CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63/351,321, filed on June 10, 2022, the disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grant no. GM118174 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in xml format and is hereby incorporated by reference in its entirety. Said .xml copy was created on June 12, 2023, is named “018617_01420_ST20.xml,” and is 86,259 bytes in size.

BACKGROUND OF THE DISCLOSURE

[0004] RNA-guided DNA/RNA degradation is not the sole mechanism for CRISPR- Cas to confer immunity against foreign genetic elements in prokaryotes. Type III CRISPR- Cas systems in particular present a plethora of alternative mechanisms, including RNA- guided secondary messenger production and signaling to activate a range of immune responses. Type III CRISPR effectors are typically assembled from multiple protein subunits. Besides conveying RNA-guided RNA cleavage, Type III effectors further synthesize secondary messenger molecule to activate ancillary proteins to induce cell dormancy, some systems further cause localized collateral DNA damage. Type III-E is a recently identified atypical Type III system. It encodes a large polypeptide as a fusion of four Cas7 domains, one Casl 1 domain, and a Big Insertion Domain (BID) of unfamiliar structural fold, but conspicuously lacks the signatures of a canonical Type III signaling system (e.g. CaslO and CARF-domain containing proteins). The effector protein in Type III-E systems was named gRAMP for ‘giant repeat-associated mysterious protein’, or ‘Cas7-11’ based on domain compositions. Subsequent studies showed that gRAMP ribonucleoprotein (RNP) complex is capable of RNA-guided RNA cleavage at two specific sites, six nucleotides (nts) apart.

Unlike the Type VI CRISPR effector Casl3, gRAMP does not cause collateral RNA cleavage, and has no cytotoxicity in eukaryotic cells. [0005] An intriguing observation is that gRAMP genes frequently associates with tpr- chat in the Type III-E operon. The resulting TPR-CHAT protein was named after its N- terminal TPR repeats and C-terminal Caspase-like domains. Caspases are a family of dimeric cysteine proteases controlling the programmed cell death (PCD) pathways in eukaryotes. They trigger apoptosis or pyroptosis through site-specific protein cleavage. Cleavage of Gasdermins, for example, triggers membrane pore formation and cause pyroptosis. An equivalent PCD pathway was recently discovered in prokaryotes, where TPR-CHAT was shown to cleave bacterial Gasdermin to induce cell death. In Type III-E systems, TPR-CHAT and gRAMP form an effector complex named Craspase, for CRISPR-guided Caspase. However, it remains unknown as to whether TPR-CHAT is a protease and whether its activity is RNA-guided. There is accordingly a need for new systems that can be used for degrading targeted proteins in a guide RNA directed manner. The disclosure is pertinent to this need.

SUMMARY OF THE DISCLOSURE

[0006] The recently discovered CRISPR-Caspase system features a Type III-E RNA- targeting effector (gRAMP/Cas7-l 1) associated with a Caspase-like protein (TPR-CHAT) to form Craspase (CRISPR-guided Caspase). Described herein is the use cryo-EM snapshots of the effector complexes to explain their RNA cleavage and protease activation mechanisms. Target-guide pairing extending into the 5’ region of the guide displaces a gating loop in gRAMP to switch on RNA cleavage. In the context of Craspase, this further triggers an extensive conformational relay that allosterically aligns the protease catalytic dyad and opens an amino acid sidechain-binding pocket. Csx30 is defined as the endogenous protein substrate that is site-specifically proteolyzed by RNA-activated Craspase. This activity is switched off by target RNA cleavage, and is not activated by self-RNA targets containing a matching protospacer flanking sequence. Compositions, methods and systems comprising modified gRAMP proteins, along with TRP-CHAT proteins and guide RNAs, are provided by this disclosure.

BRIEF DESCRIPTION OF THE FIGURES

[0007] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures. [0008] Figure 1. Structural snapshots of 56-gRAMP RNP in different functional states. (A) Native gene cluster of A7i-gRAMP-TPR-CHAT operon. Each domain is highlighted with the color shown in the panel. (B) Snapshots of A7i-gRAMP at apo state, (C) non-matching PFS RNA bound state, (D) matching PFS RNA bound state and (E) nonmatching PFS RNA post-cleavage state with MgCh. Top images are cryo-EM densities and bottom images are structural models. (F) Comparison of 5 ’-handle and first segment region of type III-E 57>-gRAMP and type III-A Csm complex. (G) Comparison of crRNA from Csm and A'A-gRAMP. (H) Extracted Cryo-EM density from non-matching PFS RNA (left), matching PFS RNA (middle) and non-matching PFS RNA post-cleavage state (right).

[0009] Figure 2. In-depth analysis of target validation and cleavage by Sb- gRAMP RNP. (A) Model depicting the gate closed structure in resting state (left) and gate open structure in target RNA bound state (middle). Superposition is shown as the right panel. (B) Sequence conservation in the gating loop region. The conserved amino acids are highlighted in red. (C) Structural comparison of resting state and non-matching PFS RNA bound state showing the hinge motion in Casl 1 domain. Vector length is proportional to the movement distance (Casl 1 region is highlighted in pink). (D) EMSA and RNA cleavage assay to compare gate disruption mutant and wild type gRAMP activities. (E) Model depicting the essential role of the gating loop in target validation. (F) Structural basis for site 1 cleavage. (G) Structural basis for site 2 cleavage. (H) Quantification of cleavage efficiency by site 1 (in blue) and site 2 (in black) active site mutants. The following sequences appear in this figure: the sequence labeled gRAMP is a portion of SEQ ID NO:2; [0010] Figure 3. Structural basis of Craspase complex. (A) 2.7 A cryoEM model and (B) structural model of Craspase (gRAMP-TPR-CHAT). (C) Molecular contacts between gRAMP and TPR. Close-up view of extracted cryoEM density is shown on the right. (D) Molecular contacts between gRAMP and CHAT. Close-up view of extracted cryoEM density of the helix in CHAT domain is shown on the right. (E) Results from co-purification of gRAMP with TPR-CHAT mutants. (F) Comparison of RNA binding and cleavage between gRAMP and Craspase complex. [0011] Figure 4. Structural basis for Craspase protease activation. (A) 3.7 A cryo- EM density (left) and structural model (right) of matching PFS RNA bound Craspase. (B) 2.6 A cryoEM density (left) and structural model (right) of non-matching PFS RNA bound Craspase. (C) Close-up view of switch helix in resting state (left), matching PFS RNA bound state (middle), and non-matching PFS RNA bound state (right). Switch helix highlighted in green and the density of crRNA and target RNA (TRNA) are shown in mesh. (D) Cartoon display of switch helix with surface display of sensor hairpins and CHAT protease center in resting state (left), matching PFS RNA bound state (middle) and non-matching PFS RNA bound state (right). (E) Peptide cleavage assay by Craspase. (F) Cleavage site mapping by mass-spectrometry. (G) Top: model depicting non-matching PFS RNA dependent Craspase activation. Bottom: AND logic gate illustrating the protease activation mechanism. (H) model depicting TPR-CHAT functional state in apo/resting state, matching PFS RNA bound state, and non-matching PFS RNA bound state.

[0012] Figure 5. Craspase proteolytically cleaves Csx30 in an RNA-dependent manner. (A) Genetic context for RpoE, Csx31 and Csx30 co-expression with Craspase wildtype (WT) or mutant (MT; H585A C627A) and a target RNA in E. coli BL21-AI. (B) SDS- PAGE protein gel showing the eluted protein content from Streptavidin purifications of Tag- RpoE, Tag-Csx31 and Tag-Csx30 after co-expression with either Craspase WT or Craspase MT (H585A C627A). Arrows indicate the expected size for full length protein. (C) Protein gels after Craspase WT or Craspase MT (H585A C627A) incubation with Csx30 in the presence of target RNA or non-target RNA. Protein cleavage products are indicated with an asterisk. (D) SDS-PAGE protein gel after Craspase WT incubation with target RNA containing either a non-matching PFS (NPFS) or matching PFS (PFS). Protein cleavage products are indicated with an asterisk and arrows. Full length Tag-Csx30 is indicated with an orange arrow. (E) Left: SDS-PAGE protein gel after incubation of Tag-Csx30 with target RNA and Craspase WT or Craspase D698A R294A, with or without prior incubation with MgCh. Right: SDS-PAGE protein gel after incubation of Tag-Csx30 with target ssDNA and Craspase D698A R294A. Full length Tag -Csx30 is indicated with the orange arrow. Protein cleavage products are indicated with red arrows. (F) Cartoon depicting the “stay-ON” Craspase variant with inactivating endonuclease mutations (D698A and R294A). (G) Cartoon showing the proposed model for Craspase functionality. Once target unbound Craspase has complementary bound a target RNA, the peptidase activity is unleashed. This results in proteolytic cleavage of Csx30, which then likely has an immune activity. Upon target RNA cleavage by Craspase, the peptidase activity is shut off, allowing it to bind new target RNAs. [0013] Figure 6. Reconstitution and biochemical characterization of the Sb- gRAMP RNP. (A) Purification profile on size-exclusion chromatography. (B) SDS-PAGE analysis of the peaks on size-exclusion. (C) Top: cleavage assay for different concentrations of 57>-gRAMP. Middle: EMSA assay using the same titration of 57>-gRAMP. Bottom: SDS- PAGE showing protein concentration in cleavage and EMSA assay.

[0014] Figure 7. Single particle cryo-EM reconstruction of 56-gRAMP / Sb- gRAMP-Non-matching PFS RNA complex. (A) Workflow of the cryo-EM image processing and 3D reconstruction for the 57>-gRAMP / A7i-gR.AMP-R.NA complex. (B) Final electron density map showing local resolution. (C) Fourier Shell Correlations (FSC) of Sb- gRAMP / A7i-gRAMP-RNA complex reconstruction, with the gold-standard cutoff (FSC = 0.143) marked with a dotted line.

[0015] Figure 8. Single particle cryo-EM reconstruction of 57>-gRAMP/Matching PFS RNA complex and A7i-gRAMP/Non-matching PFS RNA post-cleavage state with MgCh. (A) Workflow of the cryo-EM image processing and 3D reconstruction for the 57>-gRAMP- Matching PFS RNA complex. (B) Final electron density map showing local resolution for the A7i-gRAMP-Matching PFS RNA complex. (C) Fourier Shell Correlations (FSC) of the Sb- gRAMP -Matching PFS RNA complex reconstruction, with the gold-standard cutoff (FSC = 0.143) marked with a dotted line. (D) Workflow of the cryo-EM image processing and 3D reconstruction for the 5Z>-gRAMP-Non-matching PFS RNA with MgCh complex. (E) Final electron density map showing local resolution for the 56-gRAMP -Non-matching PFS RNA with MgCh. (F) Fourier Shell Correlations (FSC) of the 56-gRAMP -Non-matching PFS RNA with MgCh complex reconstruction, with the gold-standard cutoff (FSC = 0.143) marked with a dotted line.

[0016] Figure 9. Representative local map density for the different functional states. (A) EM densities for representative protein regions inside 57>-gRAMP and Sb- gRAMP/RNA complex. (B) EM densities for the 5' handle region of the crRNA. The number indicates the base order. (C) EM densities for guide RNA region inside 57>-gRAMP. (D) EM densities for the duplex of target RNA-guide RNA.

[0017] Figure 10. Structural comparison between type III-E A6-gRAMP and type III-A Csm complex. (A) Side by side over all structure comparison between type III-E Sb- gRAMP and type III-A Csm (PDB: 6IFN) complex. (B) Structural alignment and comparison of crRNAs from 57>-gRAMP and the Csm complex. (C) Side by side Cas7 comparison among Csm and the four Cas7 subunits of 57>-gRAMP. (D) Structural alignment showing that Cas7.3 has the highest structure conservation with Csm3. (E) Side by side comparison between Sb- gRAMP Casl 1 and its equivalent Csm2 in the Csm complex. (F) Side by side comparison between 5Z>-gRAMP BID and Csm5 in the Csm complex. (G) Snapshots of four Cas7 subunits bound to crRNA. Cas7.1, Cas7.2, and Cas7.3 all have an obvious thumb structure to flip out a base of crRNA. (H) Cartoon model showing the architecture of the 57>-gRAMP RNP complex.

[0018] Figure 11. Structural basis for the zinc-finger of 56-gRAMP and 5' handle recognition. (A) Close-up view of the cryoEM density of zinc-fingers from Cas7.1 to Cas7.4 subunits. (B) Close-up view of Cas7.1 and Cas7.2 contacting the -1 to -3 bases of 5' handle. (C) Close-up view of Cas7.1 and Cas7.2 contacting the -4 to -9 bases of 5' handle.

[0019] Figure 12. Mutagenesis analysis of 56-gRAMP RNP protein. (A) Model depicting the workflow of in vivo RNA knock down assay in E.coli. (B) Transformation plates of RNA knockdown assay from different mutations (C501A/C503 is the zinc-finger from Cas7.2; R37 from Cas7.1; C-l and G-5 from the 5' handle region of crRNA). (C) Bacteria solution dot assay to show the GFP signal and cell density. (D) Normalized knock down efficiency from different mutagenesis.

[0020] Figure 13. Functional characterization of the gating loop. (A) close-up view of the gating loop located in a deep cavity and blocking the “seed region” of crRNA. (B) A model showing the direction of base-pairing formation between target RNA and guide RNA. Base-pairings that can only happen after gating loop displacement likely takes place at the end. (C) A model showing that without gating loop protection, base-pairing can take place stochastically. Off-targeting may be rampant. (D) Cartoon model depicting mutagenesis of the gating loop. (E) SDS-PAGE showing the quality of the wild type and gating loop mutant 57>-gRAMP samples. Sequences shown in this figure are: RILGDTEYY (SEQ ID NO:72) and

[0021] Figure 14. RNA cleavage experiments using varying lengths of complementarity in the target RNA. (A) Cartoon model to showing the full-length Sb- gRAMP architecture and crRNA region. (B) Model of RNA substrates used in assay showing varying lengths of complementarity with crRNA. (C) Cleavage assay of RNA substrates. (D) Normalized cleavage efficiency seen in panel C.

[0022] Figure 15. Characterization of N/>-gRAMP RNase activity. (A) Structural alignment showing the main conformational change is in Casl 1 region. (B) A model showing the RNA cleavage pattern in wild type, site 1, and site 2 mutations of 57>-gRAMP. (C) RNA substrate binding and cleavage assay for 57>-gRAMP mutations. (D) Cartoon model depicting the 57>-gRAMP domains involved in each cleavage site. [0023] Figure 16. Purification and CryoEM single particle reconstruction of Sb- gRAMP-TPR-CHAT complex (Craspase). (A) Model of method used for co-expression and purification for 57>-gRAMP Craspase. (B) Purification profile comparison between Sb- gRAMP and 57>-gRAMP Craspase on size-exclusion chromatography. (C) Representative SDS-PAGE comparison between 57>-gRAMP and 57>-gRAMP Craspase. (D) Workflow of the cryo-EM image processing and 3D reconstruction for 57>-gRAMP Craspase. (E) Final electron density map showing local resolution for 57>-gRAMP Craspase. (F) Fourier Shell Correlations (FSC) of the 5Z>-gRAMP -Matching PFS RNA complex reconstruction, with the gold-standard cutoff (FSC = 0.143) marked with a dotted line.

[0024] Figure 17. CryoEM single particle reconstruction of 56-gRAMP-TPR- CHAT (Craspase)/matching PFS RNA complex. (A) Workflow of the cryo-EM image processing and 3D reconstruction for the kA-gRAMP-TPR-CHAT/matching PFS RNA complex. (B) Final electron density map with the density from each chain colored separately. (C) Final electron density map showing local resolution at different contour level. (D) Fourier Shell Correlations (FSC) of the 57>-gRAMP-TPR-CHAT/ matching PFS RNA complex reconstruction, with the gold-standard cutoff (FSC = 0.143) marked with a dotted line.

[0025] Figure 18. CryoEM single particle reconstruction of 57>-gRAMP-TPR- CHAT/Non-matching PFS RNA complex. (A) Workflow of the cryo-EM image processing and 3D reconstruction for the A'A-gRAMP-TPR-CHAT/Non-matching PFS RNA complex.

(B) Left: Final electron density map with the density from each chain colored separately. Right: Final electron density map showing local resolution. (C) Fourier Shell Correlations (FSC) of the A'A-gRAMP-TPR-CHAT/Non-matching PFS RNA complex reconstruction, with the gold-standard cutoff (FSC = 0.143) marked with a dotted line.

[0026] Figure 19. Structural analysis of TPR-CHAT effector. (A) Left: structural model of Csm complex with cOA synthetase. Middle: structural model of 57>-gRAMP with TPR-CHAT protease. Right: structural superposition of Csm complex and 57>-gRAMP-TPR- CHAT complex. (B) Right and middle: overall structure of TPR-CHAT component from Sb- gRAMP-TPR-CHAT complex. Right: Cartoon model showing the architecture of TPR- CHAT. (C) Structural comparison between Separase-Securin complex (PDB: 5MZ6) and TPR-CHAT. Left: Separase-Securin complex structure. Middle: TPR-CHAT structure. Right: structural superposition of TPR-CHAT with Separase-Securin. (D) Close-up view of the structural alignment of protease domain from Separase and CHAT. (E) Close-up view of “switch helix” and “sensor hairpins”. [0027] Figure 20. Biochemistry and structure analysis of Craspase complex. (A) RNA cleavage activity comparison between 57>-gRAMP and 5Z>-gRAMP-TPR-CHAT complex. (B) Quantification of cleaved products from site 1 and site 2 from panel A. (C) Structure model revealing that TPR-CHAT binds to the gating loop and limits gating loop dynamic movement.

[0028] Figure 21. The structural dynamics of the gating loop in Craspase. (A) Cartoon model highlighting the gating loop conformation in apo Craspase (left), matching PFS RNA bound (middle) and non-matching PFS RNA bound Craspase complex (right). (B) Structural alignment of the gating loop in apo, matching PFS RNA bound and non-matching PFS RNA bound state. (C) Model depicting the gating loop dynamics when bound to different substrates.

[0029] Figure 22. CryoEM density and conservation analyses of protease center. The extracted local density of protease center from apo state (A), matching PFS target RNA bound state (B) and non-matching PFS target bound state (C). Highlight the protease center residues H585, C627, E628 and D630. The protease center just only active in non-matching PFS target bound state. (D) Amino sequence alignment of protease center from 25 TPR- CHAT homologs.

[0030] Figure 23. The structural dynamics of switch helix in TPR-CHAT. (A) Cartoon model highlighting the switch helix conformation in apo Craspase (left), nonmatching PFS RNA bound state (middle), and superposition (right). (B) Amino acid sequence alignment of switch helix region in TPR-CHAT and five homologues. (C) Structural comparison of Craspase/matching PFS RNA complex and Craspase/non-matching PFS RNA complex showing the large conformational change in switch helix region and CHAT protease center region. Vector length correlates with the domain motion scale (color-coded as defined in the figure). The following sequences are in this figure: ID NO:79); NO:80); NO:81); NO: 82); (SEQ ID NO:83); and [0031] Figure 24. The biochemical characterization of Craspase protease activity. (A) Peptide substrate screening cleavage assay and separated on thin layer chromatography (TLC). TLC results indicate substrates AK1 and AK2 are candidates for Craspase. (B) Zoomed in view of substrate AK2 TLC results. (C) Cartoon model showing AK2 linked GST-P3C fusion protein and cleavage assay with Craspase. (D) SDS-PAGE of Craspase cleavage assay using AK2 linked GST-P3C fusion protein. (E) Cartoon model showing Cy3 labeled AK2 linked GST-P3C fusion protein and Craspase cleavage assay. (F) Time course to track the cleavage activity by Cy3 labeled AK2 linked GST-P3C fusion protein with Craspase. The following sequence is in the figure: ID NO: 74)

[0032] Figure 25. Biochemical validation of Craspase protease activity. (A) Workflow depicting the mass spectrometry analysis of the cleavage product. (B) Mass spectrometry results for the cleaved peptides. Sequences in this figure are:

[0033] Figure 26. In vitro biochemistry showing the engineered Gasdermine- Csx30 is cleaved by Craspase in RNA substrate dependent fashion, guided by the crRNA. MBP-GSDMD N -C sx3 O C GSDMD C : Csx30_CTD is inserted into the mouse Gasdermin-D protein. A portion of Gasdermin is removed (AA 243-276) for engineering purposes. This particular construct further contains a N-terminal Maltose-binding protein tag for solubility reasons. GSDMD_N: NTD of mouse Gasdermin-D, AA(l-242). Csx30_C: AA(383-570), serves as the substrate for Craspase. GSDMD C: CTD of mouse Gasdermin-D (277-487aa). Lanes 1-4: various negative controls. Lane 4 shows protein substrate is not cleaved even when RNA substrate is not present. Lanes 5 shows the protein substrate MBP- GSMD_N-Csx30_C-GSDMD_C is cleaved by Craspase in RNA substrate dependent fashion. Lanes 6, 7: In contrast, dead Craspase (gRAMP + TPR-CHAT(C627A) cannot cleave the protein substrate, without or with the RNA target present, respectively. Lane 8: Molecular markers for protein sizes.

[0034] Figure 27. Ex vivo data showing plasmid delivery of Craspase and engineered Gasdermine-Csx30 triggers human cell death in RNA substrate dependent fashion, guided by the crRNA. pGSDMD N-Csx30 C GSDMD C/TPR-CHAT/crRNA: This plasmid expressing the protein substrate (Csx30_CTD inserted in the middle of mGSDMD), the protease component of Craspase (TPR-CHAT), and the crRNA (guide) for the gRAMP component of Craspase. The two proteins are encoded in a single ORF, but translate into different proteins due to the presence of P2A and T2A tags. This ORF further contains mCherry at the end for transfection and expression efficiency controls. pgRAMP: This plasmid only encodes the full length gRAMP protein, expressed from a CMV promoter, and contains a Nuclear Export Signal (NES). GSDMD_N: NTD of mouse Gasdermin-D, AA 1-242. Csx30_C: AA 383-570, serves as the substrate for Craspase. GSDMD_C: CTD of mouse Gasdermin-D AA 277-487aa. gRAMP: full length protein, AA 1-1722. crRNA cluster: controlled by U6 promoter, contains 5 CRISPR repeats and 4 regularly-spaced spacers in between. The spacer is programmed to be complementary to a stretch of the GFP mRNA, which is expressed in HEK293-GFP cells from a chromosomal locus.

[0035] Figure 28. Plasmid map of GSDMD_N-Csx30_C_GSDMD_C.

[0036] Figure 29. Plasmid map of gRAMP.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0037] Although claimed subject matter will be described in terms of certain embodiments, other embodiments, including embodiments that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure.

[0001] Unless specified to the contrary, it is intended that every maximum numerical limitation given throughout this description includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0002] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. [0003] As used in the specification and the appended claims, the singular forms “a” "and” and “the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/-10%.

[0004] All nucleotide sequences described herein include the RNA and DNA equivalents of such sequences, i.e., an RNA sequence includes its cDNA. All nucleotide sequences include their complementary sequences.

[0005] This disclosure includes every amino acid sequence described herein and all nucleotide sequences encoding the amino acid sequences, and all other polynucleotide sequences described herein. Polynucleotide and amino acid sequences having from 80-99% similarity, inclusive, and including and all numbers and ranges of numbers there between, with the sequences provided here are included in the invention. All of the amino acid sequences described herein can include amino acid substitutions, such as conservative substitutions, that do not adversely affect the function of the protein that comprises the amino acid sequences.

[0006] All temperatures and ranges of temperatures, all buffers, and other reagents, and all combinations thereof, are included in this disclosure.

[0007] All nucleotide and amino acid sequences identified by reference to a database, such as a GenBank database reference number, are incorporated herein by reference as the sequence exists on the filing date of this application or patent.

[0038] The disclosure includes all embodiments illustrated in the Figures provided with this disclosure. The steps of the method described in the various embodiments and examples disclosed herein are sufficient to carry out the methods of the present invention. Thus, in an embodiment, the method consists essentially of a combination of the steps of the methods disclosed herein. In another embodiment, the method consists of such steps.

[0008] Any component of the proteins and systems described herein can be provided on the same or different polynucleotides, such as plasmids, which encode described proteins and/or polynucleotides. In embodiments, at least one component of the system is heterologous to the cells. In eukaryotic cells, all components of the system can be heterologous. [0009] Any protein described herein can be modified to improve its intended or actual use. In embodiments, the protein may be modified to include a linker amino acid sequence, including but not limited to a GS or glycine rich linker sequence, or a ribosomal skipping sequence. In embodiments, two or more of the described proteins may be provided as a fusion protein. In embodiments, the disclosure relates to improved gRAMP that function with TPR-CHAT proteins, and uses thereof. Examples of previously described gRAMP and TPR- CHAT proteins and their uses with guide RNAs are described in PCT publication WO 2022255865, from which the entire description is incorporated herein by reference.

[0010] In this disclosure, gRAMP proteins with mutations not described in the referenced PCT publication are provided. The presently described mutations can be combined with any previously described mutations. In embodiments, mutations in the gRAMP protein are made with respect to the amino acid sequence of gRAMP of Candidatus Scalindua, including Candidates Scalindua brodae. The described mutations provide modified gRAMP proteins. The modified gRAMP protein can comprise or consist of the described mutations. In embodiments, only 1, 2, 3, 4, 5 or 6 mutations of the gRAMP protein are made. The disclosure includes the proviso such that the modified gRAMP proteins may exclude one or both changes to amino acid positions 294 and 698 of the native gRAMP protein sequence. In embodiments, a modified RAMP protein of this disclosure comprises one or more mutations relative to the amino acid sequence of an unmodified Candidates Scalindua gRAMP protein, wherein the one or more mutations are selected from a change of amino acid at positions 806, 547, 298, 328, 367, 371, and combinations thereof, in the unmodified Candidates Scalindua gRAMP protein. gRAMP comprises four Cas7-like domains, Cas7.1, Cas7.2, Cas7.3, Cas7.4, and one Casl 1 domain. These Cas domains are naturally fused together in naturally occurring gRAMP.

[0011] In embodiments a modified gRAMP protein of this disclosure comprises a change in at least amino acid residue 806 in the sequence of the unmodified gRAMP Candidatus Scalindua gRAMP protein, or at least a change of amino acid at position 547 in the sequence of unmodified Candidatus Scalindua gRAMP protein, or at least a combination thereof. In embodiments, a modified gRAMP protein comprises or consists of mutations that are at positions 806 and 847 of the modified gRAMP protein. Without intending to be constrained by any particular theory, it is considered that the modified gRAMP proteins of this disclosure have reduced endonuclease activity, which in turn amplifies the protease activity of the associated TPR-CHAT protein. In embodiments, the disclosure includes a modified gRAMP protein that is a component of a fusion protein that also comprises a TPR- CHAT protein. TPR-CHAT may also be referred to herein as Craspase. Without intended to be bound by any particular theory, it is considered that the double mutation of D806A and D547A is expected to have the same effect or superior effects relative to the D698A and R294A construct.

[0012] In various embodiments, a protein of the present disclosure does not have the D698A and R294A mutations in the absence of any other mutations. In various embodiments, if the protein has one or the combination of D698A and R294A mutations, there may be other mutations present in the protein.

[0013] The described gRAMP- TPR-CHAT proteins function with a guide RNA. The guide RNA may be targeted to any RNA polynucleotide that is in a cell, and may be used in in vitro conditions where a targeted RNA may be present. In embodiments, recognition of the targeted RNA polynucleotide by the guide RNA activates the TPR-CHAT protease function, which may be amplified due to the described mutations which reduce degradation of the targeted RNA, thereby extending the TPR-CHAT protease function to cleave more protein. [0014] In embodiments, cleavage of a protein changes one or more properties of the cell in which the cleaved protein is present. In embodiments, the change in a property comprises a change in growth, development, or cell phenotype. In one embodiment, the changed property is lethal to the cell. As such, cleavage of the protein may induce programmed cell death (PCD) pathways in eukaryotes via apoptosis or pyroptosis through site-specific protein cleavage.

[0015] In embodiments, a described system of this disclosure is introduced into one or more prokaryotic or eukaryotic cells. In embodiments, the prokaryotic cells comprise or consist of gram positive, or gram negative bacteria. The bacteria may be non-pathogenic, or pathogenic. In embodiments, a described system is introduced into prokaryotic cells (e.g., bacterial or archaeal cells) in the context of a host, e.g., a human, animal, or plant host, e.g., the bacteria are a component of a host’s microbiome or are an abnormal component of a microbiome, e.g., a pathogen. In some embodiments, delivery of a system described herein results in the inactivation of virulence determinants of a microorganism, e.g., antibiotic resistance or toxin production. In some embodiments, delivery of a system described herein results in killing of the recipient cell. The system may kill some or all of the cells, or render the cells non-pathogenic and/or sensitive to one or more antibiotics. In embodiments, the described systems are introduced into eukaryotic cells. Such cells include but are not necessarily limited to animal cells, fungi such as yeasts, protists, algae, and plant cells. [0016] In embodiments, the eukaryotic cells into which a described system is introduced comprise animal cells, which may comprise mammalian or avian cells, or insect cells. In embodiments, the mammalian cells are human or non-human mammalian cells. In embodiments, compositions of this disclosure are administered to avian animals, or to a canine, a feline, an equine animal, or to cattle, including but not limited to dairy cattle.

[0039] As discussed above, the type of cells that comprise an RNA targeted by the guide RNA of a described system is not particularly limited. In embodiments, the RNA is any cytoplasmic RNA. In embodiments, the RNA is an mRNA. In embodiments, the mRNA is expressed in a particular cell type or tissue. In embodiments, mRNA is selectively expressed in cells that are at a particular developmental stage. Thus, the cells can be totipotent, pluripotent, multipotent, or oligopotent stem cells, or hematopoietic stem cells, or differentiated cells. In embodiments, the mRNA is selectively expressed in embryonic stem cells, or adult stem cells. In embodiments, the mRNA is selectively expressed in leukocytes. In embodiments, the leukocytes are of a myeloid or lymphoid lineage. In embodiments, the mRNA is selectively expressed in epidermal stem cells or epithelial stem cells. In embodiments, the mRNA is selectively expressed in cancer stem cells. In embodiments, the mRNA encodes a protein that participates in developing or maintaining any type of cancer. In embodiments, the mRNA encodes a mutated protein that participates in developing or maintaining cancer, or promotes metastasis, or confers resistance to an anti-cancer agent. In embodiments, the mRNA encodes a mutated KRas protein, or a mutated p53 protein, or any other protein that is expressed from an oncogene. In embodiments, the mRNA that is targeted is expressed as a result of chromosomal translocation, or is a viral-specific transcript, or is within any diseased cell that overexpresses a unique transcript, such that the unique transcript can be targeted. In embodiments, the mRNA that is targeted allows tissue engineering through cell killing, such as the selected killing of adipose tissue or senescent cells by targeting unique transcripts expressed in such cells. In embodiments, the mRNA is selectively expressed in human or are non-human animal cells.

[0040] The TPR-CHAT protease may be specific for a particular sequence. In embodiments, the sequence targeted by the TPR-CHAT comprises segment of a Csx30 protein. The segment of the Csx30 that is cleaved may be provided as a component of another protein, and as such, the Csx30 may be present in a fusion protein. In embodiments, cleavage of a fusion protein comprising the Csx30 sequence activates a product of the cleavage such that the product has an effector function, and as such may be referred to as an effector protein. In embodiments, the effector protein comprises the Csx30 segment as an intact or divided protein that, once cleaved, becomes lethal to the cell. In a non-limiting embodiment, the effector protein comprises Gasdermin. Configurations of effector proteins that comprise Gasdermin amino acids are described in the figures and are demonstrated to kill eukaryotic cells. The disclosure include Gasdermin proteins that comprise a Csx30 amino acid segment as components of systems of the disclosure that also include the TPR-CHAT, the gRAMP, and the guide RNA. Without intending to be constrained by any particular theory, it is considered that signal amplification as described herein is a function between the expression level of the mRNA target and the turnover number of Craspase (i.e., TPR-CHAT), meaning how many protein molecules, such as Gasdermin, are cleaved each time the Craspase binds to an mRNA target. For the purpose of illustration only, it may be assumed the turnover number of the wild type Craspase is 2, and that of a particular described mutant is 100. Described single mutants or their combinations may have numbers in between 2 and 100. As such, the disclosure includes mutations of the Craspase when normal cells (i.e., cells not intended to be killed) also expresses the target mRNA at a background level. In such cases, attenuated versions comprising one or a combination of described mutations may lead to a preferred dosing to kill the diseased cells selectively, while not killing the normal cells.

[0017] In embodiments, a system of this disclosure is administered to an individual in a therapeutically effective amount. In embodiments, a therapeutically effective amount of a composition of this disclosure is used. The term “therapeutically effective amount” as used herein refers to an amount of an agent sufficient to achieve, in a single or multiple doses, the intended purpose of treatment. The amount desired or required will vary depending on the particular compound or composition used, its mode of administration, patient specifics and the like. Appropriate effective amounts can be determined by one of ordinary skill in the art informed by the instant disclosure using routine experimentation. For example, a therapeutically effective amount, e.g., a dose, can be estimated initially either in cell culture assays or in animal models. An animal model can also be used to determine a suitable concentration range, and route of administration. Such information can then be used to determine useful doses and routes for administration in humans, or to non-human animals. A precise dosage can be selected by in view of the patient to be treated. Dosage and administration can be adjusted to provide sufficient levels of components to achieve a desired effect, such as a modification in a threshold number of cells. Additional factors which may be taken into account include the particular gene or other genetic element involved, the type of condition, the age, weight and gender of the patient, desired duration of treatment, method of administration, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. In certain embodiments, a therapeutically effective amount is an amount that reduces one or more signs or symptoms of a disease, and/or reduces the severity of the disease. A therapeutically effective amount may also inhibit or prevent the onset of a disease, or a disease relapse.

[0018] The disclosure includes guide RNA-containing TPR-CHAT and gRAMP complexes, which can be either produced in a cell using DNA or RNA encoding for the protein and/or RNA components or delivered in the form of one or more vectors for expression or delivered in the form of RNA encoding for the proteins and/or RNA components or delivered in the form of fully-formed protein-RNA complexes through mechanisms including but not limited to electroporation, injection, or transfection. The described systems can be recombinantly expressed and purified through known purification technologies. These systems can be used in various delivery mechanisms including but not limited to electroporation, injection, or transfection for whole-protein delivery to eukaryotic organisms or can be used for in-vitro applications for sequence targeting of nucleic acid substrates or modification of substrates, and as such, the disclosure includes diagnostic assays that are modifications of the assay known in the art as the SHERLOCK assay. In one embodiment, a protein comprising a Csx30 segment produces a detectable signal when cleaved by a described system.

[0041] The disclosure provides the following sequences, which independently include the entire sequences, and all contiguous amino acid segments that are at least 5 amino acids or 5 nucleotides long, within the sequences:

MBP-GSDMD_N-Csx30_C_GSDMD_C, protein substrate of Craspase.

CRISPR array used in the in vivo GFP-targeting experiment. Its transcription is controlled by a U6 promoter. G NO:4)

Underlined font: CRISPR repeat.

Bold font: CRISPR spacer programmed against GFP mRNA. A

Bold font: sequences that upon mRNA transcription is targeted by the crRNA spacer in the Craspase.

[0019] In embodiments, a system of this disclosure is introduced into eukaryotic cells using, for example, one or more expression vectors, or by direct introduction of ribonucleoproteins (RNPs). In embodiments, expression vectors comprise plasmids, or viral vectors. One or more plasmids or other types of expression vectors can be used. In embodiments, a viral expression vector is used. Viral expression vectors may be used as naked polynucleotides, or may comprises any of viral particles, including but not limited to defective interfering particles or other replication defective viral constructs, and virus-like particles. In embodiments, the expression vector comprises a modified viral polynucleotide, such as from an adenovirus, a herpesvirus, or a retrovirus, such as a lentiviral vector. In embodiments, a baculovirus vector may be used. In embodiments, any type of a recombinant adeno-associated virus (rAAV) vector may be used. In embodiments, a recombinant adeno- associated virus (rAAV) vector may be used. rAAV vectors are commercially available, such as from TAKARA BIO® and other commercial vendors, and may be adapted for use with the described systems, given the benefit of the present disclosure. In embodiments, for producing rAAV vectors, plasmid vectors may encode all or some of the well-known rep, cap and adeno-helper components. In certain embodiments, the expression vector is a self- complementary adeno-associated virus (scAAV). Suitable ssAAV vectors are commercially available, such as from CELL BIOLABS, INC.® and can be adapted for use in the presently provided embodiments when given the benefit of this disclosure

[0020] The following Statements provide various embodiments of the present disclosure.

Statement 1. A modified gRAMP protein comprising one or more mutations relative to the amino acid sequence of an unmodified Candidates Scalindua gRAMP protein, and wherein the one or more mutations in the modified gRAMP protein are selected from a change of amino acid at position 806, 547, 367, 371, 328, and a combination thereof.

Statement 2. The modified gRAMP protein of Statement 1, wherein the combination of mutations is present in the modified gRAMP protein.

Statement 3. The modified gRAMP protein of Statement 2, wherein the combination of mutations comprises D806A and D547A.

Statement 4. A system comprising a modified gRAMP of Statement 1, the system further comprising a TPR-CHAT protein, wherein optionally the TPR-CHAT protein is fused to the modified gRAMP protein.

Statement 5. A system comprising a modified gRAMP of Statement 2, the system further comprising a TPR-CHAT protein, wherein optionally the TPR-CHAT protein is fused to the modified gRAMP protein.

Statement 6. A system comprising a modified gRAMP of Statement 3, the system further comprising a TPR-CHAT protein, wherein optionally the TPR-CHAT protein is fused to the modified gRAMP protein.

Statement 7. The system of any one of Statements 4-6, the system further comprising a guide RNA targeted to an RNA polynucleotide within a cell, and wherein the RNA within the cell is optionally an mRNA. Statement 8. A method for modulating the function of a cell, the method comprising introducing the system of Statement 7 into the cell such that the function of the cell is modified.

Statement 9. The method of Statement 8, further comprising introducing into the cell a protein comprising an amino acid sequence that is recognized and cleaved by the TPR-CHAT protein, and wherein protein cleavage function of the TPR-CHAT protein is activated by the modified gRAMP protein associating with an RNA that is targeted by the guide RNA.

Statement 10. The method of Statement 8, wherein the amino acid sequence that is recognized and cleaved by the TPR-CHAT protein comprises a segment of a Csx30 protein.

Statement 11. The method of Statement 11, wherein the segment of the Cxs30 protein is inserted within an effector protein that affects the function of the cell after the effector protein is cleaved.

Statement 12. The method of Statement 11, wherein the effector protein that is cleaved kills the cell.

Statement 13. The method of Statement 12, wherein the effector protein is Gasdermin.

Statement 14. The method of Statement 13, wherein the cell is a eukaryotic cell.

Statement 15. One or more expression vectors that encode the gRAMP protein and the TPR- CHAT protein as in any one of Statement 4-6.

Statement 16. The one or more expression vectors of Statement 15, wherein the one or more expression vectors further encode the guide RNA.

Statement 17. The one or more expression vectors of Statement 16, wherein the one or more expression vectors further encode an effector protein comprising a segment of a Cxs30 protein that can be recognized and cleaved by the TPR-CHAT protein.

Statement 18. The one or more expression vectors of Statement 17, wherein the effector protein is Gasdermin.

Statement 19. A cell that is not Candidates Scalindua comprising a system of Statement 7.

Statement 20. The cell of Statement 19, wherein the cell is a eukaryotic cell.

[0042] The following example is presented to illustrate the present disclosure. It is not intended to be limiting in any matter. EXAMPLE

[0043] This example provides a description of a protein and method of the present disclosure.

[0044] gRAMP/Cas7-ll structures in resting, RNA-bound, and post-cleavage states. To analyze RNA-guided target RNA cleavage mechanisms inside gRAMP, we reconstituted Candidates “Scalindua brodae" gRAMP and determined its cryo-electron microscopy (cryo-EM) structures in different functional states (Figs. 1A, 6). A'A-gRAMP bound to the complementary RNA target with better than 25 nM affinity and cleaved it at two distinct locations (Fig. 6A-C). Single-particle three-dimensional reconstruction produced gRAMP RNP in four different functional states: a 3.81 A structure of the resting/qpo state, 3.65 A structure of the non-matching protospacer flanking sequence (PFS) target bound state, 3.76 A structure of the matching PFS target bound state, and 3.62 A structure of the postcleavage state (Fig. 1B-H, 7-9).

[0045] The overall architecture of 57>-gRAMP is similar to that of D. ishimotonii Cas7-11, recently reported in the substrate-bound form (no direct comparisons as the Cas7-11 PDB has been released). 57>-gRAMP shares some similarity with the canonical Type III-A effector Csm in overall architecture, guide RNA display, and target RNA binding mode using a six nucleotide segment (Figs. IB and 10). 57>-gRAMP backbone consists four idiosyncratic Cas7 domains fused together, instead of three identical Cas7 subunits in Csm (Fig. 10). A Zn ²⁺ knuckle is present in each of the four Cas7s, which appears to be a shared hallmark among Type III effectors (Fig. 11 A). Csm further contains one copy of Csm4 for 5 ’-handle recognition, two copies of Csm2 as part of the backbone, and one copy of Csm5 for continued guide-target pairing. In contrast, 57>-gRAMP is streamlined: its Cas7.1 has been repurposed for 5 ’-handle recognition, the single-copy Casl 1 domain has been repurposed for target cleavage, and a structurally distinct BID replaces Csm5 (Figs. 1B-C and 10A-H). On the guide RNA side, the 18-nt 5 ’-handle of crRNA in 57>-gRAMP is twice as long as that in Type III-A. Majority of the handle residues are bound by Cas7.1 and shielded on the top by the linker from Casl 1 to Cas7.2 and the Zn-knuckle in Cas7.2 (Fig. 10, 11B-C). Mutagenesis of Zn-knuckle structure or sequence-specific contacts to 5 ’-handle abolished the in vivo RNA silencing activity of 57>-gRAMP, presumably through disruption of RNP assembly (Fig. 12). Notably, the last two handle nucleotides (5’-A-2C-I-3’) are base-pairing competent because they are displayed like a guide (Fig. IF, 11B, H). Type I, III, and IV effectors display the crRNA spacer (guide region) in 6-nt segments, with the 6 ^th nucleotide pinned down by the thumb loop of Cas7; target is hence recognized in 5-nt segments with the 6 ^th nucleotide unspecified. 5Z>-gRAMP contains major exceptions. The first displayed 5-nt segment contains the last two nucleotides of the 5 ’-handle and the first three nucleotides of the spacer, a scenario only observed in Type III-E (Fig. 1F-G, 10H-G). The third segment deviates from the normal again, as an unconventional knocked protein loop from Cas7.4 divides the displayed based to a 3-nt segment and a 6-nt segment. Base-pairing in the 3 ^rd segment is not interrupted; a local base buckling accommodates the peptide crossover. The following crRNA nucleotides are displayed by the dynamic BID domain (aa 1031-1385) which is only resolved to low-resolution and docked with an AlphaFold predicted model (Fig. IB).

[0046] Off-targeting prevention and RNA cleavage mechanisms in Sb- gRAMP/Cas7-ll. By capturing three additional functional states, we have the temporal resolution to interpret the target recognition and cleavage mechanisms by A'A-gRAMP. The long linker from Casl 1 to Cas7.2 (G375-P408, here named the gating loop) has acquired multiple functions during evolution (Fig. 2A-B, 13). Its N-terminal portion (G375-G497) senses RNA substrate binding and controls RNase activities, whereas the C-terminal portion is part of the TPR-CHAT binding interface (explained later). In resting state, the gating loop blocks the first segment of the guide RNA and the nearby Site 1 cleavage center. This conformation is incompatible with target-guide pairing at the first segment and the gating loop has to be displaced to enable cleavage at Site 1 (Fig. 2A). It is therefore contemplated that the target-guide pairing initiates from the third and second segments and propagates into the first segment (Fig. 13), as observed for other Type III systems. In follow-up experiments, we found gRAMP’s RNase activity was optimal against a target with 18-nt complementarity from the 5' -end of the spacer portion; 12-nt or shorter complementarity abolished cleavage and 24-nt or longer complementarity attenuated cleavage (Fig. 14). This suggests that at least some base-pairing along all three segments of the guide RNA, displayed by Cas7.2-Cas7.4, is required for efficient RNA cleavage. In contrast, additional base-paring with crRNA at the BID is not required or may even be counterproductive (Fig. 14). This is consistent with the previous observation that the 3 '-end of the crRNA in the endogenous 57>-gRAMP is often as short as 20 nt, and that the BID is dispensable for Cas7-11 activity in human cells.

[0047] 57>-gRAMP was further incubated with two kinds of RNA targets whose PFS was either matching (complementary) or non-matching with the 5 '-handle in the crRNA, as complementarity in these region may be indicative of a self-target (i.e. anti-sense transcript from the CRISPR locus) and thus perhaps leads to alternative structural configurations in Sb- gRAMP. However, the described structures reveal that regardless of the PFS status, RNA binding induces the same set of conformational changes in 5Z>-gRAMP. Where the guide nucleotides are pinned down by the Cas7 thumbs, the corresponding target nucleotides (4 ^th and 10 ^th ) flip outwards. Rotation of the backbone orients their 2'-OH towards the previous phosphate, forming the so-called “in-line” conformation, which is necessary for RNA cleavage. For target RNA with a matching PFS, the first segment consists of five base-pairs, starting from the last two nucleotides of the 5 '-handle and ending with the 3 ^rd nucleotide in the spacer portion (Fig. 1H). The rest of the PFS is not traceable in the EM map. For a nonmatching PFS containing RNA target, only three base-pairs are found between the target RNA and the spacer portion of the guide. The first two nucleotides of PFS do not form hydrogen bonds with the handle residues on the opposite side, however, they remain stacked to complete the first target-guide segment (Fig. 1H). In both PFS matched and non-matched conditions, the impinging gating loop in 57>-gRAMP is pushed away from the first segment and becomes entirely disordered (Fig. 2A). Concurrently, the cleavage center at site 1 is exposed and further enhanced by a hinge motion in Casl 1 (Fig. 2C, 15A), which aligns catalytic residues among Casl 1 and Cas7.2. Stacking from the additional 2-nt PFS is not a prerequisite to activate gRAMP, as RNA substrate lacking PFS was found to be cleaved efficiently. To validate these structural findings, we replaced the tip of the gating loop with a flexible linker to evaluate its importance in target RNA recognition. Wild-type 57>-gRAMP did not bind and cleave RNA that only base-pairs with the first 9-nt of the crRNA guide. In contrast, the gating loop mutant bound target RNA efficiently to subsequently cleaved it (Fig. 2D) These experiments suggest the gating loop plays a pivotal role in preventing off- targeting. Overall, our RNA-bound 57>-gRAMP structures support a mechanistic model in which the resting 57>-gRAMP exists in an autoinhibited state to avoid sequence-unspecific RNA binding and cleavage. Target RNA is validated via crRNA-pairing in a directional fashion from 3' to the 5' region of the guide. Upon completion, movement of the gating loop initiates a chain of allosteric events to switch on the RNase centers in gRAMP (Fig. 2E).

[0048] Comparison of the pre- and post-cleavage states allowed us to interpret the cleavage mechanism with confidence (Fig. 1, 2F-G). EM densities suggest the RNA substrate was cleaved after the 3 ^rd and 9 ^th nucleotides (site 1 and site 2, respectively) (Fig. 1H), consistent with the reported biochemistry results. Since cleavage is metal-dependent, we identified multiple candidate residues around the cleavage sites that could be responsible for metal coordination (generally acidic residues), proton shuttling (generally polar residues), and transition state stabilization (generally positively charged residues) (Fig. 2F-G). Their functional importance was evaluated through mutagenesis (Fig. 15B-D). RNA cleavage at Site 1 was abolished by alanine substitutions to D547 in Cas7.2 and R294, D298, Y367, and K371 in Casl 1 (Fig. 2H). Since Site 1 is assembled from residues in both Casl 1 and Cas7.2, it may only become active after target binding-induced hinge motion in Casl 1. This would present the second layer of off-targeting avoidance, with gating loop blockage being the first layer. Cleavage at Site 2 was abolished by Cas7.3 mutations D698A and D806A, but not by Casl 1 mutations R323 A and H328A (Fig. 2H). An interesting allosteric effect was noticed: Site 1 disruptive mutations D547A and D298A impaired Site 2 cleavage as well, and Site 2 mutation H328A impaired Site 1 cleavage instead. These mutants appeared to weaken or alter the RNA-binding mode of sb -gRAMP, as revealed by EMSA (Fig. 2H, 15C). 57>-gRAMP containing the double mutations R294A/D698A or D547/D806A was efficient in RNA binding but completely inactive in RNA cleavage (Fig. 15C), effectively presenting dead- gRAMP variants that may be used in RNA editing, tagging, or tracing applications.

[0049] Craspase architecture and component interfaces. One aspect of the Type III-E system is its further association with the possible protease TPR-CHAT. To gain mechanistic insights into how this putative RNA-guided protease system may work, we reconstituted Craspase in its apo (resting) state, the matching PFS-containing RNA target bound state, and the non-matching PFS-containing target bound state and generated their corresponding cryo-EM structures at 3.7 A, 2.6 A, and 2.7 A resolutions, respectively (Fig. 16-18). The TPR-CHAT binding surface is on top of the buried crRNA 5 '-handle in Sb- gRAMP, architecturally similar to where the cOA synthetase (Csml/CaslO) binds in canonical Type III-A effector complexes (Fig. 3A-B, 19A). TPR-CHAT consists of an N- terminal TPR domain (aa 1-323), a dynamic mid-region containing the ‘switch helix ¹ (explained later, aa 324-399), and a C-terminal cysteine protease from the caspase family (aa 400-717). The domain arrangement of TPR-CHAT resembles that of Separase, an essential eukaryotic protein that cleaves the cohesin ring to allow chromosome segregation. However, the structural similarity is only strong in the CHAT domain, but not in the TPR domain (Fig. 19B-D) Like Separase, the CHAT domain contains a N-terminal pseudo-Caspase domain, a long dimeric coiled coil mid-insertion, and a C-terminal active-protease domain. Although structurally quite distinct, the two Caspase domains pack in a similar fashion as authentic Caspase dimers do in eukaryotes. In TPR-CHAT, the P-sheet structure in the pseudo-Caspase domain interact with the TPR domain, the mid-region serves as the sole anchoring point of CHAT onto 57>-gRAMP. The TPR repeats belong to the so-called solenoid domains, which are assembled from repeating structural units and mediate protein-protein or protein-ligand interactions. The seven antiparallel a-helical TPR repeats in TPR-CHAT pack side-by-side to form a C-shaped architecture, with the 7 ^th TPR repeat packing against the P-sheet of the globular CHAT domain. Together, TPR-CHAT adopts the rough shape of a padlock, with TPR being the shackle and CHAT the body (Fig. 19B). In the Craspase structure without target RNA (apo-Craspase), the shackle of the padlock captures the long switch helix (aa338- 362) in the middle. This helix is captured by the molecular contacts from the inward facing loops in the TPR repeats. When wedged in the shackle, the switch helix pins down a loophelix-loop structure underneath (aa 324-337). Together, they mediate extensive set of molecular contacts to various regions inside the padlock (Fig. 19B). A particularly area of interest is the contact to the tips of two long P-hairpins (sensor hairpins) that further extend all the way to the protease center in CHAT (Fig. 19E). These molecular structures form a long-range conformational relay to control protease activity.

[0050] An area of -75X35 A ² of the Cas7.1 surface in A'A-gRAMP is overshadowed by TPR-CHAT (Fig. 3C-D). The actual physical contacts between TPR-CHAT and gRAMP are limited to two surface patches 50 A apart. On the TPR side, a hydrophobic patch in the first and second TPR repeats makes hydrophobic and mainchain hydrogen bond contacts to a portion of the gating loop (F381, 1383, and L384), and a nearby Cas7.2 loop (L450, V451) (Fig. 3C). A more extensive and mostly hydrophobic interface is found between one of the coiled coil helix in the CHAT domain (aa 434-450) and two regions of 57>-gRAMP, namely the C-terminal portion of the gating loop (aa 396-403) and the Zn-knuckle of Cas7.2 (Fig. 3D) In particular, Y450 and L499 of CHAT insert into a hydrophobic pocket on the Sb- gRAMP surface, promoting shape complementarity at the interface. The Craspase complex was completely disrupted by Y75A and F103A mutations in the TPR interface and by A445R and L449A/Y450A mutations in the CHAT interface (Fig. 3E). Gating helix of 57>-gRAMP, which we show to play a role in regulating the RNase activity of 57>-gRAMP through conformational changes, is sandwiched between 57>-gRAMP and TPR-CHAT (Fig. 20A). Whereas the entire gating loop becomes unstructured in the RNA-bound 57>-gRAMP structure, only the tip of it is rearranged to accommodate the base-paired target in the ternary complex of Craspase with non-matching PFS-containing target RNA (Fig. 2A, 21). Given this conformational restriction, we speculated that the energetic barrier for RNase activation may be higher in Craspase compared to A'A-gRAMP. Indeed, RNA binding was consistently weaker at different temperatures and the cleavage was slowly in Craspase than in 57>-gRAMP (Fig. 3F, 20B-C)

[0051] RNA-guided protease activation mechanism in Craspase. When Craspase is in the resting state, the catalytic dyad in the TPR-CHAT protease center, Cys627 and His585, are 6.6 A apart (Fig. 22). As this distance is too far to allow H585 to assist the catalysis through hydrogen bonding mediated C627 deprotonation, which is required to initiate the nucleophilic attack on the peptide substrate, we reasoned that TPR-CHAT exists in an inactive state when Craspase has no target RNA bound.

[0052] When Craspase is bound to a target RNA with a matching PFS (Fig. 4A), a perfectly base-paired first segment is formed between guide and target. Constrained by the base-pairing from the first two PFS residues to the guide, the remaining PFS nucleotides point towards the bottom of TPR. Their densities are difficult to model. Residual phosphate densities suggest that one possible scenario is for PFS to travel underneath TPR (Fig. 4C). This path perturbs the conformation dynamics of the sensing hairpin loop 1 in CHAT such that the loop density disappears due to elevated conformational flexibility. The signal propagates along the P-strand, triggering a local backbone twitch at the protease center (aa 626-631) (Fig. 4D). This includes the catalytic C627 residue in the protease center. The allosteric change shortens the distance between C627 andH585 from 6.6 to 5.2 A (Fig. 21B). However, no sidechain-binding pocket can be found nearby the catalytic dyad (Fig. 4D). Therefore, the partial rearrangement is not expected to switch on the protease activity in CHAT.

[0053] A greater set of conformational changes take place when RNA target containing a non-matching PFS is bound by Craspase (Fig. 4B). Lacking sequence complementarity to the first 2-nt of PFS, the base-pairing in the first guide-target segment is incomplete and the gating loop is only partially pushed away (Fig. 22). While the first nucleotide of PFS forms a partially frayed A*C pair, the rest of PFS completely curl toward the surface of TPR (Fig. 4C). It clashes with the switch helix and dislodges it from the shackle of the padlock. The entire switch helix and the preceding loop-helix-loop connection rotates 90 degree and packs against CHAT as a coiled coil structure (Fig. 4D, 23). This in turn perturbs both sensor hairpins, allosterically altering the conformation of two P-strands in CHAT active site, one harbors H585 and the other C627. Consequently, the two residues are oriented within hydrogen bonding distance (3.3 A) (Fig. 4D), and a hydrophobic pocket opens up nearby (Fig. 21C). The entire CHAT domain further undergoes a rigid-body movement. The cleft between 57>-gRAMP and TPR-CHAT widens, which may enable the peptide substrate to access binding surfaces (Fig. 4D).

[0054] Based on the observed structural features in the protease center, we designed candidate peptides to probe for potential RNA-guided peptidase activity in Craspase. At least one designed peptide showed Craspase-dependent cleavage in thin-layer chromatography assays (Fig. 24A-B, 4E) Consistent with our mechanistic predictions, the activity was stronger in the presence of a non-matching PFS RNA substrate, but not a matching PFS substrate (Fig. 4E). This peptide could also be cleaved by Caspase in the context of an interdomain protein linker, and the cleavage was stimulated by non-PFS target RNA (Fig. 24C- F). The cleavage site was mapped using mass spectrometry (Fig. 25). Consistent with the structural prediction, Craspase cleaves after a medium-sized hydrophobic leucine residue. Judging by the fact that only one of the two leucine residues in the peptide were selectively cleaved (Fig. 4F), and that the cleavage activity was low and only partially RNA-regulated, we speculate that Craspase must be specifying additional sequences nearby, which remain to be fully deciphered.

[0055] A consistent mechanistic model emerges from the structural and docking analysis, in which Craspase is tightly regulated in a logic circuit fashion (Fig. 4G). Sequence complementarity in the target RNA is a prerequisite, which is indirectly read out from the gating loop movement. A NOT logic gate is in place to avoid self-RNA, with a matching PFS. Craspase is only activated when both conditions are true, that is, target RNA is complementary in the spacer portion and non-matching in the PFS. The structural feature performing the AND logic calculation is the switch helix: its movement triggers a stepwise conformational relay that allosterically unlocks the TPR-CHAT padlock and switches on the protease activity (Fig. 4H)

[0056] Craspase proteolytically cleaves Csx30 in an RNA-dependent manner.

Besides the TPR-CHAT and gRAMP, the type III-E loci contain three other well-conserved proteins: the putative sigma-factor RpoE and two unknown proteins, denoted Csx30 and Csx31. As a protease and its target are often conserved in the same genomic neighborhood, we tested if we could observe Craspase protease activity against these proteins in coexpression experiments (Fig. 5A). Full-length Csx30 was strongly reduced in the presence of target bound Craspase, whereas full-length RpoE and Csx31 were not (Fig. 5B). This effect was alleviated when Craspase carried inactivated cysteine-histidine residues (H585A and C627A) (Fig. 5B), strongly suggesting that Craspase possesses proteolytic activity against Csx30. This observation was confirmed in vitro, where purified Craspase processes Csx30 in two distinct fragments (Fig. 5C), demonstrating that Csx30 is the natural protein target of Craspase. Corroborating our structural insights, proteolytic digestion could only be observed in the presence of RNA complementary to the crRNA in Craspase while non-matching the PFS, whereas no cleavage fragments accumulated upon incubation with non-target RNA or target RNA with matching PFS (Fig. 5C; Fig. 5D). As Craspase cleaves bound RNA only under bivalent cation conditions, we reasoned that the peptidase in target bound Craspase would be in a “stay on”-state in the absence of magnesium ions. We observed a large increase in Csx30 processing under magnesium poor conditions compared to prior incubation with magnesium (Fig. 5E), suggesting that target RNA cleavage switches off the peptidase. This is further supported by the finding that the peptidase activity of a nuclease-dead variant of Craspase is not aborted by the presence of magnesium ions (Fig. 5E), rendering Craspase R294A D698A a “stay on” variant (Fig. 5F). Binding of a complementary ssDNA, which is not cleaved by Craspase, does not activate the peptidase (Fig. 5E). These findings combined propose a model (Fig. 5G) in which the peptidase activity of Craspase is switched-on upon target RNA detection to cleave Csx30 in two fragments, likely enabling an immune response. Craspase then self-regulates through target RNA cleavage to switch back to a peptidase off- state, likely allowing the binding of a new target RNA to recycle the Craspase complex.

[0057] Discussion. The present disclosure demonstrates that the Craspase protease is allosterically activated by target RNA recognition event. The disclosure provides experimental and high-resolution mechanisms to explain its allosteric activation and shutoff mechanisms. The disclosure demonstrates tuning its dynamic response range using mechanism-inspired mutants. Thus the present disclosure demonstrates the RNA-activated protease activity of Craspase on its physiology substrate as well as artificial peptide substrates. RNA controls Craspase activity in an almost binary fashion towards the native substrate, but in a more gradual fashion towards the artificial substrates. The disclosure supports the intepration that the cleavage sequence in the native protein substrate is read out in the context of the 3D structure, which has been demonstrated for the molecular recognition between eukaryotic Caspases and Gasdermins.

[0058] Despite structural distinctions, the disclosure reveals that Type III-E systems share mechanistic similarities with canonical CRISPR-Cas Type III systems. For example, whereas Craspase does not differentiate self and non-self RNA targets at the RNA-guided RNA cleavage level, it only turns on the protease activity in response to non-self RNA targets. Craspase switches off protease activity upon target RNA cleavage, suggesting that the activity may only be needed temporarily in the cell. The stringent regulation of the protease activity by the gating loop provides that RNA activation is only achieved by target RNA that match the majority of the guide sequence. Because the present disclosure shows that the Craspase peptidase is only active in the presence of a specific RNA species renders it useful for both in vivo (e.g. gene expression profiling by transcript detection) and in vitro (e.g. RNA diagnostics) biotechnological applications, and will further transform the range of biomolecular engineering possibilities of Cas effector proteins.

Materials and Methods

[0059] Cloning, Expression, and Purification. Plasmids used in this study are listed in table 2. Primers described in table 2 were used for PCR amplification using the Q5 high- fidelity Polymerase (New England Biolabs). Ordered DNA sequences are listed in table 2. All plasmids were verified by Sanger-sequencing (Macrogen Europe, Amsterdam, The Netherlands). Cloning was performed using NEBuilder HiFi DNA Assembly (New England Biolabs) unless stated otherwise. Bacterial transformations for cloning were performed using NEB® 5-alpha Competent A. coll (New England Biolabs) and carried out by electroporation using ECM630 Electrocell Manipulator (2.5 kV, 200 Q, 25 pF).

[0060] For Craspase expression, pMLGR AMP, pCRISPRl , pMLGR AMP CRISPR I , p5B-GRAMP_CRISPRl target 1 and TPR-CHAT were used. For SZ>-gRAMP-crRNA expression, the p5B-GRAMP_CRISPR was transformed into BL21(DE3) cells. A single colony was picked and grown for overexpression in 4 liters of LB media supplemented with streptomycin (add concentration here). Over expression was induced by adding isopropyl-P- D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and incubation at 16°C overnight. Cells were collected by centrifugation at 5000 rpm on J6 centrifuge and lysed by sonication in buffer A (200 mM NaCl, 50 mM HEPES pH 7.5, 2 mM TCEP, 10% glycerol) with 1 mM phenylmethyl sulfonyl fluoride (PMSF). The lysate was centrifuged at 12,000 rpm for 60 minutes at 4°C, and the supernatant was filtered through a 0.45 pm syringe filter and loaded onto 5 ml of pre-equilibrated Strep-Tactin ®XT Affinity Resin (IB A Lifesciences GmbH). The loaded resin was washed with 25 ml of buffer A and the protein eluted by 20 ml buffer A with 2.5 mM dethio-biotin. The elution was concentrated and further purified by size-exclusion chromatography (Superdex 200 Increase 10/300 GL; Cytiva) equilibrated with buffer B (175 mM NaCl, 25 mM HEPES pH 7.5). The second size-exclusion chromatography peak was collected, concentrated, and flash frozen with liquid nitrogen.

[0061] For Craspase expression, the p5B-GRAMP-CRISPR plasmid was cotransformed into B121(DE3) with pTPR-CHAT plasmid. A single colony was picked and grown for expression in 4 liters of LB media. Expression was induced by adding isopropyl-P- D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and incubation at 16 °C overnight. Cells were collected by centrifugation and lysed by sonication in buffer A (200 mM NaCl, 50 mM HEPES pH 7.5, 2 mM TCEP, 10% glycerol) with 1 mM phenylmethyl sulfonyl fluoride (PMSF). The lysate was centrifuged at 12,000 rpm. for 60 minutes at 4 °C, and the supernatant was filtered through a 0.45 pm syringe filter and loaded onto 5 ml of pre-equilibrated Strep-Tactin ®XT Affinity Resin (IBA Lifesciences GmbH). The loaded resin was washed with 20 ml buffer A and eluted by 25 ml buffer A with 2.5 mM dethio-biotin. The elution was then loaded onto 3 ml of pre-equilibrated Ni-NTA resin (Qiagen). After washing with 15 ml of buffer A, the sample was eluted by 15 ml buffer A with 300 mM imidazole. This eluted sample was concentrated and further purified by sizeexclusion chromatography (Superdex 200 Increase 10/300 GL; Cytiva) equilibrated with buffer B (175 mM NaCl, 25 mM HEPES pH 7.5). The peak was collected, concentrated, and flash frozen with liquid nitrogen.

[0062] To construct pTag -Csx30, pTag-Csx31 and pTag-RpoE, a coding sequence corresponding to an E. coll codon-optimized Csx30, Csx31 and RpoE proteins were designed, ordered (Life Technologies Europe BV) and cloned into the plasmid pGFPuv using NEBuilder HiFi DNA Assembly (New England Biolabs), combined with a cloned fragment encoding the LacI repressed T7 promoter followed by a N-terminal Twin-Strep-Tag II and a SUMO-tag.

[0063] To construct pTPR-CHAT-targetl, pTPR-CHAT was enriched with a sequence encoding for target RNA complementary to crRNAl under control of the IPTG inducible T7 promoter, using NEBuilder HiFi DNA Assembly (New England Biolabs).

[0064] Purification of Csx30, Csx31 and RpoE overexpressed together with Craspase. Plasmid p5B-GRAMP_CRISPRl, pTPR-CHAT and one of the following three (pTag-RpoE, pTag-Csx31 or pTag-Csx30) were transformed in electrocompetent E. coll BL21(AI) cells and grown overnight on selection media (100 pg/mL spectinomycin, 25 pg/mL chloramphenicol, 100 pg/mL ampicillin). Colonies were streaked from the plate and grown in 200 mL LB medium containing antibiotics (100 pg/mL spectinomycin, 25 pg/mL chloramphenicol, 100 pg/mL ampicillin) in baffled flasks at 37 °C and 150 rpm until they reached exponential phase (ODeoo 0.3-0.5). The grown cultures were incubated on ice for 1 hour and protein overexpression was induced with a final concentration of 0.2% L-arabinose and 0.5 mM IPTG followed by overnight incubation at 20 °C and 150 rpm. Cells were collected by centrifugation at 16000 rpm at 4 °C. The supernatant was discarded and the pellets were resuspended in PBS (50 mL PBS/initial 1 L culture) and harvested by centrifugation (30 minutes, 3900 rpm 4 °C). The supernatant was discarded, and the pellets stored at -80 °C until further use. Bacterial cell pellets of each 200 mL culture were resuspended in 10 mL of ice-cold lysis buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5% glycerol, pH 7.5). The cells were lysed by sonication (2 minutes, 30 sec on 30 sec off, amplitude 30%). The lysate was centrifuged at 16,000 rpm for 30 minutes at 4 °C, and the supernatant was filtered through a 0.45 pm syringe filter and loaded onto 0.25 ml (column bed volume) of pre-equilibrated Strep-Tactin ®XT Affinity Resin (IBA Lifesciences GmbH). The loaded resin was washed with with 10 column volumes of ice-cold wash buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5% glycerol, pH 7.5) and eluted in 750 pL of elution buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5% glycerol, 50 mM Biotin, pH 7.5).

[0065] Purification of Csx30 for cleavage experiments. The plasmid pTag-Csx30 was transformed in electrocompetent E. coll BL21(AI) cells and grown overnight on selection media (100 pg/mL ampicillin). Colonies were streaked from the plate and grown in 4L or 8L of LB medium containing antibiotics (100 pg/mL ampicillin) in baffled flasks at 37 °C and 150 rpm until they reached exponential phase (ODeoo 0.3-0.5). The grown cultures were incubated on ice for 1 hour and protein overexpression was induced with a final concentration of 0.2% L-arabinose and 0.5 mM IPTG followed by overnight incubation at 20 °C and 150 rpm. Cells were collected by centrifugation at 16000 rpm at 4°C. The supernatant was discarded and the pellets were resuspended in PBS (50 mL PBS/initial 1 L culture) and harvested by centrifugation (30 minutes, 3900 rpm 4 °C). The supernatant was discarded, and the pellets stored at -80 °C until further use. Bacterial cell pellets of were resuspended in ice-cold lysis buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5% glycerol, pH 7.5), 50 ml of buffer/lL initial culture. 1 tablet of cOmplete™ EDTA-free Protease Inhibitor Cocktail was added per 50 mL resuspended pellet. The cells were lysed with 3 runs at 1000 bar in a cooled French press. The lysate was centrifuged at 16,000 rpm for 30 minutes at 4 °C, and the supernatant was filtered through a 0.45 pm syringe filter and loaded onto pre-equilibrated Strep-Tactin ®XT Affinity Resin (IBA Lifesciences GmbH), 1.5 mL column bed volume/4L initial volume. The loaded resin was washed with 10 column volumes of ice-cold wash buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5% glycerol, pH 7.5) and eluted in 750 pL of elution buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5% glycerol, 50 mM Biotin, pH 7.5).

[0066] For removal of the Csx30 purification Strep-SUMO tag, Tobacco Etch Virus (TEV) protease (Sigma Aldrich; T4455) was added for overnight incubation at 4 °C, followed by affinity chromatography containing HIS-Select Nickel Affinity Gel (Sigma-Aldrich) to remove the TEV protease. The collected flow-through was subjected to size exclusion chromatography using Superdex 200 Increase 10/300 GL (Cytiva) column equilibrated with running buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM DTT, 5% glycerol, pH 7.5) with 0.3 mL/min flow rate using running buffer as mobile phase. Pooled fractions were concentrated, flash frozen in liquid nitrogen and stored at -80 °C until further use.

[0067] In vitro target RNA and non-target RNA generation. gBlocks (Table 2) containing the T7 promoter and target RNA (complementary to CRISPR1) or non-target RNA (not complementary to CRISPR1) were synthesized (IDT) and PCR amplified with 5’- (SEQ ID NO: 6) and 5’- (SEQ ID NO:7). -500 ng of purified PCR fragment was in vitro transcribed overnight using the HiScribeTM T7 High Yield RNA Synthesis Kit (NEB) and subsequently treated with DNase I according to the manufacturer’s protocol. For RNA extraction, acidic phenol (pH 4.5, phenol: chloroform = 5: 1, Invitrogen) was added to the sample in a 1 :1 ratio, vortexed for 1 minute and centrifuged for 10 minutes at 13,200 rpm at room temperature. The aqueous phase was collected and subjected to RNA precipitation (20 pL 3M NaAcetate and 500 pL 100% ethanol per 200 pL of sample) for 1 hour at -20 °C. Samples were centrifuged at 13,200 rpm at 4 °C for 2 hours, washed twice with ice-cold 70% ethanol and centrifuged at 13,200 rpm at 4 °C for 10 minutes. The pellet was dried in a SpeedVac concentrator (Thermo Fisher Scientific) for 30 minutes at 60 °C and resuspended in RNA grade water. The four variants of target RNA were mixed in an equimolar ratio before usage in Csx30 protein cleavage reactions.

[0068] RNA cleavage Assays. 20pL reactions were prepared where 20 nM Cy5 labeled target RNA (table 2) was incubated with ,S7i-gRAMP RNP in cleavage buffer (100 mM KC1, 50 mM HEPES pH 7.5, 2 mM PME, 5 mM MgCh) and incubated at room temperature for 30 min (unless otherwise specified). The 20 pL reactions were quenched with the addition of EDTA to 150 mM (final concentration) and 1 : 1 volume of 100% formamide. Samples were heated to 95 °C for 10 minutes and run on 12% urea-PAGE gel. Fluorescent signals were imaged using ChemiDoc (BioRad) and quantified using Image Lab.

[0069] RNA knock down assay in E.coli. p5B-GRAMP wild type or mutants (Spec ^R ), pCRISPR GFP guide wild type or repeat region mutants (Kana ^R ) and pGFP (Amp ^R ) were co-transformed into E. coli BL21(DE3) cells and plated on LB-agar plates with appropriate antibiotics. Transformation plates were incubated for 20 hours at 37 °C and then scanned via GFP channel using a ChemiDoc (BioRad) (Figure 6D-E). For the dot assay, single colonies were picked after transformation and cultured in 3 ml LB media with appropriate antibiotics for 18 hours in 37 °C. Then adjusted all cultures to the same OD at 0.1. 10 pL from each culture was pipetted on a plastic membrane. Fluorescent signals were imaged using a ChemiDoc (BioRad) via 515 nm to monitor GFP signal and 600 nm to monitor cell density.

[0070] Electrophoretic mobility shift assay. 5 nM final concentration of fluorescently labeled target RNA was incubated with specified concentration of 57>-gRAMP or Craspase complex in a 20 pL reaction containing binding buffer (50 mM Tris pH 8.0, 150 mM NaCl, 10% glycerol). After a 20-minute incubation on ice (unless otherwise specified), 10 pL of each sample was loaded onto 1% agarose gel equilibrated in 0.5 x TBE buffer. Electrophoresis was performed at 60 V for 30 min at 4 °C. Fluorescent signals were recorded using a ChemiDoc (Bio-Rad).

[0071] Peptide cleavage assay. 500 ng FAM labeled synthesized peptides (GenScript) were dissolved in 500 pL buffer A (150 mM NaCl, 25 mM HEPES pH 7.5, 5% glycerol) to a final concentration of 1 mM. Peptides were further diluted to 100 pM before performing the assay. 2 pL of the peptide was mixed with 3 pL of Craspase RNP (1 pM final concentration) with preincubated target RNA (4 pM final RNA concentration when added) and incubated at 37 °C for 1 hour. 2 pL of reaction was applied to a thin layer chromatography plate (K6 silica gel, catalogue No: 4860-820). Thin layer chromatography (TLC) plate was put in a sealing glass tank with 3 cm of acetone for 30 min. After chromatography, FAM signals on the plate were imaged using a ChemicDoc (Bio-Rad).

[0072] For GST-AK2-P3C fusion protein cleavage assay, mixed 3 pg fusion protein with 2.5 pg Craspase (if with target RNA pre-incubated complex, RNA concentration is 4 pM) and incubated at 37 °C for 2 hours. Next, 4 pL of 5x SDS-loading dye was added and reaction was heated in 95 °C for 10 min. 8 pL of reaction was loaded and run on a 12% SDS- PAGE gel for analysis.

[0073] Cryo-EM sample preparation, data acquisition, and processing. Sb- gRAMP was incubated with the specified target RNA in cryoEMbuffer (150 mM NaCl, 25 mM HEPES pH 7.5) for 15 minutes at room temperature. RNA was supplied at a 3-fold molar excess to 57>-gRAMP (0.5mg/mL final concentration). 3.5pL of the incubation was applied to a Quantifoil holey carbon grid (1.2/1.3, 200 mesh) which had been glow- discharged with 20 mA at 0.39 mBar for 30 seconds (PELCO easiGlow). Grids were blotted with Vitrobot blotting paper (Electron Microscopy Sciences) for 3 seconds at 4 °C, 100% humidity, and plunge-frozen in liquid ethane using a Mark IV FEI/Thermo Fisher Vitrobot. Data was collected on a 200 kV Talos Arctica or Krios G3i Cryo Transmission Electron Microscope (Thermo Scientific) with a Ceta 16M CMOS camera 300kV, Gatan K3 direct electron detector. The total exposure time of each movie stack led to a total accumulated dose of 50 electrons per A2 which fractionated into 50 frames. Dose-fractionated super-resolution movie stacks collected from the Gatan K3 direct electron detector were binned to a pixel size of 1.1 A. The defocus value was set between -1.0 pm to -2.5 pm.

[0074] Motion correction, CTF-estimation, blob particle picking, 2D classification, 3D classification and non-uniform 3D refinement were performed in cryoSPARC v.2. Refinements followed a standard procedure, a series of 2D and 3D classifications with Cl symmetry were performed as shown herein to generate the final maps. A solvent mask was generated and was used for all subsequent local refinement steps. CTF post refinement was conducted to refine the beam-induced motion of the particle set, resulting in the final maps. The detailed data processing and refinement statistics for cryo-EM structures are summarized herein and Table 1.

[0075] In vitro Csx30 cleavage reactions. Csx30 cleavage reactions were performed in 10 pL reaction volume, containing purified 2225 nM Craspase (WT, MT (TPR-CHAT H585A C627A) or MT (D698A R294A), without SUMO tag (Fig. 5C) or with SUMO tag (Fig. 5E and 5F), 5 pM (for incubation with Craspase D698A R294A) or 11 pM (for incubation with target RNA containing matching or non-matching PFS) Dual Strep- SUMO- TEV-Csx30 protein (Fig. 5E and 5F) or 4 pM tag-less Csx30 protein (Fig. 5C, 900 ng in vitro generated target RNA or non-target RNA or 3 pM target RNA containing matching or nonmatching PFS, 100 mM Tris, 150 mM NaCl, 10 mM DTT, 0 mM or 2 mM MgC12 (Fig. 5C reactions contain 2 mM MgC12, Fig. 5E was incubated with 90 ng of target RNA and 2 mM MgC12 for 2 hours at 37 °C prior to addition of 11 pM DualStrep-SUMO-TEV-Csx30 protein). Reactions were run for 1 hour at 37 °C. Afterwards, the reactions were supplemented with 10 pL MilliQ water, 5 pL of 5X Laemmli buffer (375 mM Tris-HCl, 9% SDS, 50% glycerol, 0.03% bromophenol blue) and 2.5 pL 1 M DTT, and incubated at 95 °C for 10 minutes before loading on a 4-20% surePAGE™ Bis-Tris protein gel (GenScript).

Gels were run in IX MOPS buffer (GenScript) at 200 V for 30-45 minutes, washed in MilliQ water and stained for at least 2 hours with BioSafe Coomassie G-250 stain (Bio-Rad) under continue shaking. Gels were washed in MilliQ water for 4 hours before imaging.

[0077] Table 2. Oligos and peptides used in this study

[0078] Figure 27 shows ex vivo data showing plasmid delivery of Craspase and engineered Gasdermine-Csx30 triggers human cell death in RNA substrate dependent fashion, guided by the crRNA. Equal amounts of HEK293-GFP cells (50,000 cells/well) were seeded on to 96-well plate. 24 h after seeding, each well were transfected with equal amount of plasmid, premixed with 0.25 pL transfection reagent JetOptimus. The cells are then allowed to settle and recover in the 37 °C incubator for 12 hrs, after which the entire plat is loaded to the Incucyte instrument for growth monitoring. The cell confluency was quantified by Incucyte from wide-view cell imaging. The transfection reagent JetOptimus is mildly toxic to the cells, causing the slight decline in cell confluency (diamond line and the inverted triangle line). They showed that incomplete Craspase does not cause cell death, neither does the uncleaved engineered GSDMD-Csx30 fusion. The positive control confirms the cytotoxicity of mouse GSDMD N-terminal fragment.

[0079] The circled line reveals the strong cytotoxicity caused by the co-expression of RNA-programmed Craspase (gRAMP-crRNA further assembled with TPR-CHAT) and the engineered cleavage cytotoxin (GSDMD-Csx30 fusion), in the presence of the RNA substrate (GFP mRNA). The data in Figure 27 were generated using a double mutant gRAMP with D698A/R294. It is expected that similar cytotoxicity will be obtained using other mutations and mutation combinations described herein, including but not necessarily limited to a mutated gRAMP protein with amino acid changes at one or both of positions 806 and 547.

[0080] Although the present disclosure has been described with respect to one or more particular embodiments and/or examples, it will be understood that other embodiments and/or examples of the present disclosure may be made without departing from the scope of the present disclosure.