Conversion of iscb and cas9 into RNA-guided RNA-editors

Abstract

Provided is a compact RNA-editing platform engineered from IscB, which has comparable or higher activity than Cas13, but with less or no cytotoxicity. IscB, has intrinsic affinity for complementary single-stranded (ss)DNA and RNA. This activity becomes dominant when its dsDNA binding activity is switched off, through the deletion or mutation of its Target Adjacent Motif domain, resulting in a modified IscB. The resulting R-IscB is comparable or better than Cas13, can efficiently alter splicing outcomes in human cells, and can mediate trans-splicing to correct mutations at the mRNA level. R-IscB also drives efficient A-to-I editing on mRNA when fused to ADAR2 and mediates cleavage-based mRNA knockdown upon HNH engineering. The same approach converts some Cas9s to RNA-targeting tools.

Description

[0001] Conversion of IscB and Cas9 into RNA-guided RNA-editors CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. provisional application no. 63 / 686,541, filed August 23, 2024, the entire disclosure of which is incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with government support under 5R35GM118174 awarded by the National Institutes of Health. The government has certain rights in the invention. RELATED INFORMATION RNA plays a central role in biology as it bridges the flow of genetic information from DNA to proteins. Significant effort has been devoted to developing therapeutics that alter the expression level, splicing pattern, or sequence content of mRNA. Because the consequence of RNA editing is not heritable, inadvertent off-targeting events will not result in heritable genetic scars. When combined with long-persistence delivery tools such as Adenovirus Associated Virus (AAV)1, a single delivery of RNA editor can lead to a long-lasting impact2. However, there remains on ongoing unmet need for improved compositions and methods that can be used to target RNA. The present disclosure is related to this need. BRIEF SUMMARY IccB RNP is an RNA-guided DNA-targeting nuclease. The present disclosure reveals that wild type IscB can also bind a complementary ssRNA target, however, when a dsDNA target is present, it strongly prefers to bind the dsDNA target. The disclosure includes introducing mutations to IscB to convert it to an exclusive ssRNA- or ssDNA-targeting complex, with little affinity for dsDNA. Because ssDNA is rarely present inside the cells, the disclosure provides an RNA-targeting complex. Previously, RNA-targeting activity has been reported for Cas13 and Cas9 RNPs. Similar to the Wildtype IscB, the RNA-targeting activity in Cas9 is likely secondary to its DNA-targeting activity, meaning that Cas9 strongly prefers dsDNA over ssRNA or ssDNA. The present disclosure is different from previous approaches at least because it is demonstrated that by removing all or most of the TAM (Target-Adjacent Motif) interaction domain (TID) is deleted, or mutated, or replaced with an RNA-binding domain, the wild type IscB the IscB is converted to an exclusively RNA-targeting molecule. The same approach in Cas9 is unlikely to be successful because removing the equivalent domain will disrupt the Cas9 structure, but the present disclosure provides modified Type II- D Cas9 (Cas9d) proteins with altered sequences, which are suitable for new uses as further described below. The disclosure includes data demonstrating: 1) Modified IscB proteins can perturb the splicing patten of mRNA inside human cells. It is expected this aspect can be used to treat genetic diseases such as the Duchenne Muscular Dystrophy, or be used for PCSK9 knock- down to prevent heart disease, as well as other therapeutic approaches. The described approach may also knock-down triplet repeat expansion transcripts.2) The disclosure demonstrates IscB variants that can cleave RNA targets. This can destroy mRNAs containing dominant negative mutations.3) The disclosure provides IscB-Adar2 fusion enzymes that can be used for RNA-guided RNA editing (change A to I, which will be interpreted by the ribosome as G). This can correct mRNA and rescue recessive mutations.4) The disclosure provides an in situ RNA detection module, by further coupling the described modified IscB proteins with enzymatic or fluorescent modules to either report the presence of mRNA, or change the cell fate based on the presence of a particular mRNA.5) The disclosure provides modified Cas9 related enzymes, such modified Type II-D Cas9, proteins, wherein the PID domain may be partially or fully deleted or otherwise modified. BRIEF DESCRIPTION OF THE FIGURES FIG.1 Wildtype IscB bound ssRNA and ssDNA but strongly preferred dsDNA. (A) Domain organization and structure of O.geu IscB (PDB: 7UTN). P1D, P1 interaction domain; TID, TAM-interaction domain. The RuvC domain is separated into three segments: RuvC I, II, and III. (B) Native agarose EMSA showing that OgeuIscB RNP bound to ssDNA and ssRNA tightly and sequence specifically. (C) Denaturing PAGE showing that the extent of dsDNA cleavage was not significantly affected by the excess amount of ssRNA competitor. (D) EMSA showing that ssRNA binding was readily competed off by the dsDNA target. See also FIG.7. FIG.2 TID removal disrupted dsDNA binding, but did not affect ssRNA and ssDNA binding. (A) Domain organization of OgeuIscB-ΔTID and OgeuIscB-ΔTID / +S1. Residues from AA433-487 were deleted in OgeuIscB-ΔTID, which were replaced by the S1 domain from E. coli PNPase. (B) Superposition of wildtype OgeuIscB RNP (PDB: 7UTN) with the Alphafold3 predicted OgeuIscB-ΔTID and OgeuIscB-ΔTID / +S1. (C) EMSA showing OgeuIscB-ΔTID’s affinity for dsDNA was disrupted to the background level. (D) Denaturing PAGE showing that OgeuIscB-ΔTID was not able to cleave dsDNA. (E) EMSA showing OgeuIscB-ΔTID’s affinity for ssDNA and ssRNA was on par with wildtype OgeuIscB. (F) EMSA showing that ssRNA binding by OgeuIscB-ΔTID could not be competed off by the dsDNA target. (G) OgeuIscB-ΔTID (hereby named R-IscB) / ssRNA binding kinetics measured from single-molecule colocalization studies. A representative trace, as well as the kon, koff, Kdparameters, are shown. (H) Bar graph documenting the impact of mismatches between guide RNA and target RNA on R-IscB mediated binding. All values are shown as mean ± s.d. (n = 3). See also FIG.8, 9. FIG.3 R-IscB mediated robust splicing perturbation in RNA-guided fashion. (A) Schematics of the expected splicing outcome in PKM. R-IscB targeting is expected to selectively downregulate the abundance of PKM2 isoform. (B) Guide binding locations. M1 to M8 were designed to target the 3’ splicing site of exon 10. M2 and M5 were highlighted in orange. (C) PKM1 and PKM2 mRNA abundance after M1-M8 guided R-IscB targeting in HEK293T cells. (D) PKM2 / PKM1 abundance ratio after targeting. M2 was the most effectiveguide in selective PKM2 knockdown; M5 was also effective. (E) Schematics of the guidesequence (K1 to K6) designs for PCSK9 knockdown. (F) PCSK9 mRNA abundance change after K1-K6 guided R-IscB targeting in HepG2 cells. K3 was the most effective guide. (G-H) PKM1 and PKM2 mRNA abundance (G) and PKM2 / PKM1 ratio (H) after R-IscB targeting with longer guides (extended from 16-nt to 23- and 30-nt). (I) The splicing pattern of the wild-type DMD, the Δexon 48-50 mutation in many DMD patients, and the functional rescue upon exon 51 skipping. (J) Guide designs (D1 and D2) to induce DMD exon 51 skipping. (K) DMD mRNA knockdown in HEK293T cells by D1, D2 guided R-IscB, or D2 guided sextuple TID-mutant IscB. For C, D, F-H, K, all values are shown as mean ± s.d. (n = 3). n.s., not significant. *, p<0.05. ***, p<0.005. a.u., arbitrary unit. See also FIG.10. FIG.4 R-IscB mediated efficient RNA sequence correction through either trans- splicing or A-to-I editing. (A) Schematics of R-IscB mediated trans-splicing to correct RNA sequence. A premature stop codon was introduced to the intron-inserted EGFP sequence. R- IscB-ωRNA appended with the correcting GFP sequence elements was programmed to target the intron. Successful trans-splicing would lead to functional GFP production. (B) Percentage of cells with green fluorescence after trans-splicing editing, with various controls to prove that editing was RNA-guided. (C) Trans-splicing efficiency by six different guides. (D) Full- length OgeuIscB was not able to mediate trans-splicing. (E) Schematics of R-IscB mediated RNA base editing. A premature stop codon mutation in mCherry would be corrected to a tryptophan upon A-to-I editing, leading to red fluorescence accumulation. The ωRNA guide was extended by 51-nt to generate an ADAR editing site upon target binding. ADAR2dd was tethered to the C-terminus of R-IscB through a flexible fusion linker. (F) Phase / red fluorescence overlayed images showing that R-IscB-ωRNA-ADAR2dd conferred efficient RNA A-to-I editing. (G) The editing efficiency was influenced by the distance between the A- C mismatch and the guide. See also FIG.11. FIG.5 Structure-guided engineering enabled ssRNA cleavage in R-IscB and improved knockdown efficiency. (A) Left: Multiple sequence alignment of the HNH domain from NmeCas9, CjCas9, SpCas9, and OgeuIscB consistency, and its five variants (VR1 to VR5). Right: active site arrangement in the HNH domains of IscB (PDB: 7UTN) and NmeCas9 (PDB: 8JA0). (B) ssRNA cleavage efficiency of IscB-WT, IscB-VR3, IscB-VR4, and R-IscB-VR4, evaluated on denaturing PAGE. (C) dsDNA cleavage by IscB-VR3, IscB- VR4, and R-IscB-VR4 on denaturing PAGE. (D) Extent of mismatched ssRNA cleavage by R-IscB-VR3, normalized against the perfect-matched control. (E) Guide designs in the cleavage-based PCSK9 knockdown. The seven guides either target individual exons or the exon-exon junctions. (F) Normalized PCSK9 mRNA abundance after each guided targeting by R-IscB-VR4 in HepG2 cells. C2 and C4 were the most efficient guides. (G) Normalized EZH2 mRNA abundance in HEK293T cells after R-IscB-VR4 targeting at three sites or sextuple TID mutant IscB targeting E3. (H) Volcano plotting showing the transcriptome-wide differentially expressed genes after R-IscB-VR4 mediated ADARB1 targeting. Relative mRNA level was normalized and compared with its counterparts in the non-targeting control. (I) Side-by-side comparison of mRNA knockdown efficiency by R-IscB, PspCas13b, and RfxCas13d across five targets. For D, F, G, and I, all values are shown as mean ± s.d. (n = 3). **, p<0.01, ***, p<0.005. ****, p<0.001. a.u., arbitrary unit. See also FIG.12. FIG.6 PID removal converted NmeCas9 to an efficient RNA-targeting tool in human cells. (A) Domain organization of NmeCas9, with and without PID deletion. An artificial linker (HPNDY) was introduced to connect the structural elements after PID (AA951-1059) removal. (B) Agarose gel showing that RNA-guided dsDNA cleavage was abolished in NmeCas9-ΔPID. (C) Denaturing PAGE showing that ssDNA and ssRNA cleavage activity in NmeCas9 was preserved upon PID removal. Cleavage sites were indicated with arrows on the ssDNA and ssRNA sequences. ssRNA cleavage was mainly confined to site2 in NmeCas9-ΔPID. (D) Top: Schematics showing our approaches to removePID from SaCas9, CjCas9, and Cas9d. Bottom: Side-by-side comparison of mRNAknockdown activity on EZH2 in HEK293T cells by RuvC-disabled NmeCas9-ΔPID (D16A), dead NmeCas9-ΔPID (D16A / 588A), SaCas9-ΔPID, CjCas9-ΔPID, and Cas9d-ΔPID. Their wildtype full-length counterparts were also assayed to reveal the impact of PID removal. All values are shown as mean ± s.d. (n = 3). See also FIG.12. FIG.7 Biochemical characterization of DNA and RNA targeting activities in thewildtype and engineered OgeuIscB. (A) EMSA showing that altering TAM sequence didnot reduce the binding of IscB for ssDNA (left) and ssRNA (right). (B) Denaturing PAGE showing that IscB lacking TID was still capable of ssDNA cleavage. (C) Denaturing PAGE showing that neither full-length IscB nor IscB-ΔTID cleaved ssRNA well, even at 1 µM concentration at 37°C for 30 min. (D) Strep-tag purification of OgeuIscB-ωRNA, OgeuIscB- ΔTID-ωRNA, and OgeuIscB-ΔTID / +S1-ωRNA, analyzed on SDS-PAGE. (E) Elution profile of OgeuIscB-WT-ωRNA and OgeuIscB-ΔTID-ωRNA on mono Q column. Higher O.D.260 reading is consistent with ωRNA presence. FIG.8. Single molecular setup to characterize R-IscB-ssRNA interactions. (A) TwinStrep-tagged R-IscB was immobilized onto the neutravidin-bound biotinylated quartz slide. For association rate (kon) measurement, Cy5-labeled ssRNA was flown into the prepared slide chamber under continuous TIRF recording to monitor the kinetics of Cy5 immobilization. For dissociation rate (koff) measurement, the Cy5-labeled ssRNA was pre- incubated with the R-IscB immobilized slide chamber, then washed out. Knowing that dwell time is very long after IscB binding, to avoid photobleaching, instead of doing continuous TIRF recording, we collected a series of burst-recordings spaced every 5 minutes for 45 minutes. On-state dwell time was inferred from the loss of immobilized Cy5 spot over time. Note that, only the fluorescence from the surface-immobilized ssRNA can be observed in the TIRF setup, and nonspecific immobilization was negligible on the quartz surface without R-IscB. (B, C) Representative traces showing that only target ssDNA bound to R-IscB stably(left), the non-complementary ssRNA only interacted with the surface transiently (right). FIG.9. Mismatch tolerance in R-IscB-mediated ssRNA binding and cleavage. (A) Left, EMSA results showing the relative affinity of R-IscB for the equimolar amount of perfectly matched and mismatched ssRNA substrates in the same tube, the former was labeled with 5´-FAM, and the latter with either 5´-Cy5 or Cy3.10 µM cold primers were present. The top band in each lane was selected to quantify binding extent, which is shown in FIG.2H. A control gel was run (right) where everything was kept the same but without R- IscB. The total fluorescence from each ssRNA was quantified from this gel to correct for the labeling efficiency in RNA synthesis. (B) The assay was repeated for R-IscB-VR4, and the cleavage extent was resolved on denaturing PAGE. The cleaved RNA band was quantified from each reaction and normalized against total fluorescence signals to generate FIG.5D. FIG.10. R-IscB outperformed PspCas13b and RfxCas13d in binding or cleavage-based knockdown applications, without discernible cytotoxicity. (A) The full- length IscB with a 30-nt guide was not able to reduce the PKM2 / PKM1 ratio, whereas R-IscB reduced it by 4-fold via the same guide. (B) Similarly, DMD mRNA abundance was only reduced slightly by the full-length IscB in HEK293T cells. (C-D) PKM1 and PKM2 mRNA abundance (C) and PKM2 / PKM1 ratio (D) after targeting by dPspCas13b and dRfxCas13d with 30-nt guides in HEK293T cells. No reduction in PKM2 / PKM1 ratio was observed, whereas R-IscB drove 4-fold reduction at the same site through splicing perturbation. (E) DMD mRNA abundance changed very modestly after dPspCas13b and dRfxCas13d targeting in HEK293T cells. The same two guides drove 6- to 8-fold reduction by R-IscB through splicing perturbation. (F) Phase contrast images showing that PspCas13b exhibited significant cytotoxicity one day after transfection, whereas R-IscB-VR4 showed no signs of cytotoxicity. (G) Cell confluency monitored over the course of 27 hours after plasmid transfection. Cells transfected with PspCas13b plasmid exhibited reduced cell confluency over the next 24 hrs whereas cells receiving NmeCas9, SpyCas9, and R-IscB-VR4 expression plasmids grew as well as those receiving the GPF plasmid. This suggests cytotoxicity is not a concern for R-IscB-VR4. The data points and the shaded areas represent the mean values and error bars, respectively, derived from three biological replicates. For A-E and G, all values are shown as mean ± s.d. (n = 3). a.u., arbitrary unit. FIG.11. Additional data characterizing R-IscB-mediated trans-splicing and A- to-I editing. (A) R-IscB revived GFP in a high percentage of cells via trans-splicing, whereas full-length IscB failed to revive any. (B) Representative views of IMR-90 lung fibroblast cells after transfection of trans-splicing plasmid. (C) A-to-I editing efficiency (%) quantified from NGS reads at the target site. All values were obtained from three biological replicates. Only the averaged values were shown. (D) Representative cell images from A-to-I editing controls.No mCherry signal was observed when ADAR2dd or the mCherry-targeting guide wasomitted from the trio. Negligible fluorescence was observed when R-IscB was omitted from the trio. The background fluorescence was presumably due to ADAR2dd editing mediated by the guide alone. FIG.12. Quantification of RNA-guided RNA and DNA cleavage by the engineered R-IscB variants. (A) Left: denaturing PAGE comparing ssRNA cleavage activity by eight different variants, in the context of the full-length IscB. VR3 (H243E, R270Q) has the strongest ssRNA cleavage activity. Right: Activity difference between VR1 and VR2, showing that H243E enabled RNA cleavage. (B) Left: the denaturing PAGE showing VR4 prevented R-IscB from cleaving ssDNA. Mw, molecular weight ladder; H, alkaline hydrolysis ladder made from a FAM-labeled ssRNA. We were able to assign the cleavage site because different panels were from the same gel. Right: denaturing PAGE to resolve the cleavage sites on ssDNA. Bottom: mapping cleavage sites onto the ssDNA and ssRNA sequences. (C) Off-target candidates were searched using either a 16-nt or an extended 25-nt guide sequence. Sequences with up to three mismatches were considered as potential off- targets. Only 6 hits were found for the 25-nt guide, and none of them are located in the up- or down-regulated genes. There were 8506 potential off-targets if we allowed up to two mismatches along the 16-nt guide. We randomly picked 181 sites for further analysis. Among them, four mapped onto the up-regulated genes and four onto the down-regulated genes. The lack of preference suggests off-targeting was unlikely to be the reason for the observed expression perturbation. (D) Left: gene ontology plot showing that the up-regulated genes (n = 358, p-value < 1e-30) were enriched in specific pathways such as RNA splicing, cell cycle regulation, and DNA replication. Right: the same plot showing that the down-regulated genes (n = 905, p-value < 1e-6) were enriched in pathways such as amide metabolism, sulfur metabolism, and glycosaminoglycan metabolism. (E) Denaturing PAGE revealing the cleavage pattern of wildtype and ΔPID NmeCas9s on two different ssRNA substrates. Cleavage site1 is consistent between two substrates and mapped to between PAM+3 and PAM+4. Cleavage site2 is only observed for ssRNA1. Variations in cleavage sites suggest the HNH nuclease may latch onto the gRNA-target RNA duplex in multiple ways. FIG.13. FIG.13 shows a cartoon in the left panel generally illustrating a split protein for which dimerization can be induced, followed by apoptosis. The right panel illustrates IscB proteins with induced Capsase9 dimerization and dual RNA-targeting, leading to cell death. DETAILED DESCRIPTION Some aspects of this disclosure are related to PCT Publication WO 2023 / 215915, published November 9, 2023, from which the entire description is incorporated herein by reference. However, in examples of this disclosure, the IscB PLMP domain may be retained. Unless defined otherwise herein, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Every numerical range given throughout this specification includes its upper and lower values, as well as every narrower numerical range that falls within it, as if such narrower numerical ranges were all expressly written herein. 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 and other values may be expressed herein as from “about” or “approximately” one particular value, and / or to “about” or “approximately” another particular value. When values are expressed as approximations by the use of the antecedent “about” or “approximately” it will be understood that the particular value forms another example. The term “about” and “approximately” in relation to a numerical value encompasses variations of + / -10%, to + / - 1%. The disclosure includes all steps and reagents such as proteins and nucleic acids, and all combinations of steps reagents, described herein, and as depicted on the accompanying figures. The steps described may be performed sequentially. Any one or combination of steps may be omitted. Every example described in this disclosure may comprise, consist essentially of, or consist of the described composition, combination, protein, nucleic acid, or any combination thereof. The disclosure includes all polynucleotide and amino acid sequences described herein. Each RNA sequence includes its DNA equivalent, and each DNA sequence includes its RNA equivalent. Complementary and anti-parallel polynucleotide sequences are included. Every DNA and RNA sequence encoding polypeptides disclosed herein is encompassed by this disclosure. Amino acids of all protein sequences and all polynucleotide sequences encoding them are also included, including but not limited to sequences included by way of sequence alignments. Sequences of from 80.00%-99.99% identical to any sequence (amino acids and nucleotide sequences) of this disclosure are included. The disclosure includes any protein having at least 80% amino acid sequence identity with a specific amino acid sequence defined herein by way of a sequence identifier or database entry. Percent amino acid sequence identity with respect proteins means the percentage of amino acid residues in another sequence that are identical with the amino acid residues in the defined sequence, after aligning the sequences in the same reading frame and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and optionally not considering any conservative substitutions as part of the sequence identity. The disclosure includes all polynucleotide and all amino acid sequences that are identified herein by way of a database entry. Such sequences are incorporated herein as they exist in the database on the filing date of this application or patent. Amino-acid residue sequences described herein are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus, unless stated differently. Additionally, a dash at the beginning or end of an amino acid sequence may indicate a peptide bond to a further sequence comprising one or more amino-acid residues. The disclosure includes all amino acid sequences that are defined by sequence identifier, but with one or more changed amino acids, relative to a native amino acid sequence. Amino acid changes include conservative changes, such as by changing an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping, and non-conservative changes, such as changing an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping. The present specification includes IscB proteins with a truncated, mutated, or a completely removed or inactivated Tam Interacting Domain (TID). In an example, the disclosure provides a described protein with an inactivated TID domain. An inactivated TID domain means that the TID has been truncated or otherwise mutated so that it does not recognize a cognate target-adjacent motif (TAM). A truncated TID means amino acids of the endogenous TID segment of the IscB have been removed. A mutated IscB means one or more of the original amino acids produced in the protein in nature have been deleted or replaced. A representative wild type sequence is provided herein. Combinations of TID truncations and mutations to the remaining TID segment are included. The modified IscB proteins may be modified by altering the TID such that they preferentially bind to single stranded (ss) nucleic acids (polynucleotides) relative to binding to double stranded (ds) nucleic acids. In examples, a modified IscB protein is a modified IscB protein that can preferentially bind to ssRNA or ss DNA relative to binding to dsDNA. In examples, a modified IscB protein preferentially binds to ssRNA. In examples, the IscB protein may be configured to bind to a ss nucleic acid but not modify it, such as by using a nuclease dead modified IscB protein. In certain examples, the modified IscB protein modifies a ss nucleic acid, including but not limited to RNA, and including but not limited to mRNA. In examples the disclosure provides modified IscB proteins, fusion proteins that contain modified IscB sequence as a component of the fusion protein, and methods and systems that use the described modified IscB proteins. “Modified IscB proteins” includes but is not limited to fusion proteins that contain a modified IscB segment. A representative full length IscB wild type protein may comprise SEQ ID NO:1. OgeuIscB Wild-type sequence: MAVVYVISKSGKPLMPTTRCGHVRILLKEGKARVVERKPFTIQLTYESAEETQPLVLGI DPGRTNIGMSVVTESGESVFNAQIETRNKDVPKLMKDRKQYRMAHRRLKRRCKRR RRAKAAGTafeegekqrllpgcfkpitcksirnkearfnnrkrpvgWLTPTANHLLVTHLNVVKKVQKILP VAKVVLELNRFSFMAMNNPKVQRWQYQRGPLYGKGSVEEAVSMQQDGHCLFCKHGI DHYHHVVPRRKNGSETLENRVGLCEEHHRLVHTDKEWEANLASKKSGMNKKYHAL SVLNQIIPYLADQLADMFPGNFCVTSGQDTYLFREEHGIPKDHYLDAYCIACSALTDAKKV SSPKGRPYMVHQFRRHDrqachkanlnrsyymggklvatnrhkamdqktdsleeyraahsaadvskltvkh psaqykdmsrimpgsilvsgegklftlsrsegrnkgqvnyfvstegikywarkcqylrnngglqiyv (SEQ ID NO:1). In SEQ ID NO:1, the following annotation is used: PLMP domain; RuvC domain italicized Bridge helix bold; Linker lowercase HNH domain bold italicized; P1-interacting domain lowercase bold TAM-interacting domain lowercase italicized. OgeuIscB delta-TID sequence: MAVVYVISKSGKPLMPTTRCGHVRILLKEGKARVVERKPFTIQLTYESAEETQPLVLGI APGRTNIGMSVVTESGESVFNAQIETRNKDVPKLMKDRKQYRRAHRRLKRRCKRRR RAKAAGTAFEEGEKQRLLPGCKKPITCKSIRNKEARFNNRKRPKGWLTPTANHLLVT HLNVVKKVQKILPVAKVVLELNRFSFMAMNNPKVQRWQYQRGPLYGKGSVEEAVS MQQDGHCLFCKHGIDHYHHVVPRRKNGSETLENRVGLCEEHARLVHTDKEWEANL ASKKSGMNKKYHALSVLNQIIPYLADQLADMFPGNFCVTSGQDTYLFREEHGIPKDH YLDAYCIACSALTDAKKVSSPKGRPYMVHQFRRHDRQACHKANLNRSYYMGGKKV ATNRHKAMDQKTDSLEEYRAAHSAADVSKLTVKHPSAQY-NGGLQIYV (SEQ ID NO:2) The sequence between SAQY (SEQ ID NO:4) and NGG in the TID was deleted. D60A, H269A were introduced to switch off the nuclease activity. M102R, F137K, V159K, L393K were introduced to enhance the nucleic acid interaction. ssRNA-cleavage enhancing variants on the basis of IscB-delta TID sequence include the following: VR1: R270Q, VR2: H243E, VR3: H243E R270Q, VR4: H243E H269N R270Q, VR5: H243E H245D H269N R270Q, VR6: H243E H269N, VR7: H269N R270Q, and VR8: H243E H245D H269N. OgeuIscB TID-mutated sequence: MAVVYVISKSGKPLMPTTRCGHVRILLKEGKARVVERKPFTIQLTYESAEETQPLVLGI APGRTNIGMSVVTESGESVFNAQIETRNKDVPKLMKDRKQYRRAHRRLKRRCKRRR RAKAAGTAFEEGEKQRLLPGCKKPITCKSIRNKEARFNNRKRPKGWLTPTANHLLVT HLNVVKKVQKILPVAKVVLELNRFSFMAMNNPKVQRWQYQRGPLYGKGSVEEAVS MQQDGHCLFCKHGIDHYHHVVPRRKNGSETLENRVGLCEEHARLVHTDKEWEANL ASKKSGMNKKYHALSVLNQIIPYLADQLADMFPGNFCVTSGQDTYLFREEHGIPKDH YLDAYCIACSALTDAKKVSSPKGRPYMVHQFRRHDRQACDAANLNRSYYMGGKKV ATNRHKAMDQKTDSLEEYRAAHSAADVSKLTVKHPSAQYEDMSQIMPGSILVSGEG KLFTLSASEGDNKGQVNYFVSTEGIKYWARKCQYLRNNGGLQIYV. (SEQ ID NO:5). In this sequence, R461D, R457A, K434E, H379D, K380A, and R438Q changes were made to disrupt the TID function. Similarly, D60A, H269A were introduced to switch off the nuclease activity. M102R, F137K, V159K, L393K were introduced to enhance the nucleic acid interaction. NmeCas9 wild-type sequence: AAFKPNSINYILGLDIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAM ARRLARSVRRLTRRRAHRLLRTRRLLKREGVlqaanfdenglikslpntpwqlraaaldrkltplewsavllhlik hrgylsqrknegetadkelgallkgvagnahalqtgdfrtpaelalnkfekesghirnqrsdyshtfsrkdlqaelillfekqkefgnp hvsgglkegietllmtqrpalsgdavqkmlghctfepaepkaakntytaerfiwltklnnlrileqgserpltdteratlmdepyrks kltyaqarkllgledtaffkglrygkdnaeastlmemkayhaisralekeglkdkksplnlspelqdeigtafslfktdeditgrlkdri qpeileallkhisfdkfvqislkalrrivplmeqgkrydeacaeiygdhygkKNTEEKIYLPPIPADEIRNPVVLRA LSQARKVINGVVRRYGSPARIHIETAREVGksfkdrkeiekrqeenrkdrekaaakfreyfpnfvgepkskdil klrlyeqqhgkclysgkeinlgrlnekgyveidhalpfsrtwddsfnnkvlvlgsenqnkgnqtpyeyfngkdnsrewqefkarvet srfprskkqrillqkfdedgfkernlNDTRYVNRFLCQFVADRMRLTGKGKKRVFASNGQITNLLRG FWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRYKEMNAFDGKTIDKETG EVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAEKLSSRPEAV HEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVLRVPLTQLKLKDLEKM VNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNRTQQVKAVRVEQV QKTGVWVRNHNGIADNATMVRVDVFEKGDKYYLVPIYSWQVAKGILPDRAVVQGK DEEDWQLIDDSFNFKFSLHPNDLVEVITKKARMFGYFASCHRGTGNINIRIHDLDHK IGKNGILEGIGVKTALSFQKYQIDELGKEIRPCRLKKRPPVR (SEQ ID NO:7). In this sequence, the following annotations are used: RuvC domain; Bridge helix italicized; recdomain lowercase; hnh domain italicized lowercase; WED domain Bold PAM-interactingdomain Bold and italicized. NmeCas9 delta-PID sequence: AAFKPNSINYILGLAIGIASVGWAMVEIDEEENPIRLIDLGVRVFERAEVPKTGDSLAM ARRLARSVRRLTRRRAHRLLRTRRLLKREGVLQAANFDENGLIKSLPNTPWQLRAAA LDRKLTPLEWSAVLLHLIKHRGYLSQRKNEGETADKELGALLKGVAGNAHALQTGD FRTPAELALNKFEKESGHIRNQRSDYSHTFSRKDLQAELILLFEKQKEFGNPHVSGGL KEGIETLLMTQRPALSGDAVQKMLGHCTFEPAEPKAAKNTYTAERFIWLTKLNNLRIL EQGSERPLTDTERATLMDEPYRKSKLTYAQARKLLGLEDTAFFKGLRYGKDNAEAST LMEMKAYHAISRALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKTDEDITGRLKDRIQ PEILEALLKHISFDKFVQISLKALRRIVPLMEQGKRYDEACAEIYGDHYGKKNTEEKI YLPPIPADEIRNPVVLRALSQARKVINGVVRRYGSPARIHIETAREVGKSFKDRKEIEK RQEENRKDREKAAAKFREYFPNFVGEPKSKDILKLRLYEQQHGKCLYSGKEINLGRL NEKGYVEIDAALPFSRTWDDSFNNKVLVLGSENQNKGNQTPYEYFNGKDNSREWQE FKARVETSRFPRSKKQRILLQKFDEDGFKERNLNDTRYVNRFLCQFVADRMRLTGKG KKRVFASNGQITNLLRGFWGLRKVRAENDRHHALDAVVVACSTVAMQQKITRFVRY KEMNAFDGKTIDKETGEVLHQKTHFPQPWEFFAQEVMIRVFGKPDGKPEFEEADTLE KLRTLLAEKLSSRPEAVHEYVTPLFVSRAPNRKMSGQGHMETVKSAKRLDEGVSVL RVPLTQLKLKDLEKMVNREREPKLYEALKARLEAHKDDPAKAFAEPFYKYDKAGNR TQQVKAVRVEQVQKTGVWVRNHNGIADNATMVRV-HPNDY- KYQIDELGKEIRPCRLKKRPPVR (SEQ ID NO:8). In this sequence, amino acids between MVRV950 and K1060YQI were deleted. An HPNDY (SEQ ID NO:9) linker was appended to promote protein folding. D16A was introduced to switch off RuvC nuclease domain activity, but this variant can still cleave ssRNA targets. D16A, H588A dual mutations were introduced to switch off HNH and RuvC domain activity for splicing perturbation applications, or other applications that involve ssRNA binding affinity while no cleavage is required. SauCas9 wild-type sequence AKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRR RRHRIQRVKKLLFDYnlltdhselsginpyearvkglsqklseeefsaallhlakrrgvhnvneveedtgnelstkeqisrns kaleekyvaelqlerlkkdgevrgsinrfktsdyvkeakqllkvqkayhqldqsfidtyidlletrrtyyegpgegspfgwkdikew yemlmghctyfpeelrsvkyaynadlynalndlnnlvitrdenekleyyekfqiienvfkqkkkptlkqiakeilvneedikgyrv tstgkpeftnlkvyhdikditarkeiienaelldqiakiltiyqssediqeeltnlnseltqeeieqisnlkgytgthnlslkainlildelw htndnqiaifnrlklvpkkvdlsqqkeIPTTLVDDFILSPVVKRSFIQSIKVINAIIKKYGLPNDIIIELA REknskdaqkminemqkrnrqtnerieeiirttgkenakyliekiklhdmqegkclysleaipledllnnpfnyevdhiiprsvsfd nsfnnkvlvkqeenskkgnrtpfqylsssdskisyetfkkhilnlakgkgrisktkkeylleerdinrfsvqkdfinrnlvdtrYATRG LMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYKHHAEDALIIANA DFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIKHIKDFKDY KYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKSP EKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSKKDNGPVI KKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLD VIKKENYYEVNSKCYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNR IEVNMIDITYREYLENMNDKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIK KG (SEQ ID NO:9). In this sequence, the following annotation is used. RuvC domain; Bridge helixitalicized; rec domain lowercase; hnh domain italicized lowercase; WED domain Bold;PAM-interacting domain bold and italicized. SauCas9 delta-PID sequence: AKRNYILGLDIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKR RRRHRIQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRR GVHNVNEVEEDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKT SDYVKEAKQLLKVQKAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWY EMLMGHCTYFPEELRSVKYAYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENV FKQKKKPTLKQIAKEILVNEEDIKGYRVTSTGKPEFTNLKVYHDIKDITARKEIIENAE LLDQIAKILTIYQSSEDIQEELTNLNSELTQEEIEQISNLKGYTGTHNLSLKAINLILDEL WHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVDDFILSPVVKRSFIQSIKVINAIIKK YGLPNDIIIELAREKNSKDAQKMINEMQKRNRQTNERIEEIIRTTGKENAKYLIEKIKL HDMQEGKCLYSLEAIPLEDLLNNPFNYEVDHIIPRSVSFDNSFNNKVLVKQEENSKKG NRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSVQKDFIN RNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGYK HHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITP HQIKHIKDFKDYKYSHRVDKKPNRELINDTLYSTRKDDKGNTLIVNNLNGLYDKDND KLKKLINKSPEKLLMYHHDPQTYQKLKLIMEQYGDEKNPLYKYYEETGNYLTKYSK KDNGPVIKKIKYYGNKLNAHLDITDDYPNSRNKVVKLSLKP (SEQ ID NO:10). Amino acids beyond (e.g., C-terminal) to P911 were removed. CjCas9 wild-type sequence ARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLARR KARLNHLKHLIANEFKLnyedyqsfdeslakaykgslispyelrfralnellskqdfarvilhiakrrgyddiknsddkek gailkaikqneeklanyqsvgeylykeyfqkfkenskeftnvrnkkesyerciaqsflkdelklifkkqrefgfsfskkfeeevlsva fykralkdfshlvgncsfftdekrapknsplafmfvaltriinllnnlkntegilytkddlnallnevlkngtltykqtkkllglsddyef kgekgtyfiefkkykefikalgehnlsqddlneiakditlikdeiklkkalakydlnqnqidslsklefkdhlnisfkalklvtplmle gkkydeacnelnlkvainEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLKKYGK VHKINIELARevgknhsqrakiekeqnenykakkdaeleceklglkinsknilklrlfkeqkefcaysgekikisdlqdekmle idhiypysrsfddsymnkvlvftkqnqeklnqtpfeafgndsakwqkievlaknlptkkqkrildknykdkeqknfkdrnlndtryia rlvlnytKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSALRHTWGFSAKDRN NHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFFEPFSG FRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKEGVLKALELGK IRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAVARSKKGEIK DWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSSTVSLIVSKHDNKFETL SKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKAEFRQREDFKK (SEQ ID NO:11). In this sequence, the following annotation is used: RuvC domain; Bridge helix italicized; rec domain lowercase; hnh domain italicized lowercase: WED domain bold; PAM-interacting domain bold and italicized. CjCas9 delta-PID sequence ARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLARSARKRLAR RKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALNELLSKQDF ARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYKEYFQKFK ENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEVLSVAFYKR ALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTKDDLN ALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKALGEHNLSQ DDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKALKLVTPLM LEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKEYRKVLN ALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEKLGLKINS KNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVLVFT KQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQKNFKD RNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGMLTSAL RHTWGFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAKKISELD YKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQSYGGKE GVLKALELGKIRKVNGKIVKNGDMFRV-HPNDY-KYIVSALGEVTKAEFRQREDFKK (SEQ ID NO:12). In this sequence, the amino acids between MFRV832 and K961YIV were removed. An HPNDY (SEQ ID NO:9) linker was appended to improve structure stability. Cas9d wild-type sequence ERELVLGIDYGGKYTGLAVVDRRHNQVLYANRLKMRDDVAGILKDRRKQRGIRRTAQ TKKKRLRELKNYLKSIGYnestatfetvyslahkrgydyadmpeektseeieamdveerkqwekekqeweetkrnsr hrkevvkdvhkamiegrateeqikrverifnkqyrpkrfnnriltkckvedcgvntplrknvrdllienivrffpieqsekdnlkda vldknrreevksffrkhktdehirkqvydiadnklsgrtvfckehilehtehskeerkvfrlAPSLKTKIENVLAVIKDEI LPKFTVNKVVMESNNFdiaaktqgkkrlakeeygkgpregketrkeallretdgrciycgksidisnahddhifprkag glnifanlvaccavcnenkkgrtplesgispkpeiiafmkndlkkkiledarnintvdfnkymshasigwrymrdrlresagnkklP IERQSGIYTAYFRRWWGFKKERGNTLHHALDAVILASRKGYSDDGLVDMTLKPKYN KGGEFDPEKHLPEPIEFKMDKGSRGSALHDRNPLSYKKGIITRRFMVTEIECGKED DVISETYREKLKEAFKRFDTKKGKCLTDKEAKEAGFCIKKNELVMSLKCSIKGT GPGQMIRINNNVFKTNVHNVGVDVYLDEKGKKKAYERKNPRLSKHFIEPPPQPNGR VSFTLKRRDMVTVEGEDAIYRIKKLGTSPTIEAVVGSDGKTRTVSATKLTKANSAE (SEQ ID NO:13) In this sequence, the following annotation was used. RuvC domain; Bridge helix italicized; rec domain lowercase; hnh domain italicized lowercase WED domain bold;PAM-interacting domain bold and italicized.Cas9d delta-PID sequence ERELVLGIDYGGKYTGLAVVDRRHNQVLYANRLKMRDDVAGILKDRRKQRGIRRTA QTKKKRLRELKNYLKSIGYNESTATFETVYSLAHKRGYDYADMPEEKTSEEIEAMDV EERKQWEKEKQEWEETKRNSRHRKEVVKDVHKAMIEGRATEEQIKRVERIFNKQYR PKRFNNRILTKCKVEDCGVNTPLRKNVRDLLIENIVRFFPIEQSEKDNLKDAVLDKNR REEVKSFFRKHKTDEHIRKQVYDIADNKLSGRTVFCKEHILEHTEHSKEERKVFRLAP SLKTKIENVLAVIKDEILPKFTVNKVVMESNNFDIAAKTQGKKRLAKEEYGKGPREG KETRKEALLRETDGRCIYCGKSIDISNAHDDHIFPRKAGGLNIFANLVACCAVCNENK KGRTPLESGISPKPEIIAFMKNDLKKKILEDARNINTVDFNKYMSHASIGWRYMRDRL RESAGNKKLPIERQSGIYTAYFRRWWGFKKERGNTLHHALDAVILASRKGYSDDGLV DMTLKPKYNKGGEFDPEKHLPEPIEFKMDKGSRGSALHDRNPLSYKKGIITRRFMVT EIECGKEDDVISETYREKLKEAFKRFDTKKGKCLTDKEAKEAGFCIKKNELVMSLKCS IKGTGPGQMIRINNNVFKTNVH-KNPRLSKHFIE (SEQ ID NO:14). In this sequence, the sequence from TNVHN654 and AYER672, and P684PPQP and beyond were removed. The sequence KNPRLSKHFIE (SEQ ID NO:15) was retained to improve structure stability. It will be recognized from the foregoing representative sequences that the disclosure provides modified IscB and Ca9d proteins that have amino acid changes, relative to naturally occurring proteins. The described modified proteins can comprise only one of the described mutations, or any combination thereof. In examples a modified IscB protein retains its PLMP domain in its endogenous position, i.e., the PLMP domain is not repositioned or removed from its naturally occurring location. The disclosure includes homologous IscB and Cas9d proteins from any other species, with commensurate modifications that will be recognized by those skilled in the art when given the benefit of this disclosure using IscB and / or Cas9d protein amino acid sequence alignment tools. An amino acid linker can be included between a modified protein and any additional amino acids that are added to the modified IscB and / or Cas9d sequence. In an example, any amino acid linker of this disclosure may comprise comprises Gly and Ser amino acids. In an example, the linker may be lengthened compared to standard linker lengths to, for example, permit more accessibility for an N-terminal nuclear localization signal (NLS). The described modified IscB proteins can be provided in systems that include the described proteins and a guide RNA, referred to herein as a ωRNA and omega RNA. The ωRNA can be provided as a single RNA polynucleotide or may be split into two RNA polynucleotides. Representative omega RNA sequences are provided in the figures. Modified IscB proteins and / or Cas9d proteins of this disclosure can be provided as fusion proteins. As such, additional amino acids can be added to the N-terminus, the C- terminus, or both, of any modified protein described herein. In one example a fusion protein of the disclosure includes a described IscB or Cas9d protein segment and a distinct protein segment. A distinct protein segment means a protein or segment of a fusion protein that is not the IscB or Cas9d protein sequence. In examples, any IscB or Cas9d protein described herein may be provided as a component of a fusion protein that further comprises a protein segment that is capable of influencing interaction of the fusion protein with nucleic acids. In examples, the fusion protein comprises an IscB or Cas9d protein segment at the N-terminus and additional amino acids at the C-terminus. In examples, the additional amino acids are an enzyme, or a non-enzymatic nucleic interaction domain. In examples, examples, a described fusion protein can comprise the amino acid sequence of a protein that can interact with ss nucleic acids, including but not necessarily limited to any type of RNA (i.e., mRNA, pre-mRNA, microRNA, and the like). In examples, a described IscB fusion protein can include a segment that can modify nucleotides or the sugar-phosphate backbone of a ss nucleic acid. In an example, the disclosure thus provides a new nucleotide modifying enzyme. Thus, in examples, the fusion protein can comprise the IscB segment and another segment with or without enzymatic activity. In this regard, in examples, a fusion protein comprises a nucleotide modifying enzyme, such as an adenosine deaminase enzyme that catalyzes the chemical conversion of adenosines to inosine (an ADAR enzyme) but on a ss nucleic acid using the modified IscB- containing protein. In examples, a fusion protein comprises any SR protein that is a member of a family structurally related RNA binding proteins. An SR protein used in a fusion protein of this disclosure may include an RNA recognition motifs (RRM), a KH domains or a zinc finger, to alter / enhance preference for ssRNA of ds nucleic acids. In examples, an S1, S2. S3, S4, S5, S6, S7 or S8 protein is used. In any fusion protein of this disclosure, the protein that is added to the modified IscB protein may replace all or a described portion of the TID, or may be located elsewhere in the modified IscB protein. In an example, an IscB protein may be modified by introducing point mutations in the HNH domain or another location to affect and / or inactivate nuclease activity on the target strand, including on dsRNA and dsDNA. In examples, a described fusion protein or other modified protein described herein can comprise any suitable cellular localization signal, including but not limited to a nuclear or other organelle localization signal. In examples, a described modified IscB protein or Cas9 protein may be provided in a split-protein configuration wherein the IscB protein or the Cas9 protein is provided as two separate polypeptides that are configured to contain interacting dimerization domains, whereupon dimerization provides a functional modified IscB or Cas9 protein. In an example, dimerization is inducible. A representative dimerization domain includes is shown as a Caspase 9 protein that is capable of inducible dimerization, as depicted on FIG.13. The same approach applies to Cas9 proteins, such as Cas9d. In examples, a described system comprising a described IscB protein and an omega RNA is used for producing an indel, which may be achieved in a DNA repair template free manner. In examples, the indel corrects a mutation in an open reading frame encoded by a selected chromosome locus or converts a sequence into an open reading frame. In examples, the selected chromosome locus comprises a mutation in a gene that is correlated with a monogenic disease. In non-limiting examples, the indel is produced within a protein coding segment of a chromosome, at a splice junction, in a promoter, in an enhancer element, or at any other location wherein generation of an indel is desirable, provided a suitable TAM is present. In examples, the indel corrects a missense mutation, a frameshift mutation, or a nonsense mutation. In examples, the indel changes a codon for at least one amino acid in a protein coding sequence, and thus may correct a mutation in an exon. In examples, the indel corrects a deleterious mutation that is a component of a monogenic disorder, e.g., a disorder caused by variation in a single gene. In examples, an indel is 1, 2, 3, 4, or more nucleotides that are deleted or inserted. In examples, modified protein as described herein is introduced into the cell as a recombinant or purified protein, or as an RNA encoding the protein that is expressed once introduced into the cell, or as an expression vector, which is expressed once in the cell. In examples, 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 examples, expression vectors comprise viral vectors. In examples, a viral expression vector is used. Viral expression vectors may be used as naked polynucleotides, or may comprise any of viral particles, including but not limited to defective interfering particles or other replication defective viral constructs, and virus-like particles. In examples, the expression vector comprises a modified viral polynucleotide, including but not limited to polynucleotides from an adenovirus, a herpesvirus, or a retrovirus, such as a lentiviral vector. In examples, any type of a recombinant adeno-associated virus (rAAV) vector may be used. In examples, 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 examples, for producing rAAV vectors, plasmid vectors may encode all or some of the well-known rep, cap and adeno-helper components. In certain examples, 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 examples when given the benefit of this disclosure. In examples, the disclosure is considered suitable for use in any eukaryotic cells, and can also be used in prokaryotic cells, such as for bioengineering prokaryotes, and for use as anti-bacterial agents. In examples, eukaryotic cells that are modified by the approaches of this disclosure are totipotent, pluripotent, multipotent, or oligopotent stem cells when the modification is made. In examples, the cells are neural stem cells. In examples, the cells are hematopoietic stem cells. In examples, the cells are leukocytes. In examples, the leukocytes are of a myeloid or lymphoid lineage. In examples, the cells are embryonic stem cells, or adult stem cells. In examples, the cells are epidermal stem cells or epithelial stem cells. In examples, the cells are cancer cells, or cancer stem cells. In examples, the cells are differentiated cells when the modification is made. In examples, the cells are mammalian cells. In examples, the cells are human, or are non-human animal cells. In examples, the non- human eukaryotic cells comprise fungal, plant or insect cells. In one approach the cells are engineered to express a detectable or selectable marker, or a combination thereof. In examples, the modification introduced into eukaryotic cells according to this disclosure is homozygous or heterozygous. In examples, the modification comprises a homozygous dominant or homozygous recessive or heterozygous dominant or heterozygous recessive mutation correlated with a phenotype or condition, and is thus useful for modeling such phenotype or condition. In examples a modification causes a malignant cell to revert to a non-malignant phenotype. In certain aspects the disclosure includes a pharmaceutical formulation comprising one or more components of a system described herein. A pharmaceutical formulation comprises one or more pharmaceutically acceptable additives, many of which are known in the art. In some examples, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for administration to humans. In some examples, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for intraocular injection. In some examples, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for topical application. In some examples, the pharmaceutical compositions comprise a pharmaceutically acceptable carrier suitable for intravenous injection. In some examples, the pharmaceutical compositions comprise and a pharmaceutically acceptable carrier suitable for injection into arteries. In some examples, the pharmaceutical composition is suitable for oral or topical administration. All of the described routes of administration are encompassed by the disclosure. In examples, expression vectors, proteins, RNPs, polynucleotides, and combinations thereof, can be provided as pharmaceutical formulations. A pharmaceutical formulation can be prepared by mixing the described components with any suitable pharmaceutical additive, buffer, and the like. Examples of pharmaceutically acceptable carriers, excipients and stabilizers can be found, for example, in Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, the disclosure of which is incorporated herein by reference. Further, any of a variety of therapeutic delivery agents can be used, and include but are not limited to nanoparticles, lipid nanoparticle (LNP), exosomes, and the like. In examples, a biodegradable material can be used. In examples, poly(lactide-co-galactide) (PLGA) is a representative biodegradable material. In examples, any biodegradable material, including but not necessarily limited to biodegradable polymers. As an alternative to PLGA, the biodegradable material can comprise poly(glycolide) (PGA), poly(L-lactide) (PLA), or poly(beta-amino esters). In examples, the biodegradable material may be a hydrogel, an alginate, or a collagen. In an example the biodegradable material can comprise a polyester a polyamide, or polyethylene glycol (PEG). In examples, lipid-stabilized micro and nanoparticles can be used. In certain approaches, compositions of this disclosure, including the described systems, or cells modified using the described systems, are used for treatment of condition or disorder in an individual in need thereof. The term “treatment” as used herein refers to alleviation of one or more symptoms or features associated with the presence of the particular condition or suspected condition being treated. Treatment does not necessarily mean complete cure or remission, nor does it preclude recurrence or relapses. Treatment can be effected over a short term, over a medium term, or can be a long-term treatment, such as, within the context of a maintenance therapy. Treatment can be continuous or intermittent. In examples, a system of this disclosure is administered to an individual in a therapeutically effective amount. In examples, 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 examples, 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. In examples, cells modified according to this disclosure are administered to an individual in need thereof in a therapeutically effective amount. In examples, the disclosure comprises providing a treatment to an individual in need thereof by introducing a therapeutically effective amount a composition of this disclosure to treat alleviate, inhibit, or prevent the formation of one or more conditions, diseases, or disorders. In examples, the described systems are introduced into eukaryotic cells that include but are not limited to non-human animal cells, or fungi or plant cells. In examples, 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. In examples, the disclosure provides an article of manufacture, which may comprise a kit. In examples, the article of manufacture may comprise one or more cloning vectors. The one or more cloning vectors may encode any one or combination of proteins and polynucleotides described herein. The cloning vectors may be adapted to include, for example, a multiple cloning site (MCS), into which a sequence encoding any protein or polynucleotide, such as any desired targeting RNA, may be introduced. An article of manufacture may include one or more sealed containers that contain any of the aforementioned components, and may further comprise packaging and / or printed material. The printed material may provide information on the contents of the article and may provide instructions or other indication of how the contents of the article may be used. In an example, the printed material provides an indication of a disease or disorder that is to be treated using the contents of the article. In examples, when polynucleotides are delivered, they may comprise modified polynucleotides or other modifications, such as phosphate backbone modifications, and modified nucleotides, such as nucleotide analogs. Suitable modifications and methods for making nucleic acid analogs are known in the art. Some examples include but are not limited to polynucleotides which comprise modified ribonucleotides or deoxyribonucleotides. For example, modified ribonucleotides may comprise methylations and / or substitutions of the 2' position of the ribose moiety with an --O-- lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an --O-aryl group having 2-6 carbon atoms, wherein such alkyl or aryl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or with a hydroxy, an amino or a halo group. In examples modified nucleotides comprise methyl-cytidine and / or pseudo-uridine. The nucleotides may be linked by phosphodiester linkages or by a synthetic linkage, i.e., a linkage other than a phosphodiester linkage. Examples of inter-nucleoside linkages in the polynucleotide agents that can be used in the disclosure include, but are not limited to, phosphodiester, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, morpholino, phosphate triester, acetamidate, carboxymethyl ester, or combinations thereof. In examples, the DNA analog may be a peptide nucleic acid (PNA). The disclosure includes making any described modified protein. In an example, a method comprises expressing a described protein within cells using an expression vector, allowing expression of the described protein, and separating the protein from the cells. The protein may be purified to any desired degree of purity using conventional techniques. In connection with the present disclosure, it will be recognized that tools developed from the Cas9 family of RNA-guided DNases have revolutionized genomic medicine3-5. Serving as the effector of the Type II CRISPR-Cas system, Cas9 utilizes the associated crRNA / tracrRNA as the guide, opens R-loop at the matching DNA target that is flanked by an interference Protospacer-Adjacent Motif (PAM), and introduces a double-strand break via its HNH and RuvC nuclease domains6-11. It was later discovered that Cas9 originated from IscB, an RNA-guided homing nuclease in the IS200 / 605 family of transposons12,13. The associated OMEGA RNA (ωRNA) is a natural fusion of crRNA and tracrRNA, twice of their combined size, and plays additional roles than driving ribonucleoprotein (RNP) assembly and serving as the guide13,14. IscB protein is one-third or half the size of a typical Cas9 and lacks the REC domain in Cas9. Nonetheless, it adopts a similar overall architecture to Cas9, searches for and cleaves at the DNA target using the same mechanism (FIG.1A)15-17. Even the built-in allosteric motion in the HNH nuclease domain is conserved between IscB and Cas917-21. IscB’s compact size and tunable DNA nicking activities make it a desirable platform for CRISPR 2.0 type of genome editing applications, such as base-editing and prime-editing22,23. The naturally occurring IscBs have marginal editing efficiency in human cells. Many DNA-targeting CRISPR effectors, including Cascade, Cas9 and Cas12f, have been shown to also bind ssRNA and ssDNA in RNA-guided fashion27-30. Some Cas9 homologs were shown to be further capable of ssRNA cleavage31-33. This activity in some, but not all cases require the assistance of a PAMer ssDNA oligo (i.e. SpCas9)27,31. A dead version of SpCas9 was named RCas9 and utilized for RNA tracking or transcript knockdown34,35. Cas7-11, the Type III-D CRISPR-Cas effector, was also shown to be capable of RNA- targeting in human cells36. However, neither RCas9 nor Cas7-11 gained popularity in in vivo applications, presumably because their performance was not robust. The Type III effectors have also been utilized for in vivo RNA knockdown in human cells37,38, however, the multi- component systems are cumbersome to use. Most researchers chose Cas13 from Type VI CRISPR systems for RNA-guided RNA editing applications39-47. However, the majority of Cas13s further cleave the bystander mRNAs, and this collateral damage was shown to be toxic to bacterial and mammalian cells42,48. Cytotoxicity was the main reservation for moving Cas13-based therapeutics into clinical trials. This disclosure demonstrates that IscB can be converted into an exclusive RNA- targeting tool when its TID is mutated or partially or fully deleted. The resulting R-IscB mediates a robust and versatile set of RNA-guided RNA targeting applications in eukaryotic cells, including splicing perturbation, mRNA cleavage, RNA-guided A-to-I editing, and RNA-guided trans-splicing. The former two activities can be used to target dominant- negative genetic diseases, whereas the latter two can find usage in curing recessive genetic diseases, through sequence correction at the mRNA level. R-IscB is more active than Cas13 and has no discernible cytotoxicity in human cells. The disclosure also shows that four different Cas9s can be converted to an exclusive RNA-targeting enzyme following the same approach. These tools are attractive alternatives to Cas13 in research and medicine. The description provides examples of the disclosure and discussion of the results. Explanations of experiments in the following Examples are not intended to be limiting or bound by any particular theory or interpretation. Example 1 Wildtype IscB also binds ssDNA and ssRNA, but strongly prefers dsDNA To explore the usage of IscB for RNA-targeting, we first tested its single-stranded nucleic acid binding and cleavage activities in vitro. In electrophoretic mobility shift assays (EMSA), O. geu IscB-ωRNA RNP bound strongly and specifically to guide-complementary ssDNA and ssRNA, but not to the non-complementary controls. The apparent dissociation constants were better than 32 nM (FIG.1B). The multiple binding species likely correspond to complexes in different conformations rather than the binding of multiple IscBs, because the substrate is shorter than the combined footprint of two IscBs. Binding did not require a TAM sequence (5'-NWRRNA) in the target (FIG.7A). ssDNA was further cleaved by OgeuIscB, and the cleavage site was consistent with that in the dsDNA (FIG.7B). In contrast, ssRNA cleavage was barely detectable even after a 30-minute incubation at 37 ˚C (FIG.7C). With such a strong affinity for ssRNA, when IscB targets a coding gene in the sense orientation, the transcribed mRNA could potentially compete for IscB and inhibit DNA editing. However, we and others never observed orientation-dependent variations in editing efficiency13,26. Consistently, in competition experiments, the extent of dsDNA cleavage was not inhibited when we titrated in increasing amounts of ssRNA competitors (FIG.1C). Conversely, titrating DNA target into a binding reaction competed IscB away from ssRNA efficiently (FIG.1D). Therefore, wildtype IscB predominantly binds and cleaves dsDNA. Example 2 TID removal switches off DNA-binding but leaves ssDNA / RNA binding intact The disclosure includes an analysis of whether IscB could be converted into an ssDNA / ssRNA-targeting platform. Without intending to be constrained by any particular theory, it is considered that to do so, it would need to disengage from dsDNA binding. Since the TAM-interacting domain (TID) is the only domain in IscB that interacts with dsDNA15-17, we analyzed whether the TID might have engaged IscB in a perpetual TAM-searching loop, preventing it from ‘discovering’ the complementary mRNAs nearby. If true, removing this domain could disengage IscB from DNA binding and unmask its RNA targeting activity. We designed a TID-deletion construct to test this idea (OgeuIscB-ΔTID, deleting amino acids 433-487; FIG.2A-B). In case TID deletion disrupted both activities, we also prepared a backup design, in which TID was replaced by a nonspecific ssRNA-binding module, the S1 domain from E. coli polynucleotide phosphorylase (FIG.2B). Example 3 Both versions of IscB RNP assembled and purified well (FIG.7D, 7E). Results showed that OgeuIscB-ΔTID behaved in the manner described herein. It lost its affinity for dsDNA targets; the apparent Kd from EMSA was at the nonspecific level (> 1 µM), and target cleavage was not detected despite intact HNH and RuvC (FIG.2C-D). In contrast, its affinity and specificity for complementary ssDNA and ssRNA were intact (FIG.2E); ssDNA cleavage activity was also preserved (FIG.7C). Unlike the wildtype counterpart, OgeuIscB- ΔTID was not displaced from ssRNA by dsDNA competitors (FIG.2F). OgeuIscB with the TID-to-S1 substitution (OgeuIscB-ΔTID / +S1) behaved similarly to the ΔTID construct in EMSA assays (FIG.2E). Example 4 R-IscB binds ssRNA through a seed sequence We named OgeuIscB-ΔTID R-IscB for its exclusive RNA-guided RNA-targeting activity and further characterized its binding kinetics using single-molecule methods (FIG. 8A). Binding of ssRNA to the surface-immobilized R-IscB was inferred from fluorescence immobilization; nonspecific surface adsorption was negligible. The association rate was very fast (kon ~0.0154 nM-1s-1), whereas the dissociation rate was too slow to be reliably quantified from a continuous recording setup because dissociation could not be reliably differentiated from photobleaching. koff(4.5 x 10-4s-1) was instead derived from a burst-recording series stretched over 45 minutes. The average half-life of the R-IscB-ssRNA complex was 37 minutes (FIG.2G). The fast konand slow kofftranslated to subnanomolar binding affinity (Kd= kon / koff = 29 pM). In contrast, R-IscB only made infrequent and transient contacts with a random ssRNA, confirming that the interaction was RNA-guided (FIG.8B-C). We further quantified R-IscB’s ability to differentiate perfectly matched and mismatched RNA. While single-nucleotide (nt) mismatches were largely tolerated by R-IscB, di-nt mismatches along the 16-nt guided region were strongly disfavored; 3- and 4-nt mismatches led to even higher discrimination (FIG.2H, 9A). The level of binding specificity is on par with that of Cas1340,41,44. The data suggest the presence of a seed sequence at the TAM-proximal region, where R-IscB is more sensitive to mismatches. The same behavior has been defined for Cas9 in DNA-binding studies3,9,49. Structures revealed that the guide of Cas9 in the seed region pre-organizes into an A-form geometry50. Target discovery nucleates from seed-mediated duplex formation and propagates directionally to the distal side. This mechanism is kinetically efficient, which might be the reason why it is also adopted by the Argonaut family of proteins51,52. Interestingly, Cas13 does not show a consistent seed sequence preference53-55, and in structural studies, its guide was found to be free-floating rather than adopting an A-form seed53,56,57. In this regard, R-IscB is superior to Cas13 in targeting-searching. Example 5 R-IscB alters the splicing outcome in an RNA-guided fashion Because R-IscB lacks RNA cleavage activity, we first probed its usage in splicing perturbation. This could conceivably be achieved through splice site (SS) sequestration. However, it is a challenging task because the tool needs to be faster in SS association and slower in SS dissociation than the spliceosome. There were only a few successful cases of Cas13-mediated splicing perturbation39, and none has been reported for RCas9. SS-targeting Anti-Sense Oligos (ASO) can be effective in splicing perturbation58,59. However, off- targeting has been a concern because ASOs are typically administered at high concentrations60,61. We analyzed the efficacy of R-IscB in systems where ASOs have been shown to be effective. The first target was the pyruvate kinase gene PKM, which undergoes mutually exclusive alternative splicing to include either exon 9 (PKM1) or 10 (PKM2) in the mature mRNA. Whereas PKM1 is the dominant isoform in normal cells, PKM2 is upregulated by as much as 10-fold in energy-demanding cells, including cancer cells62. Because of this, there have been efforts to knock down PKM2 using ASO and siRNA63,64. We designed eight guides to program R-IscB to target either the 5' SS or 3' SS of exon 10 (M1 to M8; FIG.3A- B). A single plasmid containing the optimized R-IscB and ωRNA was transfected into HEK293T cells26. Total mRNA was extracted from cells 48 hours later, and the PKM isoforms were quantified using quantitative polymerase chain reaction (qPCR) andnormalized against the internal control GAPDH. Among the eight guides, M2-targetingclearly knocked down the PKM2 isoform (33.0±6.3% of the non-targeting control (NTC))without affecting the PKM1 level (94.5±12.2%; FIG. 3C). M5 was also mildly effective,whereas the rest of the guides were either indiscriminative (M7 and M8) or ineffective (FIG. 3C-D). Positional dependency was also observed for ASOs65, either because the target sites were not equally accessible or because targeting was not equally impactful. M2-mediated ~2.8-fold reduction in PKM2 / PKM1 ratio rivals the performance of leading ASO-mediated interventions (FIG.3D)63. Next, we tested whether R-IscB can be used to knock down the proprotein convertase subtilisin kexin type 9 (PCSK9) level through splicing perturbation in hepatoblastoma (HepG2) cells. We designed six guides (K1 to K6) to target the 5' or 3' SS of PCSK9 exons 1, 2, 4, or 10. Exon skipping in these regions would introduce premature stop codons and possibly trigger nonsense-mediated mRNA decay (FIG.3E). Among the six guides, the best- performing guide K3 reduced PCSK9 mRNA levels by ~2-fold (45.1±18.2% of NTC; FIG. 3F), matching the efficacy achieved in RNAi-based clinical trials66. We analyzed if longer guides may lead to more efficient splicing perturbation, either by decreasing koff or by sequestering more cis-elements from the spliceosome. To test this, we extended the length of PKM2-guides M2 and M5, from 16 to 23 and 30 nt. They indeed resulted in more pronounced PKM2 knockdowns (i.e.33.0±6.3% for M2-16nt and 23.6±1.2% for M2-30nt) and better PKM2 / PKM1 ratio (i.e.35.4±9.0% for M2-16nt and 24.9±2.9% for M2-30nt). This improvement was more pronounced when the guide length was increased from 16 to 23 nt, but less so when it was further extended from 23 nt to 30 nt (FIG.3G, 3H). Using the longer guide design, we explored the feasibility of R-IscB-mediated exon 51 skipping in the DMD gene, mutations of which cause Duchenne muscular dystrophy (DMD). Deletions in exons 48-50 are a frequent cause of DMD, which can be partially rescued through further skipping of exon 51 (FIG.3I)67,68. We designed two 30-nt R-IscB guides to target exon 51 in HEK293T cells. D1 was derived from an effective antisense oligonucleotide (ASO)69, whereas D2 targeted the vicinity of the 5' splice site (FIG.3J). Both guides were highly efficient in causing exon 51 skipping. D1 reduced the exon 51- containing mRNA level by 8-fold, while D2 reduced it by 6-fold (12.1 ± 1.7% and 15.0 ± 4.2% of NTC, respectively; FIG.3K). The stronger impact on splicing may be correlated with the lower DMD expression level in HEK293T cells. However, even a two-fold expression increase has been found to be therapeutically beneficial in muscular dystrophy diseases70,71. We investigated whether TID deletion was necessary for splicing perturbation. The better-performing PKM2 and DMD knockdown guides (M2_30 and D2, respectively) were tested again in the context of the full-length OgeuIscB, bearing the same inactivating HNH and RuvC mutations as in R-IscB. No significant knockdown was observed in either PKM2 or DMD knockdown experiments (FIG.10A-B), suggesting that TID removal is the cause for the observed RNA-targeting activities in vivo. We further investigated whether RNA- targeting can be achieved by disrupting the dsDNA binding function of TID rather than deleting the whole domain. Six TID mutations (R461D, R457A, K434E, H379D, K380A, and R438Q) were introduced to disrupt PAM recognition and DNA backbone contacts. When programmed with the D2 guide, this sextuple IscB variant was quite efficient in DMD knockdown (29.2^2.8% of NTC; FIG.3K), albeit less so than the ΔTID variant R-IscB. Example 6 Benchmarking against Cas13 We next benchmarked R-IscB against two popular RNA-targeting tools, PspCas13b and RfxCas13d39,47. PKM2-M2_30 guide drove a 4-fold knockdown by R-IscB. When the same guide was programmed into the catalytically dead versions of PspCas13b (dPspCas13b) and RfxCas13d (dRfxCas13d), neither one was able to reduce the PKM2 isoform level, nor the PKM2 / PKM1 ratio (FIG.10C-D). DMD-D1 and D2 on R-IscB reduced exon 51 level by 6-8 fold. When programmed with the same guides, neither dPspCas13b nor dRfxCas13d was able to induce even a 2-fold reduction in exon 51 level (FIG.10E). Importantly, PspCas13b transfection led to severe cytotoxicity in HEK293T cells. A large percentage of cells lost vitality within 24 hours, and the growth curve was significantly stunned. In contrast, cytotoxicity was not discernible in R-IscB-treated groups in terms of growth curve and cell morphology (FIG.10F-G). Example 7 R-IscB mediates efficient trans-splicing, allowing gene correction at mRNA level Trans-splicing is a versatile approach to correcting gene sequences at the mRNA level. We tested the efficacy of R-IscB in mediating 5' trans-splicing using a GFP-reporter assay72. The target in our assay was an EGFP gene carrying a human LMNA intron in the middle. A premature stop codon in the upstream exon rendered the EGFP nonfunctional. The functional version of this exon and part of an intron were fused to the guide region of ωRNA, which was programmed to target the parental intron. Successful targeting would bring the fused exon and intron to the vicinity of the downstream exon, tricking the spliceosome to perform a trans-splicing reaction to generate functional EGFP mRNAs and green HEK293T cells (FIG.4A). Six guides targeting various regions of the LMNA intron were tested; all of them were effective in mediating trans-splicing in vivo. Based on the percentage of cells displaying green fluorescence ranged from 40.9±1.9% (guide-T6) to 23.5±5.0% (T3) (FIG. 4B-C). No GFP+cells were observed when we omitted R-IscB, the GFP-targeting guide, or both in the targeting experiments, confirming that R-IscB-guided trans-splicing indeed took place (FIG.4B-C). The full-length IscB was incapable of mediating trans-splicing, proving again that TID removal drives RNA-based applications (FIG.4D, FIG.11A). The trans- splicing efficiency by R-IscB is comparable with the reported values in Cas13 studies72. Using the same setup, we observed GFP signals from IMR-90 human lung fibroblast, suggesting that R-IscB is capable of mediating trans-splicing in primary cells (FIG.11B). Given the small size, R-IscB and its guide can be easily packaged in an AAV vector, while leaving an additional 2.5 kb of payload for the trans-splicing donor. Example 8 ADAR-fusion enables RNA-guided RNA editing for R-IscB We further explored the efficacy of R-IscB in mediating RNA-guided adenosine-to- inosine (A-to-I) editing in mRNA. The guide region of the ωRNA was extended at the 5' end to create an ADAR recognition site, which contained an A-C mismatch in the duplexed region. The adenosine (A) in the mismatch can be enzymatically converted to inosine (I) by ADAR2dd47, which is fused to R-IscB through a flexible linker. A fluorescence-rescue assay was set up to detect A-to-I editing in HEK293T cells by an all-in-one plasmid (FIG.4E)44. Successful editing would correct a premature stop codon to a tryptophan (UAG to UIG, decoded as UGG by the ribosome), reviving mCherry fluorescence. NLS and NES (nuclear localization / export signal) were appended to the N- and C-termini of R-IscB, respectively, to shuttle R-IscB-ADAR2dd RNP from nucleus to cytoplasm. We systematically varied the distance between the R-IscB target site and the ADAR2dd editing sites to define a preferred spacing for A-to-I editing. Results showed that R-IscB-ADAR2dd rescued mCherry fluorescence robustly (FIG. 4F). Red fluorescence was readily detectable 24 hours after plasmid transfection, and it remained strong till the end of the experiments two days later. After normalization for transfection efficiency, we found that the percentage of mCherry-positive cells was the highest (71.1±2.7%) when the spacing between R-IscB and the ADAR2dd editing sites was 30 nt apart. Efficiency decreased steeply with longer spacing, more gradually with shorter spacing, and became undetectable with 10 nt and 5 nt spacing. We harvested cells from one experiment, reverse-transcribed a region of mCherry bracketing the editing site. The next- generation sequencing reads revealed that the correctly edited reads accounted for ~12% of the total reads. This efficiency matches the performance of the reported Cas13-ADAR systems43,47. Bystander A-to-I editing events were only detected at two nearby sites, and the off-targeting frequency was 60-fold lower than the on-targeting frequency (FIG.11C). No mCherry+cells were observed when we omitted either ADAR2dd or the mCherry-targeting guide; only negligible fluorescence was observed when R-IscB was omitted from the trio combination (FIG.11D). These controls suggest that the observed A-to-I editing was indeed guided by R-IscB-ωRNA. Example 9 Structure-guided engineering enables RNA-guided RNA cleavage in R-IscB RNA-guided RNA cleavage was hardly detectable in R-IscB (FIG.12A); enabling it may lead to more efficient knockdowns. In contrast, NmeCas9 and CjCas9 are naturally capable of ssRNA cleavage, mediated by the HNH nuclease32,33. We analyzed whether only minor adjustments in the HNH active site are needed to enable RNA cleavage in R-IscB. To achieve it, we aligned the HNH domain sequences to identify active site residues that were common in RNA-cleaving Cas9s but distinct in OgeuIscB. We then introduced these residues into R-IscB individually or in combinations (FIG.5A). A total of eight variants were constructed and biochemically tested (FIG.12A). Results showed that ssRNA cleavage can be enabled in IscB by a single amino acid substitution (Variant 2, H243E). RNA cleavage was the strongest in a two-residue substitution (Variant 3, H243E / R270Q). This variant, however, was still capable of DNA cleavage in the context of the full-length IscB (FIG.5B-C). A third substitution completely abolished DNA cleavage, with a small compromise in RNA cleavage (Variant 4, H243E / R270Q / H269N) (FIG.5B-C, 12A-B). Interestingly, while R-IscB cleaved ssDNA at the same location as wildtype IscB did, it cleaved ssRNA 4-nt further downstream, between TAM+7 and +8 (FIG.12B). A potential reason is that the gRNA-target RNA duplex inside R-IscB adopts an ideal A-form conformation rather than a distorted A-form inside the DNA-targeting IscB (PDB: 7UTN). The difference in substrate conformation may have led to the altered cleavage site. Overall, Variant 4 represented the best compromise between activity and specificity. Before in vivo experiments, we profiled its off-targeting activity by mixing it with equimolar amounts of perfectly matched and mismatched ssRNA targets (FIG.5D) and quantifying their relative cleavage extent (FIG.9B). While singly mismatched ssRNAs were not adequately differentiated at the binding level (FIG.2H), they were strongly discriminated against at the cleavage level, with cleavage extent reduced to only 25~28% of the perfectly matched level (FIG.5D, 9B). Additional mismatches further reduced the cleavage extent to 4~15% of the perfectly matched level (FIG. s 5D, 9B). The higher stringency in cleavage is within expectation, as IscB undergoes a conformational change after substrate binding before committing to cleavage17. Example 10 RNA cleavage further enhances R-IscB-mediated mRNA knockdown The PCSK9 knockdown experiment was repeated using the cleavage-enabled R-IscB- VR4. To distinguish the impact of mRNA cleavage from splicing perturbation, we designed seven 16-nt guides directed against either internal regions of exons 1, 2, and 5, or the exon1- exon2 junction (FIG.5E). Guides C2 and C4 demonstrated high efficacy, reducing PCSK9 mRNA abundance to 15.3±5.8% and 13.7±4.8% of non-targeting control levels, respectively. (FIG.5F); C5, C6, and C7 also achieved statistically significant knockdowns. Overall, the cleavage-based knockdown was more efficient than splicing-based knockdown, which is expected given that splicing perturbation requires sustained splice site occupation. While less effective, splicing-based knockdowns may be safer because off-targeting is unlikely to be consequential. To check how robust R-IscB-VR4 was, we further programmed it with 30-nt guides to target three sites in EZH2, which has been targeted in Cas13 studies44,47. R-IscB-VR4 mediated targeting was highly efficient, reducing EZH2 mRNA abundance to 19.0±4.0% (E1), 6.5±1.0% (E2), and 3.4%±1.5% (E3) of the non-targeting control level (FIG.5G). Next, we introduced the VR4 combination into the TID-substitution variant and programmed it with the same E3 guide. This sextuple-VR4 variant knocked EZH2 expression down to 14.6^7.5% of the non-targeting control level (FIG.5G). This result is consistent with the DMD knockdown data, confirming that the TID-substitution variant is functional but less efficient than the TID-deletion variant in RNA editing. We further characterized the off-targeting behavior of R-IscB-VR4 by programming the guide to target the endogenous ADARB1 gene, which encodes the ADAR2 deaminase. Cells were harvested 48 hours after R-IscB-VR4 mediated targeting, and the total mRNAs were subject to RNA-seq based transcriptome analysis. A volcano plot was generated to display differentially expressed genes, with settings including a false discovery rate of 0.05 and a fold change threshold of 2 (FIG.5H). The on-target, ADARB1, was knocked down ~64-fold relative to the non-targeting control. The majority of differentially expressed genes were upregulated rather than downregulated (FIG.5H), and possibly off-targets were not enriched in the downregulated genes (FIG.12C). Both observations pointed to expression level changes due to the loss of ADAR2 regulation rather than R-IscB mediated off-targeting. Consistently, the differentially expressed genes showed clear clustering in gene ontology analysis (FIG.12D). Upregulated genes clustered into RNA splicing, cell cycle regulation, and DNA replication pathways, while downregulated genes clustered into metabolic pathways (FIG.12D). These results are consistent with reported regulatory roles of ADARB1 in RNA stability, alternative splicing, translation regulation, and cell proliferation73-75. Collectively, these data fail to reveal a clear pattern of R-IscB-VR4-mediated off-targeting. Instead, they reveal the potential of R-IscB-VR4 for knockdown-based functional screening, paralleling RNAi approaches. We benchmarked the knockdown efficiency of R-IscB-VR4 against PspCas13b and RfxCas13d in HEK293T cells at five characterized sites in Cas13 studies44,47. Consistent vector backbones, identical 30-nt guide sequences, and the same assay conditions were used. R-IscB significantly outperformed both Cas13s at three targets, with a winning margin of over 60-fold at NF2 (R-IscB-VR4:PspCas13b:RfxCas13d, 0.88±0.2%:62.9±4.2%:79.7±15.4%). R-IscB performed similarly to two Cas13s at the SMARCA4 site and only underperformed PspCas13b at the STAT3 site (FIG.5I). Considering that RNA-cleavage has not been extensively optimized for R-IscB, our results suggest R-IscB is comparable or better than Cas13 in mediating RNA knockdowns. Example 11 Removing PID converts multiple Cas9s to exclusive RNA-targeting tools Given the success with IscB, we next explored whether Cas9s can also be converted into exclusive RNA-targeting tools using the same strategy. The challenge was that Cas9's equivalent PAM-interaction domain (PID) is more extensively integrated into surrounding structures, making the deletion constructs more prone to misfolding. This is especially true for SpCas9, the most widely used homolog, as it has evolved additional structural elements around PID, making a clean PID excision difficult7. We instead focused on the N. meningitidis Cas9 (NmeCas9), where a 1:1 domain correlation with IscB could be established. An empirical sequence (HPNDY) was introduced to preserve structural integrity between adjacent domains after PID removal (FIG.6A). Consistent with previous findings33, the wildtype NmeCas9 was capable of cleaving complementary dsDNA, ssDNA, and ssRNA (FIG.6B-C). However, it is not capable of RNA knockdown in vivo (FIG.6D). Upon PID removal, NmeCas9-ΔPID was no longer capable of dsDNA cleavage but still capable of ssDNA and ssRNA cleavage (FIG.6B-C). It cleaved ssDNA at the canonical site (Site1, between PAM+3 and PAM+4). NmeCas9 cleaved ssRNA1 at an additional site, and the cleavage extent varied depending on whether the wildtype or ΔPID enzyme was used (FIG. 6C). When tested on ssRNA2, cleavage converged onto the canonical site (FIG.12E). Next, we tested the mRNA knockdown efficacy of NmeCas9-ΔPID in HET293T cells. When programmed to target the 3’ splice site of EZH2 exon 9, dNmeCas9-ΔPID (D16A / H588A) reduced the EZH2 expression to 24.4±0.8% of the non-targeting control level. The HNH-active version of NmeCas9-ΔPID (D16A) reduced the EZH2 expression by another 2-fold, to 13.1±0.8% (FIG.6D). These results align with prior observations that ssRNA cleavage yields better mRNA knockdown efficiency. We expanded the PID-deletion effort to SaCas9, CjCas9, and Cas9d. Knockdown efficiency was evaluated again at the EZH2 site. All three ΔPID Cas9s mediated efficient EZH2 knockdowns that were comparable (SaCas9: 13.8±1.2%) or even better than that by NmeCas9 (CjCas9: 9.8±0.9%; Cas9d: 9.6±1.2%) (FIG.6D). Notably, without the PID deletion, only CjCas9 was mildly effective; SaCas9 and Cas9d failed to knock down EZH2 at the RNA level (FIG.6D). DNA targeting was unlikely because their PAMs are not found at the target site. Cas9d is particularly attractive because it is only ~700 amino acids in size and specifies a 20-nt R-loop, 4-nt longer than IscB. We conclude that PID-deletion can enable RNA targeting in many Cas9s. We collectively name them R-Cas9s. Discussion of Examples There has been a need for new RNA-guided RNA-targeting tools because the current tool of choice, Cas13, has undesirable cytotoxicity in eukaryotic cells. This prevented Cas13- based applications from moving into clinics. In vitro RNA-targeting activity has been reported for Cas9 and others. However, robust in vivo RNA-targeting applications have yet to emerge from them. Through in-depth mechanistic studies on the IscB system, we identified the root cause of the problem and found a solution to unmask its RNA-targeting activity. By removing its TAM-interaction domain, which likely locked IscB into a constant TAM-search mode on dsDNA, we converted IscB into an exclusive RNA-targeting tool. TID-disruption enabled the same activity, albeit less effective than TID-deletion. We expect that other IscB homologs can be similarly engineered for RNA-targeting, as we successfully extended the PID-deletion approach to four distantly related Cas9 enzyme. Extending the PID-deletion to more sophisticated Cas9s (i.e., SpCsa9 and FnCas9) involved their having evolved additional PAM-recognition elements that are intricately woven with PID. Removing these elements may compromise surrounding structural integrity and cause protein misfolding. In such cases, surface substitutions to disrupt DNA binding in PID - following the sextuple TID substitution approach for IscB (FIG.3K, 5G) is encompassed by the disclosure, as is converting other popular RNA-guided DNases such as Cas12, Fanzor, and TnpB to RNA editors. This surprising solution shows that sometimes, less is more. By removing structural elements essential for the native function of an enzyme, we enabled a whole new line of activities from it. The absence of discernible cytotoxicity is a clear advantage for R-IscB. An additional advantage is the surprising finding that R-IscB is superior to Cas13 in mediating RNA- targeting in human cells. Unlike Cas13, R-IscB has the ability to screen for candidate RNA targets through seed duplex formation. Related Cas9 studies show that the target is then validated through directional duplex formation20. Once a perfectly matched target is found, the off-rate is very slow, presumably because IscB undergoes additional conformational changes to completely encapsulate the gRNA / target RNA duplex, similar to what it does for dsDNA17. If Cas13 is so easily outcompeted, why didn’t nature evolve a Cas13 with better RNA-binding kinetics? It is possible that the target-searching property of Cas13 is sufficient for its role in RNA-guided defense, and that the evolutionary pressure has been instead on its ability to trigger a wider anti-viral response76, including collateral RNA damage that has been shown to send bacterial cells into dormancy77. Consistent with this, it was recently found that Cas13 originated from a toxin-antitoxin system78, where instead of serving as a guide, RNA functions as the antitoxin to repress the nonspecific RNase activity in the HEPN domains. We demonstrated robust usage of R-IscB in a variety of settings, from splicing perturbation to strand cleavage, from base correction to long sequence correction via trans- splicing. This supports therapeutic and research uses of the described systems. With respect to therapeutic uses, the disclosure includes the described trans-splicing based approaches. Many recessive genetic diseases result from a diverse set of loss-of-function mutations. Correcting them via base editing or prime editing requires treating each mutation as a distinct molecular disease, each requiring individual efficacy and safety evaluation. This is both time-consuming and resource-demanding. RNA-guided trans-splicing as described herein offers the potential to dramatically simplify therapeutic strategies by correcting multiple mutations with a single trans-splicing guide. Given R-IscB’s small footprint, as much as 2.5 kb of the AAV payload can be allocated for the gene-correction content, enabling correction of notoriously long disease genes such as DMD. Supplemental information Supplemental Table 1: primers, plasmids, and synthesized gBlocks used in the experiments. Supplemental Table 2: Variant sequences of IscB and Cas9s.

[0002] Supplemental Table 1

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[0005] Supplemental Table 2

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Shen, H., An, O., Ren, X., Song, Y., Tang, S.J., Ke, X.Y., Han, J., Tay, D.J.T., Ng, V.H.E., Molias, F.B., et al. (2022). ADARs act as potent regulators of circular transcriptome in cancer. Nat Commun 13, 1508.10.1038 / s41467-022-29138-2. 75. Jiang, Q., Isquith, J., Zipeto, M.A., Diep, R.H., Pham, J., Delos Santos, N., Reynoso, E., Chau, J., Leu, H., Lazzari, E., et al. (2019). Hyper-Editing of Cell-Cycle Regulatory and Tumor Suppressor RNA Promotes Malignant Progenitor Propagation. Cancer Cell 35, 81-94 e87. 10.1016 / j.ccell.2018.11.017. 76. VanderWal, A.R., Park, J.U., Polevoda, B., Nicosia, J.K., Molina Vargas, A.M., Kellogg, E.H., and O'Connell, M.R. (2023). Csx28 is a membrane pore that enhances CRISPR-Cas13b-dependent antiphage defense. Science 380, 410-415.10.1126 / science.abm1184. 77. Meeske, A.J., Nakandakari-Higa, S., and Marraffini, L.A. (2019). Cas13-induced cellular dormancy prevents the rise of CRISPR-resistant bacteriophage. 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Budhathoki, J.B., Xiao, Y., Schuler, G., Hu, C., Cheng, A., Ding, F., and Ke, A. (2020). Real-time observation of CRISPR spacer acquisition by Cas1-Cas2 integrase. Nat Struct Mol Biol 27, 489- 499. 10.1038 / s41594-020-0415-7. STAR Methods EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS Escherichia coli DH5 alpha This strain was purchased from New England Biolab (NEB) and used for cloning. Cells were grown at 37°C in LB supplemented with appropriate antibiotics Escherichia coli T7 Express lysY This strain was purchased from NEB and used for RNP purification. Cells were grown at 37°C in LB supplemented with appropriate antibiotics METHOD DETAILS Plasmid construction Oligonucleotides were ordered from Integrated DNA Technologies. Plasmid encoding wild-type NmeCas9 and sgRNA was a gift from E. Sontheimer (Addgene plasmid 71474)28. SaCas9 with sgRNA was a gift from Y. Huang (Addgene plasmids 99735)79. Dead PspCas13b with ADAR2dd (E488Q / T375G) was a gift from F. Zhang (Addgene plasmid 103871)47. RfxCas13d was a gift from H. Yang (Addgene plasmid 171380)44. OgeuIscB plasmid for RNP purification has been described previously17. ωRNA-V2 uses the optimized scaffold sequence26and has a 16-nt guide at the 5´ side (5´-AAAAGAGTGAACGAGA). ωRNA was cloned into pUC57, in between HindIII and EcoRI, and under the control of an lpp promoter and a T7 terminator. IscB-ΔTID expression vector was constructed by doing PCR mutagenesis on the full-length construct using primers remove_TID_F (aatggtggcctgcagatttatgttgggtccaaaaggccggcg) and remove_TID_R (taaatctgcaggccaccattatactgtgcgctcggatgtttaacgg). For in vivo editing experiments, R-IscB contained TID deletion, nuclease-dead mutations (D60A, H269A), a subset (M102R, F137K, V159K, L393K) of the previously described eight editing-enhancing mutations26, N- and C-terminal NLS for nucleus localization, and in some cases a T2A-tagged mCherry for transfection efficiency control. Cleavage-enabled R-IscBs contained mutational combinations in the HNH domain as specified in the text. This construct was cloned into pcDNA3.1, under the control of a human cytomegalovirus (CMV) promoter and a bGH polyadenylation signal. The ωRNA-V2 was cloned into the same plasmid under the control of a U6 promoter and a polyU terminator. In trans-splicing experiments, the trans-splicing guide was put under the control of a U1A promoter for pol II transcription, 5’ capping, and 3’ polyadenylation. Its guide was varied via PCR mutagenesis according to the intended target site. See Supplementary Tables for key sequence information. R-IscB-ADAR2dd construct has ADAR2dd fused to the C-terminus of R-IscB through a flexible linker. ADAR2dd refers to the engineered high-fidelity version of adenosine deaminase acting on RNA type 2, with the E488Q / T375G mutation47. Protein expression and purification Plasmids encoding IscB (WT, ΔTID, and ΔTID / +S1) and ωRNA were co-transformed into E. coli T7 Express lysY cells. The cell culture was grown in an LB medium supplemented with 0.75g L-cysteine / L at 37°C until the optical density at 600 nm reached 0.8. Expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM at 16°C overnight. Cells were collected by centrifugation and lysed by French press in buffer A (50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP, 5% glycerol, 2.5 mM MgCl2) with 1 mM phenylmethylsulfonyl fluoride (PMSF). The supernatant after centrifugation was applied onto strep-tactin XT resin (IBA lifesciences) preequilibrated in buffer A. Resin was then washed with 15mL of buffer A plus 0.1 mM CaCl2 and 10U DNase I (Thermo Scientific), 20mL of buffer B (50 mM HEPES pH 7.5, 1 M NaCl, 0.5 mM TCEP, 2.5 mM MgCl2), and 40mL of buffer A. Proteins were eluted from the resin using 10 mL of buffer A plus 50mM Biotin. The IscB-containing fractions were pooled, desalted, and further purified on MonoQ 5 / 50GL (Cytiva) with a NaCl gradient. The desired IscB fractions were pooled, concentrated, and snap-frozen in liquid nitrogen and stored at -80 ˚C. Electrophoretic mobility shift assays DNA substrates were PCR-amplified from plasmid templates using a common set of fluorescent primers (F-Cy5: Cy5-CTGTCTCTTATACACATCT; R-FAM: FAM- CAAGCAGAAGACGGCATAC) and purified on an agarose gel to remove primers. ssDNA and ssRNA substrates were chemically synthesized from IDT in PAGE purification grade. Native EMSA was carried out in agarose gels. A typical 20 μL reaction contained 10 nM of nucleic acid substrate and the specified amount of IscB RNP in a binding buffer containing 50 mM HEPES pH 7.5, 50 mM NaCl, 0.5 mM TCEP, 2.5 mM MgCl2. After a 20-minute incubation at 37°C, each sample was mixed with 2.5 μL of 60% glycerol and immediately loaded onto a 1.5% 0.5XTBE agarose gel. Electrophoresis was performed in the cold room at 35 V for 120 min. The fluorescent signals from the gel were recorded either on ChemiDoc (Bio-Rad) or Typhoon (Cytiva) and quantified using ImageJ or ImageQuant (for Typhoon scanned images). In vitro cleavage assays The cleavage assays were typically set as 20 μL reactions, containing 20 nM final concentration of labeled target DNA, ssDNA, or ssRNA, specified concentration of IscB RNPs, in a cleavage buffer containing 50 mM HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 0.5 mM TCEP, and incubated at 37°C for 30 minutes. The reactions were quenched with 1 μL of 0.5 M EDTA, treated first with Proteinase K at 37°C for 30 min to digest away the IscB protein, and then with phenol extraction to further remove the IscB protein. The nucleic acids were precipitated from the aqueous phase, and the dried pellet was dissolved in 20μL of formamide, heated to 95°C for 10 minutes, and then separated on 14% urea-PAGE. The fluorescent signals from the gel were documented using either ChemiDoc (Bio-Rad) or Typhoon (Cytiva). Cleavage assays for NmeCas9 used the same setup, with 200 nM of NmeCas9-WT / ΔPID-sgRNA RNP and 20 nM of fluorescently labeled ssRNA or ssDNA, at 37 °C for 30 minutes. Cell culture and transfection HEK293T cells were cultured in DMEM (Corning, 10-013-CV) supplemented with 10% FBS (Corning, 35-010-CV) and penicillin / streptomycin (MP Biomedicals). HepG2 cells were cultured in EMEM (ATCC, 30-2003) with 10% FBS and penicillin / streptomycin. Human primary fibroblast IMR-90 cells were purchased from ATCC (CCL-186). The cells were cultured in DMEM with high glucose, sodium pyruvate, and L-glutamine (Corning, 10- 013-CV) supplemented with 10% FBS (Corning, 35-010-CV). Cells were maintained in 5% CO2 at 37 °C and passaged regularly upon reaching 80% confluency. Cells were seeded in antibiotic-free media 24 hrs before transfection to aim for ~60% confluency at the time of transfection. Transfection was performed with either jetOPTIMUS (Polyplus, 101000006) or Lipofectamine 3000 (Invitrogen, L3000015) following the manufacturer’s instructions. Plasmid DNA was delivered to IMR-90 cells by nucleofection (Lonza, V4XC-1032) with a Lonza 4D-Nucleofector X unit following the manufacturer’s protocol. Briefly, 2 × 105cells were transfected with program CM-120 in a 20-µl Nucleocuvette containing plasmid DNA (400 ng each). Cells were resuspended in 500 µl of DMEM and incubated in a 24-well plate. Cells were imaged using an Olympus BX53 microscope 48 h post-transfection. Cell confluency measurement Cell confluency measurements in HEK293T cells were carried out in 24-well format. Each well contained ~1.3x105cells. Specifically, 500 ng of the EGFP plasmid, NmeCas9- sgRNA with a non-targeting sequence, SpCas9-sgRNA with a non-targeting sequence, R- IscB-ωRNA sgRNA with a non-targeting sequence, or PspCas13b-crRNA with a non- targeting sequence was diluted into 50 μL of jetOPTIMUS buffer plus 0.5 μL of jetOPTIMUS, then introduced into each well. After transfection, cell confluency was monitored for 27 hours using the Incucyte Live-Cell Analysis System (Sartorius) and quantified using the built-in software. Default parameters were used for image processing. Cellular mRNA knockdown Knockdown experiments in HEK293T cells were carried out in 24-well format. Each well contained ~1.3x105cells. Specifically, 500 ng of the R-IscB-ωRNA all-in-one plasmid was diluted into 50 μL of jetOPTIMUS buffer plus 0.5 μL of jetOPTIMUS, then introduced into each well. After transfection, cells were incubated for 48 hours before harvest. Cas9 homolog experiments were carried out in the same procedure. Knockdown experiments in HepG2 cells were carried out in 24-well format. Transfection reagent for each well was prepared by two-step mixing. First, 800 ng of the R- IscB-ωRNA all-in-one plasmid was mixed with 25μL of OptiMEM media (Gibco) and 1.6μL of P3000 reagent, then the mixture was further mixed with premixed 25μL of OptiMEM media (Gibco) and 0.8μL of Lipofectamine 3000 reagent before delivery. After a 15-minute incubation, the transfection reagent was introduced to a single well. Cells were incubated for 48 hours before harvest. Quantitative PCR, RNA-seq and analysis. To quantify RNA knockdown efficiency of R-IscBs, RNAs were extracted from transfected cells using Trizol (Invitrogen) and reverse-transcribed to cDNAs with ProtoScript II First Strand cDNA Synthesis Kit (NEB, E6560). Quantitative PCR (qPCR) was performed with the cDNA for each sample on a CFX96 real-time system (Biorad) using Luna Universal qPCR Master Mix (NEB, M3003) following the manufacturer’s suggestion. QPCR data were processed using Maestro. QPCR results were analyzed with the −ΔΔCTmethod, in which differences between average CT values of target genes and reference gene GAPDH for three biological replicates were used to calculate the relative expression level of the target gene and normalized against the corresponding non-targeting controls. RNA samples were further purified using oligo-dT beads, followed by random priming. Then, they were fragmented, reverse transcribed, and prepared for sequencing using an NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, E7760L).150-bp paired-end sequencing was performed on Illumina NovaSeq6000 instrument. The RNA-seq data were first filtered with SolexaQA (v.3.1.7.1)80to remove the low- quality reads. RNA-seq reads of RNA knockdown experiments were aligned to the hg38 reference genome using Hisat2 (v.2.0.4)81. All uniquely mapped reads were processed by HTSeq-count82to generate a read count matrix. DESeq283was used to calculate differentially expressed genes. Genes with fold change >2 and false discovery rate (FDR) < 0.05 were labeled as differentially expressed genes and used for volcano plotting visualization. Gene ontology analysis was conducted based on ClusterProfiler84and a customized script in RStudio. RNA trans-splicing, A-to-I editing and sequencing analysis RNA trans-splicing was performed following the previously described transfection protocol. EGFP fluorescence was monitored for 72 hours using the Incucyte Live-Cell Analysis System (Sartorius) and quantified using the built-in software. Default parameters were used for image processing, with the exception of the GFP sensitivity parameter, which was adjusted to 0.8. The percentage of GFP-positive cells was calculated as the green fluorescence area normalized against the phase-contrast area, which reflects cell confluency. RNA editing was performed following the previously described transfection protocol. mCherry fluorescence was monitored for 48 hours using the Incucyte Live-Cell Analysis System and quantified with the Incucyte software. The ratio of mCherry-positive cells was calculated as the red fluorescence area normalized against the phase-contrast area. To quantify the A-to-I base editing efficiency from the deep-sequencing reads, the amplicon bracketing the edited region was RT-PCRed from Trizol-extracted total RNA using specific primers. Deep sequencing libraries were prepared by appending i5 / i7 index sequences via a second round of PCR, then underwent 150-bp paired-end sequencing on an Illumina MiSeq instrument. Sequencing data were first demultiplexed by Cutadapt (v.2.8) based on index sequences. The demultiplexed reads were then processed by CRISPResso285for the quantification of A-to-I conversion efficiency at target sites. Single-molecule RNA binding kinetic assays. Quartz slides for single-molecule experiments were first sonicated in acetone for 25 min, in 1M KOH aqueous solution for 25 min, and then in ethanol for 10 min. As-prepared slides were coated with 1% 3-Aminopropyltriethoxysilane (APTES) in Methanol overnight at room temperature, followed by rinsing them with Milli-Q water. Then, the slides were incubated in 80μL 0.1M NaHCO3aqueous solution with 1mg of PEG-5000-Biotin and 20mg of PEG-5000 (Laysan Bio Inc.) at room temperature for 2 hrs, followed by rinsing them with Milli-Q water and blow-drying with nitrogen. The reaction chamber was assembled manually from a treated quartz slide further containing drilled holes for inlet and outlet, a hand-carved tape to specify the flow path, and a cover slide as described before86. Single-molecule assays were performed using a home-built prism-type TIRF microscope. The TIRF reaction chamber was filled with 40μL of buffer S (50 mM HEPES pH 7.5, 100 mM NaCl, 2.5 mM MgCl2) plus 0.1mg / mL neutravidin for 5 min. Unbound neutravidin was washed away by 40μL of buffer S. R-IscB-ωRNA RNP was introduced in 40μL of buffer S containing 400 nM R-IscB-ωRNA RNP for 10 min, followed by 40μL of buffer S to wash away unbound RNP. The TIRF experiment was initiated by flowing in 40μL of 1 nM Cy5-labelled ssDNA / ssRNA dissolved in buffer S plus oxygen scavenging reagents (1 mM TROLOX, 30% glucose, catalase, glucose oxidase). Surface-bound ssDNA / ssRNA molecules were visible in the TIRF setup under direct excitation by the red laser (640 nm). To capture binding kinetics, 10-min movies were recorded at a 100ms / frame refresh rate. To capture dissociation kinetics, unbound Cy5-ssRNA was washed away, and a series of burst- recordings were recorded every 5 minutes for 45 minutes. On-state dwell time was inferred from the loss of immobilized Cy5 spot over time. Control experiments done in the absence of IscB RNP showed that long-lasting nonspecific binding to the quartz surface was not a concern. QUANTIFICATION AND STATISTICAL ANALYSIS Data analysis was performed using Origin 2024b (OriginLab Software) by student t- test. P-values are indicated in the FIG. legends.

Claims

What is claimed is:

1. A modified IscB protein from which at least a portion of its Tam Interacting Domain (TID) has been removed to inhibit binding to the Tam, and / or wherein the IscB protein comprises one or more mutations relative to a wild type IscB protein, such that binding of the modified IscB to double-stranded nucleic acids is reduced, relative to binding to double stranded nucleic acids by an IscB protein that includes an intact TID.

2. The modified IscB of claim 1, wherein the modified IscB exhibits preferential binding to single stranded DNA (ssDNA) and / or to single stranded RNA (ssRNA), relative to binding to ssDNA or RNA binding of an IscB protein that includes an intact TID or no mutations .

3. The modified IscB protein of claim 1, wherein the modified IscB protein is a component of a fusion protein.

4. The modified IscB protein of any one claim 1, comprising mutations of SEQ ID NO:1 (OgeuIscB Wild-type sequence), wherein said modifications comprise one or more of the following: a) mutation of the TAM-interacting segment of SEQ ID NO:1, said mutation comprisinga deletion of the sequence KDMSRIMPGSILVSGEGKLFTLSRSEGRNKGQVNYFVSTEGIKYWARKCQYLR, and / or R461D, R457A, K434E, H379D, K380A, R438Q, or a combination thereof; and / or b) a mutation to reduce nuclease activity of the modified protein, wherein the mutationis D60A, H269A, or a combination thereof; and / or c) a mutation to enhance nucleic acid interaction of the modified protein, wherein themutation is M102R, F137K, V159K, L393K, or a combination thereof; and / or d) a mutation to enhance ssRNA-cleavage activity of the modified protein, wherein themutation is selection from: VR1: R270Q, VR2: H243E, VR3: H243E and R270Q,VR4: H243E and H269N and R270Q, VR5: H243E and H245D and H269N and R270Q, VR6: H243E and H269N, VR7: H269N and R270Q, or VR8: H243E and H245D and H269N.

5. The modified IscB protein of claim 3, comprising the sequence: MAVVYVISKSGKPLMPTTRCGHVRILLKEGKARVVERKPFTIQLTYESAEETQPLVLGI APGRTNIGMSVVTESGESVFNAQIETRNKDVPKLMKDRKQYRRAHRRLKRRCKRRR RAKAAGTAFEEGEKQRLLPGCKKPITCKSIRNKEARFNNRKRPKGWLTPTANHLLVTH LNVVKKVQKILPVAKVVLELNRFSFMAMNNPKVQRWQYQRGPLYGKGSVEEAVSM QQDGHCLFCKHGIDHYHHVVPRRKNGSETLENRVGLCEEHARLVHTDKEWEANLAS KKSGMNKKYHALSVLNQIIPYLADQLADMFPGNFCVTSGQDTYLFREEHGIPKDHYL DAYCIACSALTDAKKVSSPKGRPYMVHQFRRHDSAQYNGGLQIYV (OgeuIscB delta- TID sequence) (SEQ ID NO:2).

6. The modified IscB protein of claim 3, comprising the sequence: MAVVYVISKSGKPLMPTTRCGHVRILLKEGKARVVERKPFTIQLTYESAEETQPLVLGI APGRTNIGMSVVTESGESVFNAQIETRNKDVPKLMKDRKQYRRAHRRLKRRCKRRR RAKAAGTAFEEGEKQRLLPGCKKPITCKSIRNKEARFNNRKRPKGWLTPTANHLLVTH LNVVKKVQKILPVAKVVLELNRFSFMAMNNPKVQRWQYQRGPLYGKGSVEEAVSM QQDGHCLFCKHGIDHYHHVVPRRKNGSETLENRVGLCEEHARLVHTDKEWEANLAS KKSGMNKKYHALSVLNQIIPYLADQLADMFPGNFCVTSGQDTYLFREEHGIPKDHYL DAYCIACSALTDAKKVSSPKGRPYMVHQFRRHDRQACDAANLNRSYYMGGKKVAT NRHKAMDQKTDSLEEYRAAHSAADVSKLTVKHPSAQYEDMSQIMPGSILVSGEGKLF TLSASEGDNKGQVNYFVSTEGIKYWARKCQYLRNNGGLQIYV (OgeuIscB TID- mutated sequence) (SEQ ID NO:5).

7. The modified IscB protein of claim 1, wherein the modified IscB protein is a component of a fusion protein.

8. The modified IscB protein of claim 7, wherein the fusion protein comprises an additional single-stranded nucleic acid interaction protein that is optionally a single stranded RNA interaction protein that is optionally an RNA nucleotide modifying protein that introduces chemical changes to either the sugar-phosphate backbone or the base of the RNA.

9. The modified IscB protein of any one of claims 1-8, wherein the IscB protein exhibits: i) improved single stranded (ss) DNA binding and / or improved ssDNA target cleavage or ssDNA nucleotide modification when used with an omega-RNA directed to the target; or ii) improved ssRNA binding, or improved ssRNA target cleavage or RNA nucleotide modification when used with an omega-RNA directed to a target; or iii) a combination if i) and iii).

10. A system comprising a modified IscB protein of claim 9 or a polynucleotide encoding the modified IscB protein, and an omega-RNA directed to a target of interest.

11. A method comprising contacting nucleic acids with a system of claim 10 such that single stranded nucleic acids are preferentially modified by the system, relative to modification of double stranded nucleic acids.

12. The method of claim 10, wherein the nucleic acids are within a cell.

13. The method of claim 12, wherein the nucleic acids comprise ssRNA.

14. The method of claim 13, wherein the wherein the nucleic acids are present in cells of an individual and the nucleic acids are associated with a disorder, and wherein modification of the nucleic acids has a therapeutic or prophylactic effect in the individual.

15. The method of claim 14, wherein the modification comprises cleavage of ssRNA, or modification of at least one RNA nucleotide in the ssRNA.

16. The method of claim 15, The method of claim 15, wherein ssRNA comprises mRNA.

17. The method of claim 16, wherein the splicing pattern of the mRNA is altered.

18. The method of claim 16, wherein the mRNA is degraded, or wherein at least one nucleotide in the mRNA is modified, and wherein if a nucleotide in the mRNA is modified, the modification optionally comprises a deamination of adenosine to convert the adenosine to inosine.

19. A polynucleotide encoding a modified IscB protein of any one of claims 1-8.

20. The polynucleotide of claim 19, wherein the polynucleotide is an mRNA.

21. A kit comprising a modified IscB protein of any one of claims 1-8, or an isolated or recombinant polynucleotide encoding the modified IscB protein, and at least one sealed or sealable container that holds the modified protein or the isolated or recombinant polynucleotide.

22. The kit of claim 21, further comprising printed material providing instructions for using or making the modified IscB protein.

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