseases and in ex vivo cell therapies.

[0009] SUMMARY OF INVENTION

[0010] In an aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:

[0011] (a) a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0012] (b) one or more type III CRISPR-Cas proteins, wherein at least one protein is capable of producing a signaling molecule; and

[0013] (c) at least one accessory protein capable of causing cell death or cell growth arrest which accessory protein is activated by the signaling molecule, wherein, the presence of the target RNA within the cell causes the gRNA and the one or more type III CRISPR-Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell. In another aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RIMA), the method comprising delivering to the cell:

[0014] (a) a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0015] (b) one or more type III-A CRISPR-Cas, type III-B CRISPR-Cas, type III-C CRISPR-Cas, type III-D CRISPR-Cas or type III-E CRISPR-Cas proteins, wherein at least one protein is capable of producing a signaling molecule; and

[0016] (c) at least one accessory protein capable of causing cell death or cell growth arrest which accessory protein is activated by the signaling molecule, wherein, the presence of the target RNA within the cell causes the gRNA and the one or more type III CRISPR-Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0017] In another aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:

[0018] (a) a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0019] (b) type III-D CRISPR-Cas proteins comprising:

[0020] (1) CaslO;

[0021] (2) Casll;

[0022] (3) Cas7;

[0023] (4) Cas5;

[0024] (5) Cas6; wherein the CaslO is capable of generating a signaling molecule, and

[0025] (c) at least one accessory protein capable of causing cell death or cell growth arrest, which accessory protein is activated by the signaling molecule; wherein, the presence of the target RNA within the cell causes the gRNA and type III-D CRISPR- Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0026] In another aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:

[0027] (a) a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0028] (b) type III-D CRISPR-Cas proteins comprising:

[0029] (1) CaslO;

[0030] (2) Casll;

[0031] (3) Cas7;

[0032] (4) Cas5; (5) Cas6; wherein the CaslO is capable of generating a signaling molecule, and wherein Cas7 comprises at least one mutation with reduces or eliminates Cas7 ribonuclease activity; and

[0033] (c) at least one accessory protein capable of causing cell death or cell growth arrest, which accessory protein is activated by the signaling molecule, wherein, the presence of the target RNA within the cell causes the gRNA and type III-D CRISPR- Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0034] In another aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:

[0035] (a) a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0036] (b) type III-Dv CRISPR-Cas proteins comprising:

[0037] (1) a Cas7-Cas5-Casll fusion protein;

[0038] (2) a Cas7-Cas7 fusion protein;

[0039] (3) a Cas7-insertion protein;

[0040] (4) a CaslO;

[0041] (5) a Csxl9; wherein the CaslO is capable of generating a signaling molecule; and

[0042] (c) at least one accessory protein capable of causing cell death or cell growth arrest, which accessory protein is activated by the signaling molecule, wherein, the presence of the target RNA within the cell causes the gRNA and type III-Dv CRISPR- Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0043] In another aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:

[0044] (a) a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0045] (b) type III-Dv CRISPR-Cas proteins comprising:

[0046] (1) a Cas7-Cas5-Casll fusion protein;

[0047] (2) a Cas7-Cas7 fusion protein;

[0048] (3) a Cas7-insertion protein;

[0049] (4) a CaslO;

[0050] (5) a Csxl9;

[0051] (6) a Cas6; wherein the CaslO is capable of generating a signaling molecule; and (c) at least one accessory protein capable of causing cell death or cell growth arrest, which accessory protein is activated by the signaling molecule, wherein, the presence of the target RNA within the cell causes the gRNA and type III-Dv CRISPR- Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0052] In another aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:

[0053] (a) a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0054] (b) type III-Dv CRISPR-Cas proteins comprising:

[0055] (1) a Cas7-Cas5-Casll fusion protein;

[0056] (2) a Cas7-Cas7 fusion protein;

[0057] (3) a Cas7-insertion protein;

[0058] (4) a CaslO;

[0059] (5) a Csxl9;

[0060] (6) optionally, a Cas6 wherein the Cas 10 is capable of generating a signaling molecule, and wherein one, two or all three of (1), (2) and (3) comprise at least one mutation in a Cas7 domain which reduces or eliminates Cas7 ribonuclease activity; and

[0061] (c) at least one accessory protein capable of causing cell death or cell growth arrest, which accessory protein is activated by the signaling molecule wherein, the presence of the target RNA within the cell causes the gRNA and type III-Dv CRISPR- Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0062] In another aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:

[0063] (a) a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0064] (b) type III-A CRISPR-Cas proteins comprising:

[0065] (1) CaslO;

[0066] (2) Casll;

[0067] (3) Cas7;

[0068] (4) Cas5;

[0069] (5) Cas6; wherein the CaslO is capable of generating a signaling molecule; and

[0070] (c) at least one accessory protein capable of causing cell death or cell growth arrest, which accessory protein is activated by the signaling molecule, wherein, the presence of the target RNA within the cell causes the gRNA and type III-D CRISPR- Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0071] In another aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:

[0072] (a) a nucleic acid encoding a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0073] (b) at least one nucleic acid encoding one or more type III CRISPR-Cas proteins, wherein at least one type III CRISPR-Cas protein is capable of producing a signaling molecule; and

[0074] (c) at least one accessory protein capable of causing cell death or cell growth arrest, which accessory protein is activated by the signaling molecule wherein (a) and (b) are encoded by the same nucleic acid or different nucleic acids and are expressed by the cell, and wherein, the presence of the target RNA within the cell causes the gRNA and the one or more type III CRISPR-Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0075] In another aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:

[0076] (a) a nucleic acid encoding a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0077] (b) one or more type III CRISPR-Cas proteins, wherein at least one type III CRISPR-Cas protein is capable of producing a signaling molecule; and

[0078] (c) a nucleic acid encoding at least one accessory protein capable of causing cell death or cell growth, which accessory protein is activated by the signaling molecule, wherein (a) and (c) are encoded by the same nucleic acid or different nucleic acids and are expressed by the cell, and wherein, the presence of the target RNA within the cell causes the gRNA and the one or more type III CRISPR-Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0079] In another aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:

[0080] (a) a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell; (b) a nucleic acid encoding one or more type III CRISPR-Cas proteins, wherein at least one type III CRISPR-Cas protein is capable of producing a signaling molecule; and

[0081] (c) a nucleic acid encoding at least one accessory protein capable of causing cell death or cell growth arrest, which accessory protein is activated by the signaling molecule, wherein (b) and (c) are encoded by the same nucleic acid or different nucleic acids and are expressed by the cell, and wherein, the presence of the target RNA within the cell causes the gRNA and the one or more type III CRISPR-Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0082] In another aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:

[0083] (a) a nucleic acid encoding a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0084] (b) a nucleic acid encoding one or more type III CRISPR-Cas proteins, wherein at least one type III CRISPR-Cas protein is capable of producing a signaling molecule; and

[0085] (c) a nucleic acid encoding at least one accessory protein capable of causing cell death or cell growth arrest, which accessory protein is activated by the signaling molecule, wherein (a), (b) and (c) are encoded by the same nucleic acid or different nucleic acids, or wherein

[0086] (a) and (b) are encoded by the same nucleic acid and (c) is encoded by a different nucleic acid, or wherein (a) and (c) are encoded by the same nucleic acid and (b) is encoded by a different nucleic acid, or wherein (b) and (c) are encoded by the same nucleic acid and (a) is encoded by a different nucleic acid and are expressed by the cell, and wherein, the presence of the target RNA within the cell causes the gRNA and the one or more type III CRISPR-Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0087] In another aspect of the present invention there is provided a test kit or article of manufacture intended for use in targeting cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the test kit or article of manufacture comprising:

[0088] (a) a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell, or a nucleic acid encoding the gRNA;

[0089] (b) one or more type III CRISPR-Cas proteins or a nucleic acid encoding the one or more type III CRISPR-Cas proteins, wherein at least one type III CRISPR-Cas protein is capable of producing a signaling molecule;

[0090] (c) at least one accessory protein capable of causing cell death or cell growth arrest, or a nucleic acid encoding the at least one accessory protein, which accessory protein is activated by the signaling molecule; and (d) instructions for how to achieve targeted cell death or targeted cell growth arrest in the cell expressing a target RNA.

[0091] BRIEF DESCRIPTION OF THE FIGURES

[0092] Figure 1 shows type III-Dv can target endogenous gene transcripts in HEK293 cells and activate the accessory nuclease NucC. A. RT-qPCR data showing ~50% reduction in RNA levels when targeting CKB, XIST and BRCA1 RNAs. Genes are shown below with CRISPR (cr) RNAs and RT-qPCR primers annotated. B. Schematic of a type III complex binding target RNA causing the CaslO domain to catalyze the conversion of ATP to cyclic oligoadenylates (cOAs), which in turn causes the activation of hexameric NucC. Once activated, NucC degrades genomic DNA. C. In vitro degradation assay demonstrating CRISPR-Cas type III-Dv / NucC coupled activity. The type III-Dv effector complex in combination with a NucC DNA nuclease specifically recognizes a target RNA and triggers cleavage of a gDNA substrates.

[0093] Figure 2 shows the design of cell killing vector pPF3816 and proposed mechanism for type III- Dv and NucC triggered cell death in mammalian cells. A. Schematic of the mammalian expression vector for targeted cell killing (pPF3816). B. Proposed mechanism of action to cause mRNA expression- and sequence-specific cell death in mammalian cells. This utilizes the production of cOAs by the CaslO Palm domain only when the target RNA is bound to the type III-Dv complex. The cOA signaling molecules activate NucC and trigger degradation of genomic DNA, resulting in cell death.

[0094] Figure 3 shows RNA targeting of type III-Dv activates NucC to cause cell death in HEK293 cells. A. Representative confocal images taken at lOOx magnification of type III-Dv plasmids transfected into HEK293 cells. From left to right: 1) Untransfected cells, 2) cells transfected with the CRISPRi plasmid (inactive Palm domain) with the addition of NucC (pPF3934) and a non-targeting spacer. 3) As for 2) but with BRCA1 -target! ng spacer. Cells transfected with cell killing plasmid that contains type III-Dv (active Palm domain) and NucC (pPF3816) with 4) a non-targeting spacer, 5) BRCA1 transcript targeting spacer, or 6) CKB transcript targeting spacer. B. Plots of grey-scale intensity variation for DAPI and RFP across selected cells in A, section of cells analyzed indicated by arrow and dashed line. C. Quantification of the number of DAPI stained nuclei, RFP+ / type III-Dv+ cells, dead cells and RFP+ cells that are dead from confocal images at lOOx from 3-6 fields of view done in technical duplicate for each condition (6- 12 total fields of view per condition). Significance in the variation of number of DAPI stained nuclei between non-targeting and BRCA1 and CKB transcript-targeting spacers was analyzed by one-way ANOVA. ****p<0.0001. D. Percentage of RFP+ / type III-Dv+ cells that are dead, determined from data in C. Statistical analysis was performed using one-way ANOVA analysis relative to the non-targeting control spacer in pPF3816. *** £><0.001; ****£><0.0001.

[0095] Figure 4 shows flow cytometry and viability assays demonstrating HEK-293 cell death caused by RNA targeting of type III-Dv and activation of NucC. A. Flow cytometry data showing representative ungated scattering data for live and dead HEK293 samples (dead cells were heat killed), gate 1 represents the scatter observed for live and dead HEK293 cells, while gate 2 represents the scatter profile for live / viable HEK293 cells. B. Side scatter for live and dead cells transfected with cell killing plasmid (pPF3816) with either non-targeting, BRCA1, or CKB transcript-targeting spacers. C. Viable cell counts determined from flow cytometry data for non-targeting, BRCA1- and CKB- targeting spacers. D. Resazurin metabolic assay done in parallel of cells analyzed by flow cytometry in B - C, showing significantly reduced metabolic activity of HEK293 cells when endogenous genes are targeted. Statistical analysis was performed using one-way ANOVA analysis relative to the non-targeting control spacer in pPF3816. **p<0.01; ****p<0.0001. E. Trypan-blue viability assay done in parallel to samples analyzed in B - D.

[0096] Figure 5 shows the type III-Dv / NucC system can be induced with tetracycline to trigger CRISPRi and cell killing. A. Schematic showing the tetracycline inducible promoter in a type III-Dv backbone, showing Bsal and Spel restriction sites and location of the type III-Dv repeat and cloning site for spacers. B. Venus median fluorescent intensity (MFI) as measured by flow cytometry of HEK293T-rtTA cells with 5 pg / mL of tetracycline for cells transfected with tetracycline-inducible CRISPRi vector (pPF3951) either empty (non -targeted) or with a Venus transcript targeting spacer. Differences in Venus MFI between the two treatments were determined using an unpaired Welch t-test. **p<0.005. C. Normalized percentage of viable RFP+ / type III-Dv+ cells, comparing cells transfected with tetracyclineinducible cell killing vector (pPF3952) containing a BRCA1 transcript-targeting spacer to empty (nontargeted) control, at increasing tetracycline concentrations.

[0097] Figure 6 shows viability assays demonstrating reduced HeLa metabolism indicating cell death caused by RNA targeting of type III-Dv and activation of NucC. Resazurin metabolic assay showing significantly reduced metabolic activity of HeLa cells when endogenous gene transcripts for BRCA1 and CKB are targeted. Statistical analysis was performed using one-way ANOVA analysis relative to the nontargeting control spacer in pPF3816. **p<0.005; ***p<0.0005.

[0098] Figure 7 shows mutations in the Cas7 domain in the type III-Dv complex retains mammalian cell killing activity observed in the type III-Dv / NucC system. A. & B. Flow cytometry data showing the total number of and mCherry+ live HEK293 cells, respectively, in an untargeted control versus the CKB RNA transcript targeting samples. Statistical significance of the differences between the two treatments was determined using an unpaired Welch t-test. *p<0.05.

[0099] Figure 8 shows that the type III-A / NucC system sequence specifically can kill mammalian cells. A. & B. Flow cytometry data showing the total number of and GFP+ live HEK293 cells, respectively, in cells transfected with a type III-A untargeted control versus the type III-A BRCA1 RNA transcript targeting samples. C. Resazurin viability assay run in parallel with flow cytometry analysis showing fluorescence at 585 nm for HEK293 cells in various conditions; untransfected, heat killed HEK293 cells, buffer only, cells transfected with an untargeted type III-A / NucC control and cells transfected with a BRCA1 targeting type III-A / NucC system. Statistical significance of the differences between the two treatments was determined using an unpaired Welch t-test. **p<0.01.

[0100] Figure 9 shows the RNA targeting and binding of type III-Dv can activate other accessory proteins such as the TIR-SAVED domain and Csxl to result in cell killing in mammalian cells. A. & B. Flow cytometry data showing the total number of and mCherry+ live HEK293 cells, respectively, in cells transfected with a type III-Dv / TIR- SAVED untargeted control versus the type III-Dv CKB RNA transcript targeting samples. C. Resazurin viability assay run in parallel with flow cytometry analysis showing fluorescence at 585 nm for HEK293 cells in various conditions; untransfected, heat killed HEK293 cells, buffer only, cells transfected with an untargeted type III-Dv / TIR-SAVED untargeted control and cells transfected with a CKB targeting type III-Dv / TIR-SAVED system. D. & E. Flow cytometry data showing the total number of and mCherry+ live HEK293 cells, respectively, in cells transfected with a type III- Dv / Csxl untargeted control versus the type III-Dv CKB RNA transcript targeting samples. C. Resazurin viability assay run in parallel with flow cytometry analysis showing fluorescence at 585 nm for HEK293 cells in various conditions; untransfected, heat killed HEK293 cells, buffer only, cells transfected with an untargeted type III-Dv / Csxl untargeted control and cells transfected with a CKB targeting type III- Dv / Csxl system. Statistical significance of the differences between treatments was determined using an unpaired Welch t-test. *p<0.05; ****p<0.0001.

[0101] GENERAL DEFINITIONS

[0102] As used herein, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0103] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0104] The term "about," as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, "about X" where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measurable value may include any other range and / or individual value therein.

[0105] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y" and phrases such as "from about X to Y" mean "from about X to about Y."

[0106] The term "comprise," "comprises" and "comprising" as used herein, specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0107] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term "consisting essentially of" when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising."

[0108] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, in immunology, immunohistochemistry, protein chemistry, molecular genetics, synthetic biology and biochemistry).

[0109] Throughout this specification, unless specifically stated otherwise, or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[0110] SELECTED DEFINITIONS

[0111] The term "accessory protein" as used herein means any protein with catalytic or DNA binding activity that is activated by a signaling molecule produced by a type III CRISPR-Cas ribonucleic acid complex in the presence of a target RIMA. Examples of accessory proteins according to any of the methods described herein include, but are not limited to, NucC nuclease, Csm6 nuclease, Csxl nuclease, Canl nuclease, Can2 nuclease, Cardl nuclease, CalpL protease, SAVED-CHAT protease, Cadi deaminase, Toll-like interleukin receptor (TIR) NADase and CorA, Caml, Cam2, Cam3, Csx23 membrane proteins. The term "cell" as used herein refers to a prokaryotic or eukaryotic cell and is not limited. A cell may be derived from any bacteria, archaea, plant, animal, or yeast. A cell may be derived from a vertebrate or non-vertebrate animal. A cell may be derived from a non-human or human animal. A cell may be mammalian or non-mammalian.

[0112] The term "adjacent" as used herein means next to a location, which may be directly next to, indirectly next to, or proximal to a location. When used with reference to a nucleic acid sequence, 'adjacent' may mean directly upstream or downstream of a location, with no nucleotide bases between the nucleic acid sequence and the location, or may mean proximal to a location with a few nucleotide bases between the nucleic acid sequence and the location, such as below 10 nucleotide bases for example.

[0113] The terms "base pairing affinity" and "complementarity" as used herein may be used interchangeably and refer to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non-traditional types. The terms "complementary" or "complementarity," as used herein, refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A." Complementarity between two single-stranded molecules may be "partial," in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0114] The term "complementary" as used herein to describe the complementarity between a guide RNA and a target RNA includes exact or partial complementarity between ribonucleic acid sequences. In certain examples according to all methods described herein, the complementarity between the guide RNA and the target RNA is exact (i.e.) 100%. In other examples according to all methods described herein, the complementarity between the guide RNA and target RNA is partial (i.e.) 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. A person skilled in the art will appreciate that any partial complementarity between the guide RNA and the target RNA may be continuous or non-continuous across the binding interaction.

[0115] The terms "CRISPR-Cas proteins" and "type III CRISPR-Cas proteins" as used herein are intended to mean the Cas proteins associated with CRISPR-Cas complexes, and in particular the Cas proteins associated with type III CRISPR-Cas complexes including, but not limited to, type III-A CRISPR-Cas complexes, type III-B CRISPR-Cas complexes, type III-C CRISPR-Cas complexes, type III-D CRISPR- Cas complexes and type III-E CRISPR-Cas complexes all of which are particularly well adapted for the cell killing applications in accordance with the methods described herein.

[0116] The CRISPR-Cas proteins typically associated with type III-A CRISPR-Cas complexes include CaslO, Casll (Csm2), Cas7 (Csm3, Csm5), Cas5 (Csm4) and Cas6. As such, the terms "one or more type III-A CRISPR-Cas proteins" and / or "at least one type III-A CRISPR-Cas proteins" as used herein includes one or more of CaslO, Casll (Csm2), Cas7 (Csm3, Csm5), Cas5 (Csm4) and Cas6.

[0117] The CRISPR-Cas proteins typically associated with type III-B CRISPR-Cas complexes include CaslO, Casll (Cmr5), Cas7 (Cmrl, Cmr4, Cmr6), Cas5 (Cmr3) and Cas6. As such, the terms "one or more type III-B CRISPR-Cas proteins" and / or "at least one type III-B CRISPR-Cas proteins" as used herein includes one or more of CaslO, Casll (Cmr5), Cas7 (Cmrl, Cmr4, Cmr6), Cas5 (Cmr3) and Cas6.

[0118] The CRISPR-Cas proteins typically associated with type III-C CRISPR-Cas complexes include CaslO, Casll (Cmr5), Cas7 (Cmrl, Cmr6, Cmr4), Cas5 (Cmr3) and Cas6. As such, the terms "one or more type III-C CRISPR-Cas proteins" and / or "at least one type III-C CRISPR-Cas proteins" as used herein includes one or more of CaslO, Casll (Cmr5), Cas7 (Cmrl, Cmr6, Cmr4), Cas5 (Cmr3) and Cas6.

[0119] The CRISPR-Cas proteins typically associated with type III-D CRISPR-Cas complexes include CaslO, Casll (Csm2), Cas7 (Csm5), Cas5 (Csxl9) and Cas6. As such, the terms "one or more type III-D CRISPR-Cas proteins" and / or "at least one type III-D CRISPR-Cas proteins" as used herein includes one or more of CaslO, Casll (Csm2), Cas7 (Csm5), Cas5 (Csxl9) and Cas6.

[0120] The CRISPR-Cas proteins typically associated with type III-Dv CRISPR-Cas complexes include (1) a Cas7-Cas5-Casll fusion subunit, (2) a Cas7-Cas7 fusion subunit, (3) a Cas7-insertion subunit, (4) a CaslO subunit, (5) a Csxl9 subunit, and, optionally, (6) a Cas6 subunit. As such, the terms "one or more type III-Dv CRISPR-Cas proteins" and / or "at least one type III-Dv CRISPR-Cas proteins" as used herein includes one or more of (1) a Cas7-Cas5-Casll fusion subunit, (2) a Cas7-Cas7 fusion subunit, (3) a Cas7-insertion subunit, (4) a CaslO subunit, (5) a Csxl9 subunit, and, optionally, (6) a Cas6 subunit.

[0121] The CRISPR-Cas protein associated with type III-E CRISPR-Cas complexes include Cas7-ll fusion (Csm3-Csm2-Csm5(3)). As such, the term "type III-E CRISPR-Cas protein" as used herein includes a Cas7-ll fusion protein (Csm3-Csm2-Csm5(3)).

[0122] The terms "CRISPR-Cas complex" and "type III CRISPR-Cas complex" as used herein is intended to mean an assembled complex between the guide RNA and (type III) CRISPR-Cas proteins when bound to the target RNA.

[0123] The terms "CRISPR-Cas system" and "type III CRISPR-Cas system" as used herein is intended to mean all components required for CRISPR-Cas function, including the guide RNA, type III CRISPR-Cas proteins and accessory proteins possessing activity sufficient to cause cell death or cell growth arrest in accordance with the methods described herein.

[0124] The term "effector complex" as used herein means any type III CRISPR-Cas complex inclusive of a CaslO domain which, when activated, produces at least one signaling molecule (e.g. cyclic oligoadenylates, S-adenosyl methionine-AMP etc) sufficient to activate accessory proteins defined herein.

[0125] The terms "percent sequence identity" or "percent identity" as used herein refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) as compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some examples, "percent identity" can refer to the percentage of identical amino acids in an amino acid sequence. As used herein "sequence identity" refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. "Identity" can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). As used herein, the phrase "substantially identical," or "substantial identity" in the context of two nucleic acid molecules, nucleotide sequences or protein sequences, refers to two or more sequences or subsequences that have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. In particular examples, substantial identity can refer to two or more sequences or subsequences that have at least about 80%, at least about 85%, at least about 90%, at least about 95, 96, 96, 97, 98, or 99% identity.

[0126] Throughout this specification in any context, optimal alignment may be determined using, for example, any of the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). For purposes of this invention "percent identity" may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0127] The term "perfectly complementary" as used herein means about 100% nucleotide or amino acid residues are complementary. Suitably that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.

[0128] The term "substantially complementary" as used herein means at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% nucleotide or amino acid residues are complementary, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection. Suitably at least a percentage proportion of the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. This may also correspond to nucleic acids that hybridize under stringent conditions.

[0129] The terms "hybridization", "hybridize", "hybridizing", and grammatical variations thereof as used herein, refer to the binding of two complementary nucleotide sequences or substantially complementary sequences in which some mismatched base pairs are present. The conditions for hybridization are well known in the art and vary based on the length of the nucleotide sequences and the degree of complementarity between the nucleotide sequences. In some examples, the conditions of hybridization can be high stringency, or they can be low stringency depending on the amount of complementarity and the length of the sequences to be hybridized.

[0130] The term "stringent conditions" for hybridization as used herein refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions surrounding the nucleic acids, temperature, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The Tm is the temperature at which more than 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

[0131] Very High Stringency (allows sequences that share at least 90% identity to hybridize) Hybridization: 5x SSC at 65 °C for 16 hours; wash twice: 2x SSC at room temperature (RT) for 15 minutes each; wash twice: 0.5x SSC at 65°C for 20 minutes each.

[0132] High Stringency (allows sequences that share at least 80%> identity to hybridize) Hybridization: 5x-6x SSC at 65°C-70°C for 16-20 hours; wash twice: 2x SSC at RT for 5-20 minutes each; wash twice: lx SSC at 55°C-70°C for 30 minutes each.

[0133] Low Stringency (allows sequences that share at least 50%> identity to hybridize); hybridization: 6x SSC at RT to 55°C for 16-20 hours; wash at least twice: 2x-3x SSC at RT to 55 °C for 20-30 minutes each.

[0134] Methods performed according to the present invention may be employed occurring ex vivo, for example in a cell or cell culture. In ex vivo treatments, diseased cells may be removed from the body, treated with the products / methods of the invention, and then transplanted back into the patient. Ex vivo modification has an advantage of allowing the target cell population to be well defined and the specific dosage of therapeutic molecules delivered to cells to be specified.

[0135] In vivo examples are also provided. In vivo modification can be used advantageously from this disclosure and the knowledge in the art.

[0136] A "fragment" or "portion" of a nucleic acid will be understood to mean a nucleotide sequence of reduced length relative (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides) to a reference nucleic acid or nucleotide sequence and comprising a nucleotide sequence of contiguous nucleotides that are identical or almost identical (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the reference nucleic acid or nucleotide sequence. Such a nucleic acid fragment or portion according to the invention may be, where appropriate, included in a larger polynucleotide of which it is a constituent. In some examples, a fragment of a polynucleotide can be a fragment that encodes a polypeptide that retains its function which may be termed a 'functional fragment'.

[0137] A "native" or "wild type" or unmodified nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence. Thus, for example, a "wild type mRNA" is a mRNA that is naturally occurring in or endogenous to the organism. A "homologous" nucleic acid is a nucleic acid naturally associated with a host cell into which it is introduced.

[0138] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleic acid construct," "nucleotide sequence" and "polynucleotide" refer to single-stranded or double-stranded nucleic acids, such as RNA or DNA that is linear or branched, single or double-stranded, or a hybrid thereof. The term also encompasses RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases, such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides that contain C-5 propyne analogues of uridine and cytidine have been shown to bind RNA with high affinity and to be potent antisense inhibitors of gene expression. Other modifications, such as modification to the phosphodiester backbone, or the 2'-hydroxy in the ribose sugar group of the RNA can also be made. The nucleic acid constructs of the present disclosure can be DNA or RNA, but are preferably DNA. Thus, although the nucleic acid constructs of this invention may be described and used in the form of DNA, depending on the intended use, they may also be described and used in the form of RNA.

[0139] As used herein, the term "nucleotide sequence" refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule and includes DNA or RNA molecules, including cDNA, a DNA fragment or portion, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA, and anti-sense RNA, any of which can be single-stranded or double-stranded. The terms "nucleotide sequence" "nucleic acid," "nucleic acid molecule," "nucleic acid construct," "oligonucleotide," and "polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides. Except as otherwise indicated, nucleic acid molecules and / or nucleotide sequences provided herein are presented herein in the 5' to 3' direction, from left to right and are represented using the standard code for representing the nucleotide characters as set forth in the U.S. sequence rules, 37 CFR §§1.821 - 1.825 and the World Intellectual Property Organization (WIPO) Standard ST.25. A "5' region" as used herein can mean the region of a polynucleotide that is nearest the 5' end. Thus, for example, an element in the 5' region of a polynucleotide can be located anywhere from the first nucleotide located at the 5' end of the polynucleotide to the nucleotide located halfway through the polynucleotide. A "3' region" as used herein can mean the region of a polynucleotide that is nearest the 3' end. Thus, for example, an element in the 3' region of a polynucleotide can be located anywhere from the first nucleotide located at the 3' end of the polynucleotide to the nucleotide located halfway through the polynucleotide. An element that is described as being "at the 5'end" or "at the 3'end" of a polynucleotide (5' to 3') refers to an element located immediately adjacent to (upstream of) the first nucleotide at the 5' end of the polynucleotide, or immediately adjacent to (downstream of) the last nucleotide located at the 3' end of the polynucleotide, respectively.

[0140] The term "identity" and "identical" and grammatical variations thereof, as used herein, mean that two or more referenced entities are the same (e.g., nucleic acid or amino acid sequences). Thus, where two sequences are identical, they have the same nucleic acid sequence or the same amino acid sequence. The identity can be over a defined area, e.g. over at least 22, 23, 24, 25 or 26 contiguous nucleic acids of the parent nucleic acid sequence, or over at least 22, 23, 24, 25 or 26 contiguous amino acid residues of a parent peptide sequence, or whichever alignment is the best fit with gaps permitted.

[0141] Identity can be determined by comparing each position in aligned sequences. A degree of identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleic acids or amino acids at positions shared by the sequences, i.e. over a specified region. Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, as are known in the art, including the Clustal Omega program available at the website location at www.ebi.ac.uk / Tools / mas / clustalo / , the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wis., U.S.A.). Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings). Software for performing BLAST analysis may be available through the National Center for Biotechnology Information (through the internet at the website located at www.ncbi.nlm.nih.gov). Such algorithms that calculate percent sequence identity (homology) generally account for sequence gaps and mismatches over the comparison region or area. For example, a BLAST (e.g., BLAST 2.0) search algorithm (see, e.g., Altschul et al., J. Mol. Biol. 215:403 (1990), publicly available through NCBI) has exemplary search parameters as follows: Mismatch -2; gap open 5; gap extension 2. For polypeptide sequence comparisons, a BLASTP algorithm is typically used in combination with a scoring matrix, such as PAM 100, PAM 250, BLOSUM 62 or BLOSUM 50.

[0142] FASTA (e.g., FASTA2 and FASTA3) and SSEARCH sequence comparison programs are also used to quantitate the extent of identity (Pearson et al., Proc. Natl. Acad. Sci. USA 85:2444 (1988); Pearson, Methods Mol Biol. 132: 185 (2000); and Smith et al., J. Mol. Biol. 147: 195 (1981). Programs for quantitating protein structural similarity using Delaunay-based topological mapping have also been developed (Bostick et al., Biochem Biophys Res Commun. 304:320 (2003)).

[0143] DETAILED DESCRIPTION

[0144] The present invention is directed to the use of type III CRISPR-Cas systems, as well as engineered variants thereof, to recognize specific RNA molecules and trigger cell killing via activation of accessory proteins (e.g.) accessory proteins which will collaterally cleave essential cellular molecules such as (e.g.) deoxyribose nucleic acids, ribose nucleic acids, cellular cofactors and peptides / proteins.

[0145] The Applicants provide the first direct evidence to demonstrate that bacteria-derived type III CRISPR-Cas complexes in combination with accessory proteins may be successfully delivered / expressed in vivo in a mammalian cell line which until now has remained undocumented. Further, the present invention is predicated, in part, on the surprising and unexpected discovery that signaling molecules (e.g. cyclic oligoadenylates) produced by CRISPR-Cas complexes in the presence of a target RNA retain signaling function in vivo in a mammalian cell line. Accordingly, these signaling molecules may be used to successfully activate accessory proteins thereby resulting in cell death or arrested growth.

[0146] Initial validation of the cell killing concept according to the present invention was performed using a type III-Dv CRISPR-Cas derived system from Synechocystis (refer to WO 2023 / 244127, incorporated herein by reference). When introduced / expressed in human mammalian cells (e.g.) HEK293 and HeLa cell lines in the presence of a guide RNA designed to target endogenous BRCA1, CKB and XIST RNAs, the type III-Dv CRISPR-Cas complex binds to the target RNA , and when specifically coupled with different accessory proteins (e.g. NucC, Csxl and TIR-SAVED) resulted in cell death or arrested cell growth. Refer to the results presented in Examples 1-3 and 5 when read in conjunction with Figures 1- 6.

[0147] Specifically, these data demonstrate ~50% reduction in mRNA levels in human embryonic kidney (HEK293) cells expressing the BRCA1, CKB and XIST genes when transformed with a vector encoding a type III-Dv CRISPR-Cas system (Figure 1A). When the type III-Dv system was coupled with NucC, a DNA nuclease activated by cOA, and used to target HEK293 cells expressing BRCA1 and CKB gene transcripts, significant cell death compared to untargeted and negative control experiments was observed as measured via 4',6-diamidino-2-phenylindole (DAPI) nuclei staining and confocal microscopy (Figures 3C and 3D). Further analysis using cell viability and metabolic activity assays (e.g. flow cytometry, resazurin and trypan blue assays) also revealed a significant reduction in total cell count and activity (Figures 4A-E), further validating the targeted cell death concept described herein.

[0148] Similar results were also achieved when a mutant form of the type III-Dv CRISPR-Cas system, in which specific domains of Cas7 fusion protein(s) had been altered in order to diminish or abolish RNA nuclease activity, was expressed in HEK293 cells. Refer to Example 3 and Figure 7.

[0149] The Applicants extended their work to validate the cell killing concept using a type III-A CRISPR- Cas system derived from Streptococcus thermophilus

[0018] . Refer to Example 4 when read in conjunction with Figure 8 in which BRCA1 transcripts were targeted in HEK293 mammalian cells using a type III- A / NucC system. These data very clearly demonstrate that the cell killing effect is not confined to type III-Dv CRISPR / Cas systems, and that the skilled person would reasonably predict based on the data and disclosure provided herein that the cell killing effect extends to other type III CRISPR-Cas systems which are capable of producing a signaling molecule following formation of ribonucleoprotein complexes in the presence of a target RNA. Finally, and in reference to Example 5 and Figure 9, the Applicants demonstrate activation of other accessory proteins by type III CRISPR-Cas systems described herein. Specifically, a Csxl and TIR-SAVED accessory proteins were shown to be activated by a type III-Dv system in the presence of a target RNA resulting in targeted death of HEK293 cells.

[0150] Collectively, these data are significant and provide the first empirical evidence of the application of a type III CRISPR-Cas system to cause targeted cell death in a eukaryotic / mammalian cell. The clinical and non-clinical implications of this discovery are far-reaching, and Applicants envision multiple applications, such as recognition of RNAs whose expression or sequence is indicative of a pathological state (e.g. cancer, auto-immune disease etc) and the resultant killing of these cells. Importantly, cell killing will be specific for cells that contain a particular target sequence that must also be expressed as an RNA. Accordingly, an advantage of the methods according to the present invention is avoidance of unnecessary targeting of healthy / non-diseased cells, while killing those with a chosen pathological sequence / expression profile.

[0151] Accordingly, in an aspect of the present invention there is provided a method for targeted cell death or cell growth arrest in a cell expressing a target RNA, the method comprising:

[0152] (i) delivering to the cell:

[0153] (a) a guide ribonucleic acid (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;

[0154] (b) one or more type III CRISPR-Cas proteins, wherein at least one type III CRISPR-Cas protein is capable of producing a signaling molecule; and

[0155] (c) at least one accessory protein capable of causing cell death or cell growth arrest, which accessory protein is activated by the signaling molecule, wherein, the presence of the target RNA within the cell causes the gRNA and type III CRISPR-Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

[0156] For any avoidance of doubt, the term "...accessory protein is activated by the signaling molecule" is intended to mean activation of the accessory protein by the signaling molecule sufficient to induce cell death or cell growth arrest.

[0157] In an example according to this and other aspects of the present invention, the type III CRISPR- Cas protein capable of producing at least one signaling molecule is CaslO.

[0158] In another example according to this and other aspects of the present invention, the cell is a eukaryote or a prokaryote. In a related example, the cell is a human cell.

[0159] In an example according to this and other aspects of the present invention, the type III CRISPR- Cas proteins are derived from type III-A CRISPR-Cas systems, type III-B CRISPR-Cas systems, type III-C CRISPR-Cas systems, type III-D CRISPR-Cas proteins and type III-E CRISPR-Cas proteins.

[0160] In another example according to this and other aspects of the present invention, the type III CRISPR-Cas proteins are derived from type III-A CRISPR-Cas systems, type III-B CRISPR-Cas systems and type III-D CRISPR-Cas systems, which are co-delivered with type III-A CRISPR-Cas guide RNAs, type III-B CRISPR-Cas guide RNAs and type III-D CRISPR-Cas guide RNAs, respectively. In yet another example according to this and other aspects of the present invention, the type III CRISPR-Cas proteins are derived from type III-A CRISPR-Cas systems and type III-D CRISPR-Cas systems.

[0161] In yet another example according to this and other aspects of the present invention, the type III CRISPR-Cas proteins are derived from type III-D CRISPR-Cas systems.

[0162] In yet a further example according to this and other aspects of the present invention, the type III CRISPR-Cas proteins are derived from type III-Dv CRISPR-Cas proteins comprising: (1) a Cas7-Cas5- Casll fusion protein, (2) a Cas7-Cas7 fusion protein, (3) a Cas7-insertion protein, (4) a CaslO, (5) a Csxl9, and, optionally, (6) a Cas6.

[0163] In a related example, the Cas7-Cas5-Casll fusion protein comprises or consists in a sequence set forth in SEQ ID NO: 4, or a functional fragment thereof, or a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% to SEQ ID NO: 4.

[0164] In a related example, the Cas7-Cas7 fusion protein comprises or consists in a sequence set forth in SEQ ID NO: 6, or a functional fragment thereof, or a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% to SEQ ID NO: 6.

[0165] In a related example, the Cas7-insertion fusion protein comprises or consists in a sequence set forth in SEQ ID NO: 10, or a functional fragment thereof, or a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% to SEQ ID NO: 10.

[0166] In a related example, the CaslO comprises or consists in a sequence set forth in SEQ ID NO: 2, or a functional fragment thereof, or a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% to SEQ ID NO: 2.

[0167] In a related example, the Csxl9 comprises or consists in a sequence set forth in SEQ ID NO: 8, or a functional fragment thereof, or a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,

[0168] 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% to SEQ ID NO: 8.

[0169] In a related example, the Cas6 comprises or consists in a sequence set forth in SEQ ID NO: 12, or a functional fragment thereof, or a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,

[0170] 93%, 94%, 95%, 96%, 97%, 98%, 99%, and / or 100% to SEQ ID NO: 12.

[0171] The type III CRISPR-Cas proteins used in the methods according to the present invention may be modified from native form. By way of illustration only, at least one Cas7 domain of a type III CRISPR- Cas protein may be modified (mutated) such that the Cas7 RNase activity is reduced or eliminated compared to the equivalent Cas7 domain(s) which remain unmodified. Without wishing to be bound by theory, it is anticipated that reducing or eliminating Cas7 RNase activity will not limit the production of signaling molecules including (e.g.) cyclic oligoadenylates and S-andenosyl methionine-AMP. For the reason that the gRNA and RNA endogenous to the cell remain in-tact, thereby allowing prolonged activation of the type III CRISPR-Cas complex and consequently the prolonged activation of at least one accessory protein(s). In a related example, the Cas7-Cas7 fusion protein comprises a mutation at positions D246 and / or D33 of SEQ ID NO: 6, or positions corresponding thereto, which mutation(s) reduce or eliminate Cas7 ribonuclease activity. This modified Cas7-Cas7 fusion protein is set forth in SEQ ID NO: 14.

[0172] In another related example, the Cas7-Cas5-Casll fusion protein comprises a mutation at position D26 of SEQ ID NO: 4, or a position corresponding thereto, which mutation reduces or eliminates Cas7 ribonuclease activity. This modified Cas7-Cas5-Casll fusion protein is set forth in SEQ ID NO: 16.

[0173] Activation of the type III CRISPR-Cas complex occurs when the gRNA recognizes and binds to its complementary sequence within the target RNA transcript. Activation of the type III CRISPR-Cas complex triggers the production of signaling molecules (e.g. cyclic oligoadenylates or SAM-AMP) via the palm domain of CaslO. In certain examples, activation of the CaslO palm domain produces a cyclic oligoadenylate selected from one or more of cyclic oligoadenylate 2 (cA2), cyclic oligoadenylate 3 (cA3), cyclic oligoadenylate 4 (cA4), and cyclic oligoadenylate 5 (cA5) and cyclic oligoadenylate 6 (cA6). In a preferred example, activation of the CaslO palm domain produces a cyclic oligoadenylate selected from one or more of cyclic oligoadenylate 3 (cA3), cyclic oligoadenylate 4 (cA4) and cyclic oligoadenylate 6 (cA6). In another example, activation of the CaslO palm domain produces S-adenosyl methionine-AMP.

[0174] In yet a further related example, activation of the type III CRISPR-Cas complex in accordance with the methods described herein triggers production of:

[0175] • cA2;

[0176] . cA3;

[0177] • cA4;

[0178] • cA5;

[0179] . cA6;

[0180] • S-adenosyl methionine-AMP;

[0181] • cA2 and cA3;

[0182] • cA2 and cA4;

[0183] • cA2 and cA5;

[0184] • cA2 and cA6;

[0185] • cA2 and S-adenosyl methionine-AMP;

[0186] • cA3 and cA4;

[0187] • cA3 and cA5;

[0188] • cA3 and cA6;

[0189] • cA3 and S-adenosyl methionine-AMP;

[0190] • cA4 and cA5;

[0191] • cA4 and cA6;

[0192] • cA4 and S-adenosyl methionine-AMP;

[0193] • cA5 and cA6;

[0194] • cA5 and S-adenosyl methionine-AMP;

[0195] • cA2, cA3 and cA4;

[0196] • cA2, cA3 and cA5;

[0197] • cA2, cA3 and cA6;

[0198] • cA2, cA3 and S-adenosyl methionine-AMP;

[0199] • cA2, cA4 and cA5; • cA2, cA4 and cA6;

[0200] • cA2, cA4 and S-adenosyl methionine-AMP;

[0201] • cA2, cA5 and cA6;

[0202] • cA2, cA5 and S-adenosyl methionine-AMP;

[0203] • cA2, cA6 and S-adenosyl methionine-AMP;

[0204] • cA3, cA4 and cA5;

[0205] • cA3, cA4 and cA6;

[0206] • cA3, cA4 and S-adenosyl methionine-AMP;

[0207] • cA3, cA5 and cA6;

[0208] • cA3, cA5 and S-adenosyl methionine-AMP;

[0209] • cA3, cA6 and S-adenosyl methionine-AMP;

[0210] • cA4, cA5 and cA6;

[0211] • cA4, cA5 and S-adenosyl methionine-AMP;

[0212] • cA4, cA6 and S-adenosyl methionine-AMP;

[0213] • cA5, cA6 and S-adenosyl methionine-AMP;

[0214] • cA2, cA3, cA4 and cA5;

[0215] • cA2, cA3, cA4 and cA6;

[0216] • cA2, cA3, cA4 and S-adenosyl methionine-AMP;

[0217] • cA2, cA3, cA5 and cA6;

[0218] • cA2, cA3, cA5 and S-adenosyl methionine-AMP;

[0219] • cA2, cA3, cA6 and S-adenosyl methionine-AMP;

[0220] • cA2, cA4, cA5 and cA6;

[0221] • cA2, cA4, cA5 and S-adenosyl methionine-AMP;

[0222] • cA2, cA4, cA6 and S-adenosyl methionine-AMP;

[0223] • cA2, cA5, cA6 and S-adenosyl methionine-AMP;

[0224] • cA3, cA4, cA5 and cA6;

[0225] • cA3, cA4, cA5 and S-adenosyl methionine-AMP;

[0226] • cA3, cA4, cA6 and S-adenosyl methionine-AMP;

[0227] • cA3, cA5, cA6 and S-adenosyl methionine-AMP;

[0228] • cA4, cA5, cA6 and S-adenosyl methionine-AMP;

[0229] • cA2, cA3, cA4, cA5 and cA6;

[0230] • cA2, cA3, cA4, cA5 and S-adenosyl methionine-AMP;

[0231] • cA2, cA3, cA4, cA6 and S-adenosyl methionine-AMP;

[0232] • cA2, cA3, cA5, cA6 and S-adenosyl methionine-AMP;

[0233] • cA2, cA4, cA5, cA6 and S-adenosyl methionine-AMP;

[0234] • cA3, cA4, cA5, cA6 and S-adenosyl methionine-AMP; and

[0235] • cA2, cA3, cA4, cA5, cA6 and S-adenosyl methionine-AMP.

[0236] The production of one or more cyclic oligoadenylates and / or S-adenosyl methionine-AMP, in turn, activates the accessory proteins defined herein. The mechanism of activation will vary depending on the accessory protein and includes, but is not limited to, activation of a catalytic function selected from ribonuclease activity, nuclease activity, protease activity, adenosine deaminase activity and NADase activity. The accessory proteins are either delivered to the cell or expressed by the cell following delivery of a nucleic acid sequence encoding the same. In certain examples, the accessory protein possesses DNA endonuclease activity. In other examples, the accessory protein possesses RNA endonuclease activity. In yet other examples, the accessory protein possesses protease activity. In yet other examples, the accessory protein possesses adenosine deaminase activity. In yet other examples, the accessory protein possesses nicotinamide adenine dinucleotide (NAD+) nucleosidase (NADase) activity.

[0237] In another example, activation of the accessory protein comprises activation of its nucleic acid binding capacity such that it binds to and prevents further translation of RNA transcripts.

[0238] Examples of accessory proteins possessing DNA endonuclease activity, which DNA endonuclease activity is activated by cOA, include, but are not limited to, NucC, Canl, Can2 and Cardl. In a related example, the NucC is derived from Serratia sp. ATCC 39006. In another related example, the NucC comprises or consists in the sequence set forth in SEQ ID NO: 29.

[0239] Examples of accessory proteins possessing RNA endonuclease activity, which RNA endonuclease activity is activated by cOA, include, but are not limited to, Csm6, Csxl, Can2, Cardl.

[0240] Examples of accessory proteins possessing protease activity, which protease activity is activated by cOA, include, but are not limited to, CalpL and SAVED-CHAT.

[0241] Examples of accessory protein possessing NADase activity include, but are not limited to, Toll / interleukin-1 receptor (TIR) NADases.

[0242] An example of an adenosine deaminase includes, but is not limited to, Cadi adenosine deaminase.

[0243] In other examples, the accessory protein according to the present invention may be a membrane protein which include, but are not limited to, Caml, Cam2, Cam3 and Csx23.

[0244] The skilled person would recognize that any accessory protein which is activated by (e.g.) a cyclic oligoadenylate or a S-adenosyl methionine-AMP could be employed in the cell killing methods described by the present appilication.

[0245] The target RNA expressed by the cell may be any target RNA of interest. In certain examples, the target RNA is known to be associated with a disease state or condition and an illustrative example includes cancer genes such as AKT3, KLLN, BCL2L11, SH2B3, MEF2B, EIF1AX-AS1, RAD50, CDK4, CDK6, OLIG2, CDK8, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, IKZF1, MAD2L2, HOXB13, SEC23B, CARMI, CEBPA, SEMA3C, CTCF, GNA13, POLQ, STAG2, MTHFD2, CFTR, HSPH1, FRS2, MALT1, CHD2, CHD4, CHEK1, PTPRT, CHEK2, RNF139, CTRC, CKS1B, C8orf34, DIS3L2, CREBBP, AMER1, CRKL, CSF1R, CSF3R, SLC47A2, CTLA4, CTNNA1, CTNNB1, CUX1, CYLD, CYP1B1, CYP2D6, CYP3A5, Cllorf65, DAXX, SPRED1, DDB2, DDX3X, ASXL1, NQO1, DKC1, DNM2, DNMT3A, DPYD, SLC26A3, APLNR, EBF1, EGF, EGFR, ARID2, EPHA2, ELF3, FLCN, TIGIT, ENG, EP300, EPHA7, EPHB1, EPHB2, EPOR, ERBB2, ERBB3, ERBB4, ERCC1, ERCC2, AKT1, ERCC3, ERCC4, ERCC5, ERCC6, ERG, AKT2, ESRI, ETS1, ETS2, ETV1, ETV4, ETV5, ETV6, EWSR1, EZH2, FANCA, FANCC, FANCD2, FANCE, FANCB, FANCF, FANCG, FAT1, FCGR2A, FCGR3A, FDPS, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF14, FGFR1, FGFR3, FGFR2, FGFR4, FH, FHIT, TFEC, DIS3, NT5C2, SPEN, PALLD, PHLPP2, FOXO1, FOXO3, ARHGAP26, KIF1B, CIC, FLT1, FLT3, TBC1D12, PHLPP1, FLT4, SYNE1, DICER1, FNTB, SF3B1, BRD4, SUZ12, CBLC, PPP1R15A, ALK, MTOR, ABL1, FUS, RICTOR, ZNF620, G6PD, GABRA6, POTI, SETBP1, PTPN22, PHGDH, GATA1, GATA2, GATA3, GATA4, GATA6, AGO1, GREM1, ABL2, MLH3, FOXP1, GPC3, GLI1, GLI2, GNA11, GNAQ, GNAS, IFNL3, H19, C3orf70, P2RY8, GPS2, GRIN2A, SETD2, UBE2T, ARHGAP35, CD274, GRM3, GSTP1, MSH6, Hl-4, H3-3A, HAS3, HDAC1, HDAC2, HGF, NRG1, HIF1A, HLA-A, HLA-B, HLA-C, HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQA2, HLA-DQB1, HLA-DQB2, HLA-DRA, HLA-DRB1, HLA-DRB5, HLA-E, HLA-F, HLA-G, F0XA1, HOXA11, APC, HRAS, HSD3B1, HSD3B2, HSD11B2, BIRC3, HSP90AA1, APOB, STING1, IDH1, IDH2, IFIT2, IFIT1, IFIT3, IFNAR1, IFNAR2, IFNGR1, ECT2L, IFNGR2, GEN1, FAS, IL2RA, IL6R, IL7R, IL10RA, IL15, TNFRSF9, IDO1, ING1, IRF1, IRF2, IRF4, AR, ARAF, ITPKB, JAK1, JAK2, JAK3, JUN, KDR, KEL, KIT, KRAS, RHOA, LAG3, LCK, LDLR, LMNA, LMO1, LYN, EPCAM, SMAD2, SMAD3, SMAD4, MAF, MAX, MC1R, MCL1, MDM2, MDM4, MAP3K1, MEN1, MET, MGMT, CIITA, MITF, MKI67, MLH1, KMT2A, MLLT3, MN1, MPL, MRE11, ASNS, MSH2, MSH3, MTAP, MTHFR, MTRR, MUTYH, MYB, MYC, MYCL, MYCN, MYD88, MYH11, ZFHX3, NBN, ATIC, ATM, NF1, NF2, NFE2L2, NFKBIA, NOTCH1, NOTCH2, NOTCH3, NOTCH4, NPM1, NRAS, NTHL1, NTRK1, NTRK2, NTRK3, DDR2, NUP98, PAK1, PRKN, PAX3, PAX5, PAX7, PCBP1, LEF1, PDCD1, PDGFRA, PIAS4, PDGFRB, PDK1, SUFU, CDK12, RSF1, ABCB1, PIK3C2B, PIK3CA, PIK3CB, PIM1, PIK3CD, PIK3CG, PIK3R1, PIK3R2, PLCG1, LRP1B, PML, PMS1, PMS2, ATP7B, ERRFI1, POLDI, POLE, POLH, ATR, POU2F2, EGLN1, ATRX, UGT1A9, PPARA, UGT1A1, PPARD, PPARG, TET2, TENT5C, BOOR, RNF43, SDHAF2, FANCL, PPP2R1A, PBRM1, PPP2R2A, FANCI, NUDT15, FBXW7, PPP6C, PRCC, NOPIO, CHD7, NHP2, TMEM127, PRKAR1A, AXL, PRKDC, MAPK1, MAP2K1, MAP2K2, PRSS1, B2M, EMSY, CYSLTR2, PTCHI, PTEN, ARID1B, RPTOR, MIB1, ZNF471, FANCM, PTPN11, PTPN13, PTPRD, BARD1, KMT2C, RAC1, RAD21, RAD51, RAD51C, RAD51B, RAD51D, RAFI, ACTA2, RANBP2, RARA, RASA1, RBI, KDM5A, CCND1, BCL2, RET, BCL2L1, RHEB, RIT1, BCL6, BCL7A, RINT1, TNFRSF17, ROS1, RPL5, BCR, RPS6KB1, RPS15, RRM1, RXRA, BCORL1, SDHA, PRDM1, SDHB, SDHC, SDHD, BLM, CRLF2, VSIR, MAP2K4, SRSF2, NSD1, SGK1, SCG5, SHH, BCL11B, WNK1, WNK2, BMPR1A, SMARCA1, SMARCA4, SMARCB1, SMARCE1, SMO, SOD2, SOX2, SOX9, SOXIO, FOXL2, SPINK1, SRC, BRCA1, BRAF, BRCA2, STAT3, STAT4, STAT5A, STAT5B, STAT6, AURKA, STK11, SYK, TAF1, MAP3K7, TAPI, TAP2, TARBP2, ELOC, TBX3, HNF1A, HNF1B, TCF3, TCF7L2, BTG1, BTK, BUB1B, TERT, TFE3, TGFBR1, TGFBR2, NKX2- 1, TMPRSS2, TNF, TNFAIP3, TOPI, TOP2A, TP53, TPM1, TPMT, TRAF3, TSC1, TSC2, TSHR, TYMS, U2AF1, UMPS, KDM6A, VEGFA, VEGFB, VHL, WEE1, NSD2, WRN, WT1, XPA, XPC, XPO1, XRCC1, XRCC2, XRCC3, ZNF217, PAX8, CXCR4, ASPSCR1, TFEB, CDC73, DYNC2H1, GALNT12, TBL1XR1, PALB2, ZNF750, TUSC3, KAT6A, L2HGDH, CTC1, FBXO11, PREX2, CCDC6, PDCD1LG2, FGF23, KMT2D, YEATS4, CALR, TCL1A, LZTR1, ZRSR2, RBM10, KDM5C, SMC1A, KDM5D, ARID1A, AXIN1, AXIN2, BAP1, H3C2, H4C5, BRIP1, SPOP, CASP8, ZNRF3, ABRAXAS1, ARID5B, TRAF7, PHF6, RAD54L, CARD11, DOT1L, SLX4, CUL4B, CUL4A, CUL3, CUL1, CASR, HAVCR2, SLC49A4, PPM1D, AJUBA, TANCI, RUNX1, RUNX1T1, TP63, PTCH2, CBFB, SOCS1, IRS2, CBLB, ABCC3, CBR3, TNFRSF14, INPP4B, FUBP1, BCL10, PHOX2B, CCND2, CCND3, CCNE1, KLHL6, ACVR1, ACVR1B, LATS1, SMC3, ZMYM3, AURKB, CD19, MS4A1, KLF4, CD22, SLIT2, NHERF1, RECQL4, FOXQ1, QKI, CD40, NCOR1, NCOR2, IKBKE, CD70, CEP57, CD79A, CD79B, KMT2B, HDAC4, BCLAF1, KEAP1, MAGI2, MAFB, MED12, CDH1 (source: https: / / www.ncbi.nlm.nih.gov / gtr / tests / 558436 / ).

[0246] If the target RNA is, for example, a gene and the nucleotide and / or protein sequence of that gene is known then the skilled person could design, as a matter of routine, any gRNA suitable to recognize and bind to that gene, thereby initiating targeted cell death or growth arrest through co-delivery of the type III CRISPR-Cas protein(s), guide RNA(s) and accessory protein(s) taught by the disclosure in this specification.

[0247] In certain examples, the methods according to the present invention are used to target cancerous cells in vivo by targeting aberrant gene expression (e.g. targeting oncogenes, including aberrant gene transcripts of translocated gene fusions such as NPM-ALK). In other examples, the methods according to the present invention could be used ex vivo to destroy specific cell populations (e.g. CAR-T cells in clinical cell engineering applications) or as a built-in kill switch for CAR-T cell therapies during cytokine storm.

[0248] As such, according to all methods described herein the cell may be any cell including a prokaryotic cell or a eukaryotic cell. In an example according to all methods described herein, the cell is a eukaryotic cell. In another example according to all methods described herein, the eukaryotic cell is derived from an animal, plant or fungus. In another example according to all methods described herein, the cell is mammalian cell derived from a human, a canine, an equine, a porcine, a rodent, a non-human primate, a bovine, an ovine, a piscine or other possible animals. In yet another example according to all methods described herein, the cell is a human cell.

[0249] In further examples according to all methods described herein, the cell is selected from a cancer cell, an immune cell, a stem cell and any cell expressing a specific RNA that is desirable to kill.

[0250] According to all methods described herein, the guide RNA(s), type III CRISPR-Cas protein(s) and accessory protein(s) may be delivered to a cell using any delivery technique known to a person skilled in the art. These include, without limitation, a delivery technique selected from electroporation, lipofection, microinjection, nanoparticle delivery, exosomes, extracellular vesicle and viral delivery.

[0251] According to the data presented in Example 2, read in conjunction with Figure 5, Applicants demonstrate that expression of nucleic acids encoding (e.g.) gRNA, type III CRISPR-Cas proteins and / or accessory proteins may be placed under the control of one or more inducible promoter sequences. Specifically, expression of the type III-Dv plasmids in transfected HEK293T-rtTA cells was shown to be inducible through the addition of tetracycline to the culture media, resulting in a reduction in fluorescence of a reporter construct. Accordingly, Applicants have also demonstrated that a type III- Dv complex for targeting RNA in the presence of an accessory protein (e.g. NucC) in a human cell line resulting in targeted cell death may be accurately controlled with inducible promoters.

[0252] Accordingly, in another example of the methods described herein, the (i) one or more nucleic acids encoding guide RNAs or crRNA(s), or (II) one or more nucleic acids encoding one or more type III CRISPR-Cas proteins or (ill) one or more nucleic acids encoding at least one accessory protein or (iv) one or more nucleic acids encoding a target RNA, or (v) any combination of (i), (ii), (iii) and (iv), may be operably linked to an inducible promoter to control RNA targeting resulting in controllable targeted cell death. In certain examples, the promoter is a tissue specific promoter (i.e.) cell killing is only induced in a specific cell type. In another example, the promoter is responsive to cellular signals indicative of a state in which cell killing would be desirable (e.g. sensing the activation of a cytokine storm in CAR-T cell therapy). In other examples, the inducible promoter is induced through administration of a drug, light, small molecule or any other inducible expression system.

[0253] As outlined in further detail below, the type III CRISPR-Cas proteins according to the methods described herein may be delivered to the cell as discrete Cas proteins which assemble once inside the cell, or as an intact complex. Alternatively, one or more nucleic acids encoding the type III CRISPR- Cas complex or type III CRISPR-Cas system are delivered to the cell. In certain examples, the one or more nucleic acids encoding the CRISPR-Cas complex or the CRISPR-Cas system may be DNA or RNA. In other examples, the one or more nucleic acids form part of an expression plasmid or delivery vector, and the one or more nucleic acids may be on the same plasmid or delivery vector, or on different plasmids or delivery vectors. The at least one accessory protein possessing catalytic activity (e.g. ribonuclease activity, deoxyribonuclease activity, protease activity, adenosine deaminase activity and NADase activity) or nucleic acid (e.g. DNA) binding activity according to the methods described herein may be delivered to the cell as an intact protein. Importantly, in the absence of signaling molecule (e.g. cOA or S-andenosyl methionine-AMP), the catalytic activity of the accessory protein remains non-activated or dormant. Alternatively, one or more nucleic acids encoding the accessory is delivered to the cell. In certain examples, the one or more nucleic acids encoding the accessory protein may be DNA or RNA.

[0254] In some examples the cell is located in vivo (e.g. in a mammalian organism), ex vivo (e.g. in a sample obtained from an organism) or in vitro (e.g. in a cell culture). Accordingly, the methods of the present invention may be performed on any cell type irrespective of its location, provided effective delivery of the essential components recited in the inventive methods described herein is achieved.

[0255] Type III CRISPR-Cas systems

[0256] In certain examples according to the present invention the type III CRISPR-Cas system is comprised of Cas proteins, a CRISPR array or guide RNA expression sequence which contains genetic information for the guide RNAs and at least one accessory protein.

[0257] The Cas proteins employed in the methods according to the present invention may be derived from any bacterial or archaeal species. Examples of suitable species include: Microcystis aeruginosa, Acetohalobium arabaticum, Ammonifex degensii, Anabaena cylindrica, Anabaena variabilis, Caldicellulosiruptor lactoaceticus, Caldilinea aerophila, Clostridium algicarnis, Crinalium epipsammum, Cyanothece sp., Cylindrospermum stagnale, Haloquadratum walsbyi, Halorubrum lacusprofundi, Methanocaldococcus vulcanius, Methanospirillum hungatei, Natrialba asiatica, Natronomonas pharaonis, Nostoc punctiforme, Phormidesmis priestleyi, Crematoria acuminata, Picrophilus torridus, Spirochaeta thermophila, Stanieria cyanosphaera, Sulfolobus acidocaldarius, Sulfolobus islandicus, Synechocystis sp., Thermacetogenium phaeum, Thermofilum pendens, etc.

[0258] In an example, the Cas proteins employed in the methods according to the present invention are derived from a cyanobacterium. In a related example, the Cas proteins used in the present invention are derived from Synechocystis sp, including Synechocystis sp. PCC 6803.

[0259] In another example, type III-A CRISPR-Cas systems are employed in any of the methods according to the present invention.

[0260] In another example, type III-B CRISPR-Cas systems are employed in any of the methods according to the present invention.

[0261] In another example, type III-C CRISPR-Cas systems are employed in any of the methods according to the present invention.

[0262] In another example, type III-D CRISPR-Cas systems, including type III-Dv CRISPR-Cas systems, are employed in any of the methods according to the present invention.

[0263] In another example, type III-E CRISPR-Cas systems are employed in any of the methods according to the present invention.

[0264] Type III-D CRISPR-Cas system In certain examples of the present invention the type III-D CRISPR-Cas system is comprised of type III-D Cas proteins, a CRISPR array or guide RNA expression sequence which contains genetic information for the guide RNAs and at least one accessory protein.

[0265] In certain examples of the present invention the type III-D CRISPR-Cas system is a variant type III-D CRISPR-Cas system or type III-Dv CRISPR-Cas system. It should, however, be appreciated that any reference herein to 'the system' in this context may refer to either of the type III-D CRISPR-Cas system or the type III-Dv CRISPR-Cas system.

[0266] The Cas proteins employed in the methods according to the present invention may be derived from any bacterial or archaeal species. Examples of suitable species include: Microcystis aeruginosa, Acetohalobium arabaticum, Ammonifex degensii, Anabaena cylindrica, Anabaena variabilis, Caldicellulosiruptor lactoaceticus, Caldilinea aerophila, Clostridium algicarnis, Crinalium epipsammum, Cyanothece sp., Cylindrospermum stagnale, Haloquadratum walsbyi, Halorubrum lacusprofundi, Methanocaldococcus vulcanius, Methanospirillum hungatei, Natrialba asiatica, Natronomonas pharaonis, Nostoc punctiforme, Phormidesmis priestleyi, Crematoria acuminata, Picrophilus torridus, Spirochaeta thermophila, Stanieria cyanosphaera, Sulfolobus acidocaldarius, Sulfolobus islandicus, Synechocystis sp., Thermacetogenium phaeum, Thermofilum pendens, etc.

[0267] In an example, the Cas proteins employed in the methods according to the present invention are derived from a cyanobacterium. In a related example, the Cas proteins used in the present invention are derived from Synechocystis sp, including Synechocystis sp. PCC 6803.

[0268] The type III-D or type III-Dv CRISPR-Cas system may be used in any of the methods herein.

[0269] Guide RNA

[0270] A 'guide RNA' in the present context refers to an RNA molecule that can bind to (form a complex with) the type III CRISPR-Cas proteins and direct it to a target (typically single stranded) nucleic acid. Typically, it forms a complex with the relevant target recognition Cas proteins of the type III-D CRISPR- Cas system.

[0271] The methods according to the present invention may comprise one or more than one guide RNA. Each guide RNA may target a different nucleic acid sequence (e.g. a complementary region on a target RNA).

[0272] Methods of producing guide RNAs are also well known in the art, including direct expression of mature crRNAs or through expression and processing of an immature or pre-crRNA form that is then processed to form mature gRNA. Any suitable approach can be used to produce a suitable guide RNA for the various aspects and examples described herein.

[0273] The guide RNA comprises a recognition sequence which is complementary to the target RNA. This may also be known as a spacer or protospacer sequence. In certain examples, the recognition sequence may be from about 20 nucleotides to about 70 nucleotides in length, (e.g.) about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 nucleotides in length). In other examples, the recognition sequence is about 20-40 nucleotides in length. A person skilled in the art would recognize that longer complementary sequences provide higher sequence specificity to the guide RNA and a higher stability. The complementarity between the recognition sequence and that target RNA is sufficient for the recognition sequence of the guide RNA to hybridise to the target RNA, direct sequence-specific binding of the CRISPR-Cas type III-D complex to the target nucleic acid and activate production of a signaling molecule (e.g. cOA and / or S-adenosyl methionine-AMP) from the palm domain of CaslO.

[0274] In certain examples, the recognition sequence (spacer) may be fully complementary to a target nucleic acid (e.g., 100% complementary to a target sequence across its full length). In some examples, the recognition sequence may be substantially complementary (e.g., at least about 80% complementary (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, or more complementary)) to a target nucleic acid. Thus, in some examples, a recognition sequence may have one, two, three, four, five or more mismatches that may be contiguous or non-contiguous as compared to a target nucleic acid.

[0275] In some examples the complementarity between the recognition sequence and the target nucleic acid is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% or 100%.

[0276] When the type III CRISPR-Cas system is a type III-D CRISPR-Cas system the guide RNA can be a mature crRNA. In some examples the mature crRNA is approximately 37 nucleotides in length. However, other lengths can also be functional, for example from 30-50 nucleotides in length, from 32- 45 nucleotides in length, for example from 35-40 nucleotides in length. However, it will be appreciated that any length of crRNA that is capable of complexing with the type III-D CRISPR-Cas proteins, guiding it to a target RNA and activating the cOA / S-adenosyl methionine-AMP production activity of the CaslO palm domain can be used.

[0277] Modifications in the 5' repeat region of the guide RNA can be tolerated to some extent. Thus, by way of example, the guide RNA may have 1, 2, 3, 4, 5 or 6 or more changes in the 5' repeat region, provided the guide RNA retains the ability to bind to the type III-D CRISPR-Cas proteins, guide it to a target RNA and activating the cOA / S-adenosyl methionine-AMP production activity of the CaslO palm domain.

[0278] It is important to note that for type III-D CRISPR-Cas systems there is generally no requirement for a protospacer adjacent motif (PAM) or protospacer flanking sequence (PFS) for target nucleic acid binding. Advantageously, this provides greater flexibility in target sequence choice than many other CRISPR-Cas systems.

[0279] For the purpose of the illustrations which follow, namely in the sections entitled "Methods of Modification", "Contacting", "Nucleic Acids", "Vectors" and "Cells" the term "the system" is taken to mean the essential structural features required to perform the inventive methods described herein and includes, without limitation, the guide RNA, the type III CRISPR-Cas proteins and at the least one accessory protein as defined herein, or nucleic acids encoding one or more of the gRNA, type III CRISPR- Cas proteins and the at least one accessory protein.

[0280] Methods of Modification

[0281] The methods according to the present invention relate to the modification of a target single stranded nucleic acid using a type III, suitably a type III-Dv, CRISPR-Cas system. Upon contacting the target single stranded nucleic acid with the type III CRISPR-Cas system, the system is cultured or incubated for a time and under conditions suitable for modification of the target nucleic acid to occur.

[0282] In an example according to all methods described herein if contacting occurs in a cell free environment, then the system and the target single stranded nucleic acid are cultured or incubated together under suitable cell free conditions for modification to occur at the target sequence.

[0283] Suitable cell free culture techniques are well known to the skilled person.

[0284] In another example if contacting occurs within a cell then after introduction of the system and optionally the target single stranded nucleic acid into the cell, the cell is cultured for a time and under conditions suitable for modification to occur at the target sequence. Suitably the target single stranded nucleic acid may already exist in the cell, and may be endogenous to the cell.

[0285] The culture conditions may be determined by the skilled person according to the type of cell and species of cell which harbours the complex. Suitable cell culture techniques are known to the skilled person as noted above.

[0286] Accordingly, the methods according to the present invention may comprise a step of culturing the system and the target nucleic acid for a time and under conditions suitable to allow modification to occur.

[0287] In an example, the modification is cleavage, and in preferred examples cleavage of the target nucleic acid. In a related example, the cleavage is single-stranded cleavage of a single-stranded nucleic acid sequence. Preferably therefore, the method is a method of cleavage. Suitably in methods directed towards modification of a single stranded nucleic acid sequence, single strand cleavage takes place. In certain examples, the cleavage is carried out by one or more accessory proteins which are activated by the type III CRISPR-Cas complex in the presence of a target RNA.

[0288] Target single stranded nucleic acid

[0289] Essentially any single stranded nucleic acid can be targeted by the type III CRISPR-Cas systems, including type III- D CRISPR-Cas systems, or modified forms thereof. Suitably the target single stranded nucleic acid is RNA and / or ssDNA. RNA is a particularly preferred target single stranded nucleic acid, particularly when the system is a type III-Dv CRISPR Cas system.

[0290] A target RNA may include mRNA, and non-coding RNAs such as tRNA, rRNA, sRNA, siRNA, iRNA, miRNA, IncRNA, genomic RNA (e.g. RNA viral genome), and synthetic RNA. In some preferred examples the target RNA is mRNA. In some examples the target RNA is in vivo, ex vivo or in vitro.

[0291] The target single stranded nucleic acid can have essentially any sequence. As will be apparent from previous disclosure, targeting specificity of the type III-D CRISPR-Cas system is determined by the guide RNA sequence. There is no requirement for a PAM or PFS motif, as previously identified.

[0292] The target site in a target single-stranded nucleic acid can be located in an intragenic region, an intergenic region, a coding region, a non-coding region or a regulatory region of a target nucleic acid.

[0293] The target site in a target single-stranded nucleic acid may be RNA specific e.g. in a mature RNA, at a splice junctions, in a polyA region, etc.

[0294] The target site in a target single-stranded nucleic acid may be located in a target gene.

[0295] Where a method is intended to cleave a target single-stranded nucleic acid, the target site may be in within gene, or within the transcript from a gene, of which it is desirable to decrease / inhibit expression. For example, the gene may be one the expression of which causes or contributes to a disease or undesirable physiological condition. The target site may be located in a sequence in vivo, ex vivo or in vitro. A target gene, or transcript thereof, may be located within a target organism or cell. The organism may be a bacterium, a virus, an archaeon, a fungus, plant, or an animal.

[0296] Contacting

[0297] The methods of the present invention comprise contacting the target nucleic acid with a type III CRISPR-Cas system. Suitably the step of contacting may comprise contacting the target nucleic acid with the complex in vitro, in vivo, or in a cell in vitro / ex vivo.

[0298] As used herein, "contact," contacting," "contacted," and grammatical variations thereof, refers to placing the components of a desired reaction together for a time and under conditions suitable for carrying out the desired reaction. The methods and conditions for carrying out such reactions are well known in the art (See, e.g., Gasiunas et al. (2012) Proc. Natl. Acad. Sci. 109:E2579-E2586; M.R. Green and J. Sambrook (2012) Molecular Cloning: A Laboratory Manual. 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0299] Suitably the methods may be performed in a cell-free system in vitro.

[0300] Alternatively, the methods may be performed in a cell, in vitro, ex vivo, or in vivo.

[0301] Suitably when the methods are performed in a cell, the method comprises introducing the type III-D CRISPR Cas system into the cell, suitably introducing the Cas proteins, the guide RNA and the at least one accessory protein into the cell. Suitably, the Cas and / or accessory proteins may be introduced into the cell as one or more proteins, or as one or more nucleic acids encoding the Cas and / or accessory proteins, suitably which may be DNA. Suitably the guide RNA may be introduced into the cell as one or more nucleic acids encoding the guide RNA, suitably which may be RNA or DNA.

[0302] In some examples, the Cas and / or accessory proteins can be introduced as a DNA sequence encoding the Cas and / or accessory proteins upon a vector, or as a protein, whereas the guide RNA can be introduced either as a DNA sequence encoding the guide RNA upon a vector, or in the form of RNA, e.g. an in vitro transcript.

[0303] Suitably the Cas and / or accessory proteins or one or more nucleic acids encoding them, or the guide RNA or one or more nucleic acids encoding it may be introduced into the cell simultaneously, separately, or sequentially.

[0304] Alternatively, the Cas and / or accessory proteins and guide RNA may be contacted in vitro which may then be introduced into the cell.

[0305] Suitably the one or more nucleic acids may be comprised on one or more vectors as described below.

[0306] In some examples, the one or more nucleic acids of the invention may be stably or transiently introduced into a cell.

[0307] The terms "introducing," "introduce," "introduced" (and grammatical variations thereof) in the context of a nucleic acid or protein and a cell means presenting the nucleic acid sequence or protein of interest to the cell (e.g., host cell) in such a manner that the nucleic acid sequence or protein gains access to the interior of a cell and includes such terms as "conjugation", "transformation," "transfection," and / or "transduction." The terms "conjugation", "transformation," "transfection," and "transduction" as used herein refer to the introduction of a heterologous nucleic acid or protein into a cell. Such introduction into a cell may be stable or transient. Thus, in some examples, a host cell or host organism is stably transformed with the nucleic acids. In other examples, a host cell or host organism is transiently transformed with the nucleic acids.

[0308] As used herein, the term "stably introduced" means that the nucleic acid sequence is stably incorporated into the genome of the cell, and thus the cell is stably transformed with the polynucleotide. When a nucleic acid is stably transformed and therefore integrated into a cell, the integrated nucleic acid is capable of being inherited by the progeny thereof, more particularly, by the progeny of multiple successive generations. "Transient transformation" in the context of a nucleic acid sequence means that a polynucleotide is introduced into the cell and does not integrate into the genome of the cell.

[0309] Suitably introducing the one or more nucleic acids into the cell may be by transformation or transduction. Suitably the one or more nucleic acid sequences can be introduced into a cell in a single transformation event, in separate transformation events.

[0310] Suitably methods of transfection or transformation may include calcium-phosphate mediated, electroporation, liposome mediated, exosome mediated, gene gun, microinjection, agrobacterium- mediated transfection or transformation, for example. Suitable methods for carrying out such transfection will be known to a person skilled in the art, and are further described below.

[0311] For comprehensive reviews about procedures for getting proteins or nucleic acids into cells in the context of this invention, see Marschall ALJ, Frenzel A, Schirrmann T, et al. "Targeting antibodies to the cytoplasm" mAbs. (2011) 3:3-16; Gu Z, Biswas A, Zhao M, Tang Y "Tailoring nanocarriers for intracellular protein delivery" Chem. Soc. Rev. (2011) 40:3638 - 3655. Du J, Jin J, Yan M, Lu Y "Synthetic nanocarriers for intracellular protein delivery" Curr. Drug Metab. (2012) 13:82-92.

[0312] Various physical methods of disrupting the cell membrane are useful, such as microinjection and electroporation (see Zhang Y, Yu L-C. "Microinjection as a tool of mechanical delivery" Curr. Opin. Biotechnol. (2008) 19:506-510) have been proposed for delivering compounds ranging from small molecules to proteins. Share! A, Zoldan J, Adamo A, et al. "A vector-free microfluidic platform for intracellular delivery" Proc. Natl. Acad. Sci. (2013) 110: 2082 - 2087 describes a microfluidic device that transiently disrupts the plasma membrane through physical constriction. Silicon "nanowires" that pierce the cell membrane have also been reported Shalek AK, Robinson JT, Karp ES, et al. "Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells" Proc. Natl. Acad. Sci. (2010) 107: 1870-1875.

[0313] There are also peptide-based strategies using cell penetrating peptides (CPP) which can enhance permeability of the nucleic acids or proteins. For example, the TAT peptide can be covalently coupled. Also, an amphiphilic CPP Pep-1 can noncovalently complex and translocate peptide and protein cargos Morris MC, Depollier J, Mery J, et al. "A peptide carrier for the delivery of biologically active proteins into mammalian cells" Nat. Biotechnol. (2001) 19: 1173-1176.

[0314] Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91 / 17424; WO 91 / 16024.

[0315] There is also for example substance P (SP), an 11-residue neuropeptide which can be conjugated to the nucleic acids or proteins (Harford-Wright E, Lewis KM, Vink R, Ghabriel MN. "Evaluating the role of substance P in the growth of brain tumors" Neuroscience (2014) 261: 85-94. There are also various pore- or channel-forming proteins of bacterial origin which may be used to translocate nucleic acids or proteins into cells. Chatterjee S, Chaudhury S, McShan AC, et al. "Structure and biophysics of type III secretion in bacteria. Biochemistry (Mose)" (2013) 52: 2508-2517 teaches a sophisticated secretion system which transport proteins directly from the bacterial cytoplasm to the eukaryotic host. Doerner JF, Febvay S, Clapham DE. "Controlled delivery of bioactive molecules into live cells using the bacterial mechanosensitive channel MscL" Nat. Commun. (2012) 3: 990 describes functional expression of an engineered bacterial channel (MscL) in mammalian cells, the opening and closing of which could be controlled chemically. Alternatively, the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins, which are capable of forming macropores up to 30nm in diameter may be useful as "reversible permeabilization" reagents for delivering nucleic acids or proteins into cells. (See Dunstone MA, Tweten RK. "Packing a punch: the mechanism of pore formation by cholesterol dependent cytolysins and membrane attack complex / perforin-like proteins" Curr. Opin. Struct. Biol. (2012) 22: 342-349; Provoda CJ, Stier EM, Lee K-D. "Tumor cell killing enabled by listeriolysin O- liposome-mediated delivery of the protein toxin gelonin." J. Biol. Chem. (2003) 278: 35102-35108; and Pirie CM, Liu DV, Wittrup KD. "Targeted cytolysins synergistically potentiate cytoplasmic delivery of gelonin immunotoxin" Mol. Cancer Ther. (2013) 12: 1774-1782.

[0316] In addition to pore- or channel-forming proteins, the membrane-translocating domains of bacterial toxins have been proposed as a modular tool that can be fused to, and enhance the intracellular delivery of, other proteins (see Sandvig K, van Deurs B. "Membrane traffic exploited by protein toxins" Annu. Rev. Cell. Dev. Biol. (2002) 18: 1-24; Johannes L, Romer W. "Shiga toxins — from cell biology to biomedical applications" Nat. Rev. Microbiol. (2010) 8: 105-116.

[0317] Additionally, Lawrence MS, Phillips KJ, Liu DR. "Supercharging proteins can impart unusual resilience" J. Am. Chem. Soc. (2007) 129: 10110-10112 provides "supercharged" GFP, a variant engineered to have high net positive charge (+36), and certain human proteins with naturally high positive charge (see Cronican JJ, Thompson DB, Beier KT, et al. "Potent delivery of functional proteins into mammalian cells in vitro and in vivo using a supercharged protein" ACS Chem. Biol. (2010) 5: 747-752; or Cronican JJ, Beier KT, Davis TN, et al. "A class of human proteins that deliver functional proteins into mammalian cells in vitro and in vivo" Chem. Biol. (2011) 18: 833-838 have been reported to translocate across the cell membrane.

[0318] There are also virus-based strategies for packaging of the proteins or nucleic acids into virus-like particles (see Kaczmarczyk SJ, Sitaraman K, Young HA, et al. Protein delivery using engineered viruslike particles. Proc. Natl. Acad. Sci. (2011) 108: 16998-17003) or attaching them to an engineered bacteriophage T4 head (see Tao P, Mahalingam M, Marasa BS, et al. "In vitro and in vivo delivery of genes and proteins using the bacteriophage T4 DNA packaging machine" Proc. Natl. Acad. Sci. (2013) 110: 5846-5851) has been reported to enhance cytosolic delivery.

[0319] Further, there are lipid and polymer-based strategies. The proteins or nucleic acids of the invention may be encapsulated in liposomes (see Torchilin V. Intracellular delivery of protein and peptide therapeutics. Drug Discov Today Technol. (2008) 5:e95-el03) or complexed with lipids. Regarding the latter strategy, lipid formulations that have been successful in the transfection of DNA may be used. For example, a formulation based on a mixture of cationic and neutral lipids.

[0320] Similarly, polymer-based formulations that have been successfully used for nucleic acid transfections have also been examined for their ability to "transfect" proteins. For example, polyethylenimine (PEI) or poly-g-amino esters (PBAEs) which may be in the form of biodegradable nanoparticles.

[0321] Also, inorganic material-based strategies may be used; for example including silica, carbon nanotubes, quantum dots, or gold nanoparticles.

[0322] Another method is available which is induced transduction by osmocytosis and propanebetaine ((iTOP) (see D'Astolfo, D. S. et al. Efficient intracellular delivery of native proteins. Cell 161, 674-690 (2015). This method allows efficient delivery of CRISPR-Cas complexes into a wide variety of primary cell types. The iTOP approach enables virus-free transduction of native proteins and does not rely on additional peptide tags, which may interfere with protein function or editing efficiency and is particularly effective for transduction of cell types that are refractory to other delivery methods. For more information see Wen Y. Wu (2018) Nature Chem Biol. 14: 642-651.

[0323] In one example, one or more nucleic acids encoding Cas proteins, guide RNA and / or at least one accessory protein of the type III CRISPR Cas systems may be introduced into the cell by conjugation. In one embodiment, conjugation is carried out by transfer of genetic material from one bacterium to another through direct contact. Suitably therefore a donor bacterium is prepared comprising the one or more nucleic acids encoding Cas proteins and comprising a nucleic acid sequence encoding the conjugative machinery. Suitably the donor bacterium delivers the one or more nucleic acids encoding Cas proteins to other cells, suitably other bacterial cells. Such conjugation techniques are described in Woodall C.A. (2003) DNA Transfer by Bacterial Conjugation. In: Casali N., Preston A. (eds) E. coli Plasmid Vectors. Methods in Molecular Biology, vol 235. Humana Press, https: / / doi.org / 10.1385 / l- 59259-409-3:61, for example.

[0324] Incubating

[0325] Upon contacting the target nucleic acid sequence with the type III CRISPR Cas system, the system is cultured or incubated for a time and under conditions sufficient for targeting to occur at the target sequence. Suitably therefore the methods may comprise step of culturing or incubating the type III CRISPR-Cas system and the target nucleic acid.

[0326] Suitably if contacting occurs in a cell free system, then the system and the target nucleic acid are cultured or incubated under suitable cell free conditions for targeting to occur at the target sequence.

[0327] Suitable cell free culture techniques are known to the skilled person. For example, using the conditions defined in commercial cell-free kits available from myTXTL, Arbor Biosciences, or PUREsystem.

[0328] Suitably if contacting occurs within a cell, then after introduction of the system and the target nucleic acid into the cell, the cell is cultured under suitable conditions for targeting to occur at the target sequence.

[0329] Suitably the culture conditions are determined by the skilled person according to the type of cell and species of cell which harbours the complex. Suitable cell culture techniques are known to the skilled person. For example, suitable mammalian cell culture conditions may be found in Phelan, K. and May, K.M. 2017. Mammalian cell tissue culture techniques. Current Protocols in Molecular Biology, 117, A.3F.1-A.3F.23. doi: 10.1002 / cpmb.31 Nucleic Acids

[0330] Nucleic acid sequences encoding the type III CRISPR-Cas complex or systems used in the present invention or the modified type III CRISPR-Cas complex or systems or components thereof (e.g. one or more Cas protein and / or one or more guide RNA and / or one or more accessory protein) are provided herein. These nucleic acid sequences may be provided for delivery to a cell in order to form the complex and in order to carry out the methods of the invention within a cell.

[0331] In certain examples, the Cas and / or accessory protein of the type III CRISPR-Cas system may be introduced into the cell as a protein, or as one or more nucleic acids encoding the or each Cas and / or accessory protein, suitably which may be DNA. In other examples, the at least one guide RNA may be introduced into the cell as one or more nucleic acids encoding the guide RNA, suitably which may be RNA or DNA. Suitably more than one Cas protein may be encoded on one nucleic acid sequence. Suitably the nucleic acid sequences encoding each Cas protein are linked to each other, suitably in any order. Suitably by a sequence encoding a cleavable linker. Suitably by a sequence encoding a cleavable peptide. Suitably the cleavable linkers are between each nucleic acid sequence encoding each Cas protein. Suitably the guide RNA and / or accessory protein may also be encoded on the same nucleic acid. Alternatively, each Cas protein may be encoded on a separate nucleic acid. Suitably the guide RNA and / or accessory protein may be encoded on a separate nucleic acid.

[0332] Suitably when methods are performed in a eukaryotic cell, the one or more nucleic acids encoding the Cas and / or accessory proteins further comprise nuclear localising sequences (NLS). Suitable nuclear localisation sequences are known in the art. Suitably the one or more nucleic acids may comprise two NLS. Suitably a first NLS at the 5' end of each nucleic acid sequence and a second NLS at the 3' end of each nucleic acid sequence.

[0333] In some examples each nucleic acid of the invention may be regarded as an "expression cassette" or may be comprised within an expression cassette. As used herein, "expression cassette" means a recombinant nucleic acid construct comprising a nucleic acid sequence of interest (e.g., the polynucleotides encoding Cas polypeptides, guide RNAs and accessory proteins of the present invention), wherein said nucleic acid sequence of interest is operably linked with at least one regulatory sequence (e.g., a promoter). Thus, some aspects of the present invention provide expression cassettes designed to express the nucleic acids of the invention. Suitably comprised on a vector. Suitably any features of the vector described below may also be regarded as features of an expression cassette. Suitable regulatory sequences are defined hereinbelow.

[0334] Vectors

[0335] Generally, the term "vector" herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.

[0336] In certain examples, one or more vectors may comprise one or more of the nucleic acids described herein which encode one or more Cas protein of the type III CRISPR-Cas complexes disclosed herein. In other examples, one or more vectors may comprise one or more nucleic acids described herein that encode the or each guide RNA. In certain other examples, the same vector may comprise one or more of the nucleic acids described herein which encode the or each accessory protein. In certain other examples, the same vector may comprise one or more of the nucleic acids described herein which encode one or more of the Cas proteins or modified Cas proteins and one or more nucleic acids described herein that encode the or each guide RNA. In certain other examples, the same vector may comprise one or more of the nucleic acids described herein which encode one or more of the Cas proteins or modified Cas proteins, one or more nucleic acids described herein that encode the or each guide RNA and one or more nucleic acids described herein that encode the or each accessory protein.

[0337] In other examples two or more of the nucleic acids encoding the Cas proteins are comprised on a single vector, and in certain examples all of the nucleic acids encoding the Cas proteins are comprised on a single vector.

[0338] In further examples when several nucleic acids encoding the Cas proteins are comprised on a single vector, they are linked to each other, suitably in any order. Suitably they may be linked by sequence encoding cleavable linkers. Suitably by cleavable peptides as described above. Suitable cleavable linkers may comprise a 2A self-cleaving peptide, T2A, P2A, E2A, F2A, for example.

[0339] Suitably the one or more nucleic acids encoding the Cas proteins and one or more nucleic acids encoding the or each guide RNA may be comprised on the same vector or comprised on separate vectors.

[0340] Some vectors are able to direct expression of genes to which they are operatively-linked. Such vectors are "expression vectors" and there will usually be regulatory elements, which may be selected on the basis of the host cells in which the expression takes place. This means the nucleic acid to be expressed is operably linked to the regulatory elements thereby resulting in expression of the nucleotide sequence whether in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell.

[0341] Suitably the one or more vectors comprising nucleic acids encoding the Cas proteins, one or more nucleic acids encoding the guide RNA and one or more nucleic acids encoding the or each accessory protein further comprise one or more regulatory sequences. Suitably the regulatory sequences are operably linked to the nucleic acids encoding the Cas proteins and to the nucleic acids encoding the or each guide RNA.

[0342] Suitably therefore the vector or vectors may comprise an expression cassette as defined hereinabove.

[0343] By "operably linked" or "operably associated" as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related. Thus, the term "operably linked" or "operably associated" as used herein, refers to nucleotide sequences on a single nucleic acid molecule that are functionally associated. Thus, a first nucleotide sequence that is operably linked to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For instance, a promoter is operably associated with a nucleotide sequence if the promoter effects the transcription or expression of said nucleotide sequence. Those skilled in the art will appreciate that the control sequences (e.g., promoter) need not be contiguous with the nucleotide sequence to which it is operably associated, as long as the control sequences function to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a nucleotide sequence, and the promoter can still be considered "operably linked" to the nucleotide sequence. Suitable regulatory sequences control expression of the nucleic acid sequence and may include promoters, enhancers, terminators, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences) UTRs, ITRs, introns etc. For more information the average skilled person would refer to, for example, in Goeddel, (1990), Gene Expression Technology in Methods in Enzymology vol 185, Academic Press. Regulatory elements include those giving direct constitutive expression in many types of host cell and those that direct expression of the nucleotide sequence only in certain cells (i.e., tissue-specific regulatory sequences).

[0344] A tissue-specific promoter directs expression primarily in a desired tissue of interest, such as blood, specific organs (e.g., liver, pancreas), or particular cell types. Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. A promoter useful with this invention can include, but is not limited to, constitutive, inducible, developmentally regulated, tissue-specific / preferred- promoters, and the like, as described herein.

[0345] A regulatory element as used herein can be endogenous or heterologous. In some examples, an endogenous regulatory element derived from the subject organism can be inserted into a genetic context in which it does not naturally occur (e.g., a different position in the genome than as found in nature), thereby producing a recombinant or non-native nucleic acid. In some examples, promoters useful with the nucleic acid sequences described herein may be any combination of heterologous and / or endogenous promoters.

[0346] Examples of suitable promoters include pol I, pol II, pol III (e.g. U6 and Hl promoters). Examples of pol II promoters include, but are not limited to, retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the SV40 promoter, the dihydrofolate reductase promoter, the (3-acting promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.

[0347] Examples of other suitable promoters may be bacterial or phage promoters, such as those described in https: / / parts.igem.org / Promoters / Catalog. In one example, the promoter may be a Synechocystis promoter, such as the psbA2 promoter for the DI subunit from Synechocystis. In another embodiment, the promoter may be an E. coli o7Q constitutive promoter.

[0348] In some examples, inducible promoters can be used. Examples of inducible promoters include, but are not limited to, tetracycline repressor system promoters, Lac repressor system promoters, arabinose-inducible, copper-inducible system promoters, salicylate-inducible system promoters (e.g., the PRla system), glucocorticoid-inducible promoters, and ecdysone-inducible system promoters. In one embodiment, the inducible promoter is araBAD arabinose inducible promoter.

[0349] Suitably the one or more nucleic acids encoding the Cas proteins are operably linked to a promoter which is a pol II promoter.

[0350] Suitably the one or more nucleic acids encoding the or each guide RNA are operably linked to a promoter which is a pol III e.g. U6 or Hl promoter.

[0351] As well as promoters, regulatory elements may include enhancer elements, such as WPRE; CMV enhancers; the R-U5' segment in LTR of HTLV-I; SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit (3-globin. Suitably some bacterial promoters may comprise binding sites for regulatory elements such as activators. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc.

[0352] Suitably the vector may also optionally include a transcriptional and / or translational termination region ( / .e., termination region) that is functional in the selected host cell. A variety of transcriptional terminators are available and are responsible for the termination of transcription beyond the heterologous nucleotide sequence of interest and correct mRNA polyadenylation. The termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleic acid sequence, may be native to the host cell, or may be derived from another source ( / .e., foreign or heterologous to the promoter, to the nucleic acid sequence, to the host, or any combination thereof).

[0353] Suitably the vector may also include a nucleotide sequence for a selectable marker, which can be used to select a transformed host cell. As used herein, "selectable marker" means a nucleotide sequence that when expressed imparts a distinct phenotype to the host cell expressing the marker and thus allows such transformed cells to be distinguished from those that do not have the marker. Such a nucleotide sequence may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g., an antibiotic and the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening (e.g., fluorescence). Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein. In some examples, a selectable marker useful with this invention includes polynucleotide encoding a polypeptide conferring resistance to an antibiotic. Non-limiting examples of antibiotics useful with this invention include ampicillin, kanamycin, streptomycin, spectinomycin, gentamicin, tetracycline, chloramphenicol, and / or erythromycin. Thus, in some examples, a polynucleotide encoding a gene for resistance to an antibiotic may be introduced into the organism, thereby conferring resistance to the antibiotic to that organism.

[0354] Non-limiting examples of general classes of vectors include but are not limited to a viral vector, a plasmid vector, a phage vector, a phagemid vector, a cosmid vector, a fosmid vector, a bacteriophage, an artificial chromosome, or an Agrobacterium binary vector in double or single-stranded linear or circular form which may or may not be self-transmissible or mobilizable. A vector as defined herein can transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication). Additionally included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast or fungal cells). A plasmid may be vector in accordance with this description, which is a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.

[0355] Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0356] Suitably the vector used is a plasmid.

[0357] Suitably the vector is selected which is suitable for the cell or organism into which vector is to be introduced. Suitably the plasmid is selected which is suitable for the cell or organism into which plasmid is to be introduced.

[0358] Suitable plasmids for bacterial expression may include: pQE80L, pACYC-Duet, pSEVA series for example. Suitable plasmids for mammalian expression may include pcDNA3.1 + .

[0359] Suitably the, or each, vector is for introducing the Cas proteins, guide RIMA and at least one accessory protein as defined herein into a cell such that the methods of the invention can take place within the cell. Suitably therefore the methods may comprise a step of introducing a vector comprising one or more nucleic acids encoding the Cas proteins or modified Cas proteins, one or more nucleic acids encoding the guide RNAs into a cell, and one or more nucleic acids encoding the at least one accessory protein wherein the cell comprises the target nucleic acid sequence.

[0360] Suitable means of introducing vectors into cells are the same as the means for introducing nucleic acids into cells as described hereinabove. For example, methods of non-viral delivery of nucleic acids may include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid: nucleic acid conjugates, naked DNA, artificial virions, conjugation, and agent-enhanced uptake of DNA.

[0361] Suitably after introduction of the, or each, vector into the cell, the Cas proteins, the guide RNA and the one or more accessory proteins are expressed in the cell. Suitably expression of the Cas proteins, the guide RNA and the one or more accessory proteins may be induced, suitably induced from the, or each, vector. Suitably therefore the, or each, vector comprises an inducible promoter operably linked to the, or each, nucleic acid sequence encoding the Cas proteins and / or the guide RNA and / or the accessory protein(s). Suitably the cell may be contacted with an inducer to induce said expression. Suitably the inducer may induce expression of the Cas proteins and / or the guide RNA and / or the accessory protein(s)from the or each vector.

[0362] Suitably upon expression of the Cas proteins, the guide RNA, the components assemble into the type III CRISPR-Cas complex of the invention, which when coupled to the one or more accessory proteins form the type III CRISPR-Cas system of the invention.

[0363] Cells

[0364] The methods of the present invention may be carried out in a cell, and the type III CRISPR-Cas system and / or sequences encoding such a system can be provided in a cell. Therefore, there is provided a cell comprising a type III system of the invention, or a modified type III CRISPR-Cas system of the invention, comprising a vector of the invention, or comprising a nucleic acid encoding any part of the type III CRISPR-Cas system of the invention. Suitably therefore the cell may be regarded as a host cell.

[0365] Suitably the cell may be ex vivo, in vitro, or in vivo.

[0366] Suitably the cell may be eukaryotic or prokaryotic. Suitably the cell may be from a bacterium, archaeon, plant, animal, insect or fungi. Suitably the cell is a cyanobacterial cell.

[0367] Suitably the cell is an animal cell. Suitably the cell is a mammalian cell. Suitably the cell may be a human or a non-human cell. Suitably the cell may be a non-human mammalian cells. Suitably the cell may be a non-human primate cell.

[0368] Suitably the cell may be part of an organism. Suitably the cell may be located within an organism. Suitably the organism may be a prokaryote or a eukaryote. Suitably the organism is a bacterium, a virus, an archaeon, a fungus, plant, or an animal. Suitably the organism may be a host organism.

[0369] Thus, the invention includes any animal or cell, produced by the present methods, or a progeny thereof. The progeny may be a clone of the produced plant or animal or may result from sexual reproduction by crossing with other individuals of the same species to introgress further desirable traits into their offspring.

[0370] The present invention will now be further described with reference to the following non-limting examples.

[0371] EXAMPLES

[0372] Example 1: Programmable cell killing by the type III CRISPR-Cas system in mammalian

[0373] HEK293 cells

[0374] Methods / Materials

[0375] Construction of plasmids for CRISPR-Cas in mammalian cells

[0376] Vector (pPF3610) used for expression of the type III-Dv complex alone in mammalian cells was previously synthetically constructed

[0015] . Vectors for expression of the type III-Dv complex with NucC were made in this study. The cas genes were codon optimized for expression in mammalian cells and ordered as gene-blocks from IDT (Table 1). Gene-blocks were PCR-amplified using the oligonucleotides listed in Table 2 using Phusion (Thermofisher). Initial plasmids were generated through Gibson assembly (NEB) of eight gene fragments. The resulting vectors were sequence-confirmed with Oxford nanopore sequencing (Plasmidsaurus). All plasmids used and produced in this work are listed in Table 3. Due to a ~2 kb deletion in Cas7-5-ll in the sequenced construct (pPF3817) a 3,279 bp fragment encoding the desired sequences for NucC, Cas6-2a and microRFP670nano was Ndel-digested from pPF3817 and then ligated with the Ndel digested 11,656 bp backbone of pPF3727 (Table 3) to form the cell killing vector pPF3816. To produce the negative control of CRISPRi plus NucC (pPF3934) the CRISPRi vector pPF3610 was Ndel digested to produce a 11,474 bp backbone and ligated with the Ndel digested 3,279 bp NucC, Cas6-2a, and microRFP670nano fragment from pPF3817. The mutated Palm domain of CaslO in pPF3610 and pPF3934 will not produce cOAs and therefore should not activate NucC. Spacers (annealed oligonucleotides in Table 2) were cloned into pPF3610 (CRISPRi), pPF3934 (CRISPRi plus NucC) and pPF3816 (cell killing) via a Bsal restriction site. Clones were confirmed by Sanger sequencing.

[0377] To study the inducibility of cell killing by the type III-Dv / NucC system, the constitutive U6 promoter on the guide RNA was replaced by a tetracycline inducible promoter (tet-on). The tetracycline promoter was amplified from a pSBtet-GP: luciferase plasmid[19,20] using PF7917 and PF7918 (sequences are shown in Table 2) with Q5 polymerase (Thermofisher). Concurrently two amplicons either side of the U6 promoter from pPF3610 were amplified using primers PF7118 and PF7920 (97 bp amplicon), and PF7915 and PF7106 (269 bp amplicon). The primers PF7920 and PF7915 have ends homologous to both the tetracycline promoter and the backbone of various III-Dv vectors. PCR was then performed with the three amplicons using primers PF7118 and PF7107, which produced a single 501 bp amplicon. The final amplicon had Bsal and Spel restrictions sites for cloning into Bsal and Spel sites from pPF3610 and pPF3816 to produce vectors pPF3951 (tet inducible CRISPRi) and pPF3952 (tet inducible cell killing), respectively. The replacement of the U6 promoter by the tetracycline promoter was confirmed by Sanger sequencing for both vectors.

[0378] Cell culture

[0379] Human embryonic kidney cells (HEK293 and HEK293T) were cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10 % (v / v) foetal calf serum (FCS; Pan Biotech Aidenbach, Germany) and Pen-Strep (100 U / mL penicillin and 100 pg / mL streptomycin; Gibco) at 37°C with 5 % CO2. For the tetracycline inducible system (tet-on) a HEK293T-rtTA WT cell line with a sleeping beauty system that encodes the reverse tetracycline activator (rtTA)[19,20] was used. One day prior to transfection, HEK293 cells were seeded into either 12 or 24-well plates at ~1 x 105or ~5 x 104cells / mL in 10 % (v / v) FCS / DMEM without Pen-Strep, respectively. HEK293 cells were then transfected with either 1000 or 500 ng total DNA using Lipofectamine 3000 (Thermofisher Scientific, Waltham, MA, USA) as per the manufacturer's protocol. The media was replaced 6 - 12 hours post-transfection, with 10 % (v / v) FCS / DMEM supplemented with Pen-Strep. For the tet-on system media was supplemented with 0, 0.1, 1 and 5 pg / mL tetracycline. Cells were then processed for flow cytometry either 48 or 72-hours post-transfection, for determination of either CRISPRi or cell killing efficacy, respectively.

[0380] Confocal microscopy

[0381] To image transfected HEK293 cells, cells were seeded onto glass coverslips in 12-well plates. After 72-hours of transfection, cells were fixed in 4 % paraformaldehyde, then washed twice with pH 7.4 PBS before being stained with DAPI (Thermofisher Scientific, Waltham, MA, USA) and washed again in pH 7.4 PBS followed by a final wash in distilled water. Coverslips were then mounted onto microscope slides using Fluorsave (Merckmillipore). Images were acquired using a CFI Plan APO Lambda lOOx 1.49 numerical aperture oil objective (Nikon Corporation) on the multimodal imaging platform Dragonfly v.505 (Oxford Instruments) equipped with 405, 488, 561 and 637 nm lasers built on a Nikon Ti2-E microscope body with Perfect Focus System (Nikon Corporation). Data was collected in Spinning Disk 40 pm pinhole mode on the iXon888 EMCCD camera using the Fusion Studio Software v.1.4 (Andor Oxford Instruments). Z stacks were collected on the z-axis using an Applied Scientific Instrumentation stage with 500 pm piezo z drive. Images were visualized and processed using Fiji Software (Windows 64-bit). Final images and gray scale plot data were generated using Fiji Software (Windows 64-bit). Statistical analysis was performed using a one-way ANOVA multiple comparison in Prism v 10.1.1 (Graphpad), comparing treatment with targeting spacers to the non-targeting spacer control.

[0382] Flow cytometry

[0383] To quantitate the efficiency of cell killing of the type III-Dv system in HEK293 cells, flow cytometry was used. After 72-hours transfection, cells were resuspended in their growth medium with 90 and 20 pL aliquots removed from each well for viability assays which were performed in parallel to flow cytometry experiments. Cells were then centrifuged at 453 x g for 5 min. Cells were washed in triplicate by resuspending them in 1 mL of pH 7.4 PBS and then centrifuging them at 453 x g for 5 min before resuspension in a final volume of 300 pL wash buffer (pH 7.4 PBS, 0.1 w / v % BSA and 2 mM EDTA) for measurement on a Cytek Aurora Flow cytometer with SpectroFlo 3.03 software (Cytek Biosciences, Fremont, CA, USA). To compare the number of viable cells across different conditions a set volume of 25 pL of cell suspension was run on the flow cytometer. For a negative control, i.e. dead cells, cells were heat killed at 65 °C for 10 min, and then washed in a similar fashion as stated above before analysing their scatter profile on the Cytek Aurora Flow cytometer. To test the efficacy of the tet-on system, first CRISPRi was tested using pPF3610 and the Venus encoding plasmid pPF3328 containing spacers. For these experiments cells were processed 48-hours after transfection. Media was removed from cell samples, and then cells were resuspended in wash buffer. Cells were washed in triplicate by centrifuging them at 453 x g for 5 min and resuspending them in 1 mL wash buffer before being resuspended in 300 pL wash buffer for analysis by flow cytometry on a Cytek Aurora flow cytometer. For CRISPRi experiments, a total of 50,000 events were recorded. Cells were gated on SSC-H vs. FSC-H, FSC-H vs. FSC-A and SSC-H vs. SSC-A to identify the singlet population of HEK293 cells. Co-transfected singlet cells that were both microRFP670nano and Venus positive had the median fluorescence intensity (MFI) of Venus fluorescence determined. Determined MFIs were plotted and analysed using Prism v. 10.1.1 (Graphpad). Analysis of recorded data was performed using FlowJo software v.10 (BD Biosciences). Statistical analysis was performed using a one-way ANOVA multiple comparison, comparing treatment with targeting spacers to the non-targeting spacer control.

[0384] FACS

[0385] A BD FACSAria Fusion cell sorter equipped with a red 640 nm excitation laser and 670 / 30 nm band-pass filter was used to sort RFP+ cells for isolation of total RNA for RT-qPCR. For further details of the instruments complete laser set up refer to Smith et a / .

[0021] . The sorter was fitted with a 70-pm nozzle (70 p.s.i.) and was operated in accordance with standard procedures. In advance of cell sorting, the BD FACSAria Fusion fluidics systems were initialized, and instrument QC checks were performed by using the Cytometer Setup and Tracking feature of the FACSDiva software interface. Drop delay was optimized before cell sorting.

[0386] RT-qPCR

[0387] Total RNA was extracted using a RNeasy kit (QIAGEN) from RFP+ cells collected from FACS. Briefly, the sorted samples had 350 pL of RLT buffer (lysis buffer) and p-mercaptoethanol added to each. Cell lysis and homogenization was achieved by passing samples ~20x through sterile RNase free hypodermic needles. The homogenate was transferred into a clean Eppendorf and 1.5x volume of 100% ethanol (RNase-free, AR-grade) was added and mixed by vortexing. The sample was transferred into the RNeasy mini spin column, centrifuged (>8000 g for 15 s) and the flow through discarded. Next, the column was washed twice with 500 pl of Buffer RPE (washing buffer) followed by centrifugation (>8000 g for 15 s). RNA was eluted by adding 30 pl of RNase free water followed by centrifugation (>8000 g for 1 min). The sample was treated with TURBO DNase (Invitrogen) following the manufacturer's instructions. After inactivating TURBO DNase with DNase Inactivating Reagent, ~0.5 - 1 pg DNase-free RNA was reverse transcribed using Superscript III Reverse Transcriptase (Thermo Fisher Scientific) with random primers (Promega) as per manufacturer's instructions. qPCR was performed using SYBR Green qPCR Mastermix (Thermofisher) in a QuantStudioTM 6 Real-Time PCR Detection System (Thermofisher). Gene-specific primer pairs used to detect mature transcripts are listed in Table 2. Relative amounts of a given target RNA under targeting versus nontargeting conditions was calculated using the formula 2A-((CtTarget-CtGAPDH)Targeting crRNA - (CtTarget-CtGAPDH)NT crRNA). No-RT and no-template controls were run alongside all RT-qPCR experiments. RNA and cDNA concentration were determined using the Invitrogen Qubit 4 Fluorometer (Thermo Fisher Science) according to the manufacturers protocol. RNA and cDNA samples were stored at -80°C.

[0388] Viability assays

[0389] To determine cell metabolic activity 2 x 90 pL aliquots of cells were removed from each condition after resuspension of cells in growth medium. Each 90 pL aliquot was pipetted into a single well of a sterile 96-well plate. Non-transfected cells, heat-killed cells, and media only wells were included as positive and negative controls, respectively. After a recovery period of ~30 minutes at 37°C with 5 % CO2 10 pL of 10 % w / v resazurin solution was added to each well and left to incubate for 1-4 h at 37°C with 5 % CO2. After conversion of resazurin to resorufin in positive control wells 80 pL was pipetted to a black 96-well plate with a multichannel pipette. Fluorescence of each well was recorded using a 540 nm excitation I 590 nm emission filter set on a Clariostar plate reader (BMG Labtech). For Trypan-blue viability assays a LUNA - II automated cell counter was used. A volume of 20 pL of cells resuspended in their culture media was mixed with 20 pL of trypan blue solution (0.4%, Gibco), 10 pL of this mixture was loaded onto a LUNA reusable slide. The cell counter counts the unstained (viable) and stained (nonviable) cells to determine the number of viable and non-viable cells per mL of culture.

[0390] Results

[0391] Type III-Dv causes knockdown of endogenous genes in HEK293 cells

[0392] To develop the type III / NucC system for cell killing, it was important to first demonstrate that the plasmids were able to recognize and target endogenous RNA in mammalian cells. Previously the Applicants have shown that the type III-Dv system from Synechocystis sp. PCC6803 can be used in both secondary (HEK293 cells) and primary cell lines (neurons) to knockdown a fluorescent reporter and endogenous gene (MAP2) expression, respectively.

[0393] The Applicants aimed to test that crRNA guides targeting endogenous genes were functional and had the potential to therefore initiate targeted cell killing. To test this, the Applicants quantified the RNA targeting effect at the transcript level by transfecting the CRISPRi type III-Dv vector (pPF3610) that contained spacers targeting BRCA1, CKB and XIST gene transcripts into HEK293 cells (Figure 1A). Forty- eight hours after transfection, cells were sorted using FACS to isolate RFP+ cells, RNA was purified and used in RT-qPCR experiments. The percentage repression data shown in Figure 1A demonstrates that the type III-Dv system lacking NucC resulted in ~50 % of the levels of mRNA relative to a non-targeting control when targeting endogenous genes in HEK293 cells. This was the case for most spacers targeting BRCA1, CKB and XIST RNAs, except for cr2 for CKB (Figure 1A). The data provided confidence that the type III-Dv system was being expressed, formed an active complex, and targeted endogenous target RNAs. Therefore, the Applicants were now well positioned to test targeting endogenous genes with type III-Dv expression plasmids coupled with a downstream nuclease (e.g. NucC) to trigger cell killing in mammalian cells.

[0394] Binding of target RNA by type III-Dv activates the accessory nuclease NucC

[0395] Applicants next determined whether the type III-Dv system could activate the accessory nuclease NucC[17,22]. Binding of type III CRISPR-Cas complexes to target RNA results in RNA cleavage by Cas7 subunits in the backbone of the complex. Following target RNA recognition and binding, CaslO is activated to cleave ssDNA through the HD domain, and the Palm domain is activated to produce signaling molecules (e.g. cOAs) from ATP molecules. cOAs are bound by accessory proteins to activate catalytic activity (e.g. (nuclease and ribonucleases that cleave DNA or RNA, respectively) in a non-specific manner[12, 13,23,24]. The type III-Dv system that the Applicants are testing was originally encoded from a megaplasmid in Synechocystis, and the NucC nuclease accessory protein being tested was originally from Serratia sp. ACC 39006. Due to the known ability of NucC to trigger massive and rapid degradation of bacterial chromosomal DNA to elicit a cell death response

[0017] , the Applicants reasoned that, if coupled to the type III-Dv system, it could cause cell killing in eukaryotic (human) cells (Figure IB). Presently, the exact distribution of cOAs (i.e. cA3, cA4 and cA6) produced by the Palm domain of CaslO in type III-Dv is unknown. It was therefore important that the Applicants tested in vitro whether activation of the CaslO Palm domain of the type III-Dv complex by binding of target RNA could activate NucC. The presence of target RNA complementary to the crRNA in the type III-Dv complex resulted in cleavage of a double stranded DNA substrate by NucC (Figure 1C). This result provided proof- of- co nee pt that the type III-Dv system could be used in conjunction with the accessory nuclease NucC to cleave DNA in response to a specific RNA target.

[0396] Design of expression constructs and proposed cell killing mechanism

[0397] To test the cell killing capacity of type III-Dv with NucC in mammalian cells, the Applicants constructed an expression vector pPF3816 (Figure 2A). A key feature of the plasmid was the bidirectional CMV promoter, with genes cloned downstream of the promoter encoding CaslO (with active Palm and HD domains), Cas7-Cas5-Casll, Cas7-Cas7, Csxl9 and Cas7-insertion (for expression of type III-Dv proteins)), and genes cloned upstream of the promoter encoding microRFP670nano (used to determine transfection of cells by the plasmid), Cas6-2a (for crRNA processing), and NucC. Genes were separated by sequences encoding 2A ribosomal skipping sites, and each end contained a poly-A motif to terminate transcription. All proteins (except microRFP670nano) possessed a nuclear localization signal and flag tag, and CaslO also contained a His-tag to enable purification of the type III-Dv complex. A type III-Dv repeat was positioned in the backbone upstream of the Bsal restriction site, and spacer-repeat units were designed to be cloned into the restriction site, which allowed for multiplexing of spacers if required. Expression of the mini CRISPR array was under a constitutive U6 promoter. The Applicants propose that targeting of endogenous RNA transcripts, such as BRCA1 and CKB, will result in the activation of NucC, leading to degradation of genomic DNA and ultimately, cell death (Figure 2B).

[0398] RNA targeting by type III-Dv activates NucC and causes human cell death

[0399] To test if type III-Dv coupled with NucC can kill human cells, the Applicants transfected HEK293 cells with a type III-Dv / NucC expression vector containing spacers targeting BRCA1 and CKB gene transcripts. Based on our earlier RT-qPCR data, the Applicants chose to use the crl spacers for both genes (Figure 1A). Seventy-two hours after transfection, cells were fixed in 4 % PFA, stained with DAPI and then imaged by confocal microscopy. The Applicants tested 6 conditions; untransfected cells, cells transfected with a plasmid with mutated Palm and HD domains (pPF3934; CRISPRi plasmid with NucC included) with non-targeting and BRCA1 targeting spacers, and cells transfected with our cell killing vector (pPF3816) with non-targeting, BRCA1- and C / <B-targeting spacers.

[0400] Untransfected cells, cells transfected with the CRISPRi plasmid with NucC (pPF3934) with nontargeting and BRC4.I -targeting spacers, and the cell killing plasmid pPF3816 with non-targeting spacers served as negative controls. There was a significant reduction in the number of DAPI stained nuclei when comparing BRCA1 and CKB targeted samples versus the untargeted control and negative controls (Figure 3A and C). DAPI stained nuclei were enumerated from gray-scale images, each single nuclei of differing sizes and intensity were counted inside each field of view.

[0401] Interestingly, the distribution of DNA in cells with and without mRNA sequence-specific type III- Dv-mediated NucC activation differed (Figure 3B). In the absence of NucC activation (negative controls), RFP typically co-localized uniformly with DAPI (DNA). In contrast, cells with NucC activation upon specific RNA recognition displayed DAPI (DNA) around the periphery of RFP, which remained within nuclei. This DAPI pattern suggests that DNA is being degraded upon NucC activation. Cells displaying this phenotype were classified as dead. Additionally, cells that showed irregular DAPI morphology or smaller than normal DAPI stained nuclei were classified as dead or undergoing cell death. Raw counts of dead cells per field of view are shown in Figure 3C. It is important to note that the dead cell count is likely underestimated as cells that died during the experiment and lost membrane integrity will have lysed and therefore will not be included. Cell lysis was observed clearly in +BRCA1 DAPI-stained panel in Figure 3A, where DAPI stained debris was present. The lack of DAPI-stained nuclei and apparent dispersity of DAPI stain in the BRCA1 and CBK targeted cells in Figure 3A is indicative of early cell death.

[0402] The number of HEK293 cells that showed microRFP670nano fluorescence was counted from grayscale images and is shown in Figure 3C. It can be seen in Figure 3A that RFP intensity varied across cells due to differences in expression level or attenuation from sample fixation. For cells to be considered expressing the type III-Dv system (RFP+), they needed to show morphology consistent with typical HEK293 cell shape and size. Cells showing variation in intensity were counted if they were apparent in the image and showed typical HEK293 cell morphology. To enumerate the number of cells that were dead the phenotypic criteria described above was applied. The raw number of RFP+ cells classified as dead is shown in Figure 2C, for ease of interpretation this has been calculated as a percentage in Figure 3D. In cells where targeting was present and resulted in activation of NucC, there was a significantly higher percentage of dead RFP+ cells than in the absence of specific RNA recognition or capacity to activate NucC. Therefore, the type III-Dv system in concert with NucC caused mRNA sequence specific cell death in HEK293 cells.

[0403] To obtain further insight into mammalian cell killing by the type III-Dv / NucC system, the Applicants analysed cells using different viability and metabolic activity assays (flow cytometry, resazurin and trypan blue). Figure 4A shows typical ungated scatter profiles of live and dead HEK293 cells observed in flow cytometry, and the gating strategies used for analysis of samples. To examine cell death, the Applicants measured a defined volume (25 pL) of each sample by flow cytometry and enumerated the number of RFP+ cells (i.e. those transfected), and the number of viable cells within each sample based on their side scatter (SSC) and forward scatter (FSC) profiles. Targeting of BRCA1 and CKB mRNAs resulted in a significant reduction in cell counts (Figure 4B). There was also an upward shift in SSC upon targeting, indicating an increase in granularity of cells, which is indicative of unhealthy or apoptotic cells

[0025] . Enumeration of viable cells in gate 2 (Figure 4C), confirmed that there was a ~50 % reduction of viable cells in the presence of targeting versus the untargeted control.

[0404] Resazurin assays measure changes in metabolic activity of cells, providing another indication of cell death

[0026] . The assay measures reduction of NADH to NAD+ which in turn converts resazurin to resorufin. The conversion of resazurin will only occur in metabolically active cells and can be detected by resorufin fluorescence. Parallel resazurin assays of cells (Figure 4D) analyzed by flow cytometry in Figure 2D show that in the presence of targeting there was a ~20-30 % decrease in fluorescence. Notably, flow measurements done in parallel indicated a transfection efficiency of ~20-30 % for these samples. When this was used to normalize resazurin data, this was indicative of ~100 % loss of cell metabolic activity in the presence of targeting. Such data correlates to what was shown in Figure 3C from imaging, where most RFP+ cells in the targeted samples showed cell morphology consistent with cell death.

[0405] In an approach complementary to resazurin assays, trypan blue was used to further validate cell death since only dead cells can take up the stain. In the presence of targeting there was a ~50 % reduction in the number of viable cells (Figure 4E). The ratio of dead cells also rose to ~50 % in the presence of targeting versus ~20-30 % in untargeted controls. Such data further validates the findings that together show that when type III-Dv is used in concert with NucC targeted cell killing of mammalian cells occurs.

[0406] Type III-Dv guide RNA can be induced with a tetracycline promoter to cause cell death.

[0407] An application of the type III-Dv / NucC cell killing technology could be in cell therapies, where the system could be employed ex vivo as an in-built kill switch when therapy termination is required. In this way the type III-Dv / NucC system could be used at an appropriate time to terminate cells that show signs of failing or triggering unwanted side-effects. As a specific example, CAR-T cell therapies can sometimes cause toxic side effects in the form of cytokine release syndrome (CRS). The ability to kill or make dormant any chosen cell in a sequence-specific and inducible manner at the level of RNA expression, would enhance the effectiveness of existing and future cell therapies. To control the timing of type III-Dv activity the Applicants replaced the constitutive U6 promoter on the guide RNA with a tetracycline inducible promoter (tet-on). In the absence of the tetracycline inducer, no guide RNA would be expressed, thereby preventing the formation of the type III-Dv complex and subsequent cell killing. The tet-promoter was designed to shuttle the tet-inducible promoter into any of the type III-Dv expression plasmids via Bsal and Spel restriction sites (Figure 5A). To validate our tet-on system, the Applicants first tested whether the constructs could express the type III-Dv complex to suppress gene expression (CRISPRi) after induction with tetracycline. The Applicants previously found the type III-Dv system, with the guide expressed from the constitutive promoter U6, silenced Venus expressed from a reporter plasmid by ~20 %

[0015] . To test the tet-on plasmids, the Applicants co-transfected HEK293T- rtTA cells with tet-inducible CRISPRi vector pPF3951 either with no spacer (non-targeting) or with a spacer targeting the fluorescent yellow reporter Venus, and a plasmid encoding Venus under a constitutive CMV promoter (pPF3328). Tetracycline was added (5 pg / mL) to induce guide RNA expression ~12 hours after transfection, then at ~48 h after transfection the median fluorescent intensity (MFI) of Venus was determined by flow cytometry (Figure 5B). When the type III-Dv complex was expressed with a spacer targeting the Venus transcript, a ~31 % reduction in Venus MFI was observed compared to the untargeted control. This data shows that the tet-on plasmid could express active type III-Dv complex in the presence of inducer tetracycline. Therefore, the Applicants moved on to testing the inducible system for its efficacy to cause targeted cell death. Similar to the conditions tested for tetracycline inducibility of type III-Dv CRISPRi, the Applicants transfected HEK293T-rtTA cells with the cell killing vector pPF3952 either containing no spacer (non-targeting) or a spacer targeting BRCA1 transcripts. To test the sensitivity of the inducible system the Applicants added 0, 0.1, 1 and 5 pg / mL concentrations of tetracycline to induce guide RNA expression. The number of viable RFP+ / type III-Dv-i- cells (i.e. cells containing the type III-Dv expression vector) were determined using flow cytometry by analysing the scattering profile of a standardized 25 pL cell suspension volume. Data obtained for the BRCAl-targeted samples was normalized to the data from the untargeted vector control for each tetracycline concentration. In the absence of tetracycline there was no difference in the number of viable cells between non-targeted and targeted samples, indicating tight control of guide RNA expression (Figure 5C). Upon addition of the lowest concentration of tetracycline, 0.1 pg / mL, an immediate drop of ~71 % of the number of RFP+ / type III-Dv-i- cells was observed when targeting was present. The reduction in the number of viable RFP+ / type III-Dv+ cells did not change with increasing tetracycline concentration, indicating 0.1 ug / mL of tetracycline was sufficient for required levels of guide RNA to complex with the type III-Dv Cas proteins. In summary, the Applicants can control RNA targeting by our type III-Dv system in an inducible manner, thereby controlling the specific killing of human cells. Such data demonstrates the utility of our system for application in cell therapies.

[0408] Gene Blocks, Oligonucleotides and Plasmids

[0409] The following gene blocks, oligonucleotides and plasmids were used in this study. Table 1: Gene blocks used to synthesize plasmids

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417] Table 2: Oligonucleotides used in this study.

[0418] Table 3: Plasmids used and produced in this study.

[0419] Example 2: Programmable cell killing by the type III CRISPR-Cas system in mammalian HeLa cells

[0420] Methods / Materials

[0421] Cell culture

[0422] HeLa cells (Abeam, ab255928) were cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10 % (v / v) foetal calf serum (FCS; Pan Biotech Aidenbach, Germany) and Pen-Strep (100 U / mL penicillin and 100 pg / mL streptomycin; Gibco) at 37°C with 5 % CO2. One day prior to transfection, HeLa cells were seeded into 6-well plates at ~3 x 105cells / mL in 10 % (v / v) FCS / DMEM without Pen-Strep, respectively. HeLa cells were then transfected with 1000 ng total DNA using Lipofectamine 3000 (Thermofisher Scientific, Waltham, MA, USA) as per the manufacturer's protocol. The media was replaced 4 - 6 hours post-transfection, with 10 % (v / v) FCS / DMEM supplemented with Pen-Strep. Cells were then processed for resazurin assays 72-hours post-transfection, for determination of cell killing efficacy.

[0423] Viability assays

[0424] To determine cell metabolic activity, 3 x 90 pL aliquots of cells were removed from each condition after resuspension of cells in growth medium. Each 90 pL aliquot was pipetted into a single well of a sterile 96-well plate. Non-transfected cells, heat-killed cells, and media only wells were included as positive and negative controls, respectively. After a recovery period of ~30 minutes at 37°C with 5 % CO2 10 pL of 10 % w / v resazurin solution was added to each well and left to incubate for 1-4 h at 37°C with 5 % CO2. After conversion of resazurin to resorufin in positive control wells, 80 pL was pipetted to a black 96-well plate with a multichannel pipette. The fluorescence of each well was recorded using a 540 nm excitation I 590 nm emission filter set on a Clariostar plate reader (BMG Labtech).

[0425] Results

[0426] RIMA targeting by type III-Dv CRISPR-Cas system activates NucC and causes cell death in HeLa cells

[0427] To determine if the type III-Dv CRISPR-Cas system activation of NucC could be observed in alternate cell lines, the HeLa cell line was transfected with pPF3816 (Table 3) containing a non-targeting spacer control or a spacer either targeting transcripts from the endogenous genes BRCA1 or CKB. Post 72-hours transfection, resazurin assays were performed on the HeLa cells to determine their metabolic activity (Figure 6). Fluorescence from cells that targeted BRCA1 and CKB transcripts was significantly reduced compared to cells transfected with the non-targeting control. Such data indicates that programmed targeting by the type III-Dv / NucC system severely affects the metabolism of HeLa cells. This data is consistent with the reduced metabolism and cell killing observed in HEK293 cells. Therefore, the type III CRISPR-Cas cell killing approach has utility in multiple cell types.

[0428] Example 3: Programmable cell killing by a modified type III CRISPR-Cas system in mammalian cells

[0429] Methods / Materials

[0430] Construction of vectors for modified type III-Dv CRISPR-Cas expression in mammalian cells

[0431] Vector pPF4217 was constructed for expression of the type III-Dv complex containing modified cas7 domains that ablated their ability to cleave bound target RNA (Table 4). Vector pPF4217 was similar to the original expression vector pPF3816, except for the modified cas7 domains and the nanoRFP gene was replaced with a gene encoding mCherry. Control guide (annealed oligonucleotides PF7303 and PF7304) and CBK targeting guide (annealed oligonucleotides PF7713 and PF7714) were cloned via a Bsal restriction site into pPF4217, creating pPF4252 and pP4253, respectively.

[0432] Cell culture

[0433] Human embryonic kidney cells (HEK293) were cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10 % (v / v) foetal calf serum (FCS; Pan Biotech Aidenbach, Germany) and Pen-Strep (100 U / mL penicillin and 100 pg / mL streptomycin; Gibco) at 37°C with 5 % CO2. One day prior to transfection, HEK293 cells were seeded into 24-well plates ~5 x 104cells / mL in 10 % (v / v) FCS / DMEM without Pen-Strep, respectively. HEK293 cells were then transfected with 500 ng total DNA using Lipofectamine 3000 (Thermofisher Scientific, Waltham, MA, USA) as per the manufacturer's protocol. The media was replaced 6 - 12 hours post-transfection, with 10 % (v / v) FCS / DMEM supplemented with Pen-Strep. Cells were then processed for flow cytometry 72-hours post-transfection, for determination of cell killing efficacy.

[0434] Flow cytometry

[0435] To quantitate the efficiency of cell killing of the type III-Dv system transfected into HEK293 cells, flow cytometry was used. After 72-hours post-transfection, cells were resuspended in their growth medium with 90 pL aliquots removed from each well for viability assays which were performed in parallel to flow cytometry experiments. Cells were then centrifuged at 453 x g for 5 min. Cells were washed in triplicate by resuspending them in 1 mL of pH 7.4 PBS and then centrifuging them at 453 x g for 5 min before resuspension in a final volume of 300 pL wash buffer (pH 7.4 PBS, 0.1 w / v % BSA and 2 mM EDTA) for measurement on a Cytek Aurora Flow cytometer with SpectroFlo 3.03 software (Cytek Biosciences, Fremont, CA, USA). To compare the number of viable cells across different conditions a set volume of 25 pL of cell suspension was run on the flow cytometer. For a negative control, i.e. dead cells, cells were heat killed at 65 °C for 10 min and then washed in a similar fashion as stated above before analysing their scatter profile on the Cytek Aurora Flow cytometer. Cells were gated on SSC-H vs. FSC- H, FSC-H vs. FSC-A and SSC-H vs. SSC-A to identify the singlet population of HEK293 cells. To assess the percentage of viable cells carrying type III-Dv plasmids, cells were gated on mCherry fluorescence (mCherry+) as pPF4217 carries the mCherry gene. Analysis of recorded data was performed using FlowJo software v.10 (BD Biosciences). Data was plotted and analysed using Prism v. 10.1.1 (Graphpad). Statistical analysis was performed using a one-way ANOVA multiple comparison, comparing treatment with targeting spacers to the non-targeting spacer control.

[0436] Table 4: Vector sequence

[0437] Results

[0438] Type III CRISPR-Cas systems have a signal amplification step where once RNA is bound by the type III effector complex, the palm domain of the CaslO subunit produces a large quantity of cOA molecules, which activate accessory proteins to complete the immune response. In addition, the Cas7 subunits in in the type III effector complex have RNase activity and cleave the substrate RNA, which results in termination of cOA production when the RNA threat from the invader is no longer present. Previous in vitro studies have shown that modification of the Cas7 subunits maintains substrate RNA binding but prevents cleavage, which results in continued cOA production and increased activity by effector proteins

[0028] . The Applicants predict that modification of Cas7 in the context of cell killing would improve the sensitivity of the system to target RNA transcripts of low abundance. To investigate if the type III-Dv complex with modified Cas7 domains maintains cell killing ability, the Applicants modified the RNase active sites of the three Cas7 domains (Cas7(D26A)-Cas5-Casl l and Cas7(D33A')-Cas7(D246A')') in vector pPF4217. Vector pPF4217 also included a gene encoding mCherry to to identify transfected cells by flow cytometry.

[0439] The Applicants found that the type III-Dv complex with abrogated Cas7-dependent RNase activity maintained cell killing ability in HEK293 cells, where flow cytometry demonstrated ~50 % reduction in total live cells when transfected with a CKB RNA targeting complex versus the untargeted control (Figure 7A). This result was similar to data shown in Example 1. In mCherry+ cells, viable cells were reduced to ~30 % levels in HEK293 cells expressing a type III-Dv complex targeting CKB transcripts versus the untargeted control (Figure 7B). In summary, such data shows that mutation of Cas7 in type III-Dv retains cell killing capability.

[0440] Example 4: Endogenous mRNA targeting by III-A CRISPR-Cas activates NucC and causes cell death in human cells

[0441] Methods / Materials

[0442] Construction of type III-A CRISPR-Cas vectors for expression in mammalian cells

[0443] The Streptococcus thermophilus type III-A expression plasmid with gene expression optimised for human expression (pDAC569; Addgene plasmid # 195241) was selected to test cell death by an alternative type III system. To engineer the type III-A expression plasmid for cell killing, gene block PF8778 encoding NucC was PCR amplified (using primers PF209 and 210) and then cloned via a BamHI restriction site into pDAC569, creating pPF4214. Refer to Table 4 for the DNA sequence of pPF4214. Control guide (annealed oligonucleotides PF8914 and PF8915) and BRCA1 targeting guide (annealed oligonucleotides PF8793 and PF8794) were cloned via a BbsI restriction site, creating pPF4236 and pP4237, respectively.

[0444] Cell culture

[0445] Human embryonic kidney cells (HEK293) were cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10 % (v / v) foetal calf serum (FCS; Pan Biotech Aidenbach, Germany) and Pen-Strep (100 U / mL penicillin and 100 pg / mL streptomycin; Gibco) at 37°C with 5 % CO2. One day prior to transfection, HEK293 cells were seeded into 24-well plates ~5 x 104cells / mL in 10 % (v / v) FCS / DMEM without Pen-Strep, respectively. HEK293 cells were then transfected with 500 ng total DNA using Lipofectamine 3000 (Thermofisher Scientific, Waltham, MA, USA) as per the manufacturer's protocol. The media was replaced 6 - 12 hours post-transfection, with 10 % (v / v) FCS / DMEM supplemented with Pen-Strep. Cells were then processed for flow cytometry 72-hours post-transfection, for determination of cell killing efficacy.

[0446] Flow cytometry

[0447] To quantitate the efficiency of cell killing of the type III-Dv system transfected into HEK293 cells, flow cytometry was used. After 72-hours transfection, cells were resuspended in their growth medium with 90 pL aliquots removed from each well for viability assays which were performed in parallel to flow cytometry experiments. Cells were then centrifuged at 453 x g for 5 min. Cells were washed in triplicate by resuspending them in 1 mL of pH 7.4 PBS and then centrifuging them at 453 x g for 5 min before resuspension in a final volume of 300 pL wash buffer (pH 7.4 PBS, 0.1 w / v % BSA and 2 mM EDTA) for measurement on a Cytek Aurora Flow cytometer with SpectroFlo 3.03 software (Cytek Biosciences, Fremont, CA, USA). To compare the number of viable cells across different conditions a set volume of 25 pL of cell suspension was run on the flow cytometer. For a negative control, i.e. dead cells, cells were heat killed at 65 °C for 10 min and then washed in a similar fashion as stated above before analysing their scatter profile on the Cytek Aurora Flow cytometer. Cells were gated on SSC-H vs. FSC-H, FSC-H vs. FSC-A and SSC-H vs. SSC-A to identify the singlet population of HEK293 cells. To assess the percentage of viable cells carrying the III-A plasmid cells were gated on GFP fluorescence (GFP+) as pPF4214 carries a GFP protein. Analysis of recorded data was performed using FlowJo software v.10 (BD Biosciences). Data was plotted and analysed using Prism v. 10.1.1 (Graphpad). Statistical analysis was performed using a one-way ANOVA multiple comparison, comparing treatment with targeting spacers to the non-targeting spacer control.

[0448] Viability assays

[0449] To determine cell metabolic activity 1 x 90 pL aliquot of cells was removed from each condition after resuspension of cells in growth medium. Each 90 pL aliquot was pipetted into a single well of a sterile 96-well plate. Non-transfected cells, heat-killed cells, and media only wells were included as positive and negative controls, respectively. After a recovery period of ~30 minutes at 37°C with 5 % CO2 10 pL of 10 % w / v resazurin solution was added to each well and left to incubate for 1-4 h at 37°C with 5 % CO2. After conversion of resazurin to resorufin in positive control wells 80 pL was pipetted to a black 96-well plate with a multichannel pipette. Fluorescence of each well was recorded using a 540 nm excitation I 590 nm emission filter set on a Clariostar plate reader (BMG Labtech).

[0450] Results

[0451] The Applicant sought further evidence that cell death can be triggered in response to specific detection of RIMA by other diverse CRISPR-Cas type III systems that activate accessory enzymes. Therefore, the Applicants engineered a type III-A system to complement their previous results with the type III-Dv system. Plasmid pDAC569

[0027] encodes the type III-A system from Streptococcus thermophilus with codon usage optimised for expression in human cells. This plasmid was previously used to demonstrate specific RNA targeting in mammalian cell lines, including BRCA1 transcripts in HEK293 cells

[0027] . The type III-A CRISPR Cas system from S. thermophilus is an active immune system

[0018] that expresses a range of cOA molecules, including cA3, which is compatible with NucC [17,29]. The Applicants cloned NucC into the type III-A expression vector, along with either a non-targeting control guide or a guide that targets BRCA1 transcripts.

[0452] The Applicants demonstrated that when the type III-A CRISPR-Cas expression vectors were transfected into HEK293 cells and analyzed by flow cytometry, targeting the BRCA1 transcript caused a ~30 % reduction in the number of total live cells compared with an untargeted control (Figure 8A). In GFP+ HEK293 cells, targeting the BRCA1 transcript caused a ~35 % reduction compared to the untargeted control (Figure 8B). To assess the viability of HEK293 cells in the presence of BRCA1 targeting versus an untargeted control, resazurin assays were performed in parallel to flow experiments. Data shown in Figure 8C shows a ~14 % reduction in fluorescence in cells where BRCA1 was targeted versus the untargeted control. This indicates a decrease in metabolism in cells when the type III-A CRISPR-Cas complex specifically binds BRCA1 RNA transcripts and the activation of NucC. Such data shows that additional type III systems, in addition to type III-Dv, can be utilized for RNA specific cell killing of mammalian cells. Example 5: Type III activation of diverse accessory effectors (Csxl and TIR-SAVED) with different cOA specificity and downstream mechanisms of action trigger mammalian cell death in response to detection of specific mRNAs

[0453] Methods / Materials

[0454] Construction of vectors for expression of modified type III-Dv CRISPR-Cas with different effectors

[0455] Vector pPF4254 for expression of type III-Dv CRISPR-Cas with accessory protein Csxl from Synechocystis sp. PCC6803 was constructed by cloning gene block PF8789 into pPF4217 via Xmal and Nsil restriction sites (Table 4). Control guide (annealed oligonucleotides PF7303 and PF7304) and CBK targeting guide (annealed oligonucleotides PF7713 and PF7714) were cloned via Bsal restriction site, creating pPF4256 and pP4257, respectively.

[0456] Vector pPF4255 for expression of type III-Dv CRISPR-Cas with accessory protein TIR-SAVED from Microbacterium ketosireducens

[0030] was constructed by cloning gene block PF8790 into pPF4217 via Xmal and Nsil restriction sites (Table 4). Control guide (annealed oligonucleotides PF7303 and PF7304) and CBK targeting guide (annealed oligonucleotides PF7713 and PF7714) were cloned via Bsal restriction site, creating pPF4258 and pP4259, respectively.

[0457] Cell culture

[0458] Human embryonic kidney cells (HEK293) were cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10 % (v / v) foetal calf serum (FCS; Pan Biotech Aidenbach, Germany) and Pen-Strep (100 U / mL penicillin and 100 pg / mL streptomycin; Gibco) at 37°C with 5 % CO2. One day prior to transfection, HEK293 cells were seeded into 24-well plates ~5 x 104cells / mL in 10 % (v / v) FCS / DMEM without Pen-Strep, respectively. HEK293 cells were then transfected with 500 ng total DNA using Lipofectamine 3000 (Thermofisher Scientific, Waltham, MA, USA) as per the manufacturer's protocol. The media was replaced 6 - 12 hours post-transfection, with 10 % (v / v) FCS / DMEM supplemented with Pen-Strep. Cells were then processed for flow cytometry 72-hours post-transfection, for determination of cell killing efficacy.

[0459] Flow cytometry

[0460] To quantitate the efficiency of cell killing of the type III-Dv CRISPR-Cas system in HEK293 cells, flow cytometry was used. After 72-hours transfection, cells were resuspended in their growth medium with 90 pL aliquots removed from each well for viability assays which were performed in parallel to flow cytometry experiments. Cells were then centrifuged at 453 x g for 5 min. Cells were washed in triplicate by resuspending them in 1 mL of pH 7.4 PBS and then centrifuging them at 453 x g for 5 min before resuspension in a final volume of 300 pL wash buffer (pH 7.4 PBS, 0.1 w / v % BSA and 2 mM EDTA) for measurement on a Cytek Aurora Flow cytometer with SpectroFlo 3.03 software (Cytek Biosciences, Fremont, CA, USA). To compare the number of viable cells across different conditions a set volume of 25 pL of cell suspension was run on the flow cytometer. For a negative control, i.e. dead cells, cells were heat killed at 65 °C for 10 min, and then washed in a similar fashion as stated above before analysing their scatter profile on the Cytek Aurora Flow cytometer. Cells were gated on SSC-H vs. FSC-H, FSC-H vs. FSC-A and SSC-H vs. SSC-A to identify the singlet population of HEK293 cells. To assess the percentage of viable cells carrying type III-Dv CRISPR-Cas plasmids, cells were gated on mCherry fluorescence (mCherry+) as both plasmids containing the accessory proteins carry an mCherry fluorophore gene. Analysis of recorded data was performed using FlowJo software v.10 (BD Biosciences). Data was plotted and analysed using Prism v. 10.1.1 (Graphpad). Statistical analysis was performed using a one-way ANOVA multiple comparison, comparing treatment with targeting spacers to the nontargeting spacer control.

[0461] Viability assays

[0462] To determine cell metabolic activity 1 x 90 pL aliquot of cells was removed from each condition after resuspension of cells in growth medium. Each 90 pL aliquot was pipetted into a single well of a sterile 96-well plate. Non-transfected cells, heat-killed cells, and media only wells were included as positive and negative controls, respectively. After a recovery period of ~30 minutes at 37°C with 5 % CO2 10 pL of 10 % w / v resazurin solution was added to each well and left to incubate for 1-4 h at 37°C with 5 % CO2. After conversion of resazurin to resorufin in positive control wells 80 pL was pipetted to a black 96-well plate with a multichannel pipette. Fluorescence of each well was recorded using a 540 nm excitation I 590 nm emission filter set on a Clariostar plate reader (BMG Labtech).

[0463] Results

[0464] The Applicants wanted to determine if 1) effectors with different mechanisms and 2) cOA preferences could trigger mammalian cell death. To test the first question, the Applicants selected an RNase (Csxl) from Synechocystis sp. PCC6803 and a NADase (TIR-SAVED) from from Microbacterium ketosireducens. These proteins also respond to different cOAs; Csxl is activated by cA4 and TIR-SAVED by cA3, while NucC is activated by cA3. Further, the mechanism these proteins bind their cOA molecules is different, where Csxl uses a CARF domain, TIR-SAVED uses a SAVED domain and NucC possess a unique mechanism.

[0465] Data obtained from flow cytometry analysis of HEK293 cells transfected with the type III-Dv CRISPR-Cas complex targeting CKB transcripts and the TIR-SAVED protein caused a ~50 % reduction in the number of live cells compared with an untargeted control Figure 9A and B. In mCherry+ cells, targeting CBK transcripts caused a ~40 % reduction in the number of live cells. In resazurin viability assays, targeting of CKB RNA transcripts caused a ~50 % drop in fluorescence compared to the untargeted control, indicative of a reduction in cellular metabolism in the presence of CRISPR-Cas type III targeting and TIR-SAVED activity. Activation of the TIR-SAVED protein, which results in cleavage of cellular NAD+, to reduce mammalian cell viability demonstrates that the CRISPR-Cas type III system can be exploited to reduce cell viability in multiple ways.

[0466] Another accessory domain, Csxl, which is an ancillary RNase that co-occurs with the type III-Dv CRISPR-Cas interference complex system in Synechocystis sp. PCC6803, was tested for ceil killing efficacy. Flow cytometry data shown in Figure 9D and E shows that in the presence of targeting of CKB RNA transcripts by the type III-Dv complex there is a ~50 and 60 % reduction in the number of viable HEK293 ceils observed in total and mCherry+ populations, respectively. In support of this data, parallel viability assays showed a ~40 % reduction in fluorescence, indicative of a loss in metabolic function of HEK293 cells in the presence of targeting. Such data illustrates that the activation of the accessory protein Csxl by type III-Dv CRISPR-Cas targeting results in toxicity and targeted ceil death. Overall, the data in Figure 9 supports the claim that multiple accessory domains, such as NucC, TIR-SAVED and Csxl can be used in concert with CRISPR-Cas type III RNA targeting systems to cause directed celi death in mammaiian celis when specific RNA transcripts are targeted.

[0467] SEQUENCES

[0468] SEO ID NO: 1. Type III-Dv caslO DNA sequence (GenBank: BAD01969.1 )

[0469] ATGTTTCTAGTTCTAATTGAGACTTCCGGTAATCAGCATTTTATTTTCTCGACTAATAAACTAAGGGAAAAT ATTGGTGCATCAGAGTTGACCTATCTTGCTACAACGGAAATATTGTTCCAGGGGGTGGATAGGGTTTTCCAGACT AACTACTATGACCAATGGTCTGACACAAACTCCCTAAATTTTTTGGCAGATAGTAAGCTTAATCCCGCCATTGATG ATCCTAAAAATAACGCTGACATTGAAATTTTATTGGCTACCTCTGGAAAGGCGATCGCCCTGGTGAAAGAAGAGG GCAAGGCTAAACAATTAATTAAAGAAGTTACCAAGCAGGCCCTAATCAATGCCCCGGGTTTAGAAATTGGTGGTA TTTATGTGAATTGTAATTGGCAAGATAAATTAGGGGTTGCCAAAGCAGTTAAAGAAGCCCATAAACAGTTCGAAG TAAATAGGGCTAAACGGGCTGGGGCTAATGGTCGCTTTTTGCGGTTACCGATCGCCGCTGGGTGCAGTGTAAGT GAATTGCCTGCCTCTGATTTTGACTATAATGCCGATGGTGACAAGATTCCTGTTTCTACAGTCAGTAAAGTTAAAC GGGAGACTGCGAAATCTGCCAAAAAACGTTTGCGGAGCGTTGATGGTCGGCTAGTTAACGACCTAGCACAATTA GAAAAGTCCTTTGACGAATTAGATTGGTTAGCAGTGGTCCATGCCGATGGTAATGGTTTGGGGCAAATTTTACTA AGTCTTGAGAAATATATTGGTGAGCAAACAAACCGCAATTATATTGATAAATATCGTAGACTTTCTTTAGCCCTGG ATAACTGCACCATCAACGCTTTTAAAATGGCGATCGCTGTCTTCAAAGAAGATTCCAAAAAAATTGATTTACCCAT TGTCCCATTGATTTTAGGTGGAGATGACCTAACGGTAATTTGTCGGGGGGACTACGCCCTAGAATTCACCAGGG AATTTCTTGAAGCATTTGAAGGGCAGACAGAAACACATGATGATATCAAAGTAATAGCCCAAAAAGCCTTTGGCG TTGATCGCCTTTCTGCCTGCGCTGGGATCAGTATTATTAAGCCCCATTTTCCCTTCTCTGTTGCCTATACTTTGGC GGAAAGATTAATTAAATCAGCTAAGGAGGTCAAACAAAAAGTTACTGTGACAAATAGTTCGCCAATAACTCCTTT TCCCTGCTCTGCCATTGATTTTCATATTCTCTATGACAGTAGCGGCATTGATTTTGACCGTATTCGTGAAAAATTA CGGCCGGAAGATAATACCGAGCTTTACAACCGTCCCTATGTGGTGACAGCAGCGGAGAACCTCAGCCAAGCCCA GGGTTATGAATGGTCCCAGGCCCACAGTTTGCAAACACTAGCGGATCGGGTTAGTTATTTACGTTCCGAAGATG GGGAAGGAAAATCTGCATTACCCAGCAGTCAAAGCCATGCCCTACGAACGGCATTGTACCTAGAGAAAAATGAA GCAGACGCTCAATATAGCTTAATTAGCCAACGCTACAAAATTCTCAAAAACTTTGCGGAGGACGGAGAGAATAAA TCACTATTTCATCTCGAAAATGGCAAGTACGTCACCAGA I I I I I AGATGCACTGGATGCCAAAGA I I I I I I I GCTA ACGCTAACCATAAAAACCAAGGAGAATAA

[0470] SEO ID NO: 2. Type III-Dv CaslO protein sequence (GenBank: BAD01969.1 ) HD and palm domains are in bold / underline

[0471] MFLVLIETSGNQHFIFSTNKLRENIGASELTYLATTEILFQGVDRVFQTNYYDQWSDTNSLNFLADSKLNPAID DPKNNADIEILLATSGKAIALVKEEGKAKQLIKEVTKQALINAPGLEIGGIYVNCNWQDKLGVAKAVKEAHKQFEVNR AKRAGANGRFLRLPIAAGCSVSELPASDFDYNADGDKIPVSTVSKVKRETAKSAKKRLRSVDGRLVNDLAQLEKSFD ELDWLAVVHADGNGLGQILLSLEKYIGEQTNRNYIDKYRRLSLALDNCTINAFKMAIAVFKEDSKKIDLPIVPLILGGD DLTVICRGDYALEFTREFLEAFEGQTETHDDIKVIAQKAFGVDRLSACAGISIIKPHFPFSVAYTLAERLIKSAKEVKQ KVTVTNSSPITPFPCSAIDFHILYDSSGIDFDRIREKLRPEDNTELYNRPYVVTAAENLSQAQGYEWSQAHSLQTLAD RVSYLRSEDGEGKSALPSSQSHALRTALYLEKNEADAQYSLISQRYKILKNFAEDGENKSLFHLENGKYVTRFLDALD AKDFFANANHKNQGE SEO ID NO: 3. Type III-Dv Cas7-5-ll DNA sequence (GenBank: BAD01968.1 )

[0472] ATGCGAGGAATTGAGATAACCATAACCATGCAGAGTGATTGGCACGTTGGCACTGGCATGGGTCGGGGG

[0473] GAACTGGACAGTGTTGTACAACGGGATGGAGATAATCTGCCCTATATTCCCGGCAAAACCTTAACAGGTATTCTG

[0474] CGGGATAGCTGTGAACAGGTTGCCCTAGGTTTAGATAATGGTCAAACCCGAGGGCTTTGGCATGGGTGGATTAA

[0475] TTTTATTTTTGGCGATCAACCTGCCCTAGCTCAAGGAGCTATTGAGCCAGAACCTAGACCTGCCCTAATCGCCAT

[0476] TGGTTCTGCACACCTTGACCCTAAGTTAAAAGCGGCTTTTCAGGGCAAAAAACAATTGCAAGAGGCGATCGCCTT

[0477] TATGAAGCCAGGGGTGGCTATCGATGCAATCACGGGCACAGCTAAGAAAGATTTTTTACGCTTTGAAGAAGTAG

[0478] TTCGTTTGGGAGCGAAATTAACTGCGGAAGTTGAGTTAAATTTACCCGATAATTTGAGCGAAACCAATAAAAAAG

[0479] TTATTGCTGGTATTTTAGCCAGTGGAGCAAAGTTAACCGAGAGATTAGGCGGTAAACGTCGCCGGGGCAATGGG

[0480] CGCTGTGAATTAAAATTTAGTGGTTATTCTGATCAACAAATTCAATGGTTGAAAGACAATTATCAATCTGTTGATC

[0481] AACCACCTAAGTATCAACAAAATAAATTACAATCTGCCGGAGATAATCCAGAACAGCAACCCCCTTGGCATATTA

[0482] TTCCCTTAACCATTAAAACCCTTTCTCCTGTTGTTTTACCAGCTCGTACAGTCGGTAACGTTGTCGAATGTTTAGA

[0483] CTATATTCCCGGGCGTTATCTACTGGGCTATATTCACAAAACCCTAGGGGAATATTTCGACGTTAGTCAGGCAAT

[0484] CGCCGCTGGGGATTTAATTATTACCAATGCCACGATAAAAATTGATGGTAAAGCAGGACGAGCTACCCCATTTTG

[0485] TTTGTTTGGGGAAAAACTAGATGGAGGATTAGGTAAAGGTAAAGGAGTTTATAACCGTTTCCAAGAATCGGAAC

[0486] CTGATGGCATTCAATTAAAGGGAGAACGGGGCGGCTATGTTGGCCAATTTGAACAGGAGCAAAGGAATCTGCCA

[0487] AATACGGGGAAAATTAATTCAGAGTTATTTACCCATAACACCATTCAAGATGATGTCCAGCGGCCCACCAGTGAT

[0488] GTGGGGGGAGTTTATAGCTATGAAGCTATTATAGCCGGACAAACATTCGTCGCTGAGTTACGTTTACCAGATAG

[0489] CTTAGTCAAGCAAATTACAAGCAAAAATAAAAATTGGCAAGCTCAACTAAAAGCTACAATTCGCATTGGTCAGTC

[0490] TAAAAAAGATCAGTATGGCAAAATCGAAGTTACGTCGGGAAACTCTGCTGATTTGCCTAAGCCTACGGGCAACA

[0491] ATAAAACTCTTTCTATTTGGTTCTTATCCGATATCCTTCTCCGAGGCGATCGCCTAAATTTTAATGCTACTCCGGA

[0492] TGATCTCAAAAAATACTTAGAAAATGCTCTGGATATCAAGCTCAAAGAACGATCAGACAATGATTTAATTTGCATT

[0493] GCTCTCCGTTCCCAGCGGACAGAATCCTGGCAAGTACGGTGGGGTTTACCCCGGCCATCTCTAGTGGGTTGGCA

[0494] AGCTGGTAGTTGTCTGATTTATGACATTGAATCTGGCACTGTTAATGCCGAAAAATTGCAAGAATTAATGATCAC

[0495] CGGCATTGGCGATCGGTGTACAGAGGGTTACGGTCAAATCGGTTTTAACGATCCATTACTTTCGGCTTCCCTAGG

[0496] AAAGTTGACAGCTAAGCCTAAAGCTTCTAACAATCAGTCCCAAAACAGCCAATCCAACCCATTACCCACTAATCAT

[0497] CCTACCCAAGATTATGCTCGATTAATTGAAAAAGCGGCTTGGCGGGAAGCAATTCAAAATAAAGCCTTAGCCTTG

[0498] GCATCTAGCCGAGCGAAACGGGAAGAAATTTTAGGCATTAAAATTATGGGAAAAGATAGTCAACCCACCATGAC

[0499] TCAATTAGGAGGATTTCGCTCCGTATTAAAACGGCTACACTCAAGAAATAATCGAGATATTGTCACAGGTTATTTA

[0500] ACAGCTCTAGAGCAGGTTTCTAATCGAAAAGAAAAATGGAGTAATACCAGCCAAGGATTAACTAAAATTCGTAAT

[0501] TTAGTCACCCAGGAAAATCTCATTTGGAATCATCTTGATATTGATTTTTCGCCGTTAACTATTACCCAAAATGGTG

[0502] TTAATCAGCTAAAGTCTGAACTTTGGGCGGAAGCAGTGCGAACCCTTGTTGACGCTATCATTCGGGGTCATAAAC

[0503] GGGACTTAGAAAAAGCTCAAGAAAACGAATCTAATCAACAGTCACAGGGAGCAGCTTAA

[0504] SEO ID NO: 4. Type III-Dv Cas7-5-l l protein sequence GenBank: BAD01968.1 ) Cleavage residue is in bold / underline

[0505] MRGIEITITMQSDWHVGTGMGRGELDSVVQRDGDNLPYIPGKTLTGILRDSCEQVALGLDNGQTRGLWHG WINFIFGDQPALAQGAIEPEPRPALIAIGSAHLDPKLKAAFQGKKQLQEAIAFMKPGVAIDAITGTAKKDFLRFEEVVR LGAKLTAEVELNLPDNLSETNKKVIAGILASGAKLTERLGGKRRRGNGRCELKFSGYSDQQIQWLKDNYQSVDQPP KYQQNKLQSAGDNPEQQPPWHIIPLTIKTLSPVVLPARTVGNVVECLDYIPGRYLLGYIHKTLGEYFDVSQAIAAGDLI

[0506] ITNATIKIDGKAGRATPFCLFGEKLDGGLGKGKGVYNRFQESEPDGIQLKGERGGYVGQFEQEQRNLPNTGKINSEL

[0507] FTHNTIQDDVQRPTSDVGGVYSYEAIIAGQTFVAELRLPDSLVKQITSKNKNWQAQLKATIRIGQSKKDQYGKIEVT

[0508] SGNSADLPKPTGNNKTLSIWFLSDILLRGDRLNFNATPDDLKKYLENALDIKLKERSDNDLICIALRSQRTESWQVR

[0509] WGLPRPSLVGWQAGSCLIYDIESGTVNAEKLQELMITGIGDRCTEGYGQIGFNDPLLSASLGKLTAKPKASNNQSQ

[0510] NSQSNPLPTNHPTQDYARLIEKAAWREAIQNKALALASSRAKREEILGIKIMGKDSQPTMTQLGGFRSVLKRLHSRN

[0511] NRDIVTGYLTALEQVSNRKEKWSNTSQGLTKIRNLVTQENLIWNHLDIDFSPLTITQNGVNQLKSELWAEAVRTLVD

[0512] AIIRGHKRDLEKAQENESNQQSQGAA

[0513] SEO ID NO: 5. Type III-Dv Cas7 2x DNA sequence (GenBank: BAD01967.1 )

[0514] ATGGCTAGAAAAGTTACTACACGCTGGAAAATTACAGGCACATTAATTGCAGAAACCCCTTTACACATTGG TGGTGTGGGTGGCGACGCTGATACGGATTTAGCCCTGGCGGTTAATGGTGCGGGTGAATATTATGTGCCAGGG ACAAGTTTAGCCGGTGCTCTGCGGGGTTGGATGACCCAGTTATTGAATAATGATGAGTCCCAAATTAAAGATCTT TGGGGTGATCATTTAGATGCAAAACGGGGAGCTAGCTTTGTTATTGTTGACGATGCGGTTATCCATATACCCAAT

[0515] AATGCTGATGTTGAAATTAGGGAGGGTGTTGGCATCGATCGCCATTTTGGAACCGCCGCCAATGGGTTTAAATA

[0516] TAGCCGAGCAGTTATTCCCAAGGGTTCTAAATTTAAATTGCCATTAACTTTTGACAGTCAAGATGATGGGCTACC GAATGCGTTGATTCAATTGTTGTGTGCCTTAGAAGCAGGGGATATTCGCCTTGGGGCCGCAAAAACCCGGGGTT TAGGTCGCATTAAACTAGATGATTTAAAGTTAAAATCCTTTGCTTTAGATAAACCAGAAGGTATTTTTTCTGCTTTA TTAGACCAAGGTAAAAAATTAGATTGGAATCAATTAAAAGCAAACGTTACCTACCAGTCTCCTCCCTATCTAGGTA

[0517] TTAGTATTACCTGGAATCCCAAAGATCCCGTCATGGTGAAAGCTGAAGGGGATGGACTGGCGATCGATATTTTG CCCCTCGTTAGTCAAGTGGGAAGTGATGTTCGATTTGTCATTCCCGGCAGTTCCATTAAGGGGATTTTACGAACC CAGGCTGAACGTATTATTCGTACTATTTGCCAGTCTAATGGTTCTGAGAAAAACTTCCTAGAACAATTACGAATCA ATCTGGTTAATGAATTATTTGGGTCTGCTTCTTTGAGCCAAAAACAAAATGGCAAGGATATAGATCTGGGTAAAA

[0518] TCGGAGCCTTGGCAGTGAATGATTGTTTTTCTAGTTTATCCATGACCCCAGATCAATGGAAAGCGGTAGAGAATG CCACGGAGATGACGGGGAATTTACAGCCTGCTCTTAAACAAGCTACGGGTTATCCCAATAATATTAGCCAAGCTT ACAAAGTACTTCAACCGGCCATGCACGTCGCTGTAGATCGGTGGACAGGGGGAGCTGCCGAAGGAATGCTTTA CAGCGTGCTCGAACCCATTGGGGTCACCTGGGAACCGATCCAAGTTCACTTGGACATTGCCCGTCTCAAAAATT

[0519] ATTACCACGGTAAGGAAGAAAAACTTAAACCGGCGATCGCCCTATTGCTTCTTGTATTGCGGGATTTAGCTAACA AAAAAATTCCCGTAGGCTATGGCACTAACCGCGGTATGGGAACGATTACTGTCAGTCAAATCACCCTCAATGGCA AAGCCCTCCCCACTGAACTTGAACCTTTAAACAAAACAATGACTTGTCCTAATCTCACCGATCTAGATGAGGCATT TCGTCAGGACTTAAGCACTGCTTGGAAAGAGTGGATTGCCGATCCCATTGATCTATGCCAGCAGGAGGCCGCCT

[0520] AA

[0521] SEO ID NO: 6. Type III-Dv Cas7 2x protein sequence (GenBank: BAD01967.1 ) Cleavage residues are in bold / underline

[0522] MARKVTTRWKITGTLIAETPLHIGGVGGDADTDLALAVNGAGEYYVPGTSLAGALRGWMTQLLNNDESQIK DLWGDHLDAKRGASFVIVDDAVIHIPNNADVEIREGVGIDRHFGTAANGFKYSRAVIPKGSKFKLPLTFDSQDDGLP NALIQLLCALEAGDIRLGAAKTRGLGRIKLDDLKLKSFALDKPEGIFSALLDQGKKLDWNQLKANVTYQSPPYLGISIT WNPKDPVMVKAEGDGLAIDILPLVSQVGSDVRFVIPGSSIKGILRTQAERIIRTICQSNGSEKNFLEQLRINLVNELFG SASLSQKQNGKDIDLGKIGALAVNDCFSSLSMTPDQWKAVENATEMTGNLQPALKQATGYPNNISQAYKVLQPAM HVAVDRWTGGAAEGMLYSVLEPIGVTWEPIQVHLDIARLKNYYHGKEEKLKPAIALLLLVLRDLANKKIPVGYGTNRG MGTITVSQITLNGKALPTELEPLNKTMTCPNLTDLDEAFRQDLSTAWKEWIADPIDLCQQEAA

[0523] SEO ID NO: 7. Type III-Dv csx!9 DNA sequence (GenBank: BAD01966.1 )

[0524] ATGCCAGCAGGAGGCCGCCTAATGAAGAACCTTTACCACTACCACCAATATGAAATTACCCTCGAATCCG

[0525] CCGTCGATTCTTGCAAAAACCATCTCCAAGCGGCGATCGGGCTGTTGTATTCTCCCCAAAAGTGTGAACTAGTCA

[0526] AACTGGATAACTCAGGCAAGTTAGTTGATTCTTACAATCGTCTTAAGTTCAATAACCTAGGCGTATTTGAAGCCC

[0527] GCTTCTTTAATCTCAATTGTGAACTGCGATGGGTCAATGAATCTAATGGTAATGGCACTGCCGTCTTGCTTTCAG

[0528] AATCGGATATTACCTTAACTGGTTTTGAGAAAGGTTTACAGGAATTTATTACGGCGATCGACCAACAGTATTTACT

[0529] CTGGGGTGAACCCGCTAAACATCCCCCTAATGCTGATGGCTGGCAACGACTAGCGGAAGCAAGGATCGGGAAA

[0530] CTCGATATTCCCCTCGATAACCCGTTAAAACCCAAAGATCGAGTTTTTCTCACCAGCGAAGAGTACATTGCTGAA GTAGATGATTTTGGTAATTGTGCCGTTATTGACGAACGTTTAATTAAATTGGAGGTTAAGTAA

[0531] SEO ID NO: 8. Type III-Dv Csxl9 protein sequence GenBank: BAD01966.1 )

[0532] MPAGGRLMKNLYHYHQYEITLESAVDSCKNHLQAAIGLLYSPQKCELVKLDNSGKLVDSYNRLKFNNLGVFE ARFFNLNCELRWVNESNGNGTAVLLSESDITLTGFEKGLQEFITAIDQQYLLWGEPAKHPPNADGWQRLAEARIGKL DIPLDNPLKPKDRVFLTSEEYIAEVDDFGNCAVIDERLIKLEVK

[0533] SEO ID NO: 9. Type III-Dv Cas7-insert DNA sequence (GenBank: BAD01965.1 )

[0534] ATGACAGTCGGAACATTGGGCGTTGTTGGCAGTGCTAAAAACCTCAAATTACAACTTAGTTTTATCAACAC AAGGCAACAGTATGTTCAAATAACACTTTTTGAGCGAAATTCTTTTAAGGTTGCTGAGGAAGAATTTTCTACTGAA CTTGTGGAAATCATTAAAACAGCACTACCAACTCTCAAAAATAAAAAAGTTGAATTTGAGGAAGATGGCGATCAA ATTAAACAAATCCGAGAAAAAGGTCAAGCTTGGGTTGGTGCCGCAGAACAGATTGCACCTTATGTTCTTCCTTCT GGAAATATTACTGAAACACCCAGAAATGTTAACGCTAGCAACTTTCATAACCCCTACAACTTTGTCCCAGCCCTAC CCCGCGATGGCATAACCGGAGATTTAGGCGACTGTGCTCCTGCTGGTCATAGCTATTACCATGGCGATAAATAC AGCGGCAGAATTGCCGTCAAACTAACAACCGTTACCCCTCTATTGATTCCTGACGCTTCAAAAGAAGAGATAAAT AACAACCATAAAACCTATCCGGTTCGTATCGGCAAAGATGGCAAGCCCTATCTACCTCCCACTTCCATTAAGGGA ATGTTGCGCTCTGCCTATGAAGCGGTCACTAATTCCCGCTTAGCCGTGTTTGAAGATCATGACTCTCGCTTGGCC TATCGAATGCCTGCCACCATGGGATTGCAAATGGTTCCTGCCCGCATTGAAGGTGATAATATTGTTCTTTACCCA GGAACCTCAAGGATAGGCAATAATGGCCGACCAGCTAACAATGATCCTATGTATGCGGCATGGCTTCCTTACTAT CAAAATCGTATTGCTTATGATGGTAGTCGTGATTATCAGATGGCTGAGCATGGTGATCATGTCAGATTTTGGGCT GAGCGATATACCAGAGGAAACTTCTGCTATTGGCGTGTCAGACAAATTGCACGACACAATCAAAATTTAGGTAAT CGGCCTGAACGAGGACGTAATTACGGTCAACATCATTCAACAGGAGTCATTGAACAATTTGAAGGATTTGTTTAC AAAACCAATAAAAATATTGGGAATAAACATGACGAACGAGTATTTATTATTGATCGAGAAAGTATCGAAATACCTC TATCTCGAGATTTACGGCGAAAATGGCGAGAATTAATTACAAGCTATCAGGAAATACACAAAAAGGAAGTTGATA GAGGTGATACTGGCCCTTCCGCTGTAAATGGGGCTGTTTGGTCACGGCAAATTATTGCAGATGAATCAGAGCGG AATTTATCGGATGGGACTCTTTGTTATGCTCATGTTAAGAAAGAAGATGGACAGTACAAAATTCTCAATCTTTATC CTGTAATGATCACACGGGGATTATATGAAATTGCGCCGGTTGACTTATTAGATGAAACCCTAAAGCCTGCGACGG

[0535] ATAAAAAGCAACTATCCCCAGCAGACCGCGTATTTGGCTGGGTCAATCAACGGGGCAATGGTTGCTACAAAGGA

[0536] CAATTACGAATTCATAGCGTAACTTGCCAACATGATGATGCCATTGATGATTTTGGTAATCAAAATTTCTCTGTTC

[0537] CCCTTGCTATTTTGGGACAACCTAAACCAGAACAGGCTCGTTTTTATTGTGCCGATGATCGAAAAGGAATTCCTTT

[0538] AGAAGATGGCTATGATCGTGACGACGGCTATAGTGATTCAGAACAAGGCTTGCGAGGACGCAAAGTCTATCCTC

[0539] ACCACAAGGGGTTACCAAATGGCTACTGGAGTAATCCAACGGAAGACCGAAGTCAACAAGCTATCCAAGGTCAT

[0540] TACCAAGAATATCGTCGTCCTAAAAAGGATGGTCTTGAACAAAGAGATGATCAAAATCGTTCTGTAAAAGGTTGG

[0541] GTAAAACCACTGACCGAGTTTACTTTTGAAATTGACGTTACTAATCTTTCGGAAGTTGAGTTAGGTGCTCTATTGT

[0542] GGTTGTTAACCTTACCTGATTTGCATTTCCACCGTCTAGGAGGAGGTAAACCGTTAGGTTTTGGTAGTGTTCGTT

[0543] TAGATATTGACCCTGACAAGACAGACCTAAGAAATGGGGCAGGATGGCGTGATTATTACGGCTCTTTACTAGAA

[0544] ACAAGTCAACCAGATTTTACAACTCTAATTAGTCAGTGGATTAATGCTTTTCAAACGGCTGTTAAAGAGGAGTATG

[0545] GTAGCAGTAGTTTTGATCAGGTTACTTTCATCAAAGCTTCTGGTCAGAGTCTCCAAGGATTTCATGATAATGCATC

[0546] TATCCATTATCCTCGTTCTACTCCTGAGCCCAAGCCAGATGGAGAAGCTTTTAAGTGGTTTGTTGCCAATGAAAA

[0547] AGGTCGACGATTAGCCTTGCCAGCGCTGGAAAAATCCCAGAGTTTTCCAATCAAACCTAGTTAA

[0548] SEO ID NO: 10. Type III-Dv Cas7-insert protein sequence (GenBank: BAD01965.1 )

[0549] MTVGTLGVVGSAKNLKLQLSFINTRQQYVQITLFERNSFKVAEEEFSTELVEIIKTALPTLKNKKVEFEEDGDQ IKQIREKGQAWVGAAEQIAPYVLPSGNITETPRNVNASNFHNPYNFVPALPRDGITGDLGDCAPAGHSYYHGDKYSG RIAVKLTTVTPLLIPDASKEEINNNHKTYPVRIGKDGKPYLPPTSIKGMLRSAYEAVTNSRLAVFEDHDSRLAYRMPAT MGLQMVPARIEGDNIVLYPGTSRIGNNGRPANNDPMYAAWLPYYQNRIAYDGSRDYQMAEHGDHVRFWAERYTRG NFCYWRVRQIARHNQNLGNRPERGRNYGQHHSTGVIEQFEGFVYKTNKNIGNKHDERVFIIDRESIEIPLSRDLRRK WRELITSYQEIHKKEVDRGDTGPSAVNGAVWSRQIIADESERNLSDGTLCYAHVKKEDGQYKILNLYPVMITRGLYE IAPVDLLDETLKPATDKKQLSPADRVFGWVNQRGNGCYKGQLRIHSVTCQHDDAIDDFGNQNFSVPLAILGQPKPE QARFYCADDRKGIPLEDGYDRDDGYSDSEQGLRGRKVYPHHKGLPNGYWSNPTEDRSQQAIQGHYQEYRRPKKD GLEQRDDQNRSVKGWVKPLTEFTFEIDVTNLSEVELGALLWLLTLPDLHFHRLGGGKPLGFGSVRLDIDPDKTDLRN GAGWRDYYGSLLETSQPDFTTLISQWINAFQTAVKEEYGSSSFDQVTFIKASGQSLQGFHDNASIHYPRSTPEPKPD GEAFKWFVANEKGRRLALPALEKSQSFPIKPS

[0550] SEO ID NO: 11. Type III-Dv Cas6-2a DNA sequence (GenBank: BAD01970.1 )

[0551] GTGGTGGATCTAAAATCCTTAGCTGGGGCCGAAATGGTGGGATTACGCTGGCAACTGCGCTTCGACCGC CCCTGTCGCCTGGAAAGTCATTACGTTAAAGGACTCCATGCTTGGTTTTTGCATCAAGTGCAGGCCATTGATCCC GATGTTTCTGCCTGGCTCCATGATGGTCAAGGGGAAAAGCCCTTCACCATTTCCCGCCTGATAGGGCCTACCCT CTGGCAAGAAGGTCATTGGCACTGGCAAATAAATAAGACCTACCATTGGCAATTAAATTTACTATCAGGGGCTTT AATCGAAGCTTTACAACCTTGGCTAGCCCGTTTGCCAAACAAAATTGTCCTAGCTCGCCAAACATTATGGGTAGA AGCCGTTGATTGTTACCTAGCCCCCCATAACTATCAACAGTTATGGCCCCAGGGTGCTTTACCCCGACGGCAAGA GTTTACTTTCACTAGCCCTACCAGTTTCCGTCGCCAAGGCAATCACTATCCGTTACCAGAGCCCCGCAATGTTCT GCAAAGTTATCTACGGCGTTGGAATGATTTTTCTGGTTTGGCGTTCGAGCCGGAGCCATTTTTGGACTATTGGGT GCCCCAAAATGTGGTGATCGATCGCCATTGGTTGGAGTCGGTGAAGACCACAGCGGGAAAACAAGGCTCAGTG GTGGGATTTGTGGGAGCAGTGTCCCTAGTCCTTACGCCCCAGGCCCGTAATGATGGGGATGATTATGGCCGCTT GTTCCATGCCCTCTGTCGATATGGACCCTACTGTGGCACTGGGCATAAAACCACCTTTGGTTTGGGGCAAACAAT GGCGGGCTGGGCTACCCCGGACCTAAAAACTTTTGCGTGCCTCCAAGAAGATTTACAGACTCAGGTGTTAACGC AACGGATAGATCAATGCGCCTCTCTCCTCCTAGCCCAGCGTCAACGGACAGGAGGGCAGAGAGCCCAGGAAAT TTGCCATACGCTAGCCACTATTTTTGTCCGCCGAGAACAGGGGGAATCATTGCAAGAAATCGCCCTGGATTTACA GTTACCTTATGAGACAGCCCGCACCTACAGCAAACGAGCTAAGCGGGCCTTAGCCAATGTTCAATAA

[0552] SEO ID NO: 12. Type III-Dv Cas6-2a protein sequence (GenBank: BAD01970.1 )

[0553] VVDLKSLAGAEMVGLRWQLRFDRPCRLESHYVKGLHAWFLHQVQAIDPDVSAWLHDGQGEKPFTISRLIGP TLWQEGHWHWQINKTYHWQLNLLSGALIEALQPWLARLPNKIVLARQTLWVEAVDCYLAPHNYQQLWPQGALPRR QEFTFTSPTSFRRQGNHYPLPEPRNVLQSYLRRWNDFSGLAFEPEPFLDYWVPQNVVIDRHWLESVKTTAGKQGSV VGFVGAVSLVLTPQARNDGDDYGRLFHALCRYGPYCGTGHKTTFGLGQTMAGWATPDLKTFACLQEDLQTQVLTQ RIDQCASLLLAQRQRTGGQRAQEICHTLATIFVRREQGESLQEIALDLQLPYETARTYSKRAKRALANVQ

[0554] SEO ID NO: 13. Type III-Dv Dead cas7 2x.l and cas7 2x.2 DNA sequence (BAD01967.1 :c.98A>C; c.737A>O modified positions are in bold / underline

[0555] ATGGCTAGAAAAGTTACTACACGCTGGAAAATTACAGGCACATTAATTGCAGAAACCCCTTTACACATTGG

[0556] TGGTGTGGGTGGCGACGCTGATACGGCTTTAGCCCTGGCGGTTAATGGTGCGGGTGAATATTATGTGCCAGGG ACAAGTTTAGCCGGTGCTCTGCGGGGTTGGATGACCCAGTTATTGAATAATGATGAGTCCCAAATTAAAGATCTT

[0557] TGGGGTGATCATTTAGATGCAAAACGGGGAGCTAGCTTTGTTATTGTTGACGATGCGGTTATCCATATACCCAAT AATGCTGATGTTGAAATTAGGGAGGGTGTTGGCATCGATCGCCATTTTGGAACCGCCGCCAATGGGTTTAAATA

[0558] TAGCCGAGCAGTTATTCCCAAGGGTTCTAAATTTAAATTGCCATTAACTTTTGACAGTCAAGATGATGGGCTACC GAATGCGTTGATTCAATTGTTGTGTGCCTTAGAAGCAGGGGATATTCGCCTTGGGGCCGCAAAAACCCGGGGTT

[0559] TAGGTCGCATTAAACTAGATGATTTAAAGTTAAAATCCTTTGCTTTAGATAAACCAGAAGGTATTTTTTCTGCTTTA

[0560] TTAGACCAAGGTAAAAAATTAGATTGGAATCAATTAAAAGCAAACGTTACCTACCAGTCTCCTCCCTATCTAGGTA

[0561] TTAGTATTACCTGGAATCCCAAAGATCCCGTCATGGTGAAAGCTGAAGGGGATGGACTGGCGATCGCTATTTTG CCCCTCGTTAGTCAAGTGGGAAGTGATGTTCGATTTGTCATTCCCGGCAGTTCCATTAAGGGGATTTTACGAACC

[0562] CAGGCTGAACGTATTATTCGTACTATTTGCCAGTCTAATGGTTCTGAGAAAAACTTCCTAGAACAATTACGAATCA ATCTGGTTAATGAATTATTTGGGTCTGCTTCTTTGAGCCAAAAACAAAATGGCAAGGATATAGATCTGGGTAAAA

[0563] TCGGAGCCTTGGCAGTGAATGATTGTTTTTCTAGTTTATCCATGACCCCAGATCAATGGAAAGCGGTAGAGAATG CCACGGAGATGACGGGGAATTTACAGCCTGCTCTTAAACAAGCTACGGGTTATCCCAATAATATTAGCCAAGCTT

[0564] ACAAAGTACTTCAACCGGCCATGCACGTCGCTGTAGATCGGTGGACAGGGGGAGCTGCCGAAGGAATGCTTTA CAGCGTGCTCGAACCCATTGGGGTCACCTGGGAACCGATCCAAGTTCACTTGGACATTGCCCGTCTCAAAAATT

[0565] ATTACCACGGTAAGGAAGAAAAACTTAAACCGGCGATCGCCCTATTGCTTCTTGTATTGCGGGATTTAGCTAACA

[0566] AAAAAATTCCCGTAGGCTATGGCACTAACCGCGGTATGGGAACGATTACTGTCAGTCAAATCACCCTCAATGGCA AAGCCCTCCCCACTGAACTTGAACCTTTAAACAAAACAATGACTTGTCCTAATCTCACCGATCTAGATGAGGCATT TCGTCAGGACTTAAGCACTGCTTGGAAAGAGTGGATTGCCGATCCCATTGATCTATGCCAGCAGGAGGCCGCCT

[0567] AA SEO ID NO: 14. Type III-Dv Dead Cas7 2x.l and Cas7 2x.2 protein sequence (BAD01967.1 :p.D33A: P.D246A) modified residue is in bold / underline

[0568] MARKVTTRWKITGTLIAETPLHIGGVGGDADTALALAVNGAGEYYVPGTSLAGALRGWMTQLLNNDESQIK DLWGDHLDAKRGASFVIVDDAVIHIPNNADVEIREGVGIDRHFGTAANGFKYSRAVIPKGSKFKLPLTFDSQDDGLP NALIQLLCALEAGDIRLGAAKTRGLGRIKLDDLKLKSFALDKPEGIFSALLDQGKKLDWNQLKANVTYQSPPYLGISIT WNPKDPVMVKAEGDGLAIAILPLVSQVGSDVRFVIPGSSIKGILRTQAERIIRTICQSNGSEKNFLEQLRINLVNELFG SASLSQKQNGKDIDLGKIGALAVNDCFSSLSMTPDQWKAVENATEMTGNLQPALKQATGYPNNISQAYKVLQPAM HVAVDRWTGGAAEGMLYSVLEPIGVTWEPIQVHLDIARLKNYYHGKEEKLKPAIALLLLVLRDLANKKIPVGYGTNRG MGTITVSQITLNGKALPTELEPLNKTMTCPNLTDLDEAFRQDLSTAWKEWIADPIDLCQQEAA

[0569] SEO ID NO: 15 Type III-Dv Dead cas7-5-ll DNA sequence (BAD01968.1 :c.77A>C) modified positions are in bold and underlined.

[0570] ATGCGAGGAATTGAGATAACCATAACCATGCAGAGTGATTGGCACGTTGGCACTGGCATGGGTCGGGGG GAACTGCCAGTGTTGTACAACGGGATGGAGATAATCTGCCCTATATTCCCGGCAAAACCTTAACAGGTATTCTGC GGGATAGCTGTGAACAGGTTGCCCTAGGTTTAGATAATGGTCAAACCCGAGGGCTTTGGCATGGGTGGATTAAT TTTATTTTTGGCGATCAACCTGCCCTAGCTCAAGGAGCTATTGAGCCAGAACCTAGACCTGCCCTAATCGCCATT GGTTCTGCACACCTTGACCCTAAGTTAAAAGCGGCTTTTCAGGGCAAAAAACAATTGCAAGAGGCGATCGCCTTT ATGAAGCCAGGGGTGGCTATCGATGCAATCACGGGCACAGCTAAGAAAGATTTTTTACGCTTTGAAGAAGTAGT TCGTTTGGGAGCGAAATTAACTGCGGAAGTTGAGTTAAATTTACCCGATAATTTGAGCGAAACCAATAAAAAAGT ATGCGAGGAATTGAGATAACCATAACCATGCAGAGTGATTGGCACGTTGGCACTGGCATGGGTCGGGGGGAAC TGCCAGTGTTGTACAACGGGATGGAGATAATCTGCCCTATATTCCCGGCAAAACCTTAACAGGTATTCTGCGGG ATAGCTGTGAACAGGTTGCCCTAGGTTTAGATAATGGTCAAACCCGAGGGCTTTGGCATGGGTGGATTAATTTTA TTTTTGGCGATCAACCTGCCCTAGCTCAAGGAGCTATTGAGCCAGAACCTAGACCTGCCCTAATCGCCATTGGTT CTGCACACCTTGACCCTAAGTTAAAAGCGGCTTTTCAGGGCAAAAAACAATTGCAAGAGGCGATCGCCTTTATGA AGCCAGGGGTGGCTATCGATGCAATCACGGGCACAGCTAAGAAAGATTTTTTACGCTTTGAAGAAGTAGTTCGT TTGGGAGCGAAATTAACTGCGGAAGTTGAGTTAAATTTACCCGATAATTTGAGCGAAACCAATAAAAAAGTTATT GCTGGTATTTTAGCCAGTGGAGCAAAGTTAACCGAGAGATTAGGCGGTAAACGTCGCCGGGGCAATGGGCGCT GTGAATTAAAATTTAGTGGTTATTCTGATCAACAAATTCAATGGTTGAAAGACAATTATCAATCTGTTGATCAACC ACCTAAGTATCAACAAAATAAATTACAATCTGCCGGAGATAATCCAGAACAGCAACCCCCTTGGCATATTATTCCC TTAACCATTAAAACCCTTTCTCCTGTTGTTTTACCAGCTCGTACAGTCGGTAACGTTGTCGAATGTTTAGACTATA TTCCCGGGCGTTATCTACTGGGCTATATTCACAAAACCCTAGGGGAATATTTCGACGTTAGTCAGGCAATCGCCG CTGGGGATTTAATTATTACCAATGCCACGATAAAAATTGATGGTAAAGCAGGACGAGCTACCCCATTTTGTTTGT TTGGGGAAAAACTAGATGGAGGATTAGGTAAAGGTAAAGGAGTTTATAACCGTTTCCAAGAATCGGAACCTGAT GGCATTCAATTAAAGGGAGAACGGGGCGGCTATGTTGGCCAATTTGAACAGGAGCAAAGGAATCTGCCAAATAC GGGGAAAATTAATTCAGAGTTATTTACCCATAACACCATTCAAGATGATGTCCAGCGGCCCACCAGTGATGTGGG GGGAGTTTATAGCTATGAAGCTATTATAGCCGGACAAACATTCGTCGCTGAGTTACGTTTACCAGATAGCTTAGT CAAGCAAATTACAAGCAAAAATAAAAATTGGCAAGCTCAACTAAAAGCTACAATTCGCATTGGTCAGTCTAAAAA AGATCAGTATGGCAAAATCGAAGTTACGTCGGGAAACTCTGCTGATTTGCCTAAGCCTACGGGCAACAATAAAA CTCTTTCTATTTGGTTCTTATCCGATATCCTTCTCCGAGGCGATCGCCTAAATTTTAATGCTACTCCGGATGATCT CAAAAAATACTTAGAAAATGCTCTGGATATCAAGCTCAAAGAACGATCAGACAATGATTTAATTTGCATTGCTCTC

[0571] CGTTCCCAGCGGACAGAATCCTGGCAAGTACGGTGGGGTTTACCCCGGCCATCTCTAGTGGGTTGGCAAGCTG

[0572] GTAGTTGTCTGATTTATGACATTGAATCTGGCACTGTTAATGCCGAAAAATTGCAAGAATTAATGATCACCGGCA

[0573] TTGGCGATCGGTGTACAGAGGGTTACGGTCAAATCGGTTTTAACGATCCATTACTTTCGGCTTCCCTAGGAAAGT

[0574] TGACAGCTAAGCCTAAAGCTTCTAACAATCAGTCCCAAAACAGCCAATCCAACCCATTACCCACTAATCATCCTAC

[0575] CCAAGATTATGCTCGATTAATTGAAAAAGCGGCTTGGCGGGAAGCAATTCAAAATAAAGCCTTAGCCTTGGCATC

[0576] TAGCCGAGCGAAACGGGAAGAAATTTTAGGCATTAAAATTATGGGAAAAGATAGTCAACCCACCATGACTCAATT

[0577] AGGAGGATTTCGCTCCGTATTAAAACGGCTACACTCAAGAAATAATCGAGATATTGTCACAGGTTATTTAACAGC

[0578] TCTAGAGCAGGTTTCTAATCGAAAAGAAAAATGGAGTAATACCAGCCAAGGATTAACTAAAATTCGTAATTTAGT

[0579] CACCCAGGAAAATCTCATTTGGAATCATCTTGATATTGATTTTTCGCCGTTAACTATTACCCAAAATGGTGTTAAT

[0580] CAGCTAAAGTCTGAACTTTGGGCGGAAGCAGTGCGAACCCTTGTTGACGCTATCATTCGGGGTCATAAACGGGA

[0581] CTTAGAAAAAGCTCAAGAAAACGAATCTAATCAACAGTCACAGGGAGCAGCTTAA

[0582] SEO ID NO: 16 Type III-Dv Dead Cas7-5-ll protein sequence (BAD01968.1:p.D26A) modified residues are in bold and underlined.

[0583] MRGIEITITMQSDWHVGTGMGRGELASVVQRDGDNLPYIPGKTLTGILRDSCEQVALGLDNGQTRGLWHGWINFI FGDQPALAQGAIEPEPRPALIAIGSAHLDPKLKAAFQGKKQLQEAIAFMKPGVAIDAITGTAKKDFLRFEEVVRLGAKL TAEVELNLPDNLSETNKKVIAGILASGAKLTERLGGKRRRGNGRCELKFSGYSDQQIQWLKDNYQSVDQPPKYQQN KLQSAGDNPEQQPPWHIIPLTIKTLSPVVLPARTVGNVVECLDYIPGRYLLGYIHKTLGEYFDVSQAIAAGDLIITNATI KIDGKAGRATPFCLFGEKLDGGLGKGKGVYNRFQESEPDGIQLKGERGGYVGQFEQEQRNLPNTGKINSELFTHNTI QDDVQRPTSDVGGVYSYEAIIAGQTFVAELRLPDSLVKQITSKNKNWQAQLKATIRIGQSKKDQYGKIEVTSGNSA DLPKPTGNNKTLSIWFLSDILLRGDRLNFNATPDDLKKYLENALDIKLKERSDNDLICIALRSQRTESWQVRWGLPRP SLVGWQAGSCLIYDIESGTVNAEKLQELMITGIGDRCTEGYGQIGFNDPLLSASLGKLTAKPKASNNQSQNSQSNPL PTNHPTQDYARLIEKAAWREAIQNKALALASSRAKREEILGIKIMGKDSQPTMTQLGGFRSVLKRLHSRNNRDIVTG

[0584] YLTALEQVSNRKEKWSNTSQGLTKIRNLVTQENLIWNHLDIDFSPLTITQNGVNQLKSELWAEAVRTLVDAIIRGHK RDLEKAQENESNQQSQGAA

[0585] SEO ID NO: 23 - Example unprocessed type III-Dv guide RNA (spacer bold / underline)

[0586] ACUGAAACUGUAGUAGAACCAAUCGGGGUCGUCAAUAACUCCCGGTTCAACACCCTCTTTTCCCCG TCAGGGG

[0587] SEO ID NO: 24 - Example mature type III-Dv guide RNA (spacer bold / underline)

[0588] ACUGAAACUGUAGUAGAACCAAUCGGGGUCGUCAAUA

[0589] SEO ID N0:25 Type III-Dv RNA seouence tested (protospacer bold / underline)

[0590] CAUGACGGAUCGCGGGAGUUAUUGACGACCCCGAUUGGUUCUACUACAAACGUGAUACUA

[0591] SEO ID NO:26 Type III-Dv CRISPR array spacer TGTAGTAGAACCAATCGGGGTCGTCAATAACTCCCG

[0592] SEO ID N0:27 Type III-Dv CRISPR array flanking repeat

[0593] GTTCAACACCCTCTTTTCCCCGTCAGGGGACTGAAAC

[0594] SEO ID NO: 28. NucC DNA sequence (GenBank: CP025084.1 )

[0595] AAGACGGACTCGGAAAGGCATTTACTGAAAATTTGCAGGCTGTTTCCAGCGATGAAAACCGAAAACTGGA

[0596] TTGCGGTCTGGCGGTGTCGGGCGCATGTTTTGATAGTTATGATGAGGAAATAAAAATCAGAAGCGGTGAAAATG

[0597] CATTAATCTTTTTTCTGTTCCGTTTGCTCGGTAAATTGCAATCATTAGGTACGGTGCCCGCAATTGACTGGCGGGT

[0598] GTATATAGATAGTCTGGAATAA

[0599] SEO ID NO: 29. NucC protein sequence (GenBank: CP025084.1 )

[0600] MTNQAKKLSRINGREFLKQSFNLQQQLLASQLNLSRTITHDGTMGEVN ESYFLSIIRQYLPERYSVDRGVV VDSEGQTSDQIDAVIFDRHYTPTLLDQQGHRFIPAEAVYAVLEVKPTINKTYLEYAADKAASVRKLYRTSTVIKNIYGT AKPVEHFPIVAGIVAIDVEWQDGLGKAFTENLQAVSSDENRKLDCGLAVSGACFDSYDEEIKIRSGENALIFFLFRLL GKLQSLGTVPAIDWRVYIDSLE

[0601] SEO ID NO: 30. csxl DNA sequence (GenBank: BADO 1963.12

[0602] ATGAAGATCATCAGCTTCCTGGGCTTCAACAACTACCAGACCACCACCTACGTGCACCCTACAAACCCTG AGATTACATGCAAGACCAGATTCTTCCAGGAGGCTCTCGTGGACTTCTACAGACCTACCGAAATCTACGTGCTGC TGACCAAGACCGTGGCCACAGTTGCTCCTAGAGGCGCTGAAGTGACCAACTGGCAGGGCCTGCAGGACTGTCT AGCAGATAAGGTCACCATCCGGCCCATCGAGGATATCCCTGAGGGAAGCGCTCCTGAGGACCTGTGGGACATC TTCGATTGCATCACAGATTGCCTGGACCACGGCGATAGAGTGATCTTCGACTTCACCCACGGCTACAGATTCCTG CCCGTGGTTGCTCTGCTGGCCATCTCCTACCTGAGAACCGTGCGACAGGTTCAGGTCGAAGGAGTGCTGTACGG AGCTTTCGATCCTAACAGCCAAGGCGAGTCGAGCCCAACATACGACCTGCTGCCTATGCTGTCTCTCCTGGATT GGCTGGCTGCTACAGATCGGTTCGTGAACCTGGGTGACGGACTTCCTCTGGCTCAGCTGCTGCAAACAGCCATC CCTGGAGCCGAAAGGCGGGACAATCCTGAGGTGCGCAATCCAGGCAGCAGACTGGATCAGGCCGGCAAGGTG ATCGCCGAGATCAGCCAGGCTATCGCTCTCGCTAGACCCATGGAAACACTGGAACTGACCGTGCAACTGGAAGA GATCATCAACAGAGCCTCCGACAGCTTCGATCAGAGAGCTAAGCCTTTCAACCTGATCAAGGACCAGCTTCTGG CTGAATACGGACAGTTCGCTCTTCCCGAAAGTTGGGAGCCTGAAAACCTGCAGAGAAACCTGTGGCTGCAGTTT CAGCTGATCAACTGGTATCTGCAAAGAGGCCAGGCTGTGCAGGCCATGACCCTGGCTTCTGAGTGGCTCATCAG TGTGGTCGCCTTTAGACTGGGCGCTAAACAGATTCTGGATCACAGAAAGCAGATCAACTTCGCCCTGAACAACG GAGGCGCTGTGCAAAAGAAGCGGGAACCCGTTGGCCCTAGCAGCTTTGATGAGCAGTTTCAGGCCCTGTCCCA GTACCAGTTCCTGAGCCAGCTGTGGAACGACCTGACAGAGATCAGAAATGATCTGGCCCACTGCGGTATGCGGA AAGACCCCAAGTCCGCCAAAAAACTGCAAGAGAAGGCCAACAAGATCTTCCCTCAGCTCGAGGAAATCGCCAAT AGCCTGCTGAAGCAGTAG SEO ID NO: 31. Csxl protein sequence (GenBank: BAD01963.1 )

[0603] MKIISFLGFNNYQTTTYVHPTNPEITCKTRFFQEALVDFYRPTEIYVLLTKTVATVAPRGAEVTNWQGLQDCLA DKVTIRPIEDIPEGSAPEDLWDIFDCITDCLDHGDRVIFDFTHGYRFLPVVALLAISYLRTVRQVQVEGVLYGAFDPNS QGESSPTYDLLPMLSLLDWLAATDRFVNLGDGLPLAQLLQTAIPGAERRDN PEVRNPGSRLDQAGKVIAEISQAIALA RPMETLELTVQLEEIINRASDSFDQRAKPFNLIKDQLLAEYGQFALPESWEPENLQRNLWLQFQLINWYLQRGQAVQ AMTLASEWLISVVAFRLGAKQILDHRKQINFALNNGGAVQKKREPVGPSSFDEQFQALSQYQFLSQLWNDLTEIRN DLAHCGMRKDPKSAKKLQEKANKIFPQLEEIANSLLKQ

[0604] SEO ID NO: 32. tir-saved DNA sequence (GenBank: JOD63 RS03545)

[0605] ATGCCTGATACCGCCATCAACCCTCGGGACCCTGTGTTCGTGTCCTACAGACACAGCGACGGTATTGCCC TGGCCGCTGAACTGACATGGCTGCTGCGAGCTGCCGGCATCCCCGTGTGGCGGGACGTGGACGACCTGCCTCC TGGCGACACAGACGCCAGACTCCAGCAGGCCATTGACGAAGGCATCAGCGGCGCTGTTATCATCATCACCCCAC AGATCGCCGATAGCAGAGTGGTCAGAGAAGTGGAAGCCCCTCGGCTGCTGCGGCTGCACAGAAGCAGCCCTCA GTTTGCCCTGGGCATCGTGAACGCCATCCAGACCAGCACAGGCGTGGTTGATTACGACGCACCCGATAGAGTG CTGGGCATGGAAAGACCGGAACTGAGGTCTGTGGACCAGAAGAGCGCCTCCAGACTGGGACTCGTGACAATGG CCAGACAGATGCTGTGGCACCGGATCGCCGCCATTAGACCCCTGCTGAGCGCTTCTGGCGGAGAACTGAGACT GTCCCTGCAAACAAGAAATACCCCTCAAGTGTACGACAGAACCGACGCCGACCTGGACATCCGGATCAGACCTT CTGCTCACGAGAAGCTGCCCAGCGCCCATGGACTCGAGGACTTCGCCGAGACAGCCCAGTTCCTGCCTGACGC TGTGACCAGAGCCGGCGCCAATGGCGTGCGGATCGAGGGCGGCGCCCACCTGAGCGTGTCCATCGCCATCGG CGCCGCCATCCCCAGCACCAGAGTGGGACCTATGACCGTGGTGGACGGCAGAGGCGTTCACTGGGTGAGCAGC ACCGAGCCTCAGCTGCCTGATGAGCCTAGACTGAGAATCGTGAGAGAGAGCACCATCCCAAGCACAGCCCCAG CCCCAGGAAGACCTGATGTGGCCGCCTACATCGACCTGCAGCATCCTAGATCCGATGCTGCCTTCGACAACTAC CTGACCGAGCACGCCGCCGAGCTGGTGGCTTGGCAGCACCTGGCTCCCACCAGAACAGGCCTGCTTGATGCCG CCGACGGAGGAACAATCGCCGCCGAAGCCGTGGCCCACATCAGAGAGCTGAGCATGACCAACGGCAACGCCGT GGTGCACCTGATGGTGAGAGGCCCTTTCGGCCTGGCCGTGCTGATCGGCCGGCTGACCAACACCCTGCGGGTG GTGGCCTATGAGTGGACCGACAGCGACGCCCCTGACGGCACCTTTATGCCCCCCCGCTACGAGCCTATCGTGCA GCTGAGAGCCTCTACACCTGCCGGCGTGATCGAGCGGGTCATCGTCGCCGATGCTGAATGA

[0606] SEO ID NO: 33. TIR-SAVED protein sequence GenBank: WP 045274410.1 )

[0607] MPDTAINPRDPVFVSYRHSDGIALAAELTWLLRAAGIPVWRDVDDLPPGDTDARLQQAIDEGISGAVIIITPQ IADSRVVREVEAPRLLRLHRSSPQFALGIVNAIQTSTGVVDYDAPDRVLGMERPELRSVDQKSASRLGLVTMARQML WHRIAAIRPLLSASGGELRLSLQTRNTPQVYDRTDADLDIRIRPSAHEKLPSAHGLEDFAETAQFLPDAVTRAGANGV RIEGGAHLSVSIAIGAAIPSTRVGPMTVVDGRGVHWVSSTEPQLPDEPRLRIVRESTIPSTAPAPGRPDVAAYIDLQH PRSDAAFDNYLTEHAAELVAWQHLAPTRTGLLDAADGGTIAAEAVAHIRELSMTNGNAVVHLMVRGPFGLAVLIGRL TNTLRVVAYEWTDSDAPDGTFMPPRYEPIVQLRASTPAGVIERVIVADAE

[0608] SEO ID NO: 34 Type III- A csml DNA sequence (GenBank: DGCC7710 RS0103910) TTGAAGAAAGAAAAGATTGATTTATTTTACGGAGCTCTTTTGCATGATATCGGTAAGGTCATTCAAAGGG

[0609] CGACAGGAGAACGAAAAAAACACGCCTTGGTAGGCGCGGATTGGTTTGATGAGATTGCTGATAATCAAGTTATT

[0610] TCCGATCAAATTAGATATCACATGGCTAACTACCAGAGTAATAAACTTGGAAATGACCATCTTGCTTACATAACTT

[0611] ATATCGCTGATAACATTGCCTCTGGTGTCGACAGAAGACAGTCAAATGAGGAGAGTGACGAGGATGCATCAGCT

[0612] AAGATTTGGGATACCTATACAAACCAGGCTGATATTTTTAACATTTTTGGGGCACAAACGGATAAACGCTACTTTA

[0613] AACCGACGGTTCTAAACTTGAAATCTAAACCTAACTTTGCGTCGGCAACATATGAACCTTTCTCAAAAGGTGATTA

[0614] TGCGGCAATTGCGACTCGTATCAAAAATGAATTGGCAGAATTTGAGTTTAATCAAGCACAAATTGACTCTTTGTTA

[0615] AATCTGTTCGAAGCAACCCTCTCTTTTGTGCCTTCTTCGACTAATACTAAAGAAATCGCTGATATTTCACTTGCTG

[0616] ATCATAGTCGTCTGACAGCAGCTTTTGCTCTAGCCATCTATGATTACTTGGAAGACAAAGGTCGTCATAACTATAA

[0617] GGAGGACTTGTTTACTAAAGTATCAGCCTTTTATGAGGAAGAAGCTTTTCTCCTAGCTAGCTTTGACTTATCAGG

[0618] GATTCAAGACTTTATCTATAATATTAATATTGCGACGAATGGTGCTGCTAAACAATTGAAGGCTAGATCTTTATAT

[0619] CTTGACTTTATGAGCGAGTATATAGCAGACAGTTTACTTGATAAACTAGGCCTCAATCGGGCTAATATGCTCTAT

[0620] GTCGGTGGGGGACATGCTTACTTTGTCCTAGCCAATACTGAAAAAACGGTAGAAACACTCGTTCAATTTGAAAAA

[0621] GATTTCAATCAATTTTTATTGGCAAATTTCCAAACCAGATTATATGTTGCCTTTGGTTGGGGAAGCTTTGCGGCTA

[0622] AGGATATCATGAGCGAACTGAACTCACCTGAAAGCTATAGACAGGTCTATCAAAAGGCTAGTCGCATGATTTCTA

[0623] AGAAAAAAATCTCAAGGTATGATTATCAAACCCTTATGTTGTTGAACAGGGGCGGTAAATCTTCTGAAAGAGAGT

[0624] GCGAGATTTGTCATTCCGTTGAGAATTTAGTTTCTTATCATGACCAAAAAGTGTGTGACATTTGTCGAGGCTTGTA

[0625] TCAATTTTCTAAAGAGATTGCCCATGACCATTTCATTATCACTGAAAATGAAGGGCTTCCTATTGGTCCGAACGCA

[0626] TGTCTTAAAGGTGTTGCATTTGAAAAGCTGAGCCAAGAAGCTTTTTCCCGTGTCTATGTCAAAAATGACTATAAG

[0627] GCTGGTACAGTTAAGGCAACGCATGTTTTTGTTGGAGATTACCAGTGTGATGAAATATACAATTATGCTGCCTTA

[0628] TCTAAAAACGAAAATGGGTTAGGTATTAAACGTTTAGCTGTTGTACGTCTTGACGTGGATGATTTGGGAGCAGCC

[0629] TTTATGGCTGGCTTCTCCCAACAAGGAAATGGGCAATATAGTACTCTATCACGCTCAGCCACTTTCTCTCGAAGC

[0630] ATGAGTCTTTTCTTCAAGGTTTATATTAACCAGTTTGCTAGTGATAAGAAGCTCTCTATCATCTATGCTGGTGGGG

[0631] ATGATGTTTTTGCTATTGGCTCTTGGCAAGATATTATTGCCTTTACTGTTGAACTTCGTGAGAACTTCATTAAATG

[0632] GACAAATGGAAAACTAACACTATCAGCTGGTATCAGTCTGTTTGCTGATAAGACCCCTATTAGATTAATGGCACA

[0633] TCAAACAGGGGAGCTAGAAGAAGCAGCTAAAGGCAATGAGAAAGATAGTATTTCACTCTTTAGTTCCGACTATAC

[0634] CTTTAAATTTGATCGGTTTATCACTAATGTTTACGACGATAAGTTAGAGCAGATTCGCTATTTCTTTAATCACCAA

[0635] GATGAACGAGGCAAGAATTTCATTTATAAATTGATTGAATTGCTTCGAAATTATGATCGTATGAATATGGCACGTT

[0636] TAGCTTATTATTTAACACGACTTGAAGAATTGACGCGTGAAACAGACAGGGATAAATTTAAAACATTTAAAAATTT

[0637] ATTCTATTCTTGGTACACAAATAAGGATGATAAGGATAGAAAAGAAGCAGAGTTAGCCTTGCTTCTCTATATCTAT

[0638] GAGATTAGAAAGGATTAG

[0639] SEO ID NO: 35 Type III-A Csml protein sequence GenBank: WP 024704102.1 )

[0640] RLAVVRLDVDDLGAAFMAGFSOOGNGOYSTLSRSATFSRSMSLFFKVYINOFASDKKLSIIYAGGDDVFAI

[0641] GSWODIIAFTVELRENFIKWTNGKLTLSAGISLFADKTPIRLMAHOTGELEEAAKGNEKDSISLFSSDYTFKFDRFITN

[0642] VYDDKLEOIRYFFNHODERGKNFIYKLIELLRNYDRMNMARLAYYLTRLEELTRETDRDKFKTFKNLFYSWYTNKDDK DRKEAELALLLYIYEIRKD

[0643] SEO ID NO: 36 Type III-A csm2 DNA sequence (GenBank: DGCC7710 RS0103915) ATGACAATCTTGACTGATGAGAATTACGTTGATATTGCAGAAAAAGCAATTCTAAAACTAGAAAGAAATA CAAGGAACAGAAAGAATCCTGATGCCTTCTTTCTTACAACAAGTAAGCTCAGAAACTTGCTGAGCTTAACTAGTA CACTTTTTGATGAGAGTAAGGTCAAAGAATATGATGATCTCCTTGATCGTATTGCTTATTTAAGAGTACAATTTGT CTATCAAGCAGGTAGAGAGATTGCAGTAAAAGATCTGATAGAAAAGGCTCAAATTCTTGAGGCTCTTAAGGAAAT CAAAGATAGAGAGACACTTCAAAGATTTTGTAGATATATGGAAGCATTAGTAGCCTATTTCAAGTTTTATGGAGG TAAAGATTAA

[0644] SEO ID NO: 37 Type III-A Csm2 protein sequence (GenBank: WP 002950682.1 )

[0645] MTILTDENYVDIAEKAILKLERNTRNRKNPDAFFLTTSKLRNLLSLTSTLFDESKVKEYDDLLDRIAYLRVOFV YOAGREIAVKDLIEKAOILEALKEIKDRETLORFCRYMEALVAYFKFYGGKD

[0646] SEO ID NO: 38 Type III-A csm3 DNA sequence (GenBank: DGCC7710 RS0103920)

[0647] GAGATGGTTTAAAACTGCTTGAACTTGATTATCTTGGTGGTTCTGGATCTCGAGGTTACGGTAAGGTTG CTTTTGAAAAACTCAAAGCTACTACCGTATTTGGTAATTATGATGTTAAAACATTAAATGAACTTTTAACTGCGGA GGTCTAA

[0648] SEO ID NO: 39 Type III-A Csm3 protein sequence (GenBank: WP 011681112.1 )

[0649] MTFAKIKFSAOIRLETGLHIGGSDAFAAIGAIDSPVIKDPITNIPIIPGSSLKGKMRTLLAKVYNEKVAEKPSD DSDILSRLFGNSKDKRFKMGRLIFRDAFLSNADELDSLGVRSYTEVKFENTIDRITAEANPROIERAIRNSTFDFELIY EITDENENOVEEDFKVIRDGLKLLELDYLGGSGSRGYGKVAFEKLKATTVFGNYDVKTLNELLTAEV

[0650] SEO ID NO: 40 Type III-A Dead csm3 DNA sequence (DGCC7710 RS0103920:c.98A>C) modified positions are in bold and underlined

[0651] ATGACATTCGCTAAGATTAAATTTTCAGCTCAAATTCGTTTAGAGACAGGCCTCCATATTGGTGGAAGC GATGCTTTTGCAGCCATTGGTGCAATCGCTTCGCCTGTTATTAAAGATCCTATTACCAACATACCGATCATTCCTG GTTCAAGTCTCAAAGGAAAAATGAGAACGCTTCTTGCCAAGGTTTATAATGAAAAGGTAGCTGAGAAACCAAGC GATGACAGTGATATTCTTAGCCGTTTATTTGGGAATAGTAAAGATAAACGATTCAAAATGGGACGCTTGATTTTTC GTGATGCCTTCTTGTCAAACGCTGATGAGCTAGACTCTCTTGGGGTAAGAAGTTATACAGAAGTAAAATTTGAAA ATACAATTGACCGTATCACTGCCGAAGCTAATCCAAGACAAATTGAACGTGCTATTCGTAACAGTACTTTTGATTT CGAGTTGATTTATGAAATTACAGATGAGAATGAAAATCAAGTCGAAGAAGATTTCAAAGTGATTCGAGATGGTTT AAAACTGCTTGAACTTGATTATCTTGGTGGTTCTGGATCTCGAGGTTACGGTAAGGTTGCTTTTGAAAAACTCAA AGCTACTACCGTATTTGGTAATTATGATGTTAAAACATTAAATGAACTTTTAACTGCGGAGGTCTAA

[0652] SEO ID NO: 41 Type III-A Dead Csm3 protein sequence (WP 011681112. l :p.D33A) modified residues are in bold and underlined MTFAKIKFSAOIRLETGLHIGGSDAFAAIGAIASPVIKDPITNIPIIPGSSLKGKMRTLLAKVYNEKVAEKPSD

[0653] DSDILSRLFGNSKDKRFKMGRLIFRDAFLSNADELDSLGVRSYTEVKFENTIDRITAEANPROIERAIRNSTFDFELIY

[0654] EITDENENOVEEDFKVIRDGLKLLELDYLGGSGSRGYGKVAFEKLKATTVFGNYDVKTLNELLTAEV

[0655] SEO ID NO: 42 Type III-A csm4 DNA sequence (GenBank: DGCC7710 RS0103925)

[0656] ATGACCTATAAACTGTATATTATGACCTTTCAGAATGCTCATTTTGGTTCGGGCACTCTTGATAGCTCAA

[0657] AATTAACATTCTCAGCAGACCGTATCTTCTCAGCACTAGTGCTAGAATCCCTAAAAATGGGAAAACTCGATGCAT

[0658] TTCTTGCGGAAGCTAACCAAGACAAGTTCACGCTCACAGATGCCTTTCCATTTCAATTTGGTCCCTTTTTGCCGAA

[0659] ACCTATTGGTTATCCCAAACATGACCAAATAGATCAATCAGTTGATGTCAAAGAGGTTCGCCGTCAAGCAAAATT

[0660] GTCTAAGAAACTGCAATTTCTTGCTCTAGAAAATGTTGACGATTATCTCAATGGAGAGTTATTTGAAAATGAAGAG

[0661] CATGCAGTCATTGATACTGTGACAAAAAATCAACCACATAAGGATGGCAATCTTTATCAGGTAGCTACAACTAGA

[0662] TTTTCAAATGATACGTCGCTTTACGTCATCGCAAACGAATCTGATTTGCTTAATGAGTTGATGTCTAGTCTTCAGT

[0663] ATTCAGGTCTTGGTGGAAAGCGTTCAAGTGGTTTTGGTCGTTTTGAGTTAGATATTCAAAATATCCCACTAGAATT

[0664] GTCAGATAGACTGACTAAGAATCATTCAGATAAAGTGATGAGTCTTACGACAGCACTTCCTGTAGATGCTGACCT

[0665] TGAAGAAGCAATGGAAGATGGACATTACTTATTAACTAAATCAAGTGGTTTTGCATTTAGTCATGCTACCAATGA

[0666] GAATTATCGTAAGCAGGATCTTTACAAATTTGCTTCTGGTTCAACTTTTAGTAAAACATTTGAAGGTCAGATTGTT

[0667] GATGTGAGACCACTTGATTTCCCTCATGCTGTTTTAAATTATGCTAAACCACTCTTCTTTAAATTGGAGGTATAA

[0668] SEO ID NO: 43 Type III-A Csm4 protein sequence (GenBank: WP 002950684.1 ).

[0669] MTYKLYIMTFONAHFGSGTLDSSKLTFSADRIFSALVLESLKMGKLDAFLAEANODKFTLTDAFPFOFGPFL

[0670] PKPIGYPKHDOIDOSVDVKEVRROAKLSKKLOFLALENVDDYLNGELFENEEHAVIDTVTKNOPHKDGNLYOVATTR

[0671] FSNDTSLYVIANESDLLNELMSSLOYSGLGGKRSSGFGRFELDIONIPLELSDRLTKNHSDKVMSLTTALPVDADLEE

[0672] AMEDGHYLLTKSSGFAFSHATNENYRKODLYKFASGSTFSKTFEGOIVDVRPLDFPHAVLNYAKPLFFKLEV

[0673] SEO ID NO: 44 Type III-A csm5 DNA sequence (GenBank: DGCC7710 RS0103930).

[0674] ATGAAAAATGACTATAGAACATTTAAATTAAGCCTCCTGACACTTGCTCCAATTCATATTGGTAATGGAG

[0675] AGAAGTATACCTCTAGAGAATTTATCTATGAAAATAAGAAGTTTTACTTTCCTGACATGGGGAAATTCTATAATAA

[0676] AATGGTGGAGAAGAGGCTTGCTGAAAAGTTTGAAGCATTTCTAATTCAAACTCGTCCAAATGCACGTAATAATCG

[0677] TCTTATTTCCTTCTTAAATGATAACCGAATTGCAGAGCGTTCTTTTGGAGGTTATAGTATCTCTGAAACAGGTTTA

[0678] GAATCGGACAGAAATCCTAATTCAGCCGGAGCTATTAACGAAGTTAATAAATTTATTCGAGATGCTTTTGGAAAT

[0679] CCCTACATTCCTGGTAGCTCACTAAAAGGTGCTATTCGTACCATTTTAATGAATACTACCCCTAAGTGGAATAATG

[0680] AAAATGCTGTAAATGACTTTGGAAGATTTCCGAAAGAGAATAAGAACCTTATCCCTTGGGGACCAAAAAAGGGAA

[0681] AAGAATACGATGATTTGTTTAACGCAATTCGTGTGAGTGATAGTAAGCCTTTTGATAATAAGAGTCTTATCTTAGT

[0682] ACAGAAATGGGATTATTCAGCGAAAACAAATAAAGCTAAACCACTTCCCTTGTATAGAGAATCAATCTCTCCATTA

[0683] ACAAAAATTGAATTTGAGATTACAACAACTACTGATGAAGCTGGAAGATTGATTGAAGAATTAGGTAAGAGAGCA

[0684] CAAGCGTTTTATAAAGACTATAAAGCATTTTTCCTATCTGAATTTCCTGATGATAAGATTCAAGCCAATCTACAATA

[0685] CCCAATTTATTTAGGTGCGGGGAGCGGTGCTTGGACAAAGACTCTATTTAAGCAAGCTGATGGTATTTTACAAAG

[0686] ACGATACAGTCGAATGAAAACTAAAATGGTTAAAAAAGGAGTTCTTAAGCTCACAAAAGCACCTCTTAAAACAGT TAAGATTCCATCTGGTAATCATTCATTAGTCAAGAACCACGAGTCCTTTTATGAAATGGGAAAAGCTAATTTCATG

[0687] ATTAAGGAGATTGATAAATGA

[0688] SEO ID NO: 45Type III-A Csm5 protein sequence (GenBank: WP 011681113.1 )

[0689] MKNDYRTFKLSLLTLAPIHIGNGEKYTSREFIYENKKFYFPDMGKFYNKMVEKRLAEKFEAFLIOTRPNARNN

[0690] RLISFLNDNRIAERSFGGYSISETGLESDRNPNSAGAINEVNKFIRDAFGNPYIPGSSLKGAIRTILMNTTPKWNNEN

[0691] AVNDFGRFPKENKNLIPWGPKKGKEYDDLFNAIRVSDSKPFDNKSLILVOKWDYSAKTNKAKPLPLYRESISPLTKIE

[0692] FEITTTTDEAGRLIEELGKRAOAFYKDYKAFFLSEFPDDKIOANLOYPIYLGAGSGAWTKTLFKOADGILORRYSRMK

[0693] TKMVKKGVLKLTKAPLKTVKIPSGNHSLVKNHESFYEMGKANFMIKEIDK

[0694] SEO ID NO: 46 Type III-A cas6 DNA sequence (GenBank: DGCC7710 RS0103905)

[0695] ATGAAAAAATTAGTATTTACTTTTAAAAGGATCGACCATCCTGCACAAGATTTGGCTGTTAAATTTCATG

[0696] GCTTCTTGATGGAGCAGTTGGATAGTGACTATGTTGATTATCTGCATCAGCAGCAAACAAATCCCTATGCGACCA

[0697] AGGTAATCCAAGGGAAAGAAAACACGCAGTGGGTTGTACATCTGCTCACAGACGACATCGAGGATAAGGTTTTT

[0698] ATGACCTTATTACAGATTAAAGAGGTGTCCTTAAACGATCTGCCTAAACTCAGTGTCGAAAAAGTTGAGATTCAG

[0699] GAGTTGGGGACAGATAAACTGTTAGAGATTTTCAATAGTGAGGAAAATCAAACCTATTTTTCAATTATTTTTGAGA

[0700] CTCCAACAGGTTTTAAATCTCAAGGTTCCTACGTCATCTTCCCGTCTATGCGTTTGATTTTTCAAAGTTTGATGCA

[0701] AAAGTATGGAAGGTTGGTTGAAAATCAACCTGAAATTGAAGAGGATACCTTAGATTACCTATCTGAACACAGCAC

[0702] TATCACGAATTATCGCTTGGAGACGAGTTATTTCAGGGTGCACAGGCAACGAATTCCTGCCTTTAGAGGAAAGTT

[0703] AACCTTTAAAGTACAAGGCGCCAAAACTCTAAAAGCTTATGTCAAAATGCTTCTAACATTCGGTGAATATTCAGGT

[0704] CTTGGCATGAAAACGAGTCTCGGTATGGGAGGGATAAAGCTTGAAGAAAGAAAAGATTGA

[0705] SEO ID NO: 47 Type III-A Cas6 protein sequence (GenBank: WP 011681111.1 )

[0706] MKKLVFTFKRIDHPAODLAVKFHGFLMEOLDSDYVDYLHOOOTNPYATKVIOGKENTOWVVHLLTDDIED

[0707] KVFMTLLOIKEVSLNDLPKLSVEKVEIOELGTDKLLEIFNSEENOTYFSIIFETPTGFKSOGSYVIFPSMRLIFOSLMOK

[0708] YGRLVENOPEIEEDTLDYLSEHSTITNYRLETSYFRVHRORIPAFRGKLTFKVOGAKTLKAYVKMLLTFGEYSGLGMKT

[0709] SLGMGGIKLEERKD

[0710] SEO ID NO: 48 - Example unprocessed type III-A guide RNA (spacer bold / underline)

[0711] ACGGAAACGGAUGUAGCCCAGGUGAGGGUUCCACAUGAACUCAUGGAUAUAAACCUAAUUACC

[0712] UCGAGAGGGGACGGAAAC

[0713] GATATAAACCTAATTACCTCGAGAGGGGACGGAAACGGATGTAGCCCAGGTGAGGGTTCCACATGAAC

[0714] TCATGATATAAACCTAATTACCTCGAGAGGGGAC

[0715] SEO ID NO: 49 - Example 1 mature type III-A guide RNA (spacer bold / underline) ACGGAAACGGAUGUAGCCCAGGUGAGGGUUCCACAUGAACUCA

[0716] SEO ID NO: 50 Type III-A RNA sequence tested (protospacer bold / underline)

[0717] AGUCTAAGGACUAUGAGUUCAUGUGGAACCCUCACCUGGGCUACAUCCUCACCUGCCCAU

[0718] SEO ID NO: 51 Type III-A CRISPR array spacer GGATGTAGCCCAGGTGAGGGTTCCACATGAACTCAT

[0719] SEO ID N0:52 Type III-A CRISPR array flanking repeat

[0720] GATATAAACCTAATTACCTCGAGAGGGGACGGAAAC

[0721] SEO ID NO: 53 - Example 2 mature type III-A guide RNA (spacer bold / underline)

[0722] ACGGAAACGGAUGUAGCCCAGGUGAGGGUUCCACAUGAA

[0723] REFERENCES

[0724] [1] Hampton HG;Watson BNJ;Fineran PC. The arms race between bacteria and their phage foes. Nature, 2020,577,327-36.

[0725] [2] Makarova KS;Wolf YI;Alkhnbashi OS;Costa F;Shah SA;Saunders SJ;et al. An updated evolutionary classification of CRISPR-Cas systems. Nat Rev Microbiol, 2015,13,722-36.

[0726] [3] Makarova KS;Wolf YI;Iranzo J;Shmakov SA;Alkhnbashi OS;Brouns SJJ;et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat Rev Microbiol, 2020,18,67-83.

[0727] [4] Makarova KS;Wolf YI;Koonin E V. Classification and Nomenclature of CRISPR-Cas Systems: Where from Here? Cris J, 2018,1,325-36.

[0728] [5] Deveau H;Garneau JE;Moineau S. CRISPR / Cas System and Its Role in Phage-Bacteria Interactions. Annu Rev Microbiol, 2010,64,475-93.

[0729] [6] Sander JD;Joung JK. CRISPR-Cas systems for editing, regulating and targeting genomes. Nat Biotechnol, 2014,32,347-55.

[0730] [7] Steens JA;Salazar CRP;Staals RHJ. The diverse arsenal of type III CRISPR-Cas-associated CARF and SAVED effectors. Biochem Soc Trans, 2022,50,1353-64.

[0731] [8] Liu TY;Doudna JA. Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation. J Biol Chem, 2020,295,14473-87.

[0732] [9] Hsu PD;Lander ES;Zhang F. Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell, 2014,157,1262-78.

[0733]

[0010] Osawa T;Inanaga H;Sato C;Numata T. Crystal Structure of the CRISPR-Cas RNA Silencing Cmr Complex Bound to a Target Analog. Mol Cell, 2015,58,418-30.

[0734]

[0011] Taylor DW;Zhu Y;Staals RHJ;Kornfeld J E; Shinka i A;van der Oost J;et al. Structures of the CRISPR-Cmr complex reveal mode of RNA target positioning. Science (80- ), 2015,348,581-5.

[0735]

[0012] Kazlauskiene M;Kostiuk G;Venclovas C;Tamulaitis G;Siksnys V. A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science (80- ), 2017,357,605-9.

[0736]

[0013] Niewoehner O;Garcia-Doval C;Rostol JT;Berk C;Schwede F; Bigler L;et al. Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature, 2017,548,543-8.

[0737]

[0014] Chi H;Hoikkala V;Gruschow S;Graham S;Shirran S;White MF. Antiviral type III CRISPR signalling via conjugation of ATP and SAM. Nature, 2023,622,826-33.

[0738]

[0015] Fagerlund RD;Fineran PC;Smith LM;Mayo-Munoz D;Taylor DW;Schwartz EA;et al. Type III-D CRISPR-Cas system and uses thereof. AU 2022901608, 2023.

[0739]

[0016] Schwartz EA;Bravo JPK;Macias LA;McCafferty CL;Dangerfield TL;Walker JN;et al. Assembly of multi-subunit fusion proteins into the RNA-targeting type III-D CRISPR-Cas effector complex. BioRxiv, 2022,2022.06.13.496011.

[0740]

[0017] Mayo-Munoz D;Smith LM;Garcia-Doval C;Malone LM;Harding KR;Jackson SA;et al. Type III CRISPR-Cas provides resistance against nucleus-forming jumbo phages via abortive infection. Mol Cell, 2022, 82, 4471-4486. e9.

[0741]

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Claims

CLAIMS1. A method for targeted cell death or cell growth arrest in a cell expressing a target ribonucleic acid (RNA), the method comprising delivering to the cell:(a) a guide RNA (gRNA) which is complementary to a recognition sequence within the target RNA expressed by the cell;(b) one or more type III CRISPR-Cas proteins, wherein at least one type III CRISPR- Cas protein is capable of producing a signaling molecule; and(c) at least one accessory protein capable of causing cell death or cell growth arrest, which accessory protein is activated by the signaling molecule, wherein, the presence of the target RNA within the cell causes the gRNA and type III CRISPR-Cas proteins to bind to the target RNA thereby producing at least one signaling molecule, and wherein production of the signaling molecule activates the accessory protein thereby causing targeted cell death or growth arrest of the cell.

2. The method according to claim 1, wherein the one or more type III CRISPR-Cas proteins is derived from a type III-A CRISPR-Cas system, a type III-B CRISPR-Cas system, a type III-C CRISPR-Cas system, a type III-D CRISPR-Cas system and / or a type III CRISPR Cas system.

3. The method according to claim 1 or claim 2, wherein the one of more type III CRISPR-Cas proteins is derived from a type III-D CRISPR-Cas system or a type III-A CRISPR-Cas system.

4. The method according to any one of claims 1 to 3, wherein the at least one type III CRISPR-Cas protein capable of producing a signaling molecule is CaslO.

5. The method according to any one of claims 1 to 4, wherein the type III CRISPR-Cas proteins comprise:(i) a Cas7-Cas5-Casll fusion protein;(ii) a Cas7-Cas7 fusion protein;(iii) a Cas7-insertion protein;(iv) a CaslO;(v) a Csxl9; and(vi) optionally, a Cas6.

6. The method according to any one of claims 1 to 5, wherein when the one or more typeIII CRISPR-Cas proteins includes a Cas7 domain, the Cas7 domain comprises at least one mutation which reduces or eliminates Cas7 ribonuclease (RNase) activity.

7. The method according to claim 5 or claim 6, wherein:(i) the Cas7-Cas7 fusion subunit comprises a mutation at positions D246 and / or D33 of SEQ ID NO: 6, or positions corresponding thereto; and / or(ii) the Cas7-Cas5-Casll fusion subunit comprises a mutation at position D26 of SEQ ID NO: 4, or a position corresponding thereto, which mutation(s) defined by (i) and (ii) reduce or eliminate Cas7 ribonuclease (RNase) activity.

8. The method according to any one of claims 1 to 7, wherein the signaling molecule produced by the at least one type III CRISPR-Cas protein is selected from cyclic oligoadenylate 3 (cA3), cyclic oligoadenylate 4 (cA4), cyclic oligoadenylate 6 (cA6) and S-adenosyl methionine-AMP.

9. The method according to any one of claims 1 to 8, wherein the accessory protein possesses a catalytic activity selected from deoxyribonuclease activity, ribonuclease activity, protease activity, adenosine deaminase activity and nicotinamide adenine dinucleotide (NAD+) nucleosidase activity or the accessory protein possesses nucleic acid binding activity.

10. The method according to any one of claims 1 to 9, wherein the at least one accessory protein is selected from NucC nuclease, Csm6 nuclease, Csxl nuclease, Canl nuclease, Can2 nuclease, Cardl nuclease, CalpL protease, SAVED-CHAT protease, Caml, Cam2, Cam3 and Csx23 membrane proteins, Cadi deaminase, TIR NADases and CorA membrane protein.

11. The method according to claim 10, wherein the NucC nuclease is derived from Serratia sp. ATCC 39006 and is set forth in SEQ ID NO: 30.

12. The method according to any one of claims 1 to 11, wherein:(I) the gRNA is encoded for by a nucleic acid;(ii) the type III CRISPR-Cas proteins are encoded for by at least one nucleic acid;(ill) the accessory protein is encoded for by a nucleic acid; and(iv) any combination of (I), (ii) and (ill) wherein the combination is encoded for by the same nucleic acid or different nucleic acids, and wherein the nucleic acid is delivered to and expressed by the cell.

13. The method according to claim 12, wherein the nucleic acid is comprised in an expression vector.

14. The method according to claim 12or claim 13, wherein the nucleic acid is under the control of an inducible promoter.

15. The method according to any one of claims 1 to 14, wherein delivering (i) to (ill) to the cell is achieved using a delivery method selected from electroporation, lipofection, microinjection, exosomes, extracellular vesicles, nanoparticles and viruses.

16. The method according to any one of claims 1 to 15, wherein the cell is a human cell.

7. The method according to any one of claims 1 to 16, wherein the cell is selected from a cancer cell, an immune cell and an engineered immune cell such as a chimeric antigen receptor (CAR) T cell.

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