RNA editing in living cells

Abstract

Non-naturally occurring systems and methods for sequence-specific RNA editing in living cells using engineered type III CRISPR complexes. Type III CRISPR complexes exclusively cleave the target RNA in six nucleotide increments. Engineered type III CRISPR-Cas systems and methods leverage this cleavage activity to excise segments from human transcripts. Resulting RNA fragments are repaired in the living cell, which produces programmable edits in target RNA. Disclosed systems and methods can be used to specifically edit RNA for therapeutic effect. For example, various engineered systems and methods may remove a premature stop codon in RNA to restore corresponding protein function. For example, various engineered systems and methods may remove a nonsense mutation common in cystic fibrosis patients, which rescues protein expression and demonstrates the therapeutic potential of this technique. More generally, disclosed is technology for RNA manipulation in living cells for therapeutic effect and a model for discovering new RNA repair pathways.

Description

RNA EDITING IN LIVING CELLSCROSS-REFERENCE TO RELATED APPLICATIONS[1] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 523,592, filed on June 27, 2023; U.S. Provisional Application No. 63 / 534,305, filed on August 23, 2023; U.S. Provisional Application No. 63 / 638,014, filed on April 24, 2024; and U.S. Provisional Application No. 63 / 651 ,936, filed on May 24, 2024; the entireties of which are each incorporated by reference herein for all purposes.GOVERNMENT RIGHTS[2] This invention was made with government support under contracts R35GM134867 and AI171893 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING[3] This application contains a Sequence Listing which has been submitted electronically inWIPO Standard ST.26 (XML format) and is hereby incorporated by reference in its entirety. Said Sequence Listing copy, created on June 27, 2024, is named “078708-0580093_SEQUENCE_LISTING.xml” and is 58,663 bytes in size.BACKGROUND[4] CRISPR-guided endonucleases have enabled programable DNA cleavage and the development of new therapeutics. The first generation of CR1SPR genome editing used Cas9 nuclease to make double- stranded DNA breaks that are repaired by the cell, leading to site- specific edits. These technologies have enriched our mechanistic understanding of DNA repair, and new insights into repair are used to improve methods for genome editing. While DNA editing has the potential to cure genetic diseases, it can result in unintended changes to the genome and toxic cellular stress responses.SUMMARY[5] The disclosure relates to RNA editing in living cells. RNA editing can alter the cellular program without changing the DNA. However, options for editing RNA are limited. For example, Cas 13, a type VI CRISPR-guided endoribonuclease, has been used for RNA knockdown, but target recognition also activates collateral nuclease activity that degrades non-target RNAs. Nuclease- inactivated mutants of Casl3 (dCasl3) have been used for exon skipping, trans- splicing of synthetic RNA payloads, or sequence- specific delivery of base-editing enzymes for adenosine-to- inosine or cytosine-to-uracil conversions. However, programmable deletions in RNA, analogous to those introduced by DNA editors, have not been described.[6] To address the need for versatile and facile RNA manipulation, disclosed is a technology for sequence- specific RNA editing in living cells. This technology uses type III CRISPR complexes for programmable cleavage of target RNA. Unlike Cas 13, type III complexes exclusively cleave the target RNA in six nucleotide increments. Disclosed systems (such as engineered type III CRISPR-Cas systems) and methods leverage this cleavage activity to excise segments from human transcripts. Resulting RNA fragments are repaired in the living cell via endogenous (and in some instances exogenous) repair mechanisms, which produces programmable deletions or other edits in target RNA. Disclosed systems and methods can be used to specifically edit RNA for a therapeutic effect. For example, various engineered systems and methods may remove a premature stop codon in RNA to restore corresponding protein function. For example, various engineered systems and methods may remove a nonsense mutation common in cystic fibrosis patients, which rescues protein expression and demonstrates the therapeutic potential of this technique. More generally, disclosed is technology for RNA manipulation in living cells for therapeutic effect and a model for discovering new RNA repair pathways.BRIEF DESCRIPTION OF THE FIGURES[71 Features of the present disclosure may be illustrated by way of example and not limited in the following figure(s), in which like numerals indicate like elements, in which:[8] FIGS. 1 A-H illustrate various aspects of programmable editing of RNA using RNA-guided CRISPR ribonucleases, according to an implementation.[9] FIG. 2 illustrates a schematic example of CRISPR-guided deletion of premature stop codons in RNA, according to an implementation.

[0010] FIGS. 3A-3E illustrate various aspects of programmable deletion of stop codons, which restores expression of the encoded protein, according to an implementation.

[0011] FIG. 4 illustrates a schematic example of engineering type III ribonucleoproteins for RNA editing, according to an implementation.

[0012] FIG. 5 illustrates a schematic example of inactivation of enzymatic activities of the CaslO subunit, which reduces toxicity and improves protein yields, according to an implementation.

[0013] FIGS. 6 A- J illustrate various aspects of RTCB ligase repair of RNA cleaved by the type III CRISPR complex, according to an implementation.

[0014] FIGS. 7A-D illustrate various aspects of repair of concurrent RNA breaks, resulting in large RNA excisions, according to an implementation.

[0015] FIGS. 8A-I illustrate various aspects of programmable excision of stop codons, which restores protein expression, according to an implementation.

[0016] FIGS. 9A-F illustrate various aspects of programmable excision of a non-sense mutation in the CFTR transcript, which restores translation, according to an implementation.

[0017] FIG. 10 illustrates a schematic example of programmable RNA editing with catalytic RNA, according to an implementation.

[0018] FIG. 11 illustrates a comparison of two methods to deliver RNA in a cell, according to an implementation.DETAILED DESCRIPTION

[0019] The disclosure relates to RNA editing using an engineered type III CRISPR complex. Many CRISPR RNA-guided nucleases are routinely used for sequence-specific manipulation of DNA. However, analogous methods for editing RNA have yet to be established. The use of CRISPR systems for editing RNA have been restricted to in vivo base editors that are incapable of deleting or inserting specific sequences by design. Thus, what is needed are RNA editing systems that enable specific RNA editing in living cells for gene therapy or other purposes.

[0020] The term “engineered”, and similar terms may refer to a deliberate generation of a system that is otherwise non-naturally occurring. Such engineering may include introducing one or more mutations to a genetic sequence, designing a genetic sequence, combining a set of components such as proteins and detection components where such combination does not occur in nature, and / or otherwise generating a non-naturally occurring system to edit nucleic acid such as RNA. “RNA editing”, “editing RNA”, and similar terms may refer to deliberately modifying RNA. For example, RNA editing may include deleting a portion of the RNA that is intended to be removed, replacing the portion of the RNA with a synthetic RNA sequence that is intended to replace the portion of the RNA, adding synthetic RNA so that the edited RNA includes the synthetic RNA without replacement (even though some examples may remove RNA then add back the removed RNA), deleting one portion of RNA while adding a synthetic RNA, and / or otherwise modifying the RNA in an intentional manner.

[0021] The engineered type III CRISPR complex may include a nuclease that cleaves RNA at specific sites guided by a CRISPR RNA (crRNA) sequence, which may be programmable. The term “programmatic”, “programmable”, and similar terms may refer to designing the engineered type III CRISPR complex to perform RNA editing. In particular, the programmable crRNA sequence refers to being designed to guide the engineered type III CRISPR complex to a specific target portion of interest in the RNA. For example, sequence specific targeting of crRNAs may be performed by designing synthetic spacer sequences. The synthetic spacer sequences may be between 20 and 60 nucleotides long that separate the repeat sequences or end with a self-cleaving ribozyme, such that the crRNA is processed into a short (20-100 nt) crRNA that is incorporated into an assembly of one or more Cas proteins, which together form a ribonucleoprotein complex that stably binds and cleaves RNAs that are complementary to the guide (spacer) sequence. The spacer sequences are designed to be complementary to a target sequence and intentionally designedto avoid complementarity to other “non-target” RNAs. In some examples, crRNA-guides are designed to include a protospacer flanking sequence (PFS) that facilitates binding, cleavage, or cyclic nucleotide synthesis. In other examples, the PFS is any sequence that is not complementary to the 5’ repeat sequence of the crRNA. The programmable crRNA sequence may therefore facilitate specific cleavage of the RNA at one or more specific sites, enabling programmatic RNA editing.

[0022] In some examples, the engineered type III CRISPR complex includes a Csm complex, which is a type III-A CRISPR complex that has RNase activity. The Csm complex may be a SthCsm, derived from Streptococcus thermophilus . In some examples, the engineered type III CRISPR complex may include an engineered type III-E effector derived from Desulfonema ishimotonii.

[0023] The engineered type III CRISPR Cas complex may be delivered inside a living cell, where the complex hybridizes with target RNA, resulting in sequence- specific cleavage of the target RNA in the living cell. The target RNA may undergo repair via endogenous (and in some instances exogenous) RNA repair mechanisms inside the living cell, resulting in the target RNA being edited and repaired.

[0024] RNA transcripts to be edited may include a premature stop codon, sometimes referred to as a nonsense mutation. These premature stop codons are often associated with a disease because they can lead to incomplete or otherwise nonfunctional proteins that would otherwise be encoded by an RNA transcript without this mutation. In these examples, RNA editing in living cells as described herein may delete all or portion of the premature stop codon to restore associated protein function. Other types of therapeutic interventions may be corrected as well.

[0025] Various examples described herein will refer to editing RNA in human cells. However, RNA molecules may be edited in other living cells using the engineered systems and methods disclosed herein.

[0026] Programmable RNA editing using a type III CRISPR complex.

[0027] FIGS. 1A-H illustrate various aspects of programmable editing of RNA using RNA-guided CRISPR ribonucleases, according to an implementation. RNA to be edited may be referred to interchangeably as target RNA, a target RNA molecule, or an RNA molecule, although it will be understood that multiple RNA molecules are edited in living cells. In particular, FIG. 1A illustrates a schematic diagram of RNA editing in eukaryotic cells through sequence- specific RNA cleavageand RNA repair. Referring to FIG. IB, human cells (293T) were transfected with plasmids encoding for NLS-tagged type III CRISPR complex of Streptococcus thermophilus (SthCsm) and RNA guides (interchangeably referred to as “guide RNAs”) targeting PPIB or PARK7 messenger RNAs. Target transcripts were quantified with RT-qPCR, and the qPCR signal was normalized to ACTB and a non-targeting guide RNA control. Data is shown as mean ± SD of three biological replicates. FIG. 1C is a zoom-in from panel FIG. IB. Deep sequencing was used to quantify the proportion of signal that is derived from edited RNA.

[0028] FIG. ID illustrates a schematic diagram of an example of a deep sequencing approach used to quantify RNA editing (top), and top five most frequent RNA editing outcomes in PARK7 transcript (guide 2) (bottom). Dotted lines indicate the positions of RNA breaks by the SthCsm complex. Data is shown as mean ± SD. FIG. IE shows an example plot of kinetics of PARK7 mRNA knockdown (guide 2) vs. NLS-tagged SthCsm complex expression. PARK7 qPCR signal was normalized to ACTB and 0 hour (h) time point. CaslO expression was normalized to ACTB and maximum expression level at 48 h. Data is shown as mean ± SD of three biological replicates. Referring to FIG. IF, PARK7 qPCR products shown in FIG. IE were sequenced, and deletion efficiency was calculated as [relative quantity] x [fraction of reads with deletions]. Illustrated is a plot of the deletion efficiency against hours post transfection. Referring to FIG. 1G, PARK7 knockdown efficiencies with the “wildtype” NLS-tagged SthCsm complex (Csmwt), catalytically inactive NLS-tagged SthCsm complex (Csmdead), crRNA expressed only with Cas6 gene (no csm genes), and cRNA alone were measured using RT-qPCR. Welch’s t-test was used to compare samples expressing targeting and non-targeting crRNA. ** - p < 0.01, *** - p < 0.001, ns - nonsignificant. Referring to FIG. 1H, RT-qPCR products shown in FIG. 1G were sequenced, and a fraction of reads with programmable deletions was quantified.

[0029] The programmable RNase activity of type IILA complexes has been used for RNA knockdown (19-21), but we hypothesized that cells repair CRISPR-guided RNA breaks, which could be leveraged for RNA editing as shown in FIG. 1 A. To test this hypothesis, we targeted two human transcripts, PPIB and PARK7, that are highly expressed in 293T cells. Mutations in PPIB are associated with severe osteogenesis imperfecta, while mutations in PARK7 are linked to Parkinson's disease. We transfected 293T cells with plasmids that express type III CRISPR- associated proteins from Streptococcus thermophilus (Csm2 - Csm5, CaslO, and Cas6) fused to a nuclear localization signal (NLS) and RNA guides complementary to PPIB or PARK7 mRNAs.To quantify target transcripts, we designed RT-qPCR primers that flank target regions. Expression of the NLS-tagged SthCsm complex results in >90% reduction of target transcript levels compared to cells transfected with a non-complementary guide RNA (Fig. IB), which is similar to the knockdown efficiencies reported for other guides. We hypothesized that the remaining target RNA was either not cleaved, cleaved and repaired without modification, or cleaved and repaired with indels at the target site (Fig. 1C).

[0030] To test this hypothesis, we deep-sequenced PPIB and PARK? amplicons. We detected 6, 12, 18, or 24 nucleotide deletions in each of the two RNA targets, which are consistent with the six-nucleotide cleavage pattern of the SthCsm complex and not seen in controls transfected with a non-targeting guide (Fig. ID). SthCsm-dependent deletions were detected in 4.4 ± 0.6% (guide 1) and 5.9 ± 0.6% (guide 2) of PPIB amplicons, and 23.9 ± 2.3% (guide 1) and 65.8 ± 3.6% (guide 2) of PARK7 amplicons (Fig. ID). These data demonstrate that most of the target RNA is degraded, but some of the RNA is repaired in human cells. To measure the kinetics of RNA repair, we extracted RNA from 293T cells at various time points after transfection with SthCsm plasmid targeting PPIB or PARK7 transcripts. RT-qPCR and amplicon-seq demonstrated that transcripts with RNA deletions were detectable at 12 h post-transfection, accumulated over time, peaked at 48 h, and decreased with decreasing expression of the SthCsm (FIGS. IE and IF). The loss of SthCsm expression is likely due to plasmid loss with cell division. The RNA knockdown plateaued at 36 h, but the quantity of edited transcripts rose until 48 h, suggesting an accumulation of edited transcripts that cannot be re- cleaved. The decrease of edited RNA after 48 h suggests that the accumulation is capped by the RNA half-life, which was estimated as 11.4 h for Ppib and 7.8 h for Park7 mouse transcripts.

[0031] To confirm that RNA deletions in PPIB and PARK7 are produced by RNA cleavage, we used an inactive SthCsm complex with D33A amino acid substitution in the Csm3 ribonuclease (18). Recent work demonstrated robust knockdown of nuclear transcripts (i.e., XIST, MALAT1, and NEAT 1 ) with the SthCsm complex. However, no knockdown is detected with inactive SthCsm complexes (19). To our surprise, targeting transcripts that are exported from the nucleus (i.e., PPIB and PARK7) with catalytically inactive NLS-tagged Csm complex (NLS-Csmdead) resulted in a robust knockdown that was identical to the knockdown with nuclease-active Csm complexes (NLS-Csmwt; p-value = 0.88) (Fig. 1G). Expression of the crRNA alone or crRNA together with crRNA-processing protein Cas6 resulted in far less knockdown, demonstrating that the Csmproteins are necessary for efficient knockdown. While knockdown efficiencies are similar for NLS-Csmdeadand NLS-Csmwt, only the nuclease active complex produced programmable RNA excision (Fig. 1H). Sequencing target RNA after knockdown identified the characteristic deletions (increments of six) only in cells transfected with the NLS-Csmwtplasmid.

[0032] CRISPR-guided deletion of premature stop codons in RNA

[0033] The ClinVar database (https(colon) / / www(.)ncbi(.)nlm(.)nih(.)gov / clinvar) documents more than 40,000 non-sense mutations (i.e., premature stop codons) in the human population that are detrimental to human health. We have developed type III CRISPR-based RNA editing technologies that can be used to delete premature stop codons from human transcripts and restore the expression of the encoded proteins.

[0034] FIG. 2 illustrates a schematic example of CRISPR-guided deletion of premature stop codons in RNA, according to an implementation. In FIG. 2, ribosomal subunits 202 are shown as with the nascent polypeptide chain 204. Type III CRISPR complexes are delivered to living cells (such as human cells or other subject cells) as a plasmid, mRNA, or ribonuclcoprotcin complexes with a guide RNA complementary to the site of the premature stop codon. CRISPR- complex makes multiple breaks in the target RNA 201 (in which breaks are illustrated as scissors for clarity) to cut out the section with the stop codon. Target RNA is then repaired by cellular RNA repair factors or RNA repair factors that are delivered together with the CRISPR complex. As a result of RNA repair, the stop codon is deleted, and expression of the encoded protein is restored.

[0035] To validate the proposed approach, we developed a reporter plasmid that encodes for the green fluorescent protein (GFP) fused to Flag-HA epitope at the N-terminus. Reference will be made to FIGS. 3A-3E, which illustrate various aspects of programmable deletion of stop codons, which restores expression of the encoded protein. FIG. 3A illustrates a schematic diagram of a reporter plasmid that encodes for a GFP protein. A stop codon 301 is inserted between the epitope tag and the GFP (Reporter A). The stop codon 301 (illustrated as uaa) is inserted upstream of the GFP gene in the same open reading frame such that it terminates the translation and prevents expression of the protein. Six crRNAs for SthCsm complex and seven crRNAs for DisCas7-l l were designed to excise the stop codon in the GFP gene (horizontal lines).

[0036] SthCsm crRNAs tile along the target in three-nucleotide increments, while the DisCas7- 11 crRNAs are shifted in one-nucleotide increments. Lines 305 defined by the outer-most verticalticks 305 indicate position, in respect to crRNA, of target RNA segments that are expected to be deleted. Vertical ticks 305 indicate predicted sites for RNA breaks.

[0037] Referring to FIG. 3B, cells transfected with reporter plasmids were imaged after 48 h incubation using fluorescence microscopy. After transfection of the STOP-GFP reporter plasmid in human cells (i.e., 293T cells), no fluorescence is detectable (FIG. 3B, left). A plasmid without the stop codon produces a robust GFP signal 48 h post-transfection (Fig 3B, right). To target the STOP-GFP reporter transcript, we used type III-A CRISPR from Streptococcus thermophilus (SthCsm complex) or type III-E effector from Desulfonema ishimotonii (DisCas7-l l; FIG. 3A, bottom). Transfection of the STOP-GFP reporter plasmid together with a type III-A CRISPR complex targeting the stop codon (SthCsm, guide #4) restores expression of the GFP protein (FIG. 3B, middle).

[0038] To quantify protein expression after RNA repair, we developed a bicistronic reporter plasmid that expresses GFP and firefly luciferase proteins that are linked with a viral 2A peptide (Reporter B; FIG. 3C, top). Referring to FIG. 3C, A stop codon 301 (uaa) is inserted at the 3’-cnd of the GFP gene such that it terminates the translation and prevents expression of the luciferase. Six crRNAs for SthCsm complex and seven crRNAs for DisCas7-l l were designed to target excision of the stop codon in the GFP gene (horizontal lines). SthCsm crRNAs tile along the target in three-nucleotide increments, while the DisCas7-l l crRNAs are shifted in one-nucleotide increments. In the 3’-end of the gfp gene, we inserted a stop codon, which allows GFP but not luciferase expression. To cut out the stop codon from the reporter transcript, we designed guide RNAs for SthCsm and DisCas7-l 1 that tile across stop codon in three- (SthCsm) or one-nucleotide (Cas7-l l) increments (FIG. 3C, bottom). 293T cells were transfected with the reporter plasmid and plasmids encoding for the CRISPR proteins and crRNAs. After 48-hour incubation, we lysed the transfected cells, measured luciferase activity, and normalized it to the luciferase activity in cells that received a reporter plasmid without the stop codon. As a baseline, we measured luciferase activity in cells transfected with a non-targeting crRNA gene, which was 0.50 + 0.06% in cells with SthCsm, 0.22 + 0.03% in cells transfected with Cas7-l l. In cells expressing SthCsm, we detected luciferase activity with five of the six tested crRNAs, which ranged from 2.0 + 0.2 % with crRNA 3 to 5.3 + 0.5% with crRNA 1 (FIG. 3D, top). With DisCas7-l l, only one of the seven tested crRNAs resulted in a statistically significant increase of the luciferase signal (0.9 + 0.2 %; p-value < 0.001), which was less efficient compared to SthCsm (FIG. 3D, bottom).Sequencing of the target RNA confirms that samples with rescued luciferase activity contain RNA deletions that eliminate the stop codon (FIG. 3E).

[0039] Lines 303 indicate position, in respect to crRNA, of target RNA segments that are expected to be deleted. Vertical ticks 305 indicate predicted sites for RNA breaks. Referring to FIG. 3D, HEK293T cells were transfected with reporter plasmid and plasmids encoding for crRNAs shown in FIG. C and SthCsm or DisCas7-l 1 proteins. Luciferase activity was measured in cell lysates 48 hours post transfection and normalized to a control transfected with reporter plasmid without stop codon and plasmids encoding for non-targeting CRISPR complexes.

[0040] Data are shown as mean + one standard deviation of three replicates. Means were compared using one-way ANOVA and samples that received targeting crRNA were compared to the nontargeting control using one-sided post hoc Dunnett’s test. * p < 0.5, ** p < 0.1, *** p < 0.001. E) Most frequent RNA editing outcomes at the target site in the reporter transcript for crRNAs that mediated the most efficient recovery of luciferase activity in (FIG. 3D). Dotted lines indicate the positions of RNA cuts introduced with the CRISPR complex. Hyphens (dashed line in sequence) depict deletions identified in the sequencing data. Editing efficiency was quantified as mean + one standard deviation of three biological replicates. Black box shows the stop codon that was targeted by type III CRISPR complexes.

[0041] Engineering CRISPR complexes to improve RNA editing efficiency and allow for edits other than RNA deletions

[0042] FIG. 4 illustrates a schematic example of engineering type III ribonucleoproteins for RNA editing, according to an implementation. Shown are approaches for improving the efficiency of RNA editing, which include the use of oligonucleotide splints that facilitate RNA ligation and fusing various RNA effectors to type III CRISPR ribonucleoproteins. Oligonucleotide splints can be co-delivered in trans (top) or in cis by linking to the guide RNA. Possible linkages might include covalent bonding or hybridization to complementary region included in the guide RNA. RNA repair proteins can be delivered to target RNA as fusions to CRISPR-associated proteins or through interactions between RNA aptamer in the guide RNA and corresponding protein fused to the RNA repair factor (e.g., MS2 aptamer and MS2 coat protein).

[0043] Hybridization of complementary oligonucleotide splints to RNA fragments facilitates the efficiency of RNA ligation. To facilitate the RNA repair in human cells, we propose to co-deliver oligonucleotide splints (RNA, DNA, chimeric or modified oligonucleotides) complementary tothe programmable RNA break sites in trans together with CRISPR complexes or in cis by fusing the splint(s) to the CRISPR complex. In one implementation, the splint is fused to the 3 ’-end of the guide RNA (as illustrated in FIG. 4), but numerous other tethering strategies can be used to link one or more splints to a single type III complex. Cis delivery of the splint is expected to increasing the local concentration of the splint and thereby facilitate the hybridization between the splint and the cleaved RNA fragments. Then, RNA breaks are repaired by RNA repair proteins.

[0044] RNA repair proteins might include endogenous RNA repair factors (e.g., RTCB ligase) that are naturally recruited to the RNA breaks or exogenous RNA repair effectors that are codelivered as fusions to the protein subunits of the CRISPR ribonucleoprotein (RNP) or recruited through an interaction between RNA aptamer in the guide RNA (e.g., MS2 aptamer) and protein fused to the RNA repair effector (e.g., MS2 coat protein). The exogenous RNA repair effectors include but are not limited to various orthologues of RNA ligases found in prokaryotes and eukaryotes, RNA end modifying enzymes, nucleotidyltransferases (NTases), RNA binding proteins for end protection, and other factors. NTases can be used for non-tcmplatcd addition of nucleotides to the ends of the RNA break (5’- or 3 ’-fragment) or addition of nucleotides, which is programmed by the template sequence in the oligonucleotide splint. Use of NTase is expected to allow templated or non-templated insertions in RNA at the RNA break site.

[0045] Inactivation of the polymerase and nuclease domains in the CaslO subunit of type III CRISPR complex reduces toxicity and improves protein yield.

[0046] FIG. 5 illustrates a schematic example of inactivation of enzymatic activities of the CaslO subunit, which reduces toxicity and improves protein yields, according to an implementation. FIG. 5A illustrates a plot of results of size-exclusion chromatography (SEC) of affinity purified wildtype SthCsm. FIG. 5B illustrates a plot of SthCsm complex containing a mutated CaslO HD- nuclease (D16 to A16, CaslOHD) and mutated polymerase (D575 and D576 to A575 and A576, CaslOPalm) active sites. FIG. 5C illustrates SDS-PAGE of purified SthCsm complexes in FIGS. 5A and 5B (~10 ug / lane). Referring to FIG. 5D, SDS-PAGE of RNAs were extracted from SthCsm complexes and were separated in 12% UREA-PAGE. Referring to FIG. 5E, protein yields and growth rates of BL21(DE3) cells transformed with expression plasmids for wildtype or mutated SthCsm complexes.

[0047] The CaslO subunit of some type III CRISPR complexes have polymerase (Palm) and / or nuclease (HD) domains that are activated upon binding to the target RNA. The former activitygenerates signal molecules that activate diverse ancillary effectors, while the latter was hypothesized to cleave DNA at the transcription bubble as the target transcript is being synthesized. Recent work demonstrates that activities of the CaslO homolog, Cmr2a, in type III- B CRISPR system can be toxic to a prokaryotic cell. The E. coli cells transformed with plasmids expressing SthCsm complex take -12 hrs to reach the mid-log growth phase (OD600 -0.5-0.6), and protein yields are low.

[0048] Mutations in either HD or Palm domains improved protein yields and bacterial growth (-8 and -10 hrs to mid- log phase, respectively). Combining mutations in the HD and Palm results in even faster growth (-4 hrs to mid-log) and better yields. These data indicate that both Palm and HD domains of CaslO play a role in the toxicity of the SthCsm-complex in E. coli cells, and mutations inactivating the nuclease (HD) and polymerase domains (Palm) of CaslO dramatically improve protein yields.

[0049] To prevent any CaslO-associated toxicity in human cells, polymerase and nuclease activities arc inactivated by mutating catalytic residues in the corresponding enzymatic sites of the protein (Palm and HD domains). Further, these mutations improve protein yields in bacteria and reduce production times, which is advantageous for applications that will require the delivery of type III complexes as purified RNP complexes rather than genetic vectors, such as plasmids, viruses, or purified mRNA.

[0050] RTCB ligase repairs type III CRISPR-mediated RNA breaks

[0051] FIGS. 6 A- J illustrate various aspects of RTCB ligase repair of RNA cleaved by the type III CRISPR complex, according to an implementation. Referring to FIG. 6A, human RNA ligase RTCB joins RNA ends that are produced in tRNA splicing (top) and non-canonical XBP1 mRNA splicing (bottom) during the unfolded protein response. We hypothesized that the ligase activity of RTCB is involved in the RNA repair of CRISPR-guided RNA breaks (middle). Referring to FIG. 6B, Western blot with anti-RTCB or anti-ACTB (loading control) antibodies was performed with lysates of 293T cells with (+) or without (-) RTCB depletion. See the uncropped images in fig. S3. Referring to FIG. 6C, the PARK7 transcript was targeted with SthCsm (guide 2) in 293T cells with (+) or without (-) RTCB depletion. The PARK7 transcript was quantified with RT-qPCR and normalized to ACTB and non-targeting guide RNA. Data are shown as the mean ± standard deviation of three biological replicates. *p-value < 0.05; Welch’s t-test. Referring to FIG. 6D, qPCR products were sequenced, and resulting reads were aligned to the reference sequence of thePARK7 transcript (NM_007262, GenBank). Graphs show sequencing depth (y-axes) at the amplified region of the transcript (x-axes). Every line shows a biological replicate (n = 3). The horizontal black bar indicates a region complementary to the guide RNA of the SthCsm complex. Vertical dotted lines mark predicted positions of RNA breaks. FIG. 6E illustrates quantification of deletions in the target region of the PARK7 transcript. Data is shown as the mean ± standard deviation of three biological replicates. Welch’s t-test was used to compare mean values. *** p < 0.001. Referring to FIG. 6F, RTCB deficient cells were transfected with a plasmid expressing RTCB (pCMV-RTCB), and the efficiency of CRISPR RNA-guided programmable deletions in PARK7 was quantified as in panels C-E. * p < 0.05; Welch’s t-test. FIG. 6G shows immuno staining of cells expressing Flag-tagged Csm complex with (NLS-Csm) or without (Csm) NLS-tag. Scale bars are 10 pm. Referring to FIGS. 6H-J, knockdown of PARK7 and XIST transcripts with cytoplasmic SthCsm (no NLS) (FIGS. 6H and 61) and knockdown of XIST with nuclear Csm (NLS-tagged) (FIG. 61) was quantified with RT-qPCR and normalized to ACT and non-targeting control, “wt” - nuclease-active SthCsm, “dead”- catalytically inactivated SthCsm (Csm3D33A mutation). *p-value < 0.05, ns - non- significant; Welch’s t-test. Data is shown as mean ± SD (n = 3).

[0052] RNA cleavage by type III CRISPR complexes leaves a 2’,3’-cyclic phosphate (2’,3’>P) and a 5 ’-hydroxyl (5 ’-OH) at each cut site. Mammalian RNA ligase RTCB joins 2',3'>P and 5'- OH termini when introns are excised during tRNA splicing and non-conventional splicing of XBP1 mRNA in the unfolded protein response (FIG. 6A). To test if RTCB ligase repairs transcripts cleaved by SthCsm in human cells, we used Cas9 nuclease to knockdown RTCB in 293T cells, and targeted PARK7 transcript in cells with or without the RTCB depletion (FIGS. 6B and 6C). In wildtype 293T cells, 2.3 ± 0.1% of the target RNA was detected after transfection with SthCsm plasmid, while target RNA was ~1.5- fold lower (1.5 ± 0.3%; p-value = 0.029) in RTCB-depleted cells.

[0053] Deep-sequencing of PARK7 amplicons showed that the proportion of reads with deletions is significantly decreased in cells with depleted RTCB (FIG. 6D). In samples with wildtype levels of RTCB, deletions in target sequence were detected in 59.7 ± 5.1% of the reads, while the frequency of these deletions was 24.4 ± 4.8% (p-value < 0.001) in cells with depleted RTCB (FIG. 6E). Complementation of RTCB with a plasmid encoding RTCB cDNA increased the abundance of ligated PARK7 transcript - 1.5-fold compared to an untransfected control (FIG. 6F; p-value =0.02). While the complementation of RNA repair was not complete (-76%), RTCB activity may be affected by the transfection efficiency and non-physiological expression levels.

[0054] Overall, the reduction of RNA repair with knockdown and complementation with RTCB overexpression support the role of RTCB in the repair of CRISPR-guided RNA breaks. To determine if repair of CRISPR-guided RNA breaks happens in the nucleus or cytoplasm, we removed the NLS tags from the SthCsm complex and targeted nuclear (XIST) or exported (PPIB and PARK7) transcripts. SthCsm complexes without the NLS tag localized in the cytoplasm (FIG. 6G) and depleted PPIB (31.2 ± 6.2% knockdown, p-value = 0.023) and PARK7 (41.9 ± 2.1% knockdown, p-value = 0.017) (FIG. 6H). However, RNA retained in the nucleus (i.e., XIST) was not depleted by the untagged Csm complex (p-value = 0.47) (FIG. 61). Deep sequencing detected programmable deletions in 1.4 ± 0.2% of PPIB and 9.1 ± 0.9% of PARK7 reads, demonstrating that transcripts cleaved in the cytoplasm are repaired in the cytoplasm (FIGS. 6C-6E). Knockdown of PPIB and PARK7 with cytoplasmic Csm complex was less efficient (-30-40%) than with NLS- taggcd Csm complex (>90%). However, this docs not mean that RNA cleavage in the cytoplasm is less efficient. In this experiment, we extracted total cellular RNA that contained both cytoplasmic RNA and nascent RNA from the nucleus. The latter is inaccessible to the cytoplasmic Csm complex and contributed to the qPCR signal.

[0055] NLS-tagged Csm complex localized in the nucleus (FIG. 6G) and knocked down 42.4 ± 11.9% of the nuclear XIST transcript (p-value = 0.009; FIG. 61). Deep sequencing of the XIST amplicons identified site-specific deletions, indicating that cleaved XIST RNA is repaired in the nucleus. Collectively, these results demonstrate that RNA repair happens both in the nucleus and in the cytoplasm of human cells.

[0056] We hypothesize that human transcripts bound by inactive SthCsm complex (-350 kDa) fail RNA quality control during nuclear export or that the bound complex blocks translation in the cytoplasm, which results in the decay of the target RNA. In agreement with the latter possibility, targeting PPIB and PARK7 in the cytoplasm with nuclease-inactive SthCsm (Csmdead) resulted in significant RNA knockdown (FIG. 6H), but targeting nuclear transcript XIST with NLS-Csmdeaddid not change its relative quantity (FIG. 6J, p-value = 0.26).

[0057] Multiple RNA breaks produce large RNA excisions

[0058] FIGS. 7A-D illustrate various aspects of repair of concurrent RNA breaks, resulting in large RNA excisions, according to an implementation. Referring to FIG. 7A, repetitive region(repeat A) in XIST transcript was targeted with SthCsm complex with four different guide RNAs. Knockdown of XIST was quantified with RT-qPCR. Data is shown as the mean of three biological replicates ± SD. Referring to FIG. 7B, Amplicon- seq was used to quantify programmed RNA deletions in XIST. Referring to FIG. 7C, repeat A was amplified and deep- sequenced. Reads were aligned to the reference sequence (NR_001564.2, GenBank). Graphs show sequencing depth (y- axes) at the amplified region of the transcript (x-axes). Every line shows a biological replicate (n = 3). Vertical light gray rectangles indicate the position of repeats targeted by Csm complexes. Referring to FIG. 7D, repeat A architecture in XIST IncRNA. Lines show binding sites for Csm complexes.

[0059] Human XIST is a long non-coding RNA that contains eight copies of a repetitive sequence (repeat A) required for XIST-mediated inactivation of the X-chromosome (29, 30). Previous work has targeted repeat A with GFP-fused nuclease-inactive SthCsm complex for live imaging of XIST RNA. We hypothesized that targeting repeat A in XIST with catalytically active SthCsm complexes will result in simultaneous cleavage at multiple locations, and repair of these cleavages will generate large deletions. We tested four different guide RNAs that target repeats in XIST (FIGS 7A and 7B). Sequencing of amplicons that span the repetitive region identified the characteristic 6, 12, and 18 nucleotide deletions but also large deletions consistent with multiple cleavages at the repeated sequence (FIGS. 7C and 7D). These data indicate that simultaneous cleavages at multiple locations within the same transcript can be used for large RNA deletions.

[0060] Programmable excision of premature stop codons in RNA restores protein expression

[0061] FIGS. 8A-I illustrate various aspects of programmable excision of stop codons, which restores protein expression, according to an implementation.

[0062] FIG. 8A illustrates a schematic representation of the proposed approach for deleting premature stop codons in human transcripts. The ClinVar database documents more than 40,000 non-sense mutations (i.e., stop codons) in the human population. We hypothesized that break-and- repair RNA editing can be used to delete premature stop codons and restore the expression of the encoded proteins (FIG. 8A). To test this, we developed a reporter plasmid that encodes for GFP fused to an N-terminal flag-HA epitope. Between the epitope and the gfp sequence, we inserted a stop codon to terminate translation (stop-GFP; FIG. 8B).

[0063] FIG. 8B illustrates a schematic diagram of the stop-GFP reporter plasmid and six guide RNAs for SthCsm complex that were designed to excise the stop codon 801 in the gfp transcript.

[0064] Referring to FIG. 8C, cells were transfected with plasmids for the stop-GFP reporter and SthCsm with a non-targeting guide (left), stop-GFP reporter and SthCsm with a targeting guide (middle), or GFP reporter and SthCsm with the non-targeting guide (right). Fluorescence microscopy was used to image cells 48 h post-transfection. Scale bars - 50 m. Transfection of the stop-GFP plasmid in 293T cells produced no detectable fluorescence. A plasmid with the same sequence but no stop codon produced a robust GFP signal 48 h post-transfection (FIG. 8C). To cut out the stop codon, we designed six guides for SthCsm complex that tile across the target RNA sequence in three-nucleotide increments (FIG. 8B). Transfection of the stop-GFP plasmid together with three of the six SthCsm complexes (crRNA 3, 4, and 6) partially restored expression of the GFP (FIG. 8C). To confirm that transcripts edited with SthCsm produce functional GFP, we used site-directed mutagenesis to make the same mutations in the stop-GFP reporter plasmid. Transfection of plasmids containing the 12 or 18 nt deletions resulted in the same levels of fluorescence as transfection of the positive control plasmid (fig. S5D).

[0065] To quantify the rescue of reporter expression, we developed a bicistronic plasmid that expresses GFP and firefly luciferase linked with a viral 2A peptide. At the 3’-end of the gfp gene, we inserted a stop codon, which permits GFP, but not luciferase expression (GFP-stop-Luc).

[0066] To cut out the stop codon, we designed six SthCsm guide RNAs that tile across the stop codon (FIG. 8D). FIG. 8D illustrates a schematic diagram of the GFP-stop-Luc reporter plasmid and six crRNAs for SthCsm complex that were designed to delete the stop codon at the 3 ’-end of the gfp gene. In cells expressing SthCsm complexes that target the stop codon, we detected luciferase activity with five of the six tested crRNAs, which ranged from 2.0 ± 0.2 % with crRNA 3 to 5.3 ± 0.5% with crRNA 1 (FIG. 8E). Underlined (dashed) sections of target RNA are expected to be deleted. Vertical lines through the sequence indicate predicted sites for RNA breaks. Sequencing of the target RNA amplicons confirmed that samples with rescued luciferase activity contain RNA deletions eliminating the stop codon (FIG. 8F).

[0067] Referring to FIG. 8E, Luciferase activity was measured in cell lysates 48 hours after transfection with GFP-stop-Luc and SthCsm plasmids. Luciferase activity is normalized to a control transfected with a reporter plasmid without the stop codon. Data are shown as mean ± SD of three replicates. Means were compared using one-way ANOVA, and samples with targeting guide RNAs were compared to the non-targeting control using one-tailed Dunnett’s test. * p < 0.5,** p < 0.1, *** p < 0.001.

[0068] Referring to FIG. 8F, most frequent RNA editing outcomes in the sample with the most efficient rescue of luciferase activity in (shown in FIG. 8B) (guide 1). Editing efficiency was quantified as mean ± SD of three biological replicates. The black box (“uaa”) shows the stop codon that was targeted by type III CRISPR complexes (FIGS. 8G-I). The same as (FIGS. 8D-F), but with eDisCas7-l 1.

[0069] Type III-E CRISPR systems encode single polyprotein effectors that process CRISPR RNA and cleave the target RNA at two sites six nucleotides apart. These effectors are simple and compact and have the potential to produce a single editing outcome, which makes them attractive for use in RNA editing. We expressed type III-E effector from Desulfonema ishitnolonii fused to the NLS tag (DisCas7-l 1) in 293T cells with seven guide RNAs tiled across the stop codon of the GFP-stop-Luc reporter (FIG. 8G). Only crRNA 7 resulted in a significant increase of the luciferase signal (0.9 ± 0.2%; p-value < 0.001), which was less efficient than SthCsm. In addition to editing, we quantified knockdown efficiencies of the target RNA using RT-qPCR. The RNA knockdown efficiency with SthCsm ranged from 36.8 ± 7.2 % to 54.0 ± 2.8 %, while knockdowns with DisCas7-l 1 were less than half as efficient (12.2 ± 5.9% with guide 6 and 16.5 ± 11.2% with guide 7) (fig. S5I). Analysis of sequenced amplicons identified six nucleotide deletions in 0.04 ± 0.01% of the reads at the site targeted with DisCas7-l 1 (fig. S5H). An enhanced version of the DisCas7- 11 complex (eDisCas7-l 1) has been recently engineered for CRISPR-assisted trans-splicing by adding positively charged amino acids to regions of the protein that are physically close to the bound target RNA (33). Targeting the GFP-stop-Luc reporter with eDisCas7-l 1 resulted in a more efficient knockdown (52.3 ± 3.8% with guide 6 and 47.7 ± 7.8% with guide 7; fig. S5I), rescue of 0.9 ± 0.1% of luciferase activity (p-value = 0.003; FIG. 8H), and programmed RNA excisions with 0.29 ± 0.03% efficiency (FIG. 81).

[0070] Programmed RNA excision with DisCas7-l l and eDisCas7-l l is less efficient compared to targeting with SthCsm. The low frequency of deletions with DisCas7-l l is consistent with the inefficiency of the DisCas7-l l nuclease in biochemical assays and the low frequency of two simultaneous breaks in the target RNA. Collectively, these data demonstrate the potential of type III-E effectors to produce a single RNA editing outcome, but further engineering of the Cas7-l l nuclease will be important to repurpose this enzyme for RNA excision.

[0071] Programmable excision of non-sense mutation in the CFTR transcript restores translation

[0072] FIGS. 9A-F illustrate various aspects of programmable excision of a non-sense mutation in the CFTR transcript, which restores translation, according to an implementation. FIG. 9A illustrates a schematic diagram of an example of a luciferase-based reporter for CFTRW1282X mutation (ILuc-CFTR, top) and guide RNA design (bottom). Firefly luciferase was genetically linked to exons 22-27 of the CFTR cDNA, and synthetic intron sequence was inserted between exons 24 and 25. Eight crRNAs (numbered 1-8) were tiled across the mutation (W 1282X) to guide the excision of the stop codon (UGA). Referring to FIG. 9B, RT-qPCR was used to quantify fLuc- CFTR transcript targeted with Csm complexes. Amplicons were deep- sequenced to quantify edited vs. unedited reporter RNA. Referring to FIG. 9C, quantification of deletions that remove stop codon (W1282X). Referring to FIG. 9D, Western blot with antibodies against CFTR amino acid residues 1204-1211 with lysates from 293T cells expressing fLuc- CFTR or fLuc-CFTRW1282X) and Csm complexes with non-targeting (nt) guide RNA or CFTR- targeting guide RNA 5.

[0073] Referring to FIG. 9E, quantification of luciferase activity in 293T cells transfected with fLuc-CFTRW1282X and Csm complexes with non-targeting guide RNA or targeting guide RNA 5. Middle bar shows mean of three biological replicates 901. Error bars show mean ± SD. *p<0.05, Welch’s t-test. (F) Left: RT-qPCR was used to quantify CFTR transcript in HBE16ge CFTRW1282X cells transfected with plasmids encoding for Csm complexes with non-targeting guide RNA (nt) or CFTR-targeting guide RNA 5. Right: qPCR amplicons were deep-sequenced, and deletions removing W1282X codon were quantified. Data is shown as the mean of three biological replicates ± SD. *p<0.05, Welch’s t-test.

[0074] After testing fluorescent and luminescent reporters, we sought to determine the therapeutic potential of CRISPR-guided RNA excision by targeting a clinically relevant nonsense mutation (c.3846G>A; W1282X) in the CFTR mRNA. The truncated CFTRW1282X protein is partially functional, but the premature stop codon triggers nonsense-mediated decay (NMD) of the CFTRW1282X mRNA in the cytoplasm. The decay of CFTR mRNA results in insufficient levels of CFTR protein, leading to the development of cystic fibrosis. We hypothesized that targeting the CFTRW1282X transcript with NLS-tagged SthCsm would remove the W1282X stop codon in the nucleus, and the repaired RNA would escape NMD in the cytoplasm, rescuing protein expression and potentially rescuing disease phenotype.

[0075] To screen for crRNAs that guide efficient excision of W 1282X in the CFTR, we created a luciferase-based NMD-reporter plasmid building off previously published designs (FIG. 9A). Wescreened eight crRNAs and identified one (crRNA 5) that guides the most efficient removal of the premature termination codon in the fLuc-CFTRW1282X reporter (FIGS. 9B and 9C). The cotransfection of fLuc-CFTRW1282X and a plasmid encoding for Csm complex with crRNA 5 restored the C-terminus of CFTR protein compared to the cells that received non-targeting Csm complex (FIG. 9D). In addition, expression of the Csm complex and crRNA 5 increased luciferase activity by 11.1% (p-value = 0.024) compared to the non-targeting control, indicating escape from the NMD (FIG. 9E).

[0076] Finally, we used immortalized human bronchial epithelium cells with the CFTRW1282X allele (16HBEge CFTRW1282X) to test if CRISPR-guided RNA excision can remove the W1282X codon in the endogenous CFTR transcript. RT-qPCR and amplicon-sequencing identified editing of 3.9 ± 0.8% CFTR transcripts (FIG. 9F). Future studies will be needed to measure the impact of this correction on cellular physiology and determine the RNA editing levels required for therapeutic outcomes.

[0077] Precise DNA manipulation has been enabled by breaking DNA at sequence-specific locations and repairing the resulting fragments. In a previous study, we applied the concept of break-and-repair to engineer viral RNA genomes in vitro. Here, we extend this approach to make RNA deletions in human cells, which is facilitated by the repair of programmable RNA breaks made using type III CRISPR complexes.

[0078] Deletion of toxic mutants, complemented by fractional repair, could be therapeutic. Alternatively, premature stop codons that trigger NMD result in low to no protein (e.g. CFTRW1282X mutation), and excision of premature stop codons could rescue protein expression. Other potential applications of site-specific RNA excision include editing non-coding RNAs, removing disease-causing trinucleotide expansions, or correcting intronic mutations that dysregulate splicing.

[0079] Repair of RNAs is understudied, and the biological significance of this process is often dismissed due to the high turnover of RNA in the cell. We hypothesize that end- modifying enzymes, additional ligases, nucleases (e.g., exosome complex), and other RNA processing factors regulate this process, and tuning RNA processing has the potential to improve the efficiency of RNA editing. We have established reporter assays for RNA repair, which enable the discovery of new RNA repair pathways and rapid screening for conditions that favor RNA repair to improve the efficiency of RNA editing.

[0080] We show that Csm-mediated knockdown of cytoplasmic transcripts does not require target RNA cleavage (FIGS. 1G and 6H). However, cleavage is required for site-specific RNA deletion (Fig. 1H). This observation suggests that the efficiency of RNA repair is an underestimate because most of the target RNA is degraded by RNA quality control pathways before it can be repaired.

[0081] Single-molecule studies of enzymatic activities of the Csm complex from Streptococcus thermophilus (used in this work) demonstrate that cleavage happens within seconds, but then the complex retains cleavage products for over ~80 minutes. These data suggest that RNA release, rather than cleavage or ligation, may be the rate-limiting step in RNA editing.

[0082] The kinetics of Csm complex dissociation is on the same timescale as mRNA transport to the cytoplasm (10-30 min for P-actin mRNA), and it is possible that the Csm complex stalls ribosomes and triggers RNA decay before it releases target RNA for repair. These observations suggest that programmable ribonucleases that cleave and release the target quickly or cleave RNA outside of the bound sequence (i.e., Casl3) will enable more efficient editing.

[0083] Orthologs of Cas9 and Casl2 cleave complementary RNA targets, enabling programmable RNA manipulation with single-subunit effectors. RNA cleavage by Cas9 or Casl2 likely produces a 5 ’-phosphate and a 3 ’-hydroxyl, similar to DNA cleavage. These ends are not compatible with RTCB-mediated ligation, but other cellular RNA repair pathways may enable Cas9- and Casl2- mediated RNA editing.

[0084] Unlike DNA editing, which often depends on adjacent sequence motifs (i.e., PAM), type III CRISPR systems only require complementarity between the RNA guide and the RNA target, which improves target site versatility. The frequency and distribution of RNA repair outcomes varied across RNA targets that we tested, which may suggest that local sequence context, secondary structure, RNA modifications, or other factors may influence the efficiency of target binding, target cleavage, or repair. Screens like those performed for Casl3 will help establish predictive models to enhance the efficiencies of RNA editing and advance this technology for therapeutic applications.

[0085] The initial efficiency of editing with Cas9 was -3-30% for indels and -0.5-0.7% efficiency for homology-directed repair. A decade later, Cas9-based technologies are precise, efficient, versatile, and clinically relevant. Here, we demonstrate programmable break- and-repair editing of RNA, which reveals an important aspect of RNA biology that has not been explored. We anticipatethat efficiencies will improve and that these technologies will lead to new insights about the RNA damage response and enable discoveries of previously unrecognized mechanisms of RNA repair.

[0086] PROGRAMMABLE RNA EDITING WITH CATALYTIC RNA

[0087] CRISPR-Cas9 technologies have enabled targeted DNA modification. Cas9 endonuclease directed by an RNA guide makes sequence- specific breaks in the DNA of a living cell, which are resolved by cellular DNA repair pathways resulting in DNA modifications. Although DNA editing has the potential to cure genetic diseases, it can cause unintended changes to the genome and activate cellular stress responses that result in toxicity. Edits to the RNA can be used to modify genetic information in human cells without introducing heritable modifications to the DNA genome and toxicity associated with DNA breaks. We have demonstrated that RNA fragments produced by cleavage with RNA-targeting type III CRISPR complexes are repaired in human cells and that this process can be applied to make programmable RNA excisions that restore gene function.

[0088] RNA editing is transient due to the RNA turnover in the cell and needs to be administered repeatedly for a durable effect. Delivery of CRISPR ribonucleoprotein complexes into human cells is challenging due to the cargo size constraints, and the need for repeated dosing limits current applications because of the adaptive immune responses to the delivery vehicle (e.g., adeno- associated virus) and the therapeutic CRISPR protein.

[0089] To address the need for RNA editing therapeutics that are easy to deliver, have durable effect, and have low immunogenicity, we propose a technology that uses programmable catalytic RNAs (i.e., ribozymes) with two active sites.

[0090] FIG. 10 illustrates a schematic example of programmable RNA editing with catalytic RNA, according to an implementation. Section A illustrates a schematic diagram of a programmable catalytic RNA with two enzymatic sites. The catalytic RNA is programmed by adding sequences complementary to the target transcript at the 5 ’-end of the catalytic site 1, between site 1 and site 2, and at the 3 ’-end of site 2. The catalytic RNA can be expressed in human cells from DNA vector, e.g., plasmid or AAV, or directly delivered as synthetic RNA. In the cell, catalytic RNA hybridizes to the complementary transcript, which activates the trans-cleavage of the target by the ribozymes. Catalytic RNAs that are used in the technology include hammerhead, twister, or hairpin ribozymes or any other catalytic RNAs with enzymatic activities, including but not limited to RNA cleavage, RNA polymerization, and RNA ligation. The tertiary structure of the active sites is reconstitutedupon binding to complementary RNA, which activates enzymatic activities, such as RNA cleavage.

[0091] Referring to section B, after catalytic RNA is activated, the RNA sequence between the active sites is cut out (‘excised sequence’). Cleavage produces 5’-hydroxyl (OH) and 2’,3’-cyclic phosphate (P<) at the termini of the RNA breaks. Referring to Section C, catalytic RNA bridges two RNA fragments, guiding the cellular repair to achieve the desired outcome, e.g., programmable RNA excision. Additional enzymatic activities can be recruited to the target RNA to insert or replace sequences at the target location. Enzymatic activity of the programmable catalytic RNA creates two RNA breaks (or possibly more in other derived designs, e.g., triple ribozyme) that excise the intervening RNA sequence. The RNA sequence between the breaks dissociates, and the catalytic RNA serves as a splint that holds 5’ and 3 ’-fragments of cleave RNA target for repair. Cellular RNA repair mechanisms ligate the RNA fragments together, creating a programmable RNA excision at the target site. Additional enzymatic activities could be targeted to the RNA fragments splinted by the catalytic RNA to insert sequences or replace sequences between the RNA breaks. In some instances, this can be achieved by using catalytic RNA as a template for polymerases that fill in the portion of the target RNA between the RNA breaks (Fig. 10, Section C).

[0092] We anticipate treatments with catalytic RNAs targeting human transcripts containing disease- causing sequences will be therapeutic. The protein-less RNA editors will have low immunogenicity and high durability and could be delivered using the same methods as for smallinterfering RNAs (siRNAs), such as lipid nanoparticles, GalNAc conjugates, viral vectors, etc., enabling the treatment of a wide variety of genetic diseases and enhancing human health.

[0093] We have demonstrated that RNA fragments with a 2’, 3 '-cyclic phosphate and 5 '-hydroxyl termini are repaired in human cells. The same end modifications may be added to synthetic RNA fragments that, upon delivery into the cell, will be repaired and combined into a single sequence. Additional modifications on RNA ends can control the directionality and correct assembly order. Potential cargoes include but are not limited to mRNA vaccines, human mRNAs for protein replacement therapies, human mRNA with health benefits, long-noncoding RNAs, or any other type of therapeutic RNA sequences. The delivery of RNA into cells using lipid nanoparticles has therapeutic potential for treating genetic diseases, developing viral and cancer vaccines, and protein replacement therapies. However, current applications arc limited by the length of RNAsequences that can be efficiently synthesized, packaged, and delivered. Liposomes with a size of less than 200 nm show increased circulation time in the blood and enhanced cellular uptake. Splitting long RNA sequences into shorter fragments that are co-delivered and assembled inside the cell could be beneficial and enable more robust delivery of large RNA cargo. To facilitate RNA ligation within a cell, we propose co-delivering splint DNA or RNA that is complementary to RNA fragments and guides the correct assembly through cellular RNA repair.

[0094] FIG. 11 illustrates a comparison of two methods to deliver RNA in a cell, according to an implementation. Upon the release, RNA is transcribed into a therapeutic protein. On the right, RNA is split into fragments that are modified to install 2’,3'-cyclic phosphate at the terminus of one fragment and 5 '-hydroxyl on the other fragment. Split RNA fragments are delivered and repaired in the cell to reconstitute the full-length transcript that serves as a template to produce therapeutic protein. In some aspects, the techniques described herein relate to an engineered system for editing target ribonucleic acid (RNA) molecules in a living cell including: an RNA guide having a sequence engineered to be complementary to target RNA molecules in the living cell; and an RNA-guided ribonuclease that introduces one or more breaks in a plurality of the target RNA molecules, wherein at least some of the plurality of the target RNA molecules are to be repaired by the living cell, resulting in one or more edited RNA molecules in the living cell based on the one or more breaks and subsequent repair by the living cell.

[0095] In some aspects, the techniques described herein relate to an engineered system, wherein the RNA-guided ribonuclease includes a type III Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas complex.

[0096] In some aspects, the techniques described herein relate to an engineered system, wherein the type III CRISPR-Cas complex includes a type III-A CRISPR-Cas complex.

[0097] In some aspects, the techniques described herein relate to an engineered system, wherein the type III-A CRISPR-Cas complex is derived from Streptococcus thermophilus.

[0098] In some aspects, the techniques described herein relate to an engineered system, further including: a CRISPR RNA (crRNA), a Csm2 protein, a Csm3 protein, a Csm4 protein, a Csm5 protein, and / or CaslO.

[0099] In some aspects, the techniques described herein relate to an engineered system, further including: a Cas6 protein.

[0100] In some aspects, the techniques described herein relate to an engineered system, further including: a Cas6 protein.

[0101] In some aspects, the techniques described herein relate to an engineered system, wherein the type III CRISPR-Cas complex includes a type III-E CRISPR-Cas complex.

[0102] In some aspects, the techniques described herein relate to an engineered system, wherein the type III-E CRISPR-Cas complex is derived from Desulfonemea ishimotonii.

[0103] In some aspects, the techniques described herein relate to an engineered system, wherein the target RNA molecules are each an RNA transcript associated with a disease.

[0104] In some aspects, the techniques described herein relate to an engineered system, wherein the RNA-guided ribonuclease includes a type III CRISPR-Cas complex that is engineered to have a programmable cut-and-repair RNA editing strategy.

[0105] In some aspects, the techniques described herein relate to an engineered system, wherein the target RNA molecules are each associated with a premature stop codon that prevents expression of an associated protein that is suspected to or actually results in the disease, and wherein the programmable cut-and-repair RNA editing strategy is configured to excise the premature stop codon from the RNA transcript to restore expression of the associated protein.

[0106] In some aspects, the techniques described herein relate to an engineered system, wherein to excise the premature stop codon, the RNA guide is engineered to tile across the premature stop codon either alone or with one or more other RNA guides.

[0107] In some aspects, the techniques described herein relate to an engineered system, wherein the programmable cut-and-repair RNA editing strategy includes a programmable RNA cleavage, a programmable RNA deletion, a programmable RNA insertion, and / or a programmable RNA excision.

[0108] In some aspects, the techniques described herein relate to an engineered system, wherein the programmable cut-and-repair RNA editing strategy is configured to generate one or more deletions in the plurality of the target RNA molecules.

[0109] In some aspects, the techniques described herein relate to an engineered system, wherein one or more deletions include a deletion of greater than 100 nucleotides from the plurality of the target RNA molecules.[HO] In some aspects, the techniques described herein relate to an engineered system, further including: an oligonucleotide splint engineered to be complementary to RNA ends, from amongthe plurality of the target RNA molecules, associated with cleavage by RNA-guided ribonuclease, wherein the oligonucleotide splint is to be inserted into the target RNA when the target RNA is repaired by one or more RNA repair proteins.[HU In some aspects, the techniques described herein relate to an engineered system, wherein the RNA-guided ribonuclease includes a type III CRISPR-Cas complex.

[0112] In some aspects, the techniques described herein relate to an engineered system, wherein the oligonucleotide splint is to be delivered in trans with the type III CRISPR-Cas complex.

[0113] In some aspects, the techniques described herein relate to an engineered system, wherein the oligonucleotide splint is fused to the RNA guide and is to be delivered in cis as part of the type III CRISPR-Cas complex.

[0114] In some aspects, the techniques described herein relate to an engineered system, wherein the oligonucleotide splint includes RNA, DNA, chimeric and / or modified oligonucleotides

[0115] In some aspects, the techniques described herein relate to an engineered system, wherein the one or more RNA repair proteins arc endogenous to the living cell.

[0116] In some aspects, the techniques described herein relate to an engineered system, wherein the one or more RNA repair proteins are exogenous to the living cell.

[0117] In some aspects, the techniques described herein relate to an engineered system, wherein the one or more exogenous RNA repair proteins are to be co-delivered as a fusion to one or more protein subunits of the type III CRISPR-Cas complex or are to be recruited through an interaction between an RNA aptamer in the guide RNA.

[0118] In some aspects, the techniques described herein relate to an engineered system, further including: a CaslO subunit having one or more catalytic residues mutated in corresponding enzymatic sites to inactivate polymerase and / or nuclease activity of the CaslO subunit, thereby reducing toxicity of the CaslO subunit in the living cell.

[0119] In some aspects, the techniques described herein relate to an engineered system, wherein a target RNA molecule, from among the plurality of the target RNA molecules, includes a repetitive region and the guide RNA is complementary to the repetitive region, resulting in repeated cleavage at the repetitive region and an RNA deletion, based on the repetitive region, that is larger than what would result from a single cleavage.

[0120] In some aspects, the techniques described herein relate to an engineered system, wherein the RNA-guided ribonuclease includes engineered ribozymes.

[0121] In some aspects, the techniques described herein relate to an engineered system, including: a first ribozyme engineered to cut target RNA at a first site when hybridized to the target RNA; a second ribozyme engineered to cut the target RNA at a second site downstream of the first site when hybridized to the target RNA, wherein a portion of the target RNA between the first site and the second site is excised from the target RNA when the first ribozyme and the second ribozyme make their respective cuts, and wherein the first ribozyme and the second ribozyme remain bound to unexcised portions of the target RNA after the portion of the target RNA between the first site and the second site is excised from the target RNA.

[0122] In some aspects, the techniques described herein relate to an engineered system, wherein the first ribozyme includes: one or more nucleotides that are upstream of the first site and are complementary to the target RNA.

[0123] In some aspects, the techniques described herein relate to an engineered system, wherein the first ribozyme includes: one or more nucleotides that are between the first site and the second site and arc complementary to the target RNA.

[0124] In some aspects, the techniques described herein relate to an engineered system, wherein the second ribozyme includes: one or more nucleotides that are between the first site and the second site and are complementary to the target RNA.

[0125] In some aspects, the techniques described herein relate to an engineered system, wherein the second ribozyme includes: one or more nucleotides that are downstream of the second site and are complementary to the target RNA.

[0126] In some aspects, the techniques described herein relate to an engineered system, wherein target RNA is modified exogenously and is repaired inside a living cell.

[0127] In some aspects, the techniques described herein relate to an engineered system, including: a first portion of the edited RNA molecule including a 2,3'-cyclic phosphate terminus; a second portion of the edited RNA molecule including a 5'-hydroxyl terminus; a first delivery vehicle including the first portion, the first delivery vehicle to transport the first portion inside a living cell; and a second delivery vehicle including the second portion, the second vehicle delivery to transport the second portion inside the living cell, wherein when delivered inside the living cell, the first portion and the second portion are repaired based on the 2,3'-cyclic phosphate terminus and the 5'- hydroxyl terminus to form the RNA molecule inside the living cell.

[0128] In some aspects, the techniques described herein relate to an engineered system, wherein the edited RNA molecule, when formed, expresses a corresponding therapeutic protein.

[0129] In some aspects, the techniques described herein relate to a ribonucleic acid (RNA) editing system, including: a first ribozyme engineered to cut target RNA at a first site when hybridized to the target RNA; and a second ribozyme engineered to cut the target RNA at a second site downstream of the first site when hybridized to the target RNA, wherein a portion of the target RNA between the first site and the second site is excised from the target RNA when the first ribozyme and the second ribozyme make their respective cuts, and wherein the first ribozyme and the second ribozyme remain bound to unexcised portions of the target RNA after the portion of the target RNA between the first site and the second site is excised from the target RNA.

[0130] In some aspects, the techniques described herein relate to a RNA editing system, wherein the first ribozyme includes: one or more nucleotides that are upstream of the first site and are complementary to the target RNA.

[0131] In some aspects, the techniques described herein relate to a RNA editing system, wherein the first ribozyme includes: one or more nucleotides that are between the first site and the second site and are complementary to the target RNA.

[0132] In some aspects, the techniques described herein relate to a RNA editing system, wherein the second ribozyme includes: one or more nucleotides that are between the first site and the second site and are complementary to the target RNA.

[0133] In some aspects, the techniques described herein relate to a RNA editing system, wherein the second ribozyme includes: one or more nucleotides that are downstream of the second site and are complementary to the target RNA.

[0134] In some aspects, the techniques described herein relate to an ribonucleic acid (RNA) delivery system, including: a first portion of an RNA molecule including a 2,3'-cyclic phosphate terminus; a second portion of the RNA molecule including a 5'-hydroxyl terminus; a first delivery vehicle including the first portion, the first delivery vehicle to transport the first portion inside a living cell; and a second delivery vehicle including the second portion, the second vehicle delivery to transport the second portion inside the living cell, wherein when delivered inside the living cell, the first portion and the second portion are repaired based on the 2,3'-cyclic phosphate terminus and the 5'-hydroxyl terminus to form the RNA molecule inside the living cell.

[0135] In some aspects, the techniques described herein relate to a RNA delivery system, wherein the RNA molecule, when formed, expresses a corresponding therapeutic protein.

[0136] In some aspects, the disclosure includes methods for providing RNA therapies by introducing the engineered CRISPR complex disclosed herein into a living cell for RNA editing. In particular, the disclosure includes methods of treating diseases such as through engineered Type III CRISPR mediated sequence-specific RNA editing in living cells. In these aspects, the engineered CRISPR complex may be delivered to living cells via the RNA delivery systems disclosed herein or other suitable techniques.

[0137] In various examples, the target RNA may be the RNA of a subject. More specifically, the target RNA may be an RNA transcript. In particular, the RNA transcript may include a mutation or other dysfunction that results in a disease. One example of such a mutation is a nonsense mutation that results in an incomplete or otherwise non-functional protein.

[0138] A subject may refer to an animal, such as a mammalian species (preferably human) or avian (c.g., bird) species, or other organism, such as a plant. More specifically, a subject can be a vertebrate, e.g., a mammal such as a mouse, a primate, a simian or a human. Animals include farm animals, sport animals, and pets. A subject can be a healthy individual, an individual that has symptoms or signs or is suspected of having a disease or a predisposition to the disease, or an individual that is in need of therapy or suspected of needing therapy.[1] A genetic modification or mutation in the context of an engineered system may refer to an alteration, variant or polymorphism in a nucleic acid that may result in altered or disabled functionality of a corresponding protein. Such alteration, variant or polymorphism can be with respect to a reference genome, the subject or other individual. Variations include one or more single nucleotide variations (SNVs), insertions, deletions, repeats, small insertions, small deletions, small repeats, structural variant junctions, variable length tandem repeats, and / or flanking sequences, CNVs, transversions, gene fusions and other rearrangements may also be considered forms of genetic variation. A variation can be a base change, insertion, deletion, repeat, copy number variation, transversion, or a combination thereof.[2] A “polynucleotide”, “nucleic acid”, “nucleic acid molecule”, or “oligonucleotide” may each refer to a polymer of nucleosides (including deoxyribonucleosides, ribonucleosides, or analogs thereof) joined by inter-nucleosidic linkages. Typically, a polynucleotide comprises at least three nucleosides. Oligonucleotides often range in size from a few monomeric units, e.g. 3-4, to hundreds of monomeric units. Whenever a polynucleotide is represented by a sequence of letters, such as “AUGCCUG,” it will be understood that the nucleotides are in 5’to 3’ order from left to right and that “A” denotes adenosine, “C” denotes cytidine, “G” denotes guanosine, and “U” denotes uracil, unless otherwise noted. The letters A, C, G, and U (or “T” denoting thymine in DNA) may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.[3] All patent filings, websites, other publications, sequence listings, accession numbers and the like cited above, below, or in any priority applications are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be so incorporated by reference.[4] If different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or filing date of a priority application referring to the accession number if applicable. Likewise, if different versions of a publication, website or the like are published at different times, the version most recently published at the effective filing date of the application is meant unless otherwise indicated. Any feature, step, element, embodiment, or aspect of the disclosure can be used in combination with any other unless specifically indicated otherwise. Although the present disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

CLAIMSWHAT IS CLAIMED IS:What is claimed is:

1. An engineered system for editing target ribonucleic acid (RNA) molecules in a living cell comprising: an RNA guide having a sequence engineered to be complementary to target RNA molecules in the living cell; and an RNA-guided ribonuclease that introduces one or more breaks in a plurality of the target RNA molecules, wherein at least some of the plurality of the target RNA molecules are to be repaired by the living cell, resulting in one or more edited RNA molecules in the living cell based on the one or more breaks and subsequent repair by the living cell.

2. The engineered system of claim 1, wherein the RNA-guided ribonuclease comprises a type III Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas complex.

3. The engineered system of claim 2, wherein the type 111 CRISPR-Cas complex comprises a type IILA CRISPR-Cas complex.

4. The engineered system of claim 3, wherein the type IILA CRISPR-Cas complex is derived from Streptococcus thermophilus.

5. The engineered system of claim 3, further comprising: a CRISPR RNA (crRNA), a Csm2 protein, a Csm3 protein, a Csm4 protein, a Csm5 protein, and / or CaslO.

6. The engineered system of claim 5, further comprising: a Cas6 protein.

7. The engineered system of claim 3, further comprising: a Cas6 protein.

8. The engineered system of claim 2, wherein the type III CRISPR-Cas complex comprises a type III-E CRISPR-Cas complex.

9. The engineered system of claim 8, wherein the type III-E CRISPR-Cas complex is derived from Desulfonemea ishimotonii.

10. The engineered system of claim 1, wherein the target RNA molecules are each an RNA transcript associated with a disease.

11. The engineered system of claim 10, wherein the RNA-guided ribonuclease comprises a type III CRISPR-Cas complex that is engineered to have a programmable cut-and- repair RNA editing strategy.

12. The engineered system of claim 11, wherein the target RNA molecules arc each associated with a premature stop codon that prevents expression of an associated protein that is suspected to or actually results in the disease, and wherein the programmable cut-and-repair RNA editing strategy is configured to excise the premature stop codon from the RNA transcript to restore expression of the associated protein.

13. The engineered system of claim 12, wherein to excise the premature stop codon, the RNA guide is engineered to tile across the premature stop codon either alone or with one or more other RNA guides.

14. The engineered system of claim 11, wherein the programmable cut-and-repair RNA editing strategy comprises a programmable RNA cleavage, a programmable RNA deletion, a programmable RNA insertion, and / or a programmable RNA excision.

15. The engineered system of claim 11, wherein the programmable cut-and-repairRNA editing strategy is configured to generate one or more deletions in the plurality of the target RNA molecules.

16. The engineered system of claim 15, wherein one or more deletions comprise a deletion of greater than 100 nucleotides from the plurality of the target RNA molecules.

17. The engineered system of claim 1, further comprising: an oligonucleotide splint engineered to be complementary to RNA ends, from among the plurality of the target RNA molecules, associated with cleavage by RNA-guided ribonuclease, wherein the oligonucleotide splint is to be inserted into the target RNA when the target RNA is repaired by one or more RNA repair proteins.

18. The engineered system of claim 17, wherein the RNA-guided ribonuclease comprises a type III CRISPR-Cas complex.

19. The engineered system of claim 18, wherein the oligonucleotide splint is to be delivered in trans with the type III CRISPR-Cas complex.

20. The engineered system of claim 18, wherein the oligonucleotide splint is fused to the RNA guide and is to be delivered in cis as part of the type III CRISPR-Cas complex.

21. The engineered system of claim 18, wherein the oligonucleotide splint comprises RNA, DNA, chimeric and / or modified oligonucleotides.

22. The engineered system of claim 18, wherein the one or more RNA repair proteins are endogenous to the living cell.

23. The engineered system of claim 18, wherein the one or more RNA repair proteins are exogenous to the living cell.

24. The engineered system of claim 23, wherein the one or more exogenous RNA repair proteins are to be co-delivered as a fusion to one or more protein subunits of the type III CRISPR-Cas complex or are to be recruited through an interaction between an RNA aptamer in the guide RNA.

25. The engineered system of claim 2, further comprising: a CaslO subunit having one or more catalytic residues mutated in corresponding enzymatic sites to inactivate polymerase and / or nuclease activity of the CaslO subunit, thereby reducing toxicity of the CaslO subunit in the living cell.

26. The engineered system of claim 1, wherein a target RNA molecule, from among the plurality of the target RNA molecules, comprises a repetitive region and the guide RNA is complementary to the repetitive region, resulting in repeated cleavage at the repetitive region and an RNA deletion, based on the repetitive region, that is larger than what would result from a single cleavage.

27. The engineered system of claim 1, wherein the RNA-guided ribonuclease comprises engineered ribozymes.

28. The engineered system of claim 27, comprising: a first ribozyme engineered to cut target RNA at a first site when hybridized to the target RNA; a second ribozyme engineered to cut the target RNA at a second site downstream of the first site when hybridized to the target RNA, wherein a portion of the target RNA between the first site and the second site is excised from the target RNA when the first ribozyme and the second ribozyme make their respective cuts, and wherein the first ribozyme and the second ribozyme remain bound to unexcised portions of the target RNA after the portion of the target RNA between the first site and the second site is excised from the target RNA.

29. The engineered system of claim 28, wherein the first ribozyme comprises: one or more nucleotides that are upstream of the first site and are complementary to the target RNA.

30. The engineered system of claim 29, wherein the first ribozyme comprises:one or more nucleotides that are between the first site and the second site and are complementary to the target RNA.

31. The engineered system of claim 28, wherein the second ribozyme comprises: one or more nucleotides that are between the first site and the second site and are complementary to the target RNA.

32. The engineered system of claim 31, wherein the second ribozyme comprises: one or more nucleotides that are downstream of the second site and are complementary to the target RNA.

33. The engineered system of claim 1, wherein target RNA is modified exogenously and is repaired inside a living cell.

34. The engineered system of claim 33, comprising: a first portion of the edited RNA molecule comprising a 2,3 ’-cyclic phosphate terminus; a second portion of the edited RNA molecule comprising a 5’-hydroxyl terminus; a first delivery vehicle comprising the first portion, the first delivery vehicle to transport the first portion inside a living cell; and a second delivery vehicle comprising the second portion, the second vehicle delivery to transport the second portion inside the living cell, wherein when delivered inside the living cell, the first portion and the second portion are repaired based on the 2,3 ’-cyclic phosphate terminus and the 5 ’-hydroxyl terminus to form the RNA molecule inside the living cell.

35. The engineered system of claim 34, wherein the edited RNA molecule, when formed, expresses a corresponding therapeutic protein.

36. A ribonucleic acid (RNA) editing system, comprising:a first ribozyme engineered to cut target RNA at a first site when hybridized to the target RNA; and a second ribozyme engineered to cut the target RNA at a second site downstream of the first site when hybridized to the target RNA, wherein a portion of the target RNA between the first site and the second site is excised from the target RNA when the first ribozyme and the second ribozyme make their respective cuts, and wherein the first ribozyme and the second ribozyme remain bound to unexcised portions of the target RNA after the portion of the target RNA between the first site and the second site is excised from the target RNA.

37. The RNA editing system of claim 36, wherein the first ribozyme comprises: one or more nucleotides that are upstream of the first site and are complementary to the target RNA.

38. The RNA editing system of claim 37, wherein the first ribozyme comprises: one or more nucleotides that are between the first site and the second site and are complementary to the target RNA.

39. The RNA editing system of claim 36, wherein the second ribozyme comprises: one or more nucleotides that are between the first site and the second site and are complementary to the target RNA.

40. The RNA editing system of claim 36, wherein the second ribozyme comprises: one or more nucleotides that are downstream of the second site and are complementary to the target RNA.

41. An ribonucleic acid (RNA) delivery system, comprising: a first portion of an RNA molecule comprising a 2,3’-cyclic phosphate terminus; a second portion of the RNA molecule comprising a 5 ’-hydroxyl terminus; a first delivery vehicle comprising the first portion, the first delivery vehicle to transport the first portion inside a living cell; anda second delivery vehicle comprising the second portion, the second vehicle delivery to transport the second portion inside the living cell, wherein when delivered inside the living cell, the first portion and the second portion are repaired based on the 2,3 ’-cyclic phosphate terminus and the 5 ’-hydroxyl terminus to form the RNA molecule inside the living cell.

42. The RNA delivery system of claim 41, wherein the RNA molecule, when formed, expresses a corresponding therapeutic protein.

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