TW202339775A - Engineered adar-recruiting rnas and methods of use thereof - Google Patents

Abstract

The present application provides methods for editing RNA by introducing a deaminase-recruiting RNA in a host cell for deamination of an adenosine in a target RNA. The present application further provides deaminase-recruiting RNAs used in the RNA editing methods and compositions and kits comprising the same.

Description

Engineered ADAR recruitment of RNA and methods of use

[ Cross-references to related applications ]

This application claims priority from International Application No. PCT/CN2022/074710, filed on January 28, 2022, and International Application No. PCT/CN2022/085144, filed on April 2, 2022, the contents of each of which are incorporated by reference. Incorporated into this article in its entirety. [ Citation from electronic sequence listing ]

The contents of the electronic sequence listing (792642002540SEQLIST.xml; size: 464,734 bytes; creation date: January 27, 2023) are incorporated herein by reference in their entirety.

The present application relates to methods and compositions for editing RNA using engineered linear or circular RNAs capable of recruiting adenosine deaminase to deaminate one or more adenosines in a target RNA.

Genome editing is a powerful tool for biomedical research and the development of disease therapeutics. Editing technologies using Cas proteins using engineered nucleases such as zinc finger nucleases (ZFNs), transcription initiation factor-like effector nucleases (TALENs), and the CRISPR system have been applied to manipulate the genomes of countless organisms. Recently, new tools for RNA editing have been developed using deaminase proteins, such as adenosine deaminase acting on RNA (ADAR). In mammalian cells, there are three types of ADAR proteins, Adar1 (two isoforms, p110 and p150), Adar2 and Adar3 (catalytically inactive). The catalytic substrate of ADAR protein is double-stranded RNA. ADAR can remove the -NH2 group on the nucleobase of adenosine (A) and change A to inosine (I). (I) is recognized as guanosine (G) and pairs with cytidine (C) during subsequent cellular transcription and translation. To achieve targeted RNA editing, the ADAR protein or its catalytic domain is fused with a λN peptide, a SNAP tag, or a Cas protein (dCas13b), and a guide RNA is designed to recruit the chimeric ADAR protein to the target site. Alternatively, it has also been reported that overexpression of ADAR1 or ADAR2 proteins and guide RNA with R/G motifs can achieve targeted RNA editing.

However, currently available ADAR-mediated RNA editing technologies have certain limitations. For example, the most efficient in vivo delivery of gene therapies is via viral vectors, but highly desirable adeno-associated virus (AAV) vectors are limited by cargo size (~4.5 kb), making it challenging to accommodate both protein and guide RNA. Furthermore, it was recently reported that overexpression of ADAR1 confers oncogenicity to multiple myeloma due to aberrant hyper-editing of RNA and generates a large number of global off-target edits. Furthermore, ectopic expression of proteins or domains thereof of non-human origin carries the potential risk of triggering immunogenicity. Additionally, pre-existing adaptive immunity and p53-mediated DNA damage responses may compromise the efficacy of therapeutic proteins such as Cas9.

Usher syndrome, also known as hereditary deafness-retinitis pigmentosa syndrome, was described and named by Charles Howard Usher in 1914. It is a rare autosomal recessive disorder caused by genetic mutations that results in congenital or progressive vision and/or hearing loss. It is estimated that more than half of the 16,000 blind and deaf people in the United States have Usher syndrome. There are tens of thousands or even hundreds of thousands of patients around the world in urgent need of treatment.

Usher syndrome can be divided into types I, II, III, and IV according to severity. Among them, type I has an early onset, is accompanied by severe congenital deafness and early visual impairment, and the window for intervention is very small; while types III and IV are relatively rare and have mild syndromes. People with Usher syndrome type II develop progressive vision loss starting between the ages of 10 and 20 years. The initial stage of the disease is night blindness, which eventually progresses to vision loss. This allows us to have a longer window for therapeutic intervention and hopefully halt the progressive loss of vision in patients with type II Usher syndrome.

In 2017, Neuhaus et al. studied 138 Usher syndrome patients through high-throughput sequencing. The results showed that USH2A gene mutations accounted for more than 90% of Usher syndrome type II patients. The results also showed that among the USH2A gene mutations sequenced, 13% were NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter), which is the most common single mutation type among all mutation types.

Currently, there are two main research and development directions for treating Usher syndrome type II through gene therapy. One is to use viruses to mediate the re-expression of the full-length USH2A gene in the eye. However, the protein sequence of the USH2A gene is very long, with more than 6,000 amino acids, which results in the corresponding coding region sequence with more than 18,000 base pairs, and common viral vectors have limitations on the length of the gene payload. Typically, lentiviruses can be used to deliver payloads of no more than 10,000 base pairs, while adeno-associated viruses can deliver payloads of no more than 4,500 base pairs. This makes it difficult to deliver the full-length USH2A gene via viral vectors. Therefore, medical and scientific workers have been left with the option of delivering a truncated version of the USH2A gene. Although this method can alleviate the progression of the disease to a certain extent, it cannot fully restore the normal physiological function of Usherin because the patient still lacks the normal full-length Usherin protein.

Another common direction for USH2A gene therapy is through exon skipping. Since some USH2A gene mutations are caused by frameshifts or nonsense mutations in a certain exon, as long as the exon can be specifically skipped during the RNA splicing process, the sequence after the exon can be translated normally. . A common approach is to introduce a short antisense nucleotide (Anti-Sense Oligo, ASO) to specifically skip the exon targeted by the translated nucleotide during splicing. This method is similar to the previous method—because the mutated exons are skipped, full-length Usherin is still not obtained.

In 2020, Boya Jiyin disclosed in CN113122577A a method for targeted editing of target RNA containing G to A mutations in USH2A gene transcripts based on linear LEAPER technology (Leaper 1.0), including using adenosine to edit the target RNA. . A deaminase-recruiting RNA (arRNA) or a construct encoding an arRNA is introduced into the cell, wherein the arRNA contains a complementary RNA sequence that hybridizes to the target RNA, and wherein the arRNA is capable of recruiting an adenosine deaminase (ADAR) to target RNA. deamination of target adenosine, thereby safely and effectively performing in vivo base editing from "A" to "I" base on RNA, repairing the pathogenic mutation site, and achieving the goal of treating diseases such as Usher syndrome Purpose.

Mucopolysaccharidosis type I (MPS I) is a condition that affects many parts of the body. The disease was once divided into three separate syndromes: Hurler syndrome (MPS I-H), Hurler-Scheie syndrome (MPS I-H/S), and Scheie syndrome (MPS I-S), listed from most severe to least severe. out. Because there is much overlap between the three syndromes, MPS I is currently divided into severe and attenuated forms.

Mutations in the IDUA gene cause MPS I. The IDUA gene provides instructions for producing an enzyme involved in the breakdown of large sugar molecules called glycosaminoglycans (GAGs). GAGs were originally called mucopolysaccharides, which is where the condition gets its name. Mutations in the IDUA gene reduce or completely eliminate the function of the IDUA enzyme. The lack of IDUA enzyme activity leads to the accumulation of GAGs within cells, especially within lysosomes. Lysosomes are compartments in cells that digest and recycle different types of molecules. Disorders that cause molecules to accumulate within lysosomes, including MPS I, are known as lysosomal storage diseases. The accumulation of GAGs increases the size of lysosomes, which is why many tissues and organs enlarge in this disease. The researchers believe that GAGs may also interfere with the function of other proteins within lysosomes and disrupt the movement of intracellular molecules.

Peptidylprolyl isomerase A (PPIA), also known as cyclophilin A (CypA) or rotamerase A, is an enzyme encoded by the PPIA gene on chromosome 7 in humans. As a member of the peptidylprolyl cis-trans isomerase (PPIase) family, this protein catalyzes the cis-trans isomerization of the proline imino peptide bond, which enables it to regulate many biological processes, including intracellular signaling, Transcription, inflammation, and apoptosis. Due to its various functions, PPIA has been implicated in numerous inflammatory diseases, including atherosclerosis and arthritis, as well as viral infections.

As a pro-inflammatory cytokine, PPIA is highly involved in acute and chronic inflammatory diseases, including sepsis, arteriosclerosis, and rheumatoid arthritis. Therefore, therapeutic targeting of PPIA with selective inhibitors could prove effective in combating such inflammatory diseases and symptoms. The correlation between plasma PPIA levels and symptoms of hyperglycemia has also promoted the utilization of PPIA as a biomarker for diabetes and vascular disease.

In addition, PPIA is involved in cerebral hypoxia-ischemia by promoting the nuclear transport of AIF, a pro-apoptotic factor, in neurons. To maintain the integrity of the blood-brain barrier and mitigate brain damage, PPIA helps recruit circulating monocytes and stimulate survival and growth pathways. In cardiomyocytes, cyclophilin has been observed to be activated by heat shock and hypoxia-reoxygenation and to complex with heat shock proteins. Therefore, cyclophilin may play a protective role in the heart during ischemia-reperfusion injury.

PPIA expression is highly related to cancer pathogenesis, but the specific mechanism remains to be elucidated. PPIA overexpression is associated with hepatocellular carcinoma, lung cancer, pancreatic cancer, endometrial cancer, esophageal squamous cell carcinoma, and melanoma.

This protein also interacts with several HIV proteins, including p55 gag, Vpr, and capsid proteins, and was shown to be required for the formation of infectious HIV viruses. Therefore, PPIA helps with viral diseases such as AIDS, hepatitis C, measles and influenza A.

Although there are different gene or RNA editing methods for specific diseases, such as those caused by Usher 2A, IDUA or PPIA mutations, identifying a method that can truly repair the pathogenic mutation and meet the standards for clinical application has been elusive and Challenging.

The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety.

The present application provides a method of editing a target adenosine in a target RNA comprising a target adenosine in a host cell, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the The dRNA includes a targeting RNA sequence that is capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to remove target adenosine in the target RNA. Deamination, wherein the RNA duplex contains one or more mismatched regions, and wherein the dRNA contains a linker nucleic acid sequence flanking the termini of the targeting RNA sequence. Also provided herein are methods of treating or preventing a disease or disorder in an individual, comprising editing a target RNA associated with the disease or disorder in cells of the individual.

In one aspect, provided herein are methods of editing a target adenosine in a target RNA comprising a target adenosine in a host cell, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding said dRNA. A host cell, wherein the dRNA comprises a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an adenosine deaminase (ADAR) enzyme acting on RNA to render the target RNA deamination of a target adenosine in, wherein the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence located 5 nucleotides to 85 nuclei upstream of the target adenosine and/or (b) a second mismatch region relative to the target RNA sequence located 20 nucleotides to 85 nucleotides downstream of the target adenosine; and wherein the dRNA Comprised is a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence, the first mismatch region being located 5 nucleotides to 25 nucleotides upstream of the target adenosine at an acid site; and/or wherein the RNA double strand comprises a second mismatch region relative to the target RNA sequence, the second mismatch region being located 20 nucleotides to 45 nuclei downstream of the target adenosine The nucleotide position. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence, the first mismatch region being located 5 nucleotides to 15 nucleotides upstream of the target adenosine at an acid site; and/or wherein the RNA double strand comprises a second mismatch region relative to the target RNA sequence, the second mismatch region being located 20 nucleotides to 45 nuclei downstream of the target adenosine The nucleotide position. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence, the first mismatch region being located 20 nucleotides to 40 nucleotides upstream of the target adenosine at an acid site; and/or wherein the RNA double strand comprises a second mismatch region relative to the target RNA sequence, the second mismatch region being located 25 nucleotides to 45 nuclei downstream of the target adenosine The nucleotide position. In some embodiments, the first mismatch region and/or the second mismatch region comprise one or more non-complementary nucleotides (mismatch) in the targeting RNA sequence.

In one aspect, provided herein are methods for editing a dRNA comprising a target RNA, wherein the dRNA comprises a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA an adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA double strand comprises: (a) a first mismatch region relative to the target RNA sequence, which is located 5 nucleotides to 85 nucleotides upstream of the target adenosine; and/or (b) a second mismatch region relative to the target RNA sequence located 20 nucleotides downstream of the target adenosine to 85 nucleotides; and wherein the dRNA comprises a linker nucleic acid sequence flanking the terminus of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form a secondary structure. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence, the first mismatch region being located 5 nucleotides to 25 nucleotides upstream of the target adenosine at an acid site; and/or wherein the RNA double strand comprises a second mismatch region relative to the target RNA sequence, the second mismatch region being located 20 nucleotides to 45 nuclei downstream of the target adenosine The nucleotide position. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence, the first mismatch region being located 5 nucleotides to 15 nucleotides upstream of the target adenosine at an acid site; and/or wherein the RNA double strand comprises a second mismatch region relative to the target RNA sequence, the second mismatch region being located 20 nucleotides to 45 nuclei downstream of the target adenosine The nucleotide position. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence, the first mismatch region being located 20 nucleotides to 40 nucleotides upstream of the target adenosine at an acid site; and/or wherein the RNA double strand comprises a second mismatch region relative to the target RNA sequence, the second mismatch region being located 25 nucleotides to 45 nuclei downstream of the target adenosine The nucleotide position. In some embodiments, the first mismatch region and/or the second mismatch region comprise one or more non-complementary nucleotides (mismatch) in the targeting RNA sequence.

In some embodiments according to any of the above methods or dRNA, the first mismatch region and/or the second mismatch region comprise one or more nucleotide deletions of the targeting RNA. In some embodiments, the first mismatch region and/or the second mismatch region comprise one or more nucleotide insertions of the targeting RNA. In some embodiments, the first mismatch region and/or the second mismatch region comprises at least one set of contiguous non-complementary nucleotides (mismatches) in the targeting RNA. In some embodiments, the first mismatch region and/or the second mismatch region comprises a deletion of at least one set of contiguous nucleotides of the targeting RNA. In some embodiments, the first mismatch region and/or the second mismatch region comprises the insertion of at least one contiguous set of nucleotides of the targeting RNA. In some embodiments, the first mismatched region is 1-50 nucleotides in length. In some embodiments, the second mismatched region is 1-50 nucleotides in length. In some embodiments, the first mismatch region is 1-10 nucleotides in length, wherein the first mismatch region comprises 1-10 contiguous non-complementary nucleotides in the targeting RNA Or deletion of 1-10 consecutive nucleotides of the targeting RNA. In some embodiments, the second mismatched region is 1-10 nucleotides in length, wherein the second mismatched region comprises 1-10 contiguous non-complementary nucleotides in the targeting RNA Or deletion of 1-10 consecutive nucleotides of the targeting RNA. In some embodiments, the first mismatch region is 4 nucleotides in length. In some embodiments, the second mismatched region is 4 nucleotides in length. In some embodiments, the first mismatch region is 4 nucleotides in length, wherein the first mismatch region includes 4 consecutive non-complementary nucleotides in the targeting RNA or the target Deletion of 4 consecutive nucleotides into the RNA. In some embodiments, the second mismatch region is 4 nucleotides in length, wherein the second mismatch region includes 4 consecutive non-complementary nucleotides in the targeting RNA or the target Deletion of 4 consecutive nucleotides into the RNA. In some embodiments, non-complementary nucleotides in the targeting RNA sequence result in bubbles in the RNA duplex. In some embodiments, deletions of nucleotides in the targeting RNA sequence result in bulges in the RNA duplex. In some embodiments, the nucleotide insertion of the targeted RNA sequence results in a bulge structure in the RNA duplex. In some embodiments, a set of contiguous non-complementary nucleotides in the targeting RNA sequence results in a bubble-like structure in the RNA duplex. In some embodiments, deletion of a contiguous set of nucleotides in the targeting RNA sequence results in a bulge structure in the RNA duplex. In some embodiments, insertion of a contiguous set of nucleotides in the targeting RNA sequence results in a bulge structure in the RNA duplex.

In some embodiments according to any of the above methods or dRNA, the target RNA comprising target adenosine encodes a PPIA protein. In some embodiments, the target RNA encoding the PPIA protein contains a target adenosine in the untranslated region (UTR). In some embodiments, the target RNA encodes wild-type PPIA.

In some embodiments according to any of the above methods or dRNA, wherein the target RNA comprising the target adenosine encodes a PPIA protein, the RNA duplex further comprises a third mismatched region relative to the target RNA, wherein the third mismatched region is located opposite between the first mismatched region and the second mismatched region of the target RNA. In some embodiments, the third mismatch region includes one or two non-complementary nucleotides in the targeting RNA and/or a deletion of one or two nucleotides in the targeting RNA sequence. In some embodiments, the third mismatch region relative to the target RNA sequence is located 5, 7, and/or 8 nucleotides downstream of the target adenosine; optionally, wherein the target RNA comprises a region located downstream of the target adenosine Adenosine at 5, 7 and/or 8 nucleotides.

In some embodiments according to any one of the above methods or dRNA, wherein the target RNA comprising a target adenosine encodes a PPIA protein, the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence, located 27 nucleotides to 38 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence located 32 nucleotides downstream of the target adenosine to 35 nucleotides. In some embodiments, the first mismatched region is located 27 nucleotides to 30 nucleotides downstream of the target adenosine relative to the target RNA sequence; optionally, wherein the first mismatched region The length is 4 nucleotides, and the second mismatched region is 4 nucleotides in length. In some embodiments, the first mismatched region comprises a deletion of 4 contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatched region comprises a deletion of 4 contiguous nucleotides of the targeting RNA sequence. Missing. In some embodiments, the first mismatched region is located 31 nucleotides to 34 nucleotides downstream of the target adenosine relative to the target RNA sequence; optionally, wherein the first mismatched region The length is 4 nucleotides, and the second mismatched region is 4 nucleotides in length. In some embodiments, the first mismatch region includes a deletion of 4 contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatch region includes a deletion of 4 contiguous nucleotides of the targeting RNA sequence. Deletion of nucleotides. In some embodiments, the first mismatched region is located 35 nucleotides to 38 nucleotides downstream of the target adenosine relative to the target RNA sequence; optionally, wherein the first mismatched region The length is 4 nucleotides, and the second mismatched region is 4 nucleotides in length. In some embodiments, the first mismatch region includes a deletion of 4 contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatch region includes a deletion of 4 contiguous nucleotides of the targeting RNA sequence. Deletion of nucleotides.

In some embodiments according to any one of the above methods or dRNA, wherein the target RNA comprising a target adenosine encodes a PPIA protein, said RNA duplex comprises: (a) a first mismatch region relative to said target RNA sequence, which is located 27 nucleotides to 38 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence which is located 41 nucleotides downstream of the target adenosine to 44 nucleotides; optionally, wherein the first mismatched region is 4 nucleotides in length and the second mismatched region is 4 nucleotides in length. In some embodiments, the first mismatch region includes a deletion of 4 contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatch region includes a deletion of 4 contiguous nucleotides of the targeting RNA sequence. Deletion of nucleotides. In some embodiments, the first mismatched region is located 27 nucleotides to 30 nucleotides downstream of the target adenosine relative to the target RNA sequence; optionally, wherein the first mismatched region has a length of 4 nucleotides, and the length of the second mismatch region is 4 nucleotides. In some embodiments, the first mismatch region includes a deletion of 4 contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatch region includes a deletion of 4 contiguous nucleotides of the targeting RNA sequence. Deletion of nucleotides. In some embodiments, the first mismatched region is located 31 nucleotides to 34 nucleotides downstream of the target adenosine relative to the target RNA sequence; optionally, wherein the first mismatched region has a length of 4 nucleotides, and the length of the second mismatch region is 4 nucleotides. In some embodiments, the first mismatch region includes a deletion of 4 contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatch region includes a deletion of 4 contiguous nucleotides of the targeting RNA sequence. Deletion of nucleotides. In some embodiments, the first mismatched region is located 35 nucleotides to 38 nucleotides downstream of the target adenosine relative to the target RNA sequence; optionally, wherein the first mismatched region has a length of 4 nucleotides, and the length of the second mismatch region is 4 nucleotides. In some embodiments, the first mismatch region includes a deletion of 4 consecutive nucleotides of the targeting RNA sequence, wherein the second mismatch region includes a deletion of 4 consecutive nucleotides of the targeting RNA sequence. Deletion of nucleotides.

In some embodiments according to any of the above methods or dRNA, the dRNA is circular. In some embodiments, the dRNA is linear and/or capable of circularization. In some embodiments, the dRNA further comprises one or more RNA recruitment domains, optionally, wherein the RNA recruitment domains are stem-loop structures. In some embodiments, the linker nucleic acid sequence is from about 5 nucleotides (nt) to about 500 nt in length. In some embodiments, at least about 50%, 60%, 70%, 80%, 85%, 90%, or 95% of any of the linker nucleic acid sequences comprise adenosine or cytidine. In some embodiments, at least 50% of the linker nucleic acid sequences comprise adenosine.

In some embodiments according to any one of the above methods, the method increases the Describe the editing level of target adenosine. In some embodiments, the method reduces one or more non-target glands compared to a corresponding method wherein the RNA duplex does not comprise one or more mismatch regions or wherein the dRNA does not comprise a linker nucleic acid sequence. Glycoside's (bystander) editing level.

In some embodiments of the method or dRNA according to any of the above, the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. Linker nucleic acid sequence. In some embodiments, the dRNA is a circular RNA, wherein a linker nucleic acid sequence connects the 5' end of the targeting RNA sequence and the 3' end of the targeting RNA sequence. In some embodiments, the dRNA is a circular RNA, wherein the dRNA further comprises a 3' exon sequence and a 5' exon sequence that can be flanked 5' of the targeting RNA sequence. The 3' catalytic Group I intronic fragment is recognized by the 3' end of the targeting RNA sequence, and the 5' exon sequence can be recognized by the 5' catalytic Group I intronic fragment flanking the 3' end of the targeting RNA sequence. In some embodiments, the dRNA also contains a 3' linker sequence and a 5' linker sequence.

In some embodiments according to any one of the methods or dRNA described above, the method includes introducing into the host cell a construct comprising a nucleic acid sequence encoding the dRNA. In some embodiments, the construct further comprises a promoter operably linked to the nucleic acid sequence encoding the dRNA.

In some embodiments, the targeting RNA sequence comprises cytidine, adenosine, or uridine directly opposite a target adenosine in the target RNA. In some embodiments, the targeting RNA sequence contains a cytidine mismatch directly opposite a target adenosine in the target RNA. In some embodiments, the cytidine mismatch is located at least 20 nucleotides from the 3' end of the targeting RNA sequence and at least 5 nucleotides from the 5' end of the targeting RNA sequence. In some embodiments, the 5' nearest neighbor of the target adenosine in the target RNA is a nucleotide selected from U, C, A, and G, preferably U>C≈A>G, and in the target RNA The 3' nearest neighbor of the target adenosine is a nucleotide selected from G, C, A and U, preferably G>C>A≈U. In some embodiments, the target adenosine is in a trihydroxyl motif of UAG, and wherein the targeting RNA sequence comprises an A directly opposite a uridine in the trihydroxyl motif, an A directly opposite a uridine in the trihydroxyl motif, Cytidine directly opposite the target adenosine, and cytidine, guanosine or uridine directly opposite the guanosine in the trisaccharyl motif. In some embodiments, the target RNA is an RNA selected from the group consisting of pre-messenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long non-coding RNA and small RNA, optionally, wherein the target RNA is pre-messenger RNA.

In some embodiments, the efficiency of editing the target RNA is at least 40%. In some embodiments, the method further includes introducing ADAR into the host cell. Edited RNA produced by any of the methods described herein and host cells having the edited RNA are also provided.

In one aspect, methods are provided for improving one or more symptoms of the following diseases in an individual: acute and chronic inflammatory diseases, cerebral hypoxia-ischemia, cancer such as hepatocellular carcinoma, lung cancer, pancreatic cancer, endometrial cancer, esophageal cancer Squamous cell carcinoma and melanoma, viral diseases such as AIDS, hepatitis C, measles and influenza A, the method comprising editing in cells of the individual according to the method of any one of claims 1-43 A target RNA related to PPIA, wherein the target RNA encodes a PPIA protein.

In some embodiments, host cells comprising any of the constructs or dRNA described above are provided. In some embodiments, a kit is provided comprising any of the constructs or dRNAs described above, wherein the kit further comprises instructions for editing a target RNA comprising a target adenosine in a host cell.

It should be understood that one, some, or all features of the various embodiments described herein may be combined to form other embodiments of the application. These and other embodiments of the present application are further described in the detailed description below.

The present application provides improved RNA editing methods and specially designed RNAs, referred to herein as deaminase-recruiting RNAs ("dRNAs") or ADAR-recruiting RNAs ("arRNAs") or constructs containing nucleic acids encoding these arRNAs, for use in a host Editing of target RNA containing target adenosine in cells. In one aspect, the present application discloses an improved Leaper-based editing method, which shows significantly higher editing efficiency and specificity, and provides a solution to the bystander off-target problem through its optimization.

The inventors of the present application have previously developed "LEAPER" (Programmable Editing of RNA Using Endogenous ADAR), which utilizes endogenous ADAR to edit target RNA by using dRNA. The LEAPER method is described in WO2021/008447 and PCT/CN2021/071292, the entire contents of which are incorporated herein by reference. Specifically, targeting RNA that is partially complementary to the target transcript is used to recruit naturally or exogenously introduced ADAR1 or ADAR2 to convert adenosine to inosine at specific sites on the target RNA. Therefore, RNA editing can be achieved in some systems without ectopic or overexpression of ADAR proteins in the host cell.

The present application provides improved LEAPER methods that allow for increased editing efficiency, reduced off-target (also referred to herein as "bystander editing") effects, and/or more precise and durable RNA editing. In some embodiments, the dRNA comprises a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex comprises one or more mismatched regions located upstream and/or downstream of the target adenosine; and wherein the dRNA A linker nucleic acid sequence flanking the terminus of the targeting RNA sequence is included, wherein the linker nucleic acid sequence in some embodiments does not hybridize to the target RNA and does not substantially form secondary structure. The mismatched region may include one or more non-complementary hydroxyl groups or one or more hydroxyl group deletions in the target RNA. The method described here has been successfully used to correct pathogenic point mutations, such as USH2A mutations. The improved LEAPER method may offer broad applicability to therapeutics and biomedical research.

Accordingly, one aspect of the present application provides a method of editing a target adenosine in a target RNA in a host cell, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding said dRNA into the host cells, wherein the dRNA contains a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate the target adenosine in the target RNA , wherein the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence, which is located 20 nucleotides to 40 nucleotides upstream of the target adenosine; and/or (b) relative to the target RNA sequence a second mismatched region of the RNA sequence located 25 nucleotides to 45 nucleotides downstream of the target adenosine; and wherein the dRNA includes a linker nucleic acid sequence flanking the end of the targeting RNA sequence, wherein the linker nucleic acid sequence is not identical to The target RNA hybridizes and essentially does not form secondary structure. In some embodiments, a method of editing a target adenosine in a target RNA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Contains a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex The strand comprises a first mismatch region relative to the target RNA sequence located 20 nucleotides to 40 nucleotides upstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the end of the targeting RNA sequence , wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure. In some embodiments, a method of editing a target adenosine in a target RNA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Contains a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex The strand comprises a second mismatch region relative to the target RNA sequence located 25 nucleotides to 45 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the end of the targeting RNA sequence , wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure. In some embodiments, a method of editing a target adenosine in a target RNA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Comprising a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex is The strand includes: (a) a first mismatch region relative to the target RNA sequence located 20 nucleotides to 40 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence. a coordination region located 25 nucleotides to 45 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the end of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and Basically no secondary structure is formed.

Another aspect of the present application provides a method of editing a target RNA associated with Usher syndrome, wherein the method includes introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into a host cell of an individual, wherein the dRNA includes a targeting RNA sequence capable of hybridizing with the target RNA to form an RNA double strand, wherein the RNA double strand can recruit adenosine deaminase (ADAR) acting on RNA to deaminate the target adenosine in the target RNA, where The RNA duplex comprises: (a) a first mismatch region comprising from 21 nucleotides to 37 nucleotides upstream of the target adenosine, relative to the target RNA sequence, from about 4 to about 10 nucleotides of the targeting RNA; a contiguous nucleotide deletion; and optionally, (b) a second mismatch region comprising 31 nucleotides to 43 nucleotides downstream of the target adenosine, relative to the target RNA sequence, of the targeting RNA About 4 to about 10 contiguous nucleotides are deleted; and optionally, (c) a third mismatch region located between the first mismatch region and the second mismatch region relative to the target RNA, wherein the third mismatch region The target RNA sequence of the coordination region contains any of the following: A, AA, U, C, CC, G, GG, or is located 3, 7 and 8 nucleotides downstream of the target adenosine, or 13 nucleotides, or upstream a nucleotide deletion ("X") at 5 nucleotides opposite the target RNA; and optionally, wherein the dRNA comprises a linker nucleic acid sequence flanking the end of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and substantially no secondary structure is formed; and wherein the length of the targeting RNA sequence in the dRNA is about 150 to about 220 nt. In some embodiments, the target RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation.

Another aspect of the present application provides a method of editing a target RNA comprising a target adenosine associated with MPS I, wherein the method comprises introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into an individual A host cell in which the dRNA contains a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to remove the target adenosine in the target RNA. Deamination, wherein the RNA duplex comprises: (a) a first mismatched region comprising approximately 6 nucleotides to 10 nucleotides upstream of the target adenosine relative to the target RNA sequence. a mismatch of 2 to about 5 contiguous nucleotides; and optionally, (b) a second mismatch region comprising 30 nucleotides to 34 nucleotides, or 40 nucleotides, downstream of the target adenosine a mismatch of about 2 to about 5 contiguous nucleotides in the targeting RNA to 44 nucleotides relative to the target RNA sequence; and optionally, (c) a third mismatch region, comprising a mismatch of about 2 to 5 contiguous nucleotides in the targeting RNA 60 nucleotides to 64 nucleotides downstream of the target adenylate relative to the target RNA sequence; and optionally, wherein The dRNA includes a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the targeting RNA sequence in the dRNA is from about 100 to about 220 nt in length. . In some embodiments, the target RNA encodes a mutant IDUA protein that includes early termination.

Another aspect of the present application provides a method of editing a target RNA comprising a target adenosine associated with a PPIA gene, wherein the method comprises introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into an individual A host cell in which the dRNA contains a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to remove the target adenosine in the target RNA. Deamination, wherein the RNA duplex includes: (a) a first mismatched region that includes a deletion of approximately 1 nucleotide of the target RNA 28 nucleotides upstream of the target adenosine relative to the target RNA sequence; and optionally, (b) a second mismatch region comprising a deletion of about 1 nucleotide of the targeting RNA 23 nucleotides upstream of the target adenosine relative to the target RNA sequence; and optionally , (c) a third mismatch region comprising a deletion of about 1 nucleotide of the targeting RNA 5 nucleotides downstream of the target adenylate relative to the target RNA sequence; and optionally, wherein the dRNA Comprised of a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 150 to about 220 nt. In some embodiments, the target RNA encodes a mutant PPIA protein.

Another aspect of the present application provides a method of editing a target RNA comprising a target adenosine associated with a PPIA gene, wherein the method comprises introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into an individual A host cell in which the dRNA contains a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to remove the target adenosine in the target RNA. Deamination, wherein the RNA duplex comprises: (a) a first mismatched region comprising 26 nucleotides to 30 nucleotides, 30 nucleotides to 34 nucleotides upstream of the target adenosine, or a mismatch of about 3 to about 5 consecutive nucleotides in the targeting RNA at nucleotides 34 to 38 relative to the target RNA sequence; and optionally, (b) a second mismatch A region comprising 31 nucleotides to 35 nucleotides, 35 nucleotides to 39 nucleotides, or 40 nucleotides to 44 nucleotides downstream of a target adenosine, relative to the target RNA sequence, a mismatch of about 3 to about 5 contiguous nucleotides in the targeting RNA; and optionally, wherein the dRNA comprises a linker nucleic acid sequence flanking the end of the targeting RNA sequence, wherein the linker nucleic acid sequence is not identical to the target RNA hybridizes and substantially does not form secondary structure; and wherein the targeting RNA sequence in the dRNA is about 150 to about 220 nt in length. In some embodiments, the target RNA encodes a mutant PPIA protein.

I. definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications and other publications mentioned herein are incorporated by reference in their entirety. To the extent that a definition set forth in this section is contrary or inconsistent with a definition set forth in a patent, application, or other publication incorporated herein by reference, the definition set forth in this section shall prevail over the definition set forth herein by reference.

It is to be understood that, for clarity, certain features of the disclosure that are described in the context of separate embodiments may also be provided combined in a single embodiment. Conversely, for the sake of brevity, various features of the disclosure that are described in the context of a single implementation may also be provided separately or in any suitable subcombination. All combinations of embodiments relating to particular method steps, reagents, or conditions are specifically included in the present disclosure and are disclosed herein as if each combination was individually and expressly disclosed.

As used herein, the term "bulge structure" refers to an asymmetric bubble in a nucleic acid duplex formed due to one or more unpaired nucleotides (e.g., non-target adenosine) in one strand of the nucleic acid duplex. structure region. The raised structures described herein may have completely unpaired regions in one strand without any corresponding complementary regions in the opposite strand. Alternatively, the bulge structures described herein may be formed from two non-complementary regions (one for each strand) with different numbers of nucleotides, which may also contain mismatched nucleotides that do not form a Watson-Crick base pair. The longer of the two non-complementary regions has at least one nucleotide (e.g., a non-target adenosine) that does not pair with any nucleotide in the non-complementary region of the opposite strand, i.e., the opposite strand contains flanking convex A nucleic acid sequence that is complementary to the nucleic acid sequence of the bulge structure, but the opposite strand does not contain at least one nucleotide opposite a nucleotide in the bulge structure (eg, a non-target adenosine). As used herein, "bulge structure" does not include regions of complete mismatch of nucleotides located within one strand of a nucleic acid duplex, i.e., the opposite strand contains nucleotides that are not complementary to every nucleotide in the bulge structure , which results in a symmetrical bubble-like structure in the nucleic acid duplex. In some examples, the bulge structure includes 1, 2, 3, 4, 5, or more than 5 nucleotides in a strand with unpaired nucleotides.

When the first nucleic acid strand and the second nucleic acid strand form a double-stranded nucleic acid region, the first nucleoside in the first nucleic acid strand that pairs with the second nucleoside base in the second nucleic acid strand is described herein as being "opposite" of each other. ” or “corresponding”, that is, the first nucleoside is opposite to the second nucleoside, and the second nucleoside is opposite to the first nucleoside.

The terms "polynucleotide," "nucleic acid," "nucleotide sequence," and "nucleic acid sequence" are used interchangeably. They refer to polymeric forms of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or their analogues.

The terms "deaminase recruiting RNA," "dRNA," "ADAR recruiting RNA," and "arRNA" are used interchangeably herein and refer to an engineered RNA capable of recruiting ADAR to deaminate target adenosine in RNA.

The terms "group I intron" and "group I catalytic intron" are used interchangeably and refer to a self-splicing ribozyme that catalyzes its own excision from an RNA precursor. Group I introns comprise two fragments, a 5' catalytic group I intron fragment and a 3' catalytic group I intron fragment, which retain their folding and catalytic functions (i.e., self-splicing activity). In its native environment, the 5' catalytic group I intronic fragment is flanked at its 5' end by a 5' exon that contains the 5' catalytic group I intronic fragment that is recognized by the 5' catalytic group I intronic fragment. 'Exon sequence; the 3' catalytic group I intronic fragment is flanked at its 3' end by a 3' exon containing the 3' catalytic group I intronic fragment that is recognized by the 3' catalytic group I intronic fragment 'Exon sequence. As used herein, the terms "5' exon sequence" and "3' exon sequence" are labeled according to the order of the exons in their natural environment relative to the Group I introns.

As used herein, the terms "adenine", "guanine", "cytosine", "thymine", "uracil" and "hypoxanthine" refer to the nucleobase itself. The terms "adenosine", "guanosine", "cytidine", "thymidine", "uridine" and "inosine" refer to a nucleobase attached to a ribose or deoxyribose moiety. The term "nucleoside" refers to a nucleoside attached to ribose or deoxyribose. The term "nucleotide" refers to the corresponding nucleobase-ribosyl-phosphate or nucleobase-deoxyribosyl-phosphate. Sometimes the terms adenosine and adenine (abbreviated as "A"), guanosine and guanine (abbreviated as "G"), cytosine and cytidine (abbreviated as "C"), uracil and uridine (abbreviated as "U"), thymine and thymidine (abbreviated as "T"), inosine and hypoxanthine (abbreviated as "I") are used interchangeably to refer to the corresponding nucleobase, nucleoside or nucleotide. Sometimes, the terms nucleoside, nucleoside and nucleotide are used interchangeably unless the context clearly requires otherwise.

The term "functional protein" refers to a naturally occurring protein, a functional variant thereof, or an engineered derivative thereof, which is functional in the treatment of a genetic disease or disorder. A disease or disorder may be caused in whole or in part by changes, such as mutations, in a wild-type naturally occurring protein that corresponds to a functional protein.

As used herein, the expression that a region is "located x to y nucleotides upstream of the target adenosine" refers to a region that may begin at any nucleotide within "x to y nucleotides upstream of the target adenosine" . For example, when the mismatched region is 4 nucleotides in length and is located 21 nucleotides to 30 nucleotides upstream of the target adenosine relative to the target RNA, the mismatched region can begin at any of the following: The target RNA is 21nt to 25nt, 22nt to 26nt, 23nt to 27nt, 24nt to 28nt, 25nt to 29nt, 26nt to 30nt, 27nt to 31nt, 28nt to 32nt, 29nt to 33nt or 30nt to 34nt upstream of adenosine. As used herein, the expression that a region is "located x to y nucleotides downstream of the target adenosine" means that the region may begin at any nucleotide within "x to y nucleotides downstream of the target adenosine." For example, when the mismatched region is 10 nucleotides in length and is located 31 nucleotides to 43 nucleotides downstream of the target adenosine relative to the target RNA, the mismatched region can begin at any of the following: The target RNA is 31nt to 40nt, 32nt to 41nt, 33nt to 42nt, 34nt to 43nt, 35nt to 44nt, 36nt to 45nt, 37nt to 46nt, 38nt to 47nt, 39nt to 48nt, 40nt to 49nt, 41nt to 50nt, downstream of adenosine. 42nt to 51nt or 43nt to 52nt.

The present disclosure provides several types of polynucleotide or polypeptide-based compositions, including variants and derivatives. These include, for example, substitutions, insertions, deletions and covalent variants and derivatives. The term "derivative" is synonymous with the term "variant" and generally refers to a molecule that has been modified and/or altered in any way relative to a reference or starting molecule.

The term "introduction" or "introduction" as used herein refers to the delivery of one or more polynucleotides, such as dRNA, or one or more constructs including a vector described herein, one or more transcripts thereof, to a host cell. The present invention serves as a basic platform for targeted editing of RNA, such as pre-messenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long non-coding RNA and small RNA (such as miRNA). The methods of the present application can use a number of delivery systems, including but not limited to viruses, liposomes, electroporation, microinjection, and conjugation, to achieve introduction of the dRNA or constructs described herein into host cells. Traditional viral and non-viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding the dRNA of the present application to cells or host organisms in culture. Non-viral vector delivery systems include DNA plasmids, RNA (eg, transcripts of the constructs described herein), naked nucleic acids, and nucleic acids complexed with delivery vehicles (eg, liposomes). Viral vector delivery systems include DNA and RNA viruses that have episomal or integrated genomes for delivery to host cells.

In the context of this application, "target RNA" refers to an RNA sequence to which the deaminase-recruiting RNA sequence is designed to have complete complementarity or substantial complementarity, and between the target sequence and the dRNA. Hybridization between the two forms a double-stranded RNA (dsRNA) region containing the target adenosine, which recruits adenosine deaminase (ADAR) that acts on RNA to deaminate the target adenosine. In some embodiments, ADARs occur naturally in host cells, such as eukaryotic cells (eg, mammalian cells, such as human cells). In some embodiments, ADAR is introduced into the host cell.

As used herein, "operably linked", when referring to a first nucleic acid sequence operably linked to a second nucleic acid sequence, refers to the situation when the first nucleic acid sequence and the second nucleic acid sequence are in a functional relationship. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription of the coding sequence. Likewise, if the signal peptide affects extracellular secretion of the polypeptide, the coding sequence for the signal peptide is operably linked to the coding sequence for the polypeptide. Typically, operably linked nucleic acid sequences are contiguous and the open reading frames are aligned where necessary to join two protein coding regions.

As used herein, "ligating" means joining nucleic acid sequences, either directly or indirectly, such as through an inserted nucleic acid sequence.

As used herein, "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through traditional Watson-Crick base pairing. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairs) with a second nucleic acid (e.g., about 5, 6, 7, 8, 9, 10/10, respectively) are approximately 50%, 60%, 70%, 80%, 90% and 100% complementary). "Perfectly complementary" means that all contiguous residues of a nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" means at least about 70%, 75%, 80% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides. The degree of complementarity of any one of %, 85%, 90%, 95%, 97%, 98%, 99% or 100%, or refers to two nucleic acids that hybridize under stringent conditions.

As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence hybridizes primarily to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are often sequence dependent and depend on many factors. In general, the longer the sequence, the higher the temperature at which the sequence will specifically hybridize to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology- Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay," Elsevier ,N,Y.

"Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between hydroxyl groups of nucleotide residues. Hydrogen bonding can occur through Watson Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. A sequence that hybridizes to a given sequence is called the "complement" of the given sequence.

"Subject", "patient" or "individual" includes mammals, such as humans or other animals, typically humans. In some embodiments, a subject, such as a patient, to which therapeutic agents and compositions are administered is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or ape. Subjects may be male or female and may be of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent, a dog, a cat, a farm animal, such as a cow or a horse, and the like.

As used herein, the term "treatment" refers to a clinical intervention designed to have a beneficial and desired effect on the natural course of the individual or cell being treated during clinical pathology. For the purposes of this disclosure, desired effects of treatment include, but are not limited to, reducing the rate of disease progression, ameliorating or alleviating disease status, and alleviating or improving prognosis. For example, if one or more symptoms associated with a progressive disease (such as Usher syndrome, MPS I, or a disease associated with PPIA) are reduced or eliminated, including but not limited to reducing the loss of host cells (e.g., reducing the loss of rod photoreceptor cells) ), reduce the symptoms caused by the disease, prevent the spread of the disease, improve the quality of life of the person suffering from the disease, reduce the dosage of other drugs needed to treat the disease, delay or prevent the progression of the disease (such as delaying or preventing the loss of vision), and /or extend the survival period of the individual.

As used herein, the term "effective amount" or "therapeutically effective amount" of a substance is at least the minimum concentration required to achieve measurable improvement or prevention of a particular disease. The effective amount herein may vary depending on factors such as the disease state, age, sex, and weight of the patient, as well as the ability of the substance to elicit the desired response in the individual. An effective amount is also an amount in which any toxic or deleterious effects of the treatment are offset by the therapeutically beneficial effects. When referring to a progressive disease (e.g., a disease manifested by progressive loss of function), an effective amount includes an amount sufficient to prevent or delay progression of the disease (e.g., prevent further loss of cells) or prevent or delay the development of symptoms of the disease (e.g., prevent loss of vision) . In some embodiments, an effective amount is an amount sufficient to delay disease progression (such as, but not limited to, delaying the loss of retinal cells). In some embodiments, an effective amount is an amount sufficient to delay the progression of disease symptoms (such as, but not limited to, delaying vision loss). With reference to cancer, an effective amount includes an amount sufficient to cause tumor shrinkage and/or reduce the rate of tumor growth (eg, inhibit tumor growth) or prevent or delay other undesirable cell proliferation in cancer. In some embodiments, an effective amount is an amount sufficient to delay the development of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay relapse. In some embodiments, an effective amount is an amount sufficient to reduce the rate of relapse in an individual. An effective amount can be administered once or multiple times. An effective amount of the drug or composition can: (i) reduce the number of cancer cells; (ii) reduce the size of the tumor; (iii) inhibit, delay, slow to a certain extent and preferably prevent the infiltration of cancer cells into peripheral organs; (iv) Inhibit (i.e., slow down and preferably stop to a certain extent) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and/or recurrence of tumors; (vii) reduce the recurrence rate of tumors, and/or (viii ) relieves one or more symptoms associated with cancer to some extent. An effective amount can be administered once or multiple times. For the purposes of this disclosure, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, preventive or therapeutic treatment. As understood in the clinical context, an effective amount of a drug, compound or pharmaceutical composition may or may not be achieved in combination with another drug, compound or pharmaceutical composition. Thus, an "effective amount" may be considered where one or more therapeutic agents are administered, and administration of a single agent may be considered in an effective amount if in combination with one or more other agents the desired result is achieved or achieved.

As used herein, the term "wild-type" is a term understood by those skilled in the art to refer to the typical form of an organism, strain, gene or trait found in nature, as opposed to a mutant or variant form.

"Host cell" as used herein refers to any cell type that can be used as a host cell so long as it can be modified as described herein. For example, the host cell may be one that has endogenously expressed RNA-acting adenosine deaminase (ADAR), or may be one in which RNA-acting adenosine deaminase (ADAR) has been introduced by methods known in the art. ADAR) host cells. For example, the host cell can be a prokaryotic cell, a eukaryotic cell, or a plant cell. In some embodiments, host cells are derived from pre-established cell lines, such as mammalian cell lines, including human cell lines or non-human cell lines. In some embodiments, the host cells are derived from an individual, such as a human individual.

"Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector comprising one or more heterologous sequences (i.e., nucleic acid sequences of non-AAV origin) flanked by at least one (in embodiments two ) AAV inverted terminal repeat (ITR). Such rAAV vectors can be replicated when they are present in a host cell that has been infected with a suitable helper virus (or a virus expressing a suitable helper) and expresses the AAV rep and cap gene products (i.e., the AAV Rep and cap proteins) and packaged into infectious virus particles. When the rAAV vector is integrated into a larger polynucleotide (e.g., in the chromosome or in another vector, such as a plasmid used for cloning or transfection), the rAAV vector is referred to as a "pro-vector" )", which can be "rescued" through replication and encapsidation in the presence of AAV packaging capabilities and appropriate assistive tools. rAAV vectors can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, encapsidated in liposomes, and encapsidated in viral particles, particularly AAV particles. rAAV vectors can be packaged into AAV viral capsids to produce "recombinant adeno-associated virus particles (rAAV particles)."

The "AAV inverted terminal repeat (ITR)" sequence is a term well known in the art and is a sequence of approximately 145 nucleotides present at both ends of the native single-stranded AAV genome. The outermost 125 nucleotides of the ITR can exist in either of two alternative orientations, leading to heterogeneity between different AAV genomes and between the ends of a single AAV genome. The outermost 125 nucleotides also contain several shorter self-complementary regions (referred to as the A, A', B, B', C, C', and D regions) that allow for intrastrand tRNA to occur within this portion of the ITR Base pairing.

Accordingly, polynucleotides encoding peptides or polypeptides comprising substitutions, insertions and/or additions, deletions and covalent modifications relative to reference sequences, particularly the polypeptide sequences disclosed herein, are included within the scope of the present disclosure. . For example, a sequence tag or amino acid, such as one or more lysines, can be added to the peptide sequence (eg, at the N-terminus or C-terminus). Sequence tags can be used for peptide detection, purification, or localization. Lysine can be used to increase peptide solubility or allow biotinylation. Alternatively, amino acid residues located in the carboxyl and amino-terminal regions of the amino acid sequence of the peptide or protein can optionally be deleted to provide a truncated sequence. Depending on the use of the sequence, e.g., expressing the sequence as part of a larger sequence soluble or linked to a solid support, certain amino acids (e.g., C-terminal residues or N-terminal residues) may selectively be delete.

The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate the involvement of human hands. When referring to a nucleic acid molecule or polypeptide, these terms mean that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which they are naturally associated in nature and as found in nature.

As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (eg, into mRNA or other RNA transcripts) and/or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides may collectively be referred to as "gene products." If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells.

As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic at the dosage and concentration employed to the cells or mammal to which it is exposed. Typically physiologically acceptable carriers are pH buffered aqueous solutions. Non-limiting examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin , gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other sugars including glucose, mannose Sugar or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, polyethylene glycol (PEG), and PLURONICS™ .

The term "package insert" is used to refer to the instructions typically included in the commercial packaging of a therapeutic product that contain information regarding the indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings regarding the use of such therapeutic product .

An "article of manufacture" is any article of manufacture (eg, package or container) or kit containing at least one agent, eg, a medicament, for treating a disease or condition. In some embodiments, articles of manufacture or kits are promoted, distributed, or sold as units for performing the methods described herein.

As used herein, the terms "includes," "contains," and "includes" are used in their open, non-limiting sense. It will also be understood that aspects and embodiments of the application described herein may include aspects and embodiments "consisting of" and/or "consisting essentially of."

It will be understood that each quantity given herein, whether or not the term "about" is expressly used, means the actual given value, and also means an estimate of such given value that can reasonably be inferred based on ordinary skill in the art. Approximations, including equivalents and approximations resulting from experimental and/or measurement conditions for such given values. Reference herein to "about" a value or parameter includes (and describes) variations with respect to the value or parameter itself. For example, descriptions that refer to "about X" include descriptions of "X."

As used herein, reference to a value or parameter that is "not" generally means and describes "different from" the value or parameter. For example, a method that is not used to treat type X disease means that the method is used to treat a disease other than type X.

The term "about X-Y" as used herein has the same meaning as "about X to about Y."

As used herein and in the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the scope of a patent application can be drafted to exclude any optional elements. Accordingly, this statement is intended to serve as a prior basis for the use of exclusive terms such as "only" and "only" when referencing elements of the claimed patent scope or using "negative" limitations.

As used herein, the term "and/or" and phrases such as "A and/or B" are intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used herein, such as the phrase "A, B and/or C" is intended to encompass each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

II.RNA Edit method

Provided herein are methods of editing a target RNA in a host cell, comprising introducing into the host cell a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA, wherein the dRNA comprises a compound capable of hybridizing to the target RNA to form an RNA duplex. A targeting RNA sequence, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate a target adenosine in the target RNA, wherein the RNA duplex includes one or more RNA duplexes relative to the target RNA A mismatch region, and wherein the dRNA includes a linker nucleic acid sequence flanking the terminus of the target RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure. The dRNA can be any of the dRNAs described in Section 3 below ("dRNA, Constructs and Libraries"). In some embodiments, the dRNA is linear. In some embodiments, the dRNA is circular. In some embodiments, the dRNA is a linear RNA capable of forming a circular RNA. In some embodiments, the methods use constructs comprising nucleic acid sequences encoding dRNA. The construct may be any of the constructs described in Section 3 below.

In some embodiments, a method of editing a target adenosine in a target RNA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Contains a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex The strand includes: (a) a first mismatch region relative to the target RNA sequence located 5 nucleotides to 85 nucleotides upstream of the target adenosine; and/or (b) a first mismatch region relative to the target RNA sequence A two-mismatch region located 20 nucleotides to 85 nucleotides downstream of the target adenosine; wherein the dRNA includes a linker nucleic acid sequence flanking the end of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and is substantially No secondary structure is formed on it. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence, the first mismatch region being: 5 nucleotides to 25 nucleotides upstream of the target adenosine, or the target adenosine 5 nucleotides to 15 nucleotides upstream of the target adenosine, or 20 nucleotides to 40 nucleotides upstream of the target adenosine. In some embodiments, the RNA duplex comprises a second mismatch region relative to the target RNA sequence, the second mismatch region being: 20 nucleotides to 65 nucleotides downstream of the target adenosine, or the target adenosine 20 nucleotides to 45 nucleotides downstream of the target adenosine, or 25 nucleotides to 45 nucleotides downstream of the target adenosine. In some embodiments, the first mismatched region is 1-50 nucleotides in length; and/or the second mismatched region is 1-50 nucleotides in length. In some embodiments, the linker nucleic acid sequence is from about 5 nucleotides (nt) to about 500 nt in length. In some embodiments, the editing rate of non-target adenosine is reduced by at least about 20%, 30%, 50%, 60%, 70%, compared to methods using dRNA comprising a targeting RNA sequence that is complementary to the target RNA. 80%, 90%, 95% or more. In some embodiments, the editing efficiency of the target adenosine is increased by at least about 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, compared to methods using dRNA that does not include a linker nucleic acid sequence. 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more.

In some embodiments, a method of editing a target adenosine in a target RNA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Contains a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex The strand includes: (a) a first mismatch region relative to the target RNA sequence located 20 nucleotides to 40 nucleotides upstream of the target adenosine; and/or (b) a first mismatch region relative to the target RNA sequence A two-mismatch region located 25 nucleotides to 45 nucleotides downstream of the target adenosine; wherein the dRNA includes a linker nucleic acid sequence flanking the end of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and is substantially No secondary structure is formed on it. In some embodiments, the first mismatched region is 1-50 nucleotides in length; and/or the second mismatched region is 1-50 nucleotides in length. In some embodiments, the linker nucleic acid sequence is from about 5 nucleotides (nt) to about 500 nt in length. In some embodiments, the editing rate of non-target adenosine is reduced by at least about 20%, 30%, 50%, 60%, 70%, compared to methods using dRNA comprising a targeting RNA sequence that is complementary to the target RNA. 80%, 90%, 95%, or more. In some embodiments, the editing efficiency of the target adenosine is increased by at least about 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, compared to methods using dRNA that does not include a linker nucleic acid sequence. 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more.

In some embodiments, a method of reducing editing of non-target adenosine in a target RNA (also referred to herein as "bystander editing") in a host cell is provided, comprising converting a deaminase recruiting RNA (dRNA) or A construct comprising a nucleic acid encoding a dRNA comprising a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex capable of recruiting an adenosine deaminase (ADAR) enzyme acting on the RNA is introduced into the host cell. Deaminating a target adenosine in a target RNA, wherein the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence located 20 nucleotides to 40 nucleotides upstream of the target adenosine ; and/or (b) a second mismatch region relative to the target RNA sequence located 25 nucleotides to 45 nucleotides downstream of the target adenosine; wherein the dRNA includes a linker flanking the end of the target RNA sequence Nucleic acid sequences wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure. In some embodiments, the first mismatched region is 1-50 nucleotides in length; and/or the second mismatched region is 1-50 nucleotides in length. In some embodiments, the linker nucleic acid sequence is from about 5 nt to about 500 nt in length. In some embodiments, the editing rate of non-target adenosine is reduced by at least about 20%, 30%, 50%, 60%, 70%, compared to methods using dRNA comprising a targeting RNA sequence that is complementary to the target RNA. 80%, 90%, 95% or more.

In some embodiments, a method for increasing the editing efficiency of a target adenosine in a target RNA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cells, wherein the dRNA contains a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate the target adenosine in the target RNA , wherein the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence, which is located 20 nucleotides to 40 nucleotides upstream of the target adenosine; and/or (b) relative to the target RNA sequence A second mismatched region of the RNA sequence located 25 nucleotides to 45 nucleotides downstream of the target adenosine; wherein the dRNA includes a linker nucleic acid sequence flanking the end of the targeting RNA sequence, wherein the linker nucleic acid sequence is not identical to the target RNA hybridizes and essentially does not form secondary structure. In some embodiments, the RNA duplex contains a mismatch region relative to the target RNA sequence located 20 nucleotides to 40 nucleotides upstream of the target adenosine. In some embodiments, the RNA duplex contains a mismatch region relative to the target RNA sequence, the mismatch region being located 26 nucleotides upstream of the target adenosine. In some embodiments, the RNA duplex contains a mismatch region relative to the target RNA sequence, the mismatch region being located 30 nucleotides upstream of the target adenosine. In some embodiments, the RNA duplex contains a mismatch region relative to the target RNA sequence, the mismatch region being located 34 nucleotides upstream of the target adenosine. In some embodiments, the RNA duplex contains a mismatch region relative to the target RNA sequence, the mismatch region being located 25 nucleotides to 45 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex contains a mismatch region relative to the target RNA sequence, the mismatch region being located 31 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex contains a mismatch region relative to the target RNA sequence, the mismatch region being located 35 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex contains a mismatch region relative to the target RNA sequence, the mismatch region being located 39 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex contains a mismatch region relative to the target RNA sequence, which is located 40 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex contains a mismatch region relative to the target RNA sequence, the mismatch region being located 41 nucleotides downstream of the target adenosine. In some embodiments, the first mismatched region is 1-50 nucleotides in length; and/or the second mismatched region is 1-50 nucleotides in length. In some embodiments, the editing efficiency of the target adenosine is increased by at least about 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, compared to methods using dRNA that does not include a linker nucleic acid sequence. 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more.

In some embodiments, the first mismatch region comprises: (a) one or more non-complementary nucleotides (mismatch) in the targeting RNA; and/or (b) one or more cores of the targeting RNA Deletion of nucleotides; and/or (c) insertion of one or more nucleotides into the targeted RNA. In some embodiments, the second mismatch region comprises: (a) one or more non-complementary nucleotides (mismatch) in the targeting RNA; and/or (b) one or more cores of the targeting RNA Deletion of nucleotides; and/or (c) insertion of one or more nucleotides into the targeting RNA. In some embodiments, the first mismatch region includes: (a) at least one contiguous set of non-complementary nucleotides (mismatch) in the targeting RNA; and/or (b) at least one contiguous set of nucleotides in the targeting RNA Deletion of nucleotides; and/or (c) insertion of at least one contiguous set of nucleotides in the targeting RNA. In some embodiments, the second mismatch region includes: (a) at least one contiguous set of non-complementary nucleotides (mismatch) in the targeting RNA; and/or (b) at least one contiguous set of nucleotides in the targeting RNA Deletion of nucleotides; and/or (c) insertion of at least one contiguous set of nucleotides in the targeting RNA. In some embodiments, targeting non-complementary nucleotides in RNA results in bubble-like structures in the RNA duplex. In some embodiments, nucleotide deletions in the targeted RNA result in bulge structures in the RNA duplex. In some embodiments, targeting nucleotide insertion in the RNA results in bulge structures in the RNA duplex. In some embodiments, targeting a set of contiguous non-complementary nucleotides in RNA results in bubble-like structures in the RNA duplex. In some embodiments, deletion of a contiguous set of nucleotides in the targeted RNA results in a bulge structure in the RNA duplex. In some embodiments, insertion of a contiguous set of nucleotides into the targeted RNA results in a bulge structure in the RNA duplex.

In some embodiments, the first mismatched region is 1-50 nucleotides in length. In some embodiments, the second mismatched region is 1-50 nucleotides in length. In some embodiments, the first mismatched region is about any one of 1-10, 10-20, 20-30, 30-40, or 40-50 nucleotides in length. In some embodiments, the length of the first mismatched region is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , any of 19 or 20 nucleotides. In some embodiments, the second mismatched region is about any one of 1-10, 10-20, 20-30, 30-40, or 40-50 nucleotides in length. In some embodiments, the length of the second mismatched region is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , any of 19 or 20 nucleotides.

In some embodiments, the first mismatched region is 1-10 nucleotides in length; and/or the second mismatched region is 1-10 nucleotides in length. In some embodiments, the first mismatched region contains 1-10 contiguous non-complementary nucleotides in the targeting RNA. In some embodiments, the first mismatched region contains a deletion of 1-10 contiguous nucleotides of the targeting RNA. In some embodiments, the second mismatched region contains 1-10 contiguous non-complementary nucleotides of the targeting RNA. In some embodiments, the second mismatched region contains a deletion of 1-10 contiguous nucleotides of the targeting RNA.

In some embodiments, the first mismatched region is 4 nucleotides in length; and/or the second mismatched region is 4 nucleotides in length. In some embodiments, the first mismatch region includes four consecutive non-complementary nucleotides in the targeting RNA. In some embodiments, the first mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA. In some embodiments, the second mismatched region comprises four consecutive non-complementary nucleotides in the targeting RNA. In some embodiments, the second mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA. In some embodiments according to any of the above methods, wherein the first mismatched region is located 20 nucleotides to 40 nucleotides upstream of the target adenosine relative to the target RNA sequence, and wherein the first mismatched region 4 nucleotides in length, the first mismatched region is any of the following: 20 to 23, 21 to 24, 22 to 25, 23 to 26, 24 to 27, 25 to 25 upstream of the target adenosine relative to the target RNA 28, 26 to 29, 27 to 30, 28 to 31, 29 to 32, 30 to 33, 31 to 34, 32 to 35, 33 to 36, 34 to 37, 35 to 38, 36 to 39, or 37 to 40 nucleotide location. In some embodiments according to any one of the above methods, wherein the second mismatched region is located 25 nucleotides to 45 nucleotides downstream of the target adenosine relative to the target RNA sequence, and wherein the second mismatched region 4 nucleotides in length, the second mismatched region is any of the following: 25 to 28, 26 to 29, 27 to 30, 28 to 31, 29 to 32, 30 to 30 downstream of the target adenosine relative to the target RNA 33, 31 to 34, 32 to 35, 33 to 36, 34 to 37, 35 to 38, 36 to 39, 37 to 40, 38 to 41, 39 to 42, 40 to 43, 41 to 44, or 42 to 45 nucleotide location.

In some embodiments, the dRNA is circular. In some embodiments, the dRNA is linear. In some embodiments, dRNA can be circularized (eg, to form a circular RNA).

In some embodiments, the target RNA encodes a mutant Usher 2A protein according to any of the methods described above for editing a target adenosine in a target RNA in a host cell. In some embodiments, mutant Usher 2A proteins comprise missense mutations, nonsense mutations, and/or frameshift mutations. In some embodiments, the mutant Usher 2A protein is a truncated Usher 2A protein. In some embodiments, the target RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation. In some embodiments, the target RNA encoding mutant Usher 2A comprises a G to A mutation relative to the target RNA encoding wild-type Usher 2A. In some embodiments, the target RNA encoding mutant Usher 2A comprises the mutation 11864G>A with reference to the target RNA encoding wild-type Usher 2A. In some embodiments, the target RNA is endogenously expressed.

In some embodiments, a method of editing a target RNA encoding mutant Usher 2A in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Contains a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex The strand includes: (a) a first mismatch region relative to the target RNA sequence located 20 nucleotides to 40 nucleotides upstream of the target adenosine; and/or (b) a first mismatch region relative to the target RNA sequence A two-mismatch region located 25 nucleotides to 45 nucleotides downstream of the target adenosine; wherein the dRNA includes a linker nucleic acid sequence flanking the end of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and is substantially No secondary structure is formed on it. In some embodiments, the target RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation. In some embodiments, the target RNA encoding mutant Usher 2A comprises a G to A mutation relative to the target RNA encoding wild-type Usher 2A. In some embodiments, the target RNA encoding mutant Usher 2A comprises the mutation 11864G>A with reference to the target RNA encoding wild-type Usher 2A. In some embodiments, with reference to SEQ ID NO: 3, the target adenosine is located at position 101.

In some embodiments, according to any of the above methods for editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes a mutant Usher 2A protein, the RNA duplex further comprises a third mismatch relative to the target RNA area. In some embodiments, the third mismatched region is located between the first mismatched region and the second mismatched region relative to the target RNA. In some embodiments, the third mismatch region includes one or two non-complementary nucleotides in the targeting RNA and/or a deletion of one or two nucleotides in the targeting RNA sequence. In some embodiments, the third mismatch region relative to the target RNA sequence is located 7 and/or 8 nucleotides downstream of the target adenosine. In some embodiments, the target RNA includes an adenosine located 7th and/or 8th nucleotide downstream of the target adenosine. In some embodiments, the target RNA comprises an "AA" sequence located 7 and 8 nucleotides downstream of the target adenosine, wherein the targeting RNA sequence comprises any of the following: A, AA, U, C, CC, G , GG, or a deletion ("X") of a nucleotide opposite the target RNA located 7 and 8 nucleotides downstream of the target adenosine.

In some embodiments, according to any of the above methods for editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes a mutant Usher 2A protein, the RNA duplex comprises: (a) relative to the target RNA sequence a first mismatch region located between 27 nucleotides and 30 nucleotides (e.g., 27 nucleotides) upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence, which Located 31 nt to 43 nt downstream of the target adenosine. In some embodiments, the second mismatch region relative to the target RNA sequence is located 32 nucleotides to 35 nucleotides (eg, 32 nucleotides) downstream of the target adenosine. In some embodiments, the second mismatch region relative to the target RNA sequence is located 36 nucleotides to 39 nucleotides (eg, 36 nucleotides) downstream of the target adenosine. In some embodiments, the second mismatch region relative to the target RNA sequence is located 40 nucleotides to 43 nucleotides (eg, 40 nucleotides) downstream of the target adenosine. In some embodiments, the first mismatched region is 4 nucleotides in length. In some embodiments, the first mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA. In some embodiments, the second mismatch region is 4 nucleotides in length. In some embodiments, the second mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA.

In some embodiments, according to any of the above methods for editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes a mutant Usher 2A protein, the RNA duplex comprises: (a) relative to the target RNA sequence a first mismatched region located 21 nucleotides to 30 nucleotides upstream of the target adenosine; and (b) a second mismatched region relative to the target RNA sequence located 36 nt downstream of the target adenosine to 43 nucleotides. In some embodiments, the second mismatch region relative to the target RNA sequence is located 36 nucleotides to 39 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 40 nucleotides to 43 nucleotides downstream of the target adenosine. In some embodiments, the first mismatched region is 10 nucleotides in length. In some embodiments, the first mismatched region contains a deletion of ten consecutive nucleotides of the targeting RNA. In some embodiments, the second mismatch region is 4 nucleotides in length. In some embodiments, the second mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA.

In some embodiments, according to any of the above methods for editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes a mutant Usher 2A protein, the RNA duplex comprises: (a) relative to the target RNA sequence a first mismatched region located 26 nucleotides to 35 nucleotides upstream of the target adenosine; and (b) a second mismatched region relative to the target RNA sequence located 35 nt downstream of the target adenosine to 44 nucleotides. In some embodiments, the first mismatched region is 1 nucleotide in length and the second mismatched region is 1 nucleotide in length. In some embodiments, the first mismatched region is 2 nucleotides in length and the second mismatched region is 2 nucleotides in length. In some embodiments, the first mismatched region is 3 nucleotides in length and the second mismatched region is 3 nucleotides in length. In some embodiments, the first mismatched region is 4 nucleotides in length and the second mismatched region is 4 nucleotides in length. In some embodiments, the first mismatched region is 7 nucleotides in length and the second mismatched region is 7 nucleotides in length. In some embodiments, the first mismatched region is 10 nucleotides in length and the second mismatched region is 10 nucleotides in length. In some embodiments, the first mismatch region includes a nucleotide insertion targeting the RNA and the second mismatch region includes a nucleotide insertion targeting the RNA. In some embodiments, the first mismatched region contains an insertion of two consecutive nucleotides into the targeting RNA and the second mismatched region contains an insertion into the targeting RNA at two consecutive nucleotides. In some embodiments, the first mismatched region contains an insertion of three contiguous nucleotides of the targeting RNA and the second mismatched region contains an insertion of three contiguous nucleotides of the targeting RNA. In some embodiments, the first mismatched region contains an insertion of four contiguous nucleotides of the targeting RNA and the second mismatched region contains an insertion of four contiguous nucleotides of the targeting RNA. In some embodiments, the first mismatched region contains an insertion of seven contiguous nucleotides of the targeting RNA and the second mismatched region contains an insertion of seven contiguous nucleotides of the targeting RNA. In some embodiments, the first mismatched region contains an insertion of ten contiguous nucleotides of the targeting RNA and the second mismatched region contains an insertion of ten contiguous nucleotides of the targeting RNA.

In some embodiments, according to any of the above methods for editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes a mutant Usher 2A protein, the RNA duplex comprises: a mismatched region relative to the target RNA sequence , which is located 26 nucleotides to 37 nucleotides upstream of the target adenosine. In some embodiments, the mismatch region relative to the target RNA sequence is located 26 nucleotides to 29 nucleotides downstream of the target adenosine. In some embodiments, the mismatch region relative to the target RNA sequence is located 30 nucleotides to 33 nucleotides downstream of the target adenosine. In some embodiments, the mismatch region relative to the target RNA sequence is located 34 nucleotides to 37 nucleotides downstream of the target adenosine. In some embodiments, the mismatch region is 4 nucleotides in length. In some embodiments, the mismatched region contains a deletion of four consecutive nucleotides of the targeting RNA.

In some embodiments, according to any of the above methods for editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes a mutant Usher 2A protein, the RNA duplex comprises: (a) relative to the target RNA sequence and (b) a second mismatched region relative to the target RNA sequence located 31 nucleotides downstream of the target adenosine. to 39 nucleotides. In some embodiments, the first mismatch region relative to the target RNA sequence is located 26 nucleotides to 29 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region relative to the target RNA sequence is located 30 nucleotides to 33 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region relative to the target RNA sequence is located 34 nucleotides to 37 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 31 nucleotides to 34 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 35 nucleotides to 38 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 39 nucleotides to 42 nucleotides downstream of the target adenosine. In some embodiments, the first mismatched region is 4 nucleotides in length. In some embodiments, the first mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA. In some embodiments, the second mismatch region is 4 nucleotides in length. In some embodiments, the second mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA.

In some embodiments, according to any of the above methods for editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes a mutant Usher 2A protein, the RNA duplex comprises: (a) relative to the target RNA sequence a first mismatched region located 26 nucleotides to 29 nucleotides upstream of the target adenosine; and (b) a second mismatched region relative to the target RNA sequence located 35 nucleotides downstream of the target adenosine to 38 nucleotides. In some embodiments, the first mismatched region is 4 nucleotides in length. In some embodiments, the first mismatched region comprises a deletion of four consecutive nucleotides in the targeting RNA. In some embodiments, the second mismatch region is 4 nucleotides in length. In some embodiments, the second mismatched region comprises a deletion of four consecutive nucleotides in the targeting RNA. In some embodiments, the RNA duplex further comprises a third mismatch region located 5 nucleotides upstream of the target adenosine relative to the target RNA sequence, and 3 nucleotides downstream of the target adenosine relative to the target RNA sequence. The fourth mismatch region at . In some embodiments, the third mismatched region contains a uracil deletion of the targeting RNA. In some embodiments, the fourth mismatched region contains a uracil deletion of the targeting RNA. In some embodiments, the RNA duplex further comprises a third mismatch region located 5 nucleotides upstream of the target adenosine relative to the target RNA sequence, and 13 nucleotides downstream of the target adenosine relative to the target RNA sequence. The fourth mismatch region at . In some embodiments, the third mismatched region contains a uracil deletion of the targeting RNA. In some embodiments, the fourth mismatched region contains a uracil deletion of the targeting RNA. In some embodiments, the RNA duplex further comprises a third mismatch region located 3 nucleotides downstream of the target adenosine relative to the target RNA sequence, and 13 nucleotides downstream of the target adenosine relative to the target RNA sequence. The fourth mismatch region at . In some embodiments, the third mismatched region contains a uracil deletion of the targeting RNA. In some embodiments, the fourth mismatched region contains a uracil deletion of the targeting RNA. In some embodiments, the RNA duplex further comprises a third mismatch region located 5 nucleotides upstream of the target adenosine relative to the target RNA sequence and 3 nucleotides downstream of the target adenosine relative to the target RNA sequence. A fourth mismatch region, and a fifth mismatch region located 13 nucleotides downstream of the target adenosine relative to the target RNA sequence. In some embodiments, the third mismatched region contains a uracil deletion of the targeting RNA. In some embodiments, the fourth mismatched region contains a uracil deletion of the targeting RNA. In some embodiments, the fifth mismatched region contains a uracil deletion of the targeting RNA.

In some embodiments, according to any of the above methods for editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes a mutant IDUA protein, the RNA duplex comprises: a mismatched region relative to the target RNA sequence, It is located 40 nucleotides to 44 nucleotides downstream of the target adenosine. In some embodiments, the mismatch region relative to the target RNA sequence is located 40 nucleotides to 44 nucleotides downstream of the target adenosine. In some embodiments, the mismatch region relative to the target RNA sequence is located 41 nucleotides to 43 nucleotides downstream of the target adenosine. In some embodiments, the mismatch region is 3 nucleotides in length. In some embodiments, the mismatched region contains a deletion of three consecutive nucleotides of the targeting RNA. In some embodiments, the mismatch region is 5 nucleotides in length. In some embodiments, the mismatched region contains a deletion of five consecutive nucleotides of the targeting RNA.

In some embodiments, according to any of the above methods for editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes a mutant IDUA protein, the RNA duplex comprises: (a) a third sequence relative to the target RNA sequence; a mismatched region located 6 nucleotides to 10 nucleotides upstream of the target adenosine; and (b) a second mismatched region relative to the target RNA sequence located 30 nucleotides downstream of the target adenosine Acid to 44 nucleotides. In some embodiments, the first mismatch region relative to the target RNA sequence is located 6 to 7 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region relative to the target RNA sequence is located 6 nucleotides to 8 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region relative to the target RNA sequence is located 6 nucleotides to 9 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region relative to the target RNA sequence is located 6 nucleotides to 10 nucleotides downstream of the target adenosine. In some embodiments, the second mismatch region relative to the target RNA sequence is located 30 nucleotides to 31 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 30 nucleotides to 32 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 30 nucleotides to 33 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 30 nucleotides to 34 nucleotides downstream of the target adenosine. In some embodiments, the second mismatch region relative to the target RNA sequence is located 40 nucleotides to 44 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 41 nucleotides to 43 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region is 2 nucleotides in length. In some embodiments, the first mismatch region comprises a mismatch of two consecutive nucleotides of the targeting RNA. In some embodiments, the first mismatched region is 3 nucleotides in length. In some embodiments, the first mismatch region comprises a mismatch of three consecutive nucleotides of the targeting RNA. In some embodiments, the first mismatched region is 4 nucleotides in length. In some embodiments, the first mismatch region comprises a mismatch of four consecutive nucleotides of the targeting RNA. In some embodiments, the first mismatched region is 5 nucleotides in length. In some embodiments, the second mismatched region is 2 nucleotides in length. In some embodiments, the second mismatch region comprises a mismatch of two consecutive nucleotides of the targeting RNA. In some embodiments, the second mismatched region is 3 nucleotides in length. In some embodiments, the second mismatch region comprises a mismatch of three consecutive nucleotides of the targeting RNA. In some embodiments, the second mismatch region is 4 nucleotides in length. In some embodiments, the second mismatch region comprises a mismatch of four consecutive nucleotides of the targeting RNA. In some embodiments, the second mismatched region is 5 nucleotides in length. In some embodiments, the RNA duplex further comprises a third mismatch region located 60 nucleotides to 64 nucleotides downstream of the target adenosine relative to the target RNA sequence. In some embodiments, the third mismatch region comprises a mismatch from two consecutive nucleotides of the targeting RNA. In some embodiments, the third mismatch region comprises a mismatch of three consecutive nucleotides of the targeting RNA. In some embodiments, the third mismatch region comprises a mismatch of four consecutive nucleotides of the targeting RNA. In some embodiments, the third mismatch region comprises a mismatch of five consecutive nucleotides of the targeting RNA.

In some embodiments, according to any of the above methods for editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes a PPIA protein, the RNA duplex comprises: (a) a first sequence relative to the target RNA a mismatched region located 26 nucleotides to 38 nucleotides upstream of the target adenosine; and (b) a second mismatched region relative to the target RNA sequence located 31 nucleotides downstream of the target adenosine to 44 nucleotides. In some embodiments, the first mismatch region relative to the target RNA sequence is located 27 nucleotides to 30 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region relative to the target RNA sequence is located 31 nucleotides to 34 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region relative to the target RNA sequence is located 35 nucleotides to 38 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 32 nucleotides to 35 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 36 nucleotides to 39 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 41 nucleotides to 44 nucleotides downstream of the target adenosine. In some embodiments, the first mismatched region is 4 nucleotides in length. In some embodiments, the second mismatch region is 4 nucleotides in length.

In some embodiments, according to any of the above methods for editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes a PPIA protein, the RNA duplex comprises: (a) a first sequence relative to the target RNA A mismatched region located 23 nucleotides to 24 nucleotides upstream of the target adenosine; (b) a second mismatched region relative to the target RNA sequence located 28 nucleotides downstream of the target adenosine 29 nucleotides, and (c) a third mismatch region relative to the target RNA sequence located 5 to 6 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region relative to the target RNA sequence is located 23 nucleotides to 24 nucleotides downstream of the target adenosine. In some embodiments, the second mismatch region relative to the target RNA sequence is located 28 nucleotides to 29 nucleotides upstream of the target adenosine. In some embodiments, the third mismatch region relative to the target RNA sequence is located 5 nucleotides to 6 nucleotides downstream of the target adenosine. In some embodiments, the first mismatched region is 1 nucleotide in length. In some embodiments, the second mismatched region is 1 nucleotide in length. In some embodiments, the third mismatch region is 1 nucleotide in length.

In some embodiments according to any of the methods described herein, the linker nucleic acid sequence is from about 5 nucleotides (nt) to about 500 nt in length. In some embodiments, the linker nucleic acid sequence is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 , 350, 400, 450 or 500 nt, or any length in between. In some embodiments, the linker nucleic acid sequence is less than or equal to 70 nt in length. In some embodiments, the length of the linker nucleic acid sequence is 10nt-50nt, 10nt-40nt, 10nt-30nt, 10nt-20nt, 20nt-50nt, 20nt-40nt, 20nt-30nt, 30nt-50nt, 30nt-40nt, or 40nt- Any integer between 50nt. In some embodiments, the linker nucleic acid sequence is from about 20 nt to about 60 nt in length. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the linker nucleic acid sequence is about 50 nt in length. In some embodiments, at least about 50%, 60%, 70%, 80%, 85%, 90%, or 95% of any one of the linker nucleic acid sequences comprise adenosine or cytidine. In some embodiments, at least about 50% to 60%, 60% to 70%, 70% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% Either of the linker nucleic acid sequences contains adenosine or cytidine. In some embodiments, all nucleic acid sequences in the linker contain adenosine or cytidine. In some embodiments, at least about 50% of the linker nucleic acids comprise adenosine. In some embodiments, at least about 50%, 60%, 70%, 80%, 85%, or 90% of any one of the linker nucleic acid sequences comprise adenosine. In some embodiments, about 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 85%, 85% to 90%, or 90% Any one to 95% of the linker nucleic acid sequences contain adenosine.

In some embodiments, the method increases the level of editing of target adenosine compared to corresponding methods in which the RNA duplex does not contain one or more mismatch regions and/or in which the dRNA does not contain a linker nucleic acid sequence. In some embodiments, the method exhibits an increase in the editing level of the target adenosine by at least about 10 compared to a corresponding method in which the RNA duplex does not comprise one or more mismatch regions and/or wherein the dRNA does not comprise a linker nucleic acid sequence. %, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2 times, 5 times, 10 times, 20 times, 50 times or more than 100 times. In some embodiments, the method reduces the binding of one or more non-target adenosines compared to a corresponding method in which the RNA duplex does not comprise one or more mismatch regions and/or wherein the dRNA does not comprise a linker nucleic acid sequence. (Spectator) Editing quality. In some embodiments, the method shows editing of one or more non-target adenosines compared to corresponding methods in which the RNA duplex does not comprise one or more mismatch regions and/or in which the dRNA does not comprise a linker nucleic acid sequence. The level is reduced by at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2x, 5x, 10x, 20x, 50x, or 100x More than times. In some embodiments, non-target adenosine is located within one or more mismatched regions. In some embodiments, the non-target adenosine is outside the mismatch region.

In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence.

In some embodiments, the first linker nucleic acid sequence is the same as the second linker nucleic acid sequence. In some embodiments, the first linker nucleic acid sequence is different from the second linker nucleic acid sequence. In some embodiments, the dRNA further comprises a 3' exon sequence and a 5' exon sequence, and the 3' exon sequence can be flanked by a 3' catalytic Group I intron targeting the 5' end of the RNA sequence. For fragment recognition, the 5' exon sequence is recognized by the 5' catalytic Group I intronic fragment flanking the 3' end of the targeting RNA sequence. In some embodiments, the dRNA also contains a 3' linker sequence and a 5' linker sequence. In some embodiments, the double-stranded RNA contains a bulge structure for each non-target adenosine in the target RNA.

In some embodiments, the targeting RNA sequence is greater than 50 nt in length. In some embodiments, the targeting RNA sequence in the dRNA is from about 100 nt to about 200 nt in length. In some embodiments, the length of the targeting RNA sequence in the dRNA is about 150 to about 220 nt. In some embodiments, the targeting RNA sequence in the dRNA is about 70 nt (eg, 71 nt) in length. In some embodiments, the length of the targeting RNA sequence in the dRNA is about 120 nt (eg, 121 nt). In some embodiments, the targeting RNA sequence in the dRNA is about 150 nt (eg, 151 nt) in length. In some embodiments, the length of the targeting RNA sequence in the dRNA is about 170 nt (eg, 171 nt). In some embodiments, the length of the targeting RNA sequence in the dRNA is about 200 nt (eg, 201 nt). In some embodiments, the length of the targeting RNA sequence in the dRNA is about 220 nt (eg, 221 nt).

In some embodiments, a method of editing a target RNA encoding mutant Usher 2A in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Contains a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex The strand comprises: (a) a first mismatch region relative to the target RNA sequence, which includes a deletion of four consecutive nucleotides of the targeting RNA located 27 nucleotides to 30 nucleotides upstream of the target adenosine; (b) A second mismatched region relative to the target RNA sequence, comprising 32 nucleotides to 35 nucleotides, or 36 nucleotides to 39 nucleotides, or 40 nucleotides downstream of the target adenosine. Deletion of four consecutive nucleotides from nucleotides to 43 of the targeting RNA; and (c) a third mismatch located between the first mismatched region and the second mismatched region relative to the target RNA A matching region, wherein the target RNA sequence of the third mismatched region contains any of the following: A, AA, U, C, CC, G, GG, or is located 7 and 8 nucleotides downstream of the target adenosine opposite the target RNA nucleotides are deleted ("X"); and wherein the dRNA includes a linker nucleic acid sequence flanking the terminus of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the dRNA The length of the targeting RNA sequence is about 150 to about 220 nt. In some embodiments, the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is from about 30 nt to about 50 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the targeting RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation.

In some embodiments, a method of editing a target RNA encoding mutant Usher 2A in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Contains a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex The strand comprises: (a) a first mismatch region relative to the target RNA sequence, which includes a deletion of ten consecutive nucleotides of the targeting RNA located 21 nucleotides to 30 nucleotides upstream of the target adenosine; (b) A second mismatched region relative to the target RNA sequence that contains the target 36 nucleotides to 39 nucleotides downstream of the target adenosine, or 40 nucleotides to 43 nucleotides downstream of the target adenosine Deletion of four consecutive nucleotides of the RNA; and (c) a third mismatch region located between the first mismatch region and the second mismatch region relative to the target RNA, wherein the third mismatch region of the targeting RNA The sequence contains any of the following: A, AA, U, C, CC, G, GG, or a deletion ("X") of nucleotides opposite the target RNA located 7 and 8 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 150 to about 220 nt. In some embodiments, the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is from about 30 nt to about 50 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the targeting RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation.

In some embodiments, a method of editing a target RNA encoding mutant Usher 2A in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Comprising a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex is The strand comprises: (a) a first mismatch region relative to the target RNA sequence, which comprises 26 nucleotides to 29 nucleotides upstream of the target adenosine, or 30 nucleotides to 33 nucleotides upstream of the target adenosine , a deletion of four consecutive nucleotides of the targeting RNA located at nucleotides 34 to 37; and (b) a second mismatched region relative to the target RNA sequence, which contains adenosine located at the target Four consecutive nucleosides of the targeting RNA 31 nt to 34 nt downstream, or 35 nt to 38 nt, or 39 nt to 42 nt deletion of acid; and wherein the dRNA includes a linker nucleic acid sequence flanking the terminus of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is About 150 to about 220 nt. In some embodiments, the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is from about 20 nt to about 50 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the targeting RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation.

In some embodiments, a method of editing a target RNA encoding mutant Usher 2A in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Contains a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex The strand contains a mismatched region relative to the target RNA sequence, the mismatched region comprising 26 nucleotides to 29 nucleotides upstream of the target adenosine, or 30 nucleotides to 33 nucleotides upstream of the target adenosine deletion of four consecutive nucleotides of the targeting RNA located 34 nucleotides to 37 nucleotides upstream of the target adenosine; and wherein the length of the targeting RNA sequence in the dRNA is about 150 to Approximately 220 nt. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation.

In some embodiments, a method of editing a target RNA encoding mutant Usher 2A in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Contains a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex The strand contains a mismatched region relative to the target RNA sequence, the mismatched region comprising 31 nucleotides to 34 nucleotides downstream of the target adenosine, or 35 nucleotides to 38 nucleotides downstream of the target adenosine deletion of four consecutive nucleotides of the targeting RNA at 39 nucleotides to 42 nucleotides downstream of the target adenosine; and wherein the length of the targeting RNA sequence in the dRNA is about 150 to approximately 220 nt. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation.

In some embodiments, a method of editing a target RNA encoding mutant Usher 2A in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA is included. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is from about 10 nt to about 50 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation.

In some embodiments, a method of editing a target RNA encoding mutant Usher 2A in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA Contains a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex The strand comprises: (a) a first mismatch region relative to the target RNA sequence, which includes a deletion of four consecutive nucleotides of the targeting RNA located 26 nucleotides to 29 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence that contains a deletion of four consecutive nucleotides of the targeting RNA located 35 nucleotides to 38 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 150 to about 220 nt. In some embodiments, the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is from about 20 nt to about 50 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation.

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of two consecutive nucleotides of the targeting RNA located 6 nucleotides to 7 nucleotides upstream of the target adenosine; (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of two consecutive nucleotides of the targeting RNA located 30 nucleotides to 31 nucleotides downstream of the target adenosine; and (c) a third mismatch region relative to the target RNA that includes a mismatch of two consecutive nucleotides of the targeting RNA located 60 to 61 nucleotides downstream of the target adenosine; and wherein the dRNA includes a flanking target a linker nucleic acid sequence toward the end of the RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220 nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of three consecutive nucleotides of the targeting RNA located 6 nucleotides to 8 nucleotides upstream of the target adenosine; (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of three consecutive nucleotides of the targeting RNA located 30 nucleotides to 32 nucleotides downstream of the target adenosine; and (c) a third mismatch region relative to the target RNA that includes a mismatch of three consecutive nucleotides of the targeting RNA located 60 to 62 nucleotides downstream of the target adenosine; and wherein the dRNA includes a flanking target a linker nucleic acid sequence toward the end of the RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220 nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of four consecutive nucleotides of the targeting RNA located 6 nucleotides to 9 nucleotides upstream of the target adenosine; (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of four consecutive nucleotides of the targeting RNA located 30 nucleotides to 33 nucleotides downstream of the target adenosine; and (c) a third mismatch region relative to the target RNA that includes a mismatch of four consecutive nucleotides of the targeting RNA located 60 to 63 nucleotides downstream of the target adenosine; and wherein the dRNA includes a flanking target a linker nucleic acid sequence toward the end of the RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220 nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of five consecutive nucleotides of the targeting RNA located 6 nucleotides to 10 nucleotides upstream of the target adenosine; (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of five consecutive nucleotides of the targeting RNA located 30 nucleotides to 34 nucleotides downstream of the target adenosine; and (c) a third mismatch region relative to the target RNA that includes a mismatch of five consecutive nucleotides of the targeting RNA located 60 to 64 nucleotides downstream of the target adenosine; and wherein the dRNA includes a flanking target a linker nucleic acid sequence toward the end of the RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220 nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of two consecutive nucleotides of the targeting RNA located 6 nucleotides to 7 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of two consecutive nucleotides of the targeting RNA located 30 nucleotides to 31 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of three consecutive nucleotides of the targeting RNA located 6 nucleotides to 8 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of three consecutive nucleotides of the targeting RNA located 30 nucleotides to 32 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of four consecutive nucleotides of the targeting RNA located 6 nucleotides to 9 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of four consecutive nucleotides of the targeting RNA located 30 nucleotides to 33 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of five consecutive nucleotides of the targeting RNA located 6 nucleotides to 10 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of five consecutive nucleotides of the targeting RNA located 30 nucleotides to 34 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of two consecutive nucleotides of the targeting RNA located 6 nucleotides to 7 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of five consecutive nucleotides of the targeting RNA located 40 nucleotides to 44 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of three consecutive nucleotides of the targeting RNA located 6 nucleotides to 8 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of five consecutive nucleotides of the targeting RNA located 40 nucleotides to 44 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of five consecutive nucleotides of the targeting RNA located 6 nucleotides to 10 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of five consecutive nucleotides of the targeting RNA located 40 nucleotides to 44 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex Comprising: (a) a first mismatch region relative to the target RNA sequence that includes a mismatch of two consecutive nucleotides of the targeting RNA located 6 nucleotides to 7 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence that includes a mismatch of three consecutive nucleotides of the targeting RNA located 41 nucleotides to 43 nucleotides downstream of the target adenosine; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex comprising a mismatch region relative to the target RNA sequence, the mismatch region comprising a mismatch of three consecutive nucleotides of the targeting RNA located 41 nucleotides to 43 nucleotides downstream of the target adenosine; and wherein the dRNA Comprised of a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220 nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, a method of editing a target RNA encoding a mutant IDUA in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein the dRNA comprises A targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, wherein the RNA duplex comprising a mismatch region relative to the target RNA sequence, the mismatch region comprising a mismatch of five consecutive nucleotides of the targeting RNA located 40 nucleotides to 44 nucleotides downstream of the target adenosine; and wherein the dRNA Comprised of a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure; and wherein the length of the targeting RNA sequence in the dRNA is from about 100 to about 220 nt. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the target RNA encodes a truncated protein at position 402 (W402X).

In some embodiments, the targeting RNA sequence comprises cytidine, adenosine, or uridine directly opposite the target adenosine residue in the target RNA. In some embodiments, the targeting RNA sequence contains a cytidine mismatch directly opposite the target adenosine residue in the target RNA. In some embodiments, the cytidine mismatch is located at least 5 nucleotides, e.g., at least 10, 15, 20, 25, 30, or more nucleotides from the 5' end of the targeting RNA sequence. In some embodiments, the cytidine mismatch is located at least 20 nucleotides, such as at least 25, 30, 35 or more nucleotides from the 3' end of the complementary RNA sequence.

In certain embodiments, the 5' nearest neighbor of the target adenosine residue is a nucleotide selected from U, C, A, and G, preferably U>C≈A>G, and the 3' nearest neighbor of the target adenosine residue is The nearest neighbor is a nucleotide selected from G, C, A and U, preferably G>C>A≈U. In some embodiments, the 5' nearest neighbor of the target adenosine residue is U. In some embodiments, the 5' nearest neighbor of the target adenosine residue is C or A. In some embodiments, the 3' nearest neighbor of the target adenosine residue is G. In some embodiments, the 3' nearest neighbor of the target adenosine residue is C.

In some embodiments, the target adenosine residue in the target RNA is selected from the group consisting of UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA, and GAU In the three-base motif. In some embodiments, the trigonyl motif is UAG and the dRNA includes an A directly opposite the U in the trigonyl motif, a C directly opposite the target A, and a G directly opposite the G in the trigonyl motif of C, G or U. In certain embodiments, the triphenyl motif is a UAG in the target RNA, and the dRNA comprises ACC, ACG, or ACU opposite the UAG of the target RNA. In certain embodiments, the triphenyl motif is a UAG in the target RNA and the dRNA comprises an ACC opposite the UAG of the target RNA.

In one aspect, the application provides a method for editing a plurality of target RNAs (e.g., at least about 2, 3, 4, 5, 10, 20 , 50, 100, 1000 or more) methods.

In some embodiments, the host cell is a prokaryotic cell. In some embodiments, the host cell is a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell. In some embodiments, the host cell is a murine cell.

In some embodiments, the host cell is a cell line, such as HEK293T, HT29, A549, HepG2, RD, SF268, SW13, and HeLa cells. In some embodiments, the host cells are primary cells, such as fibroblasts, epithelial cells, or immune cells. In some embodiments, the host cell is a T cell. In some embodiments, the host cell is a postmitotic cell. In some embodiments, the host cell is a cell of the central nervous system (CNS), such as a brain cell, such as a cerebellar cell. In some embodiments, the cells are neural cells. In some embodiments, the nerve cells are sensory nerve cells. In some embodiments, the sensory nerve cells are selected from the group consisting of optic nerve cells and auditory nerve cells. In some embodiments, the optic nerve cells are cones and/or rods. In some embodiments, the host cells are cells in or adjacent to the vitreous cavity. In some embodiments, the host cells are cells in or adjacent to the subretinal space. In some embodiments, the host cell is in a retinal epithelial cell. In some embodiments, the host cell is a retinal cell.

In some embodiments, ADARs are endogenous to the host cell. In some embodiments, an RNA-acting adenosine deaminase (ADAR) is naturally or endogenously present in the host cell, eg, naturally or endogenously present in a eukaryotic cell. In some embodiments, ADARs are expressed endogenously by the host cell. In some embodiments, the ADAR is an endogenously encoded ADAR of the host cell, wherein introducing the ADAR includes overexpressing the ADAR in the host cell. In some embodiments, ADARs are introduced exogenously into the host cell. In some embodiments, ADAR is exogenous to the host cell. In some embodiments, the construct comprising a nucleic acid encoding an ADAR is a vector, such as a plasmid, or a viral vector (eg, AAV, such as scAAV). In some embodiments, ADAR is ADAR1 and/or ADAR2. In some embodiments, the ADAR is one or more ADARs selected from hADAR1, hADAR2, mouse ADAR1, and ADAR2. In some embodiments, the ADAR is ADAR1, such as the p110 isoform of ADAR1 ("ADAR1 ^p110 ") and/or the p150 isoform of ADAR1 ("ADAR1 ^p150 "). In some embodiments, the ADAR is ADAR2. In some embodiments, the ADAR is ADAR2 expressed by a host cell, such as ADAR2 expressed by a cerebellar cell.

In some embodiments, the ADAR is an ADAR that is exogenous to the host cell. In some embodiments, the ADAR is a hyperactive mutant of a naturally occurring ADAR. In some embodiments, the ADAR is ADAR1 comprising the E1008Q mutation. In some embodiments, the ADAR is not a fusion protein comprising a binding domain. In some embodiments, the ADAR does not comprise an engineered double-stranded nucleic acid binding domain. In some embodiments, the ADAR does not comprise an MCP domain that binds to the MS2 hairpin structure of the complementary RNA sequence fused to the dRNA.

In some embodiments, the host cell has a high expression level of ADAR1 (e.g., ADAR1 ^p110 and/or ADAR1 ^p150 ), e.g., at least about 10%, 20%, 50%, relative to the protein expression level of β-tubulin. Any of 100%, 2x, 3x, 5x or more. In some embodiments, the host cell has a high expression level of ADAR2, e.g., at least about 10%, 20%, 50%, 100%, 2-fold, 3-fold, 5-fold relative to the protein expression level of β-tubulin. Any of times or more. In some embodiments, the host cell has a low expression level of ADAR3, e.g., at least about 10%, 20%, 50%, 100%, 2-fold, 3-fold, 5-fold relative to the protein expression level of β-tubulin. Any of times or more.

In certain embodiments, the methods do not induce an immune response, such as an innate immune response. In some embodiments, the methods do not induce interferon and/or interleukin expression in the host cell. In some embodiments, the methods do not induce IFN-β and/or IL-6 expression in the host cell.

Nucleic acids, including dRNA, constructs thereof, and nucleic acids encoding ADARs may be delivered using any method known in the art, including viral or non-viral delivery.

Non-viral delivery methods of nucleic acids include lipofection, nucleofection, microinjection, gene gun, virions, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, electroporation, nanoparticles, exocrine bodies, microbubbles or gene guns, naked DNA and artificial virus particles.

Delivery of nucleic acids using RNA or DNA virus-based systems is highly efficient in targeting viruses to specific cells and transporting viral payloads to the nucleus. In certain embodiments, the methods include introducing a dRNA-encoding viral vector (eg, AAV, eg, scAAV, or a lentiviral vector) into the host cell. For example, the constructs described herein can be any of the viral vectors described in Section III "dRNA, Constructs and Libraries" below.

In some embodiments, the method includes introducing a plasmid encoding a dRNA into the host cell. In some embodiments, the methods include electroporating dRNA (eg, synthetic dRNA) into the host cell. In some embodiments, the method includes transfecting dRNA into the host cell.

In certain embodiments, the target RNA has an editing efficiency of at least about 10%, such as at least about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90% or any of the above. In some embodiments, the editing efficiency of the target RNA is at least about 40%. In some embodiments, editing efficiency is determined by Sanger sequencing. In some embodiments, editing efficiency is determined by next generation sequencing. In some embodiments, editing efficiency is determined by evaluating the expression of a reporter gene, such as a fluorescent reporter gene, such as EGFP.

In certain embodiments, the methods have low off-target editing rates. In some embodiments, the method has less than about 1% (e.g., no more than any one of about 0.5%, 0.1%, 0.05%, 0.01%, 0.001%, or less) non-target A in the target RNA. ) editing efficiency. In some embodiments, the method does not edit non-target A in the target RNA. In some embodiments, the method has less than about 0.1% (e.g., no more than any one of about 0.05%, 0.01%, 0.005%, 0.001%, 0.0001%, or less) A in the non-target RNA editing efficiency.

Following deamination, modification of the target RNA and/or the protein encoded by the target RNA can be determined using different methods depending on the position of the target adenosine in the target RNA. For example, to determine whether an "A" in a target RNA has been edited to an "I", RNA sequencing methods known in the art can be used to detect modifications to the RNA sequence. When the target adenosine is located in the coding region of the mRNA, RNA editing can lead to changes in the amino acid sequence encoded by the mRNA. For example, point mutations can be introduced into the mRNA, and congenital or acquired point mutations in the mRNA can be reversed to produce a wild-type gene product due to an "A" to "I" conversion. Amino acid sequencing by methods known in the art can be used to discover any changes in the amino acid residues in the encoded protein. Modification of the stop codon can be determined by assessing the presence of functional, extended, truncated, full-length, and/or wild-type proteins. For example, when the target adenosine is located in a UGA, UAG, or UAA stop codon, modification of the target adenosine residue (UGA or UAG) or A (UAA) can produce readthrough mutations and/or elongated proteins, or encoded by the target RNA Truncated proteins can be reversed to produce functional, full-length and/or wild-type proteins. Editing of target RNA can also create aberrant splice sites and/or alternative splice sites in the target RNA, resulting in elongated, truncated, or misfolded proteins, or aberrant or alternative splice sites encoded in the target RNA. Spots may be reversed to produce functional, correctly folded, full-length and/or wild-type proteins. In some embodiments, the present application contemplates editing of innate and acquired genetic changes, such as missense mutations, early stop codons, aberrant splicing, or alternative splice sites encoded by the target RNA. Using known methods to assess the function of the protein encoded by the target RNA can reveal whether the RNA editing has achieved the desired effect. Because deamination of adenosine (A) to inosine (I) corrects mutated A at the target site in the mutated RNA encoding the protein, identification of inosine deamination can assess whether a functional protein is present or is caused by the presence of mutated adenosine. Whether the disease-causing or drug-resistance-associated RNA is reversed or partially reversed. Similarly, because deamination of adenosine (A) to inosine (I) can introduce point mutations in the resulting protein, identification of deamination to inosine can provide functional indications for identifying disease causes or disease-associated factors.

Effects of target adenosine deamination include, for example, point mutations, early stop codons, aberrant splice sites, alternative splice sites, and misfolding of the resulting protein. These effects may induce structural and functional changes in RNA and/or proteins that are associated with disease, whether they are inherited or caused by acquired genetic mutations, or may induce structural and functional changes in RNA and/or proteins associated with the development of drug resistance. and functional changes. Therefore, the dRNA, dRNA-encoding constructs and RNA editing methods of the present application can be used to prevent or treat genetic diseases or disorders, or those related to acquired diseases, by changing the structure and/or function of disease-related RNA and/or proteins. Diseases or conditions related to genetic mutations.

In some embodiments, the target RNA is pre-messenger RNA. In some embodiments, the target RNA is messenger RNA. In some embodiments, the target RNA is regulatory RNA. In some embodiments, the target RNA is ribosomal RNA, transfer RNA, long non-coding RNA, or small RNA (eg, miRNA, pri-miRNA, pre-miRNA, piRNA, siRNA, snoRNA, snRNA, exRNA, or scaRNA). The effects of target adenosine deamination include, for example, structural and functional changes in ribosomal RNA, transfer RNA, long non-coding RNA or small RNA (such as miRNA), including changes in the three-dimensional structure of the target RNA and/or loss of function or Functional gain. In some embodiments, deamination of a target adenosine in a target RNA changes the expression level of one or more downstream molecules of the target RNA (eg, protein, RNA, and/or metabolites). Changes in expression levels of downstream molecules can be increases or decreases in expression levels.

Some embodiments of the present application relate to multiplex editing of a target RNA in a host cell, which can be used to edit different variants of a target gene or different genes in the host cell. In some embodiments, wherein the method includes introducing a plurality of dRNAs into the host cell, at least two dRNAs of the plurality of dRNAs have different sequences and/or have different target RNAs. In some embodiments, each dRNA has a different sequence and/or a different target RNA. In some embodiments, the methods produce multiple (eg, at least 2, 3, 5, 10, 50, 100, 1000, or more) modifications in a single target RNA in the host cell. In some embodiments, the methods produce multiple (eg, at least 2, 3, 5, 10, 50, 100, 1000, or more) modifications of the target RNA in the host cell. In some embodiments, the methods include editing multiple target RNAs in multiple host cell populations. In some embodiments, each host cell population receives a different dRNA or a dRNA with a different target RNA than the other host cell population.

Edited RNA or host cells having edited RNA produced by any of the methods described herein are also provided. In some embodiments, the edited RNA contains inosine. In some embodiments, the host cell contains a target RNA having a missense mutation, an early stop codon, an alternative splice site, or an aberrant splice site. In some embodiments, the host cell contains a mutated, truncated, or misfolded protein. In some embodiments, the method restores the function of the target RNA.

III.dRNA , constructs and libraries

The present application also provides dRNAs, constructs encoding dRNAs, and libraries containing multiple dRNAs or constructs thereof, which may be used in any of the RNA editing methods or therapeutic methods described herein. It is intended that any features and parameters of the dRNAs or constructs described herein be combined with one another as if each combination were individually described.

In one aspect, the application provides a dRNA for editing a target RNA, which includes a targeting RNA sequence capable of hybridizing with the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an adenosine deaminase that acts on the RNA. (ADAR) to deaminate a target adenosine in a target RNA, wherein the RNA duplex contains one or more regions of mismatch relative to the target RNA, and wherein the dRNA contains a linker nucleic acid sequence flanking the termini of the targeting RNA sequence, wherein The linker nucleic acid sequence does not hybridize to the target RNA and forms substantially no secondary structure. The dRNA can be any of the dRNAs described in Section 3 below ("dRNA, Constructs and Libraries"). In some embodiments, the dRNA is linear. In some embodiments, the dRNA is circular. In some embodiments, the dRNA is a linear RNA capable of forming a circular RNA. In some embodiments, the methods use constructs comprising nucleic acid sequences encoding dRNA. The construct may be any of the constructs described in Section 3 below.

In some embodiments, a dRNA for editing a target RNA is provided, comprising a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an adenosine deaminase that acts on the RNA (ADAR) to deaminate a target adenosine in a target RNA, wherein the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence located 5 nucleotides to 85 nucleotides upstream of the target adenosine nucleotide; and/or (b) a second mismatched region relative to the target RNA sequence located 20 nucleotides to 85 nucleotides downstream of the target adenosine; and wherein the dRNA contains flanking targeting A linker nucleic acid sequence at the end of the RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence, the first mismatch region being: 5 nucleotides to 25 nucleotides upstream of the target adenosine, or the target adenosine 5 nucleotides to 15 nucleotides upstream of the target adenosine, or 20 nucleotides to 40 nucleotides upstream of the target adenosine. In some embodiments, the RNA duplex comprises a second mismatch region relative to the target RNA sequence, the second mismatch region being: 20 nucleotides to 65 nucleotides downstream of the target adenosine, or the target adenosine 20 nucleotides to 45 nucleotides downstream of the target adenosine, or 25 nucleotides to 45 nucleotides downstream of the target adenosine. In some embodiments, the first mismatched region is 1-50 nucleotides in length; and/or the second mismatched region is 1-50 nucleotides in length. In some embodiments, the linker nucleic acid sequence is from about 5 nucleotides (nt) to about 500 nt in length.

In some embodiments, a dRNA for editing a target RNA is provided, comprising a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an adenosine deaminase that acts on the RNA (ADAR) to deaminate a target adenosine in a target RNA, wherein the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence located 20 nucleotides to 40 nucleotides upstream of the target adenosine nucleotide; and/or (b) a second mismatch region relative to the target RNA sequence located 25 nucleotides to 45 nucleotides downstream of the target adenosine; and wherein the dRNA includes the flanking target A linker nucleic acid sequence toward the end of the RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure. In some embodiments, the first mismatched region is 1-50 nucleotides in length; and/or the second mismatched region is 1-50 nucleotides in length. In some embodiments, the linker nucleic acid sequence is from about 5 nucleotides (nt) to about 500 nt in length.

In some embodiments, the first mismatch region comprises: (a) one or more non-complementary nucleotides (mismatch) in the targeting RNA; and/or (b) one or more cores of the targeting RNA Deletion of nucleotides; and/or (c) insertion of one or more nucleotides into the targeting RNA. In some embodiments, the second mismatch region comprises: (a) one or more non-complementary nucleotides (mismatch) in the targeting RNA; and/or (b) one or more cores of the targeting RNA Deletion of nucleotides; and/or (c) insertion of one or more nucleotides into the targeting RNA. In some embodiments, the first mismatch region includes: (a) at least one contiguous set of non-complementary nucleotides (mismatch) in the targeting RNA; and/or (b) at least one contiguous set of nucleotides in the targeting RNA Deletion of nucleotides; and/or (c) insertion of at least one contiguous set of nucleotides in the targeting RNA. In some embodiments, the second mismatch region includes: (a) at least one contiguous set of non-complementary nucleotides (mismatch) in the targeting RNA; and/or (b) at least one contiguous set of nucleotides in the targeting RNA Deletion of nucleotides; and/or (c) insertion of at least one contiguous set of nucleotides in the targeting RNA. In some embodiments, targeting non-complementary nucleotides in RNA results in bubble-like structures in the RNA duplex. In some embodiments, nucleotide deletions in the targeted RNA result in bulge structures in the RNA duplex. In some embodiments, targeting nucleotide insertion in the RNA results in a bulged structure of the RNA duplex. In some embodiments, targeting a set of contiguous non-complementary nucleotides in RNA results in bubble-like structures in the RNA duplex. In some embodiments, deletion of a contiguous set of nucleotides in the targeted RNA results in a bulge structure in the RNA duplex. In some embodiments, insertion of a contiguous set of nucleotides into the targeted RNA results in a bulge structure in the RNA duplex.

In some embodiments, the first mismatched region is 1-50 nucleotides in length. In some embodiments, the second mismatched region is 1-50 nucleotides in length. In some embodiments, the first mismatched region is about any one of: 1-10, 10-20, 20-30, 30-40, or 40-50 nucleotides in length. In some embodiments, the length of the first mismatched region is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , any of 19 or 20 nucleotides. In some embodiments, the second mismatched region is about any one of: 1-10, 10-20, 20-30, 30-40, or 40-50 nucleotides in length. In some embodiments, the length of the second mismatched region is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , any of 19 or 20 nucleotides.

In some embodiments, the first mismatched region is 1-10 nucleotides in length; and/or the second mismatched region is 1-10 nucleotides in length. In some embodiments, the first mismatched region contains 1-10 contiguous non-complementary nucleotides in the targeting RNA. In some embodiments, the first mismatched region contains a deletion of 1-10 contiguous nucleotides of the targeting RNA. In some embodiments, the second mismatched region contains 1-10 contiguous non-complementary nucleotides of the targeting RNA. In some embodiments, the second mismatched region contains a deletion of 1-10 contiguous nucleotides of the targeting RNA.

In some embodiments, the first mismatched region is 4 nucleotides in length; and/or the second mismatched region is 4 nucleotides in length. In some embodiments, the first mismatch region includes four consecutive non-complementary nucleotides in the targeting RNA. In some embodiments, the first mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA. In some embodiments, the second mismatched region comprises four consecutive non-complementary nucleotides in the targeting RNA. In some embodiments, the second mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA.

In some embodiments, the dRNA is circular. In some embodiments, the dRNA is linear. In some embodiments, dRNA can be circularized (eg, to form a circular RNA).

In some embodiments according to any one of the dRNAs described herein, the target RNA encodes a mutant Usher 2A protein. In some embodiments, mutant Usher 2A proteins include missense mutations, nonsense mutations, and/or frameshift mutations. In some embodiments, the mutant Usher 2A protein is a truncated Usher 2A protein. In some embodiments, the target RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation. In some embodiments, the target RNA encoding mutant Usher 2A comprises a G to A mutation relative to the target RNA encoding wild-type Usher 2A. In some embodiments, the target RNA encoding mutant Usher 2A comprises the mutation 11864G>A with reference to the target RNA encoding wild-type Usher 2A.

In some embodiments according to any one of the dRNAs described herein, the target RNA encodes a Usher 2A protein. In some embodiments, the target RNA encoding Usher 2A is wild-type Usher 2A RNA, which contains the target adenosine at position 12183 (XM_005540847.1:c.12183A).

In some embodiments according to any one of the dRNAs described herein, the target RNA encodes a mutant IDUA protein. In some embodiments, mutant IDUA proteins include missense mutations, nonsense mutations, and/or frameshift mutations. In some embodiments, the mutant IDUA protein is a truncated IDUA protein. In some embodiments, the target RNA encodes a mutant IDUA protein comprising the W402X mutation. In some embodiments, a target RNA encoding a mutant IDUA comprises a G to A mutation with respect to a target RNA encoding wild-type IDUA. In some embodiments, the target RNA encoding mutant IDUA comprises a 1205G>A mutation relative to the target RNA encoding wild-type IDUA.

In some embodiments according to any one of the dRNAs described herein, the target RNA encodes a PPIA protein. In some embodiments, the target RNA encoding PPIA is wild-type PPIA RNA, which contains the target adenosine in the untranslated region (UTR). In some embodiments, the target RNA encoding PPIA is a wild-type PPIA RNA that contains the target adenosine located 45 nucleotides downstream of position 498 in the PPIA cDNA (NM_001284774.1:c.498+45A>A). In some embodiments, the target RNA encoding PPIA is a wild-type PPIA RNA comprising a target adenosine located 308 nucleotides downstream of position 498 in the PPIA cDNA (NM_021130.5:c.498+ 308A>A).

In some embodiments, a dRNA for editing a target RNA is provided, comprising a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an adenosine deaminase that acts on the RNA (ADAR) to deaminate a target adenosine in a target RNA, wherein the target RNA encodes a mutant Usher 2A protein, wherein the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence that is located in the target adenosine 20 nucleotides to 40 nucleotides upstream; and/or (b) a second mismatched region relative to the target RNA sequence located 25 nucleotides to 45 nucleotides downstream of the target adenosine ; and wherein the dRNA comprises a linker nucleic acid sequence flanking the terminus of the target RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure. In some embodiments, the target RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation. In some embodiments, the target RNA encoding mutant Usher 2A comprises a G to A mutation relative to the target RNA encoding wild-type Usher 2A. In some embodiments, the target RNA encoding mutant Usher 2A comprises the mutation 11864G>A with reference to the target RNA encoding wild-type Usher 2A. In some embodiments, with reference to SEQ ID NO: 3, the target adenosine is located at position 101.

In some embodiments according to any one of the dRNAs described herein, wherein the target RNA encodes a mutant Usher 2A protein, the RNA duplex further comprises a third mismatched region relative to the target RNA. In some embodiments, the third mismatched region is located between the first mismatched region and the second mismatched region relative to the target RNA. In some embodiments, the third mismatch region includes one or two non-complementary nucleotides in the targeting RNA and/or a deletion of one or two nucleotides in the targeting RNA sequence. In some embodiments, the third mismatch region relative to the target RNA sequence is located 7 and/or 8 nucleotides downstream of the target adenosine. In some embodiments, the target RNA includes an adenosine at the 7th and/or 8th nucleotide downstream of the target adenosine. In some embodiments, the target RNA comprises an "AA" sequence 7 and 8 nucleotides downstream of the target adenosine, wherein the targeting RNA sequence comprises any of the following: A, AA, U, C, CC, G, GG, or a deletion of nucleotides ("X") located 7 and 8 nucleotides downstream of the target adenosine opposite the target RNA.

In some embodiments according to any one of the dRNAs described herein, wherein the target RNA encodes a mutant Usher 2A protein, the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence located at the target adenosine 27 nucleotides to 30 nucleotides upstream; and (b) a second mismatch region relative to the target RNA sequence located 31 nucleotides to 43 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 32 nucleotides to 35 nucleotides downstream of the target adenosine. In some embodiments, the second mismatch region relative to the target RNA sequence is located 36 nucleotides to 39 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 40 nucleotides to 43 nucleotides downstream of the target adenosine. In some embodiments, the first mismatched region is 4 nucleotides in length. In some embodiments, the first mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA. In some embodiments, the second mismatch region is 4 nucleotides in length. In some embodiments, the second mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA.

In some embodiments according to any one of the dRNAs described herein, wherein the target RNA encodes a mutant Usher 2A protein, the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence located at the target adenosine 21 nucleotides to 30 nucleotides upstream; and (b) a second mismatch region relative to the target RNA sequence located 36 nucleotides to 43 nucleotides downstream of the target adenosine. In some embodiments, the second mismatch region relative to the target RNA sequence is located 36 nucleotides to 39 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region relative to the target RNA sequence is located 40 nucleotides to 43 nucleotides downstream of the target adenosine. In some embodiments, the first mismatched region is 10 nucleotides in length. In some embodiments, the first mismatched region contains a deletion of ten consecutive nucleotides of the targeting RNA. In some embodiments, the second mismatch region is 4 nucleotides in length. In some embodiments, the second mismatched region comprises a deletion of four consecutive nucleotides of the targeting RNA.

In some embodiments, the dRNA is circular. In some embodiments, dRNA can be circularized. In some embodiments, the dRNA is encoded in a construct comprising any one of the sequences in Table A (any of SEQ ID NOs: 15-314). In some embodiments, the dRNA is encoded in a construct comprising a variant of any of the sequences in Table A (any of SEQ ID NOs: 15-314), wherein the variant differs from the parental sequence by no more than 10, Any of 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide. In some embodiments, wherein the dRNA is circular or capable of being circularized, the circularized dRNA includes a sequence encoded by any of the circularizing sequences in Table A (the non-bold sequence of any of SEQ ID NOs: 15-314) Nucleotides. In some embodiments, wherein the dRNA is circular or capable of being circularized, the circularized dRNA comprises the non-bold sequence in any of Table A (any of SEQ ID NOs: 15-314) ), where the variant differs from the non-bold sequence by no more than any of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide . In some embodiments, the targeting RNA sequence is encoded by any one of the targeting sequences in Table A (the sequence in small capital letters of any of SEQ ID NOs: 15-314). In some embodiments, the targeting RNA sequence is encoded by a variant of any of the targeting sequences in Table A (the small capital letter sequence in any of SEQ ID NOs: 15-314), wherein the variant is identical to the parent sequence Does not differ by more than any of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide.

In some embodiments according to any one of the dRNAs described herein, the linker nucleic acid sequence is from about 5 nucleotides (nt) to about 500 nt in length. In some embodiments, the linker nucleic acid sequence is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 , 350, 400, 450 or 500 nt, or any length in between. In some embodiments, the linker nucleic acid sequence is less than or equal to 70 nt in length. In some embodiments, the linker nucleic acid sequence is 10nt-50nt, 10nt-40nt, 10nt-30nt, 10nt-20nt, 20nt-50nt, 20nt-40nt, 20nt-30nt, 30nt-50nt, 30nt-40nt, or 40nt- in length. Any integer between 50nt. In some embodiments, the linker nucleic acid sequence is from about 20 nt to about 60 nt in length. In some embodiments, the linker nucleic acid sequence is about 30 nt in length. In some embodiments, the linker nucleic acid sequence is about 50 nt in length. In some embodiments, at least about 50%, 60%, 70%, 80%, 85%, 90%, or 95% of any one of the linker nucleic acid sequences comprise adenosine or cytidine. In some embodiments, at least about 50% to 60%, 60% to 70%, 70% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% Either of the linker nucleic acid sequences contains adenosine or cytidine. In some embodiments, all nucleic acid sequences in the linker contain adenosine or cytidine. In some embodiments, at least about 50% of the linker nucleic acids comprise adenosine. In some embodiments, at least about 50%, 60%, 70%, 80%, 85%, or 90% of any one of the linker nucleic acid sequences comprise adenosine. In some embodiments, about 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 85%, 85% to 90%, or 90% Any one to 95% of the linker nucleic acid sequences contain adenosine.

In some embodiments, the dRNA is flanked by an adapter nucleic acid sequence targeting the 5' end of the RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence.

In some embodiments, the first linker nucleic acid sequence is the same as the second linker nucleic acid sequence. In some embodiments, the first linker nucleic acid sequence is different from the second linker nucleic acid sequence. In some embodiments, the dRNA further comprises a 3' exon sequence and a 5' exon sequence, and the 3' exon sequence can be flanked by a 3' catalytic Group I intron targeting the 5' end of the RNA sequence. For fragment recognition, the 5' exon sequence is recognized by the 5' catalytic Group I intronic fragment flanking the 3' end of the targeting RNA sequence. In some embodiments, the dRNA also contains a 3' linker sequence and a 5' linker sequence. In some embodiments, the double-stranded RNA contains a bulge structure for each non-target adenosine in the target RNA.

In some embodiments, the targeting RNA sequence is greater than 50 nt in length. In some embodiments, the targeting RNA sequence in the dRNA is from about 100 nt to about 200 nt in length. In some embodiments, the length of the targeting RNA sequence in the dRNA is about 150 to about 220 nt. In some embodiments, the targeting RNA sequence in the dRNA is about 70 nt (eg, 71 nt) in length. In some embodiments, the length of the targeting RNA sequence in the dRNA is about 120 nt (eg, 121 nt). In some embodiments, the targeting RNA sequence in the dRNA is about 150 nt (eg, 151 nt) in length. In some embodiments, the length of the targeting RNA sequence in the dRNA is about 170 nt (eg, 171 nt). In some embodiments, the length of the targeting RNA sequence in the dRNA is about 200 nt (eg, 201 nt). In some embodiments, the length of the targeting RNA sequence in the dRNA is about 220 nt (eg, 221 nt).

In some embodiments, a dRNA for editing a target RNA is provided, comprising a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an adenosine deaminase that acts on the RNA (ADAR) to deaminate a target adenosine in a target RNA, wherein the target RNA encodes a mutant Usher 2A protein, wherein the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence, which includes a region located in the target adenosine; Deletion of ten consecutive nucleotides of the target RNA from 21 nucleotides to 30 nucleotides upstream of the adenosine; (b) a second mismatch region relative to the target RNA sequence, which contains a region downstream of the target adenosine Deletion of four consecutive nucleotides of the target RNA at nucleotides 36 to 39, or nucleotides 40 to 43; and (c) located at the first offset relative to the target RNA A third mismatched region between the matched region and the second mismatched region, wherein the target RNA sequence of the third mismatched region contains any of the following: A, AA, U, C, CC, G, GG, or is located in the target gland nucleotides 7 and 8 nucleotides downstream of the glycoside opposite the target RNA are deleted ("X"); and wherein the dRNA includes a linker nucleic acid sequence flanking the terminus of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and substantially no secondary structure is formed; and wherein the length of the targeting RNA sequence in the dRNA is about 150 to about 220 nt. In some embodiments, the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the targeting RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation.

In some embodiments, a dRNA for editing a target RNA is provided, comprising a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting an adenosine deaminase that acts on the RNA (ADAR) to deaminate a target adenosine in a target RNA, wherein the target RNA encodes a mutant Usher 2A protein, wherein the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence, which includes a region located in the target adenosine; Deletion of ten consecutive nucleotides of the target RNA from 21 nucleotides to 30 nucleotides upstream of the adenosine; (b) a second mismatch region relative to the target RNA sequence, which contains a region downstream of the target adenosine Deletion of four consecutive nucleotides of the target RNA at nucleotides 36 to 39, or nucleotides 40 to 43; and (c) located at the first offset relative to the target RNA A third mismatched region between the matched region and the second mismatched region, wherein the target RNA sequence of the third mismatched region contains any of the following: A, AA, U, C, CC, G, GG, or is located in the target gland nucleotides 7 and 8 nucleotides downstream of the glycoside opposite the target RNA are deleted ("X"); and wherein the dRNA includes a linker nucleic acid sequence flanking the terminus of the targeting RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and substantially no secondary structure is formed; and wherein the length of the targeting RNA sequence in the dRNA is about 150 to about 220 nt. In some embodiments, the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. In some embodiments, the dRNA is circular or capable of being circularized. In some embodiments, the targeting RNA encodes a mutant Usher 2A protein comprising the Trp3955Ter mutation.

In one aspect, the application provides a dRNA for editing a target RNA, which includes a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the double-stranded RNA includes a dRNA that includes a non-target adenosine in the target RNA. structure. In some embodiments, the targeting RNA sequence has a deletion of one or more uridine residues opposite one or more non-targeting adenosines in the sequence complementary to the target RNA. In some embodiments, the dRNA is linear RNA. In some embodiments, the dRNA is circular RNA. In some embodiments, the dRNA is a linear RNA capable of forming a circular RNA.

In some embodiments, the linker nucleic acid sequence is from about 5 nt to about 500 nt in length, such as from about 50 nt to 200 nt. In some embodiments, the linker nucleic acid sequence comprises a polyadenosine (polyA), polyguanosine (polyG), or polycytosine (polyC) sequence. In some embodiments, the linker nucleic acid sequence comprises a dinucleotide repeat sequence, such as (AT) _n , where n is an integer greater than or equal to 3. In some embodiments, the linker nucleic acid sequence comprises any one of SEQ ID NOs: 8-14. In some embodiments, the dRNA is circular RNA. In some embodiments, the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence.

In one aspect, the application provides a library comprising a plurality of dRNAs or a plurality of constructs described herein.

In one aspect, the application provides a composition or host cell comprising a deaminase recruiting RNA or any of the constructs described herein. In certain embodiments, the host cell is a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is a mammalian cell. In some embodiments, the host cell is a human cell.

dRNA

The dRNA of the present application includes a targeting RNA sequence that hybridizes to a target RNA. The targeting RNA sequence is substantially complementary to the target RNA to allow hybridization of the targeting RNA sequence to the target RNA. In some embodiments, the targeting RNA sequence is at least about 70%, 80% contiguous with any one of at least about 20, 40, 60, 80, 100, 150, 200, or more nucleotides in the target RNA. , 85%, 90%, 95%, 96%, 97%, 98% or 99% or more complementary. In some embodiments, the dsRNA formed by hybridization between the targeting RNA sequence and the target RNA has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more Many) non-Watson-Crick base pairs (i.e., mismatches).

In some embodiments, a dsRNA (also referred to herein as "double-stranded RNA" or "RNA duplex") formed by hybridization between a targeting RNA sequence and a target RNA has one or more unpaired (e.g. , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleotides. In some embodiments, the dsRNA formed by hybridization between the targeting RNA sequence and the target RNA has one or more unpaired non-target adenosines in the target RNA. In some embodiments, the dRNA lacks one or more nucleotides opposite one or more non-target adenosines in the target RNA. In some embodiments, the targeting RNA sequence in the dRNA lacks the nucleotide opposite each non-target adenosine in the target RNA. In some embodiments, the targeting RNA sequence in the dRNA has two or more (e.g., 2, 3, 4 or more) contiguous nucleotides opposite the region in the target RNA that contains non-target adenosine missing. In some embodiments, the targeting RNA sequence in the dRNA is substantially complementary to the target RNA and lacks one or more nucleotides opposite one or more non-target adenosines in the target RNA. In some embodiments, the targeting RNA sequence in the dRNA is substantially complementary to the target RNA, lacking the nucleotide opposite each non-target adenosine in the target RNA.

Unpaired nucleotides in dsRNA lead to bulge structures. In some embodiments, target RNA hybridizes to dRNA to form dsRNA, which includes a bulge structure containing non-target adenosine in the target RNA. The bulge structures in dsRNA formed by hybridization of dRNA to target RNA contain non-target adenosines in the target RNA. The bulge structure can be a mononucleotide bulge structure, i.e., containing an unpaired non-target adenosine, or a polynucleotide bulge structure, i.e., containing additional unpaired or mismatched flanking unpaired, non-target adenosine. Nucleotides. In some embodiments, the bulge structure can comprise more than one (e.g., 2, 3, 4, 5, or more) unpaired nucleotides in the target RNA, i.e., the bulge structure consists of directly flanking non-target It consists of unpaired nucleotides on the 5' and/or 3' side of the adenosine residue. In some embodiments, the bulge structure may comprise one or more (e.g., 2, 3, 4, 5, or more) mismatched nucleotides directly flanking the 5' and 5' of the non-target adenosine residue. /or 3' side. In some embodiments, the bulge structure includes an unpaired non-target adenosine, one or more unpaired nucleotides flanking the 5' and/or 3' side of the non-target adenosine residue, and one or more mismatched nucleotides flanking the 5' and/or 3' side of the non-target adenosine residue. In some embodiments, the raised structures are 1nt, 2nt, 3nt or longer.

In some embodiments, the double-stranded RNA comprises two or more bulge structures, such as any of 2, 3, 4, 5, 6 or more bulge structures, wherein each bulge structure contains a target Non-target adenosine in RNA. In some embodiments, the double-stranded RNA contains a bulge structure at each non-target adenosine in the target RNA.

In some embodiments, the dsRNA formed by hybridization between dRNA and target RNA contains one or more, such as 1, 2, 3, 4, 5, 6, 7 or more mismatches (e.g., the same type or any of the different types of mismatches). In some embodiments, the dsRNA formed by hybridizing the dRNA to the target RNA contains one or more mismatches, such as 1, 2, 3, 4, 5, 6, 7 types of mismatches.

In some embodiments, in addition to the mismatch region, the targeting RNA sequence may also include one or more guanosines, such as 1, 2, 3, 4, 5, 6, or more Gs, each of which is identical to a G in the target RNA. Non-target adenosine is directly opposite. In some embodiments, in addition to the mismatched region, the targeting RNA sequence may also include two or more consecutive mismatched nucleotides opposite non-target adenosines in the target RNA (e.g., 2, 3, 4, 5 or more mismatched nucleotides).

In some embodiments, the dRNA contains a single linker nucleic acid sequence. In some embodiments, the dRNA includes a linker nucleic acid sequence at the 5' end of the targeting RNA sequence. In some embodiments, the dRNA includes a linker nucleic acid sequence at the 3' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a first linker nucleic acid sequence at the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence at the 3' end of the targeting RNA sequence. In some embodiments, the dRNA is a circular RNA comprising a linker nucleic acid sequence that directly or indirectly connects the 5' and 3' ends of the targeting RNA sequence. The first linker nucleic acid sequence and the second linker nucleic acid sequence may have the same or different sequences.

In some embodiments, the length of the linker nucleic acid sequence (including the first linker nucleic acid sequence and the second linker nucleic acid sequence) is at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, Any of 150, 200, 250, 300, 350, 400, 450 or 500 nt. In some embodiments, the length of the linker nucleic acid sequence (including the first linker nucleic acid sequence and the second linker nucleic acid sequence) does not exceed about 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, Any of 70, 60, 50, 40, 30, 20, 10 or 5 nt. In some embodiments, the length of the linker nucleic acid sequence (including the first linker nucleic acid sequence and the second linker nucleic acid sequence) is about 5-10, 10-20, 20-50, 5-50, 10-100, 5-50 ,50-100,100-200,200-300,300-400,400-500,5-100,5-200,5-300,5-400,5-500,50-200,50-300,50 -400 or 50-500nt. In some embodiments, the linker nucleic acid sequence (including the first linker nucleic acid sequence and the second linker nucleic acid sequence) is about 50 nt in length. In some embodiments, the first linker nucleic acid sequence and the second linker nucleic acid sequence are the same length. In some embodiments, the first linker nucleic acid sequence and the second linker nucleic acid sequence are different lengths.

In some embodiments, the linker nucleic acid sequence (including the first linker nucleic acid sequence and the second linker nucleic acid sequence) does not form substantially any secondary structure with any portion of the dRNA. Computational tools are those known in the art for predicting the secondary structure of RNA, including, for example, RNA folding. In some embodiments, the linker nucleic acid sequence does not form a double-stranded region with a portion of the targeting RNA sequence that is greater than any one of about 3, 4, 5, 6, or more base pairs in length. In some embodiments, the linker nucleic acid sequence does not comprise a complementary region greater than 3, 4, 5, or 6 nucleotides in length. In some embodiments, the first linker nucleic acid sequence does not have a complementary region relative to the second linker nucleic acid sequence that is greater than 3, 4, 5, or 6 nucleotides in length.

The linker nucleic acid sequence (including the first linker nucleic acid sequence and the second linker nucleic acid sequence) can be a mono- or di-nucleotide repeat sequence, or a random sequence. In some embodiments, the linker nucleic acid sequence comprises a polyadenosine (polyA), polyguanosine (polyG), or polycytosine (polyC) sequence. In some embodiments, the linker nucleic acid sequence comprises a dinucleotide repeat sequence, such as an AC or CA repeat sequence. In some embodiments, the linker nucleic acid sequence comprises (AC) _n , where n is an integer greater than or equal to 3.

In some embodiments, the linker nucleic acid sequence serves as a linker sequence connecting the 5' and 3' ends of the targeting RNA sequence in the circular dRNA.

ADAR, for example, human ADAR enzymes edit double-stranded RNA (dsRNA) structures with different specificities depending on many factors. An important factor is the degree of complementarity of the two strands that make up the dsRNA sequence. Perfect complementarity between dRNA and target RNA usually results in the catalytic domain of ADARs deaminating adenosine in a non-discriminatory manner. The specificity and efficiency of ADAR can be modified by introducing mismatches in the dsRNA region. For example, A-C mismatches are preferably recommended to increase the specificity and efficiency of adenosine deamination to be edited. Perfect complementarity does not necessarily require dsRNA formation between the dRNA and its target RNA, as long as there is sufficient complementarity for dsRNA hybridization and formation between the dRNA and target RNA. In some embodiments, the dRNA sequence or single-stranded RNA region thereof has at least about 70%, 80%, 85%, 90%, or 95% sequence complementarity with the target RNA when optimally aligned. Any suitable algorithm for aligning sequences may be used to determine the optimal alignment, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., Burrows Wheeler Aligner).

The nucleotides adjacent to the target adenosine also affect the specificity and efficiency of deamination. For example, from the perspective of the specificity and efficiency of adenosine deamination, the 5' nearest neighbor of the target adenosine to be edited in the target RNA sequence is preferably U>C≈A>G, while the target adenosine to be edited in the target RNA sequence is preferably The 3' nearest neighbor of the glycoside is preferably G>C>A≈U. In some embodiments, when the target adenosine may be in the target RNA selected from UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA, and GAU When in the trisaccharyl motif, the specificity and efficiency of adenosine deamination is higher than that of adenosine in other trisaccharyl motifs. In some embodiments, when the target adenosine to be edited is in the three-aminoglycan motif UAG, UAC, UAA, UAU, CAG, CAC, AAG, AAC, or AAA, the deamination efficiency of adenosine is much higher than that of other bases. adenosine in the sequence. For the same trisaccharide motif, different dRNA designs may also lead to different deamination efficiencies. Taking the three-alkyl motif UAG as an example, in some embodiments, when the dRNA includes cytidine (C) directly opposite to the target adenosine to be edited, adenosine (A) directly opposite to uridine, and adenosine (A) directly opposite to the target adenosine to be edited, and adenosine (A) directly opposite to the target adenosine to be edited. When the glycoside is directly opposite cytidine (C), guanosine (G), or uridine (U), the deamination efficiency of the target adenosine is higher than when using other dRNA sequences. In some embodiments, when the dRNA includes an ACC, ACG, or ACU opposite a UAG in the target RNA, the editing efficiency of the A in the UAG of the target RNA can reach about 25%-90% (e.g., about 25%-80 %, 25%-70%, 25%-60%, 25%-50%, 25%-40% or 25%-30%).

In addition to the target adenosine, one or more adenosines (referred to herein as "non-target A") may be present in the target RNA, which are not intended to be edited. For these adenosines, it is best to reduce their editing efficiency as much as possible. In some embodiments, provided herein is the introduction of a mismatched region into a dRNA, wherein the mismatched region comprises a first mismatched region relative to the target RNA sequence, the first mismatched region being located 20 nuclei upstream of the target adenosine adenosine to 40 nucleotides; and/or a second mismatch region relative to the target RNA sequence, the second mismatch region being located 25 nucleotides to 45 nucleotides downstream of the target adenosine, This results in bulges and/or bleb-like structures at mismatched regions where editing of target adenosines can be increased and off-target editing of non-target adenosines can be reduced. The dRNA may also comprise one or more unpaired nucleotides and/or one or more mismatched nucleotides directly flanking the 5' or 3' side of the non-target adenosine. As used herein, the term "mismatch" means that a nucleotide in the first strand of a double-stranded nucleic acid does not pair with any nucleotide base in the second strand of the double-stranded nucleic acid. In some embodiments, deamination efficiency is significantly reduced when guanosine is directly opposite adenosine in the target RNA. In some embodiments, the dRNA can be designed to delete one or more nucleotides (e.g., U) opposite the first non-target adenosine, and/or to have a second non-target adenosine corresponding to the target RNA to be edited. Glycoside is directly opposite to guanosine.

The desired level of specificity and efficiency in editing a target RNA sequence may depend on the different applications. Following the instructions in this patent application, those skilled in the art will be able to design dRNAs with complementary or substantially complementary sequences to the target RNA sequence according to their own needs, and obtain the results they want through some trial and error. As used herein, the term "mismatch" may refer to opposite nucleotides in double-stranded RNA (dsRNA) that do not form a perfect base pair according to the Watson-Crick base pairing rules. Mismatched hydroxyl pairs include, for example, G-A, C-A, U-C, A-A, G-G, C-C, U-U hydroxyl pairs. Taking A-C matching as an example, when a target adenosine residue is to be edited in the target RNA, the dRNA is designed to contain a C opposite to the A to be edited, thereby producing an A-C error in the dsRNA formed by hybridization between the target RNA and dRNA. match. As used herein, the term "mismatch" may also refer to a deletion of a nucleotide on one strand of a double-stranded RNA (dsRNA), thereby resulting in an unpaired nucleic acid on the opposite strand to the deleted nucleotide.

The dRNA described herein includes a targeting RNA sequence that is at least partially complementary to the target RNA. In certain embodiments, the length of the targeting RNA sequence in the dRNA comprises at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150 , any of 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 nucleotides (nt). In certain embodiments, the targeting RNA sequence in the dRNA includes no more than about 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, 140, 150, Any of 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 nucleotides. In certain embodiments, the length of the targeting RNA sequence in the dRNA is about 40-260, 45-250, 50-240, 60-230, 65-220, 70-220, 70-210, 70-200, 70- 190, 70-180, 70-170, 70-160, 70-150, 70-140, 70-130, 70-120, 70-110, 70-100, 70-90, 70-80, 75- 200, 80-190, 85-180, 90-170, 95-160, 100-200, 100-150, 100-175, 110-200, 110-160, 110-175, 110-150, 140-160, Any of 105-140 or 105-155 nucleotides. In some embodiments, the targeting RNA sequence in the dRNA is from about 100 to about 200 nt in length. In some embodiments, the targeting RNA sequence in the dRNA is about 70 nt (eg, 71 nt) in length. In some embodiments, the targeting RNA sequence in the dRNA is about 120 nt (eg, 121 nt) in length. In some embodiments, the targeting RNA sequence in the dRNA is about 150 nt (eg, 151 nt) in length. In some embodiments, the length of the targeting RNA sequence in the dRNA is about 170 nt (eg, 171 nt). In some embodiments, the targeting RNA sequence in the dRNA is about 200 nt (eg, 201 nt) in length. In some embodiments, the targeting RNA sequence in the dRNA is about 220 nt (eg, 221 nt) in length.

In some embodiments, the targeting RNA sequence comprises cytidine, adenosine, or uridine directly opposite the target adenosine residue in the target RNA. In some embodiments, the targeting RNA sequence contains a cytidine mismatch directly opposite the target adenosine residue in the target RNA. In some embodiments, the cytidine mismatch is located at least 5 nucleotides, such as at least 10, 15, 20, 25, 30 or more nucleotides from the 5' end of the targeting RNA sequence. In some embodiments, the cytidine mismatch is located at least 20 nucleotides, such as at least 25, 30, 35 or more nucleotides from the 3' end of the complementary RNA sequence. In some embodiments, the cytidine mismatch is not located within 20 (e.g., 15, 10, 5, or less) nucleotides from the 3' end of the targeting RNA sequence. In some embodiments, the cytidine mismatch is located at least 20 nucleotides (e.g., at least 25, 30, 35 or more nucleotides) from the 3' end of the targeting RNA sequence and at least 5' from the 5' end of the targeting RNA sequence. nucleotides (e.g., at least 10, 15, 20, 25, 30 or more nucleotides). In some embodiments, the cytidine mismatch is located in the center of the targeted RNA sequence. In some embodiments, the cytidine mismatch is located within the central 20 nucleotides (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide) of the targeting sequence in the dRNA. within the nucleotide).

In certain embodiments, the 5' nearest neighbor of the target adenosine residue is a nucleotide selected from U, C, A, and G, preferably U>C≈A>G, and the 3' nearest neighbor of the target adenosine residue is Neighbor is a nucleotide selected from G, C, A and U, preferably G>C>A≈U. In some embodiments, the 5' nearest neighbor of the target adenosine residue is U. In some embodiments, the 5' nearest neighbor of the target adenosine residue is C or A. In some embodiments, the 3' nearest neighbor of the target adenosine residue is G. In some embodiments, the 3' nearest neighbor of the target adenosine residue is C.

In some embodiments, the target adenosine residue is located in a triphenyl motif selected from UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA and GAU. In some embodiments, the trigonyl motif is UAG, and the dRNA includes an A directly opposite the U in the trigonyl motif, a C directly opposite the target A, and a G directly opposite the G in the trigonyl motif. Relative to C, G or U. In certain embodiments, the triphenyl motif is a UAG in the target RNA, and the dRNA comprises ACC, ACG, or ACU opposite the UAG of the target RNA. In certain embodiments, the triphenyl motif is a UAG in the target RNA and the dRNA comprises an ACC opposite the UAG of the target RNA.

In some embodiments, the targeting RNA sequence in the dRNA is single-stranded or essentially single-stranded. A dRNA can be completely single-stranded or have one or more (eg, 1, 2, 3 or more) double-stranded regions and/or one or more stem-loop regions.

In some embodiments, in addition to the targeting RNA sequence, the dRNA also contains regions for stabilizing the dRNA, for example, one or more double-stranded regions and/or stem-loop regions. In some embodiments, the double-stranded region or stem-loop region of the dRNA contains no more than about any one of 200, 150, 100, 50, 40, 30, 20, 10, or fewer base pairs. RNA editing oligonucleotides including double-stranded regions and/or stem-loop regions are disclosed in WO 2016/097212, WO2018/161032, WO2020/051555, WO2021/113264, WO2021/211894, US20190093098, US20220073915 and Katrekar et al., Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs, Nature Biotechnology (2022), the contents of which are incorporated herein by reference in their entirety. In some embodiments, the dRNA does not contain stem loops or double-stranded regions. In some embodiments, the dRNA comprises an ADAR recruitment domain. In some embodiments, the dRNA does not comprise an ADAR recruitment domain.

dRNA can contain one or more modifications. In some embodiments, the dRNA has one or more modified nucleotides, including nucleobase modifications and/or backbone modifications. Exemplary modifications of dRNA include, but are not limited to, phosphorothioate backbone modifications, 2'-substitutions in ribose (such as 2'-O-methyl and 2'-fluoro substitutions), LNA and L-RNA. In some embodiments, chemical modifications can increase the stability and efficacy of editing facilitated by dRNA. Chemical modifications are described in WO2017220751, WO2018/041973, WO2018/134301, WO2019/158475, WO2021/242870 and Vogel, P., et al., Improving site-directed RNA editing in vitro and in cell culture by chemical modification of the guideRNA. Angew Chem Int Ed Engl 53 , 6267-6271 (2014), which is incorporated herein in its entirety. In some embodiments, the dRNA contains no chemical modifications. In some embodiments, the dRNA does not comprise chemically modified nucleotides, such as 2'-O-methyl nucleotides, 2'-fluoronucleotides, or nucleotides with phosphorothioate linkages. In some embodiments, the dRNA does not contain chemically modified nucleotides. In some embodiments, the dRNA does not contain 2'-fluoronucleotides. In some embodiments, the dRNA does not contain 2'-O-methyl nucleotides. In some embodiments, the dRNA does not contain nucleotides with phosphorothioate bonds. In some embodiments, the dRNA does not comprise any of 2'-fluoronucleotides, 2'-O-methyl nucleotides, or nucleotides with phosphorothioate linkages. In some embodiments, dRNA contains one or more chemical modifications. In some embodiments, dRNA contains one or more chemically modified nucleotides. In some embodiments, the dRNA contains one or more 2'-fluoronucleotides. In some embodiments, the dRNA contains one or more 2'-O-methyl nucleotides. In some embodiments, the dRNA contains one or more nucleotides having a phosphorothioate bond. In some embodiments, the dRNA contains 2'-O-methyl and phosphorothioate bond modifications at only the first three and last three residues. In some embodiments, the dRNA is not an antisense oligonucleotide (ASO).

The dRNA may also contain one or more additional expression elements that facilitate expression and/or circularization of the dRNA.

In some embodiments, the dRNA further comprises a 3' exon sequence and a 5' exon sequence that can be flanked within the 3' catalytic I group at the 5' end of the targeting RNA sequence. Intron fragment recognition, the 5' exon sequence is recognized by a 5' catalytic Group I intron fragment flanking the 3' end of the targeting RNA sequence. In some embodiments, the Group I catalytic intron of the T4 phage Td gene is bisected in such a manner as to preserve structural elements critical to ribozyme folding. Exon fragment 2 is then ligated upstream of exon fragment 1 and the targeting RNA sequence (optionally with adapter nucleic acids flanking the 5' and/or 3' ends) is inserted between the exon-exon junctions sequence).

In some embodiments, the dRNA is a linear RNA capable of forming a circular RNA. In some embodiments, circularization is performed using the Tornado expression system ("Twister-optimized RNA for durable overexpression"), as described in Litke, JL & Jaffrey, SR Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nat Described in Biotechnol 37 , 667-675 (2019), which is incorporated herein by reference in its entirety. Briefly, Tornado-expressed transcripts contain target RNA flanked by torsion ribozymes. A twist ribozyme is any catalytic RNA sequence capable of self-cleaving. The ribozyme rapidly undergoes autocatalytic cleavage, leaving the ends ligated by RNA ligase.

In some embodiments, the dRNA comprises a targeting RNA sequence flanked (directly or indirectly) by 5' and/or 3' linker sequences. In some embodiments, the dRNA contains a 3' linker sequence. In some embodiments, the dRNA contains a 5' linker sequence. In some embodiments, the dRNA includes a 3' linker sequence and a 5' linker sequence. In some embodiments, the 3' linker sequence and the 5' linker sequence are at least partially complementary to each other. In some embodiments, the 3' linker sequence and the 5' linker sequence are at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80% , at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% complementary to each other. In some embodiments, the 3' linker sequence and the 5' linker sequence are completely complementary to each other. In some embodiments, the 5' and/or 3' linker sequences are also flanked by a 5'-twist ribozyme and/or a 3'-twist ribozyme.

In some embodiments, the dRNA is a linear RNA capable of forming a circular RNA, wherein the dRNA includes from 5' to 3': a 5' linker sequence, a first linker nucleic acid sequence, a targeting RNA sequence, a second linker nucleic acid sequence, and a 3' connection sequence. In some embodiments, the dRNA is a linear RNA capable of forming a circular RNA, wherein the dRNA includes from 5' to 3': a 5' linker sequence, a linker nucleic acid sequence, a targeting RNA sequence, and a 3' linker sequence. In some embodiments, the dRNA is a linear RNA capable of forming a circular RNA, wherein the dRNA includes from 5' to 3': a 5' linker sequence, a targeting RNA sequence, a linker nucleic acid sequence, and a 3' linker sequence. In some embodiments, the dRNA is a linear RNA capable of forming a circular RNA, wherein the dRNA includes from 5' to 3': a 5' linker sequence, a targeting RNA sequence, and a 3' linker sequence. In some embodiments, the 3' linker sequence comprises the sequence CTGCCATCAGTCGGCGTGGACTGTAG. In some embodiments, the 5' linker sequence includes the sequence AACCATGCCGACTGATGGCAG.

In some embodiments, the dRNA is a circular RNA that contains a linker sequence that directly or indirectly connects the 5' and 3' ends of the targeting RNA sequence. In some embodiments, the linker sequence comprises a 5' linker sequence and a 3' linker sequence linked to each other by a ligase, such as a T4 RNA ligase (e.g., Rnll or Rnl2).

In some embodiments, the dRNA is a circular RNA comprising in a clockwise direction: a linker sequence, a first linker nucleic acid sequence, a targeting RNA sequence, and a second linker nucleic acid sequence, wherein the linker sequence is 5' to the first linker nucleic acid sequence. The end is directly connected to the 3' end of the second adapter nucleic acid sequence. In some embodiments, the dRNA is a circular RNA that includes in a clockwise direction: a linker sequence, a linker nucleic acid sequence, and a targeting RNA sequence, wherein the linking sequence directly connects the 5' end of the linker nucleic acid sequence to the 3' end of the targeting RNA sequence. 'end. In some embodiments, the dRNA is a circular RNA comprising in a clockwise direction: a linker sequence, a targeting RNA sequence, and an adapter nucleic acid sequence, wherein the linker sequence directly connects the 5' end of the targeting RNA sequence to the 3' end of the adapter nucleic acid sequence. 'end. In some embodiments, the dRNA is a circular RNA comprising a linker sequence and a targeting RNA sequence, wherein the linker sequence directly connects the 5' end of the targeting RNA sequence to the 3' end of the targeting RNA sequence.

In some embodiments, the 3' linker sequence and the 5' linker sequence are independently at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleosides in length. acid, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, At least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, or at least about 100 nucleotides. In some embodiments, the 3' linker sequence and the 5' linker sequence are independently about 20-30 nucleotides, about 30-40 nucleotides, about 40-50 nucleotides, about 50-50 nucleotides in length. 60 nucleotides, about 60-70 nucleotides, about 70-80 nucleotides, about 80-90 nucleotides, about 90-100 nucleotides, about 100-125 nucleotides , about 125-150 nucleotides, about 20-50 nucleotides, about 50-100 nucleotides, or about 100-150 nucleotides.

In some embodiments, dRNA is circularized by RNA ligase. Non-limiting examples of RNA ligases include: RtcB, T4 RNA ligase 1 (Rnl1), T4 RNA ligase 2 (Rnl2), Rnl3, and Trl1. In some embodiments, the RNA ligase is endogenously expressed in the host cell. In some embodiments, the RNA ligase is RNA ligase RtcB. In some embodiments, the method further comprises introducing an RNA ligase (eg, RtcB) into the host cell.

In some embodiments, the dRNA is circularized prior to introduction into the host cell. In some embodiments, dRNA is chemically synthesized. In some embodiments, dRNA is cyclized by in vitro enzymatic ligation (eg, using RNA or DNA ligase) or chemical ligation (eg, using cyanogen bromide or similar condensing agents).

The dRNA described herein does not include tracrRNA, crRNA or gRNA used in CRISPR/Cas systems. In some embodiments, the dRNA does not comprise an ADAR recruitment domain. In some embodiments, the dRNA comprises an ADAR recruitment domain. An "ADAR recruitment domain" may be a nucleotide sequence or structure that binds with high affinity to an ADAR, or a nucleotide sequence that binds to a binding partner fused to an ADAR in an engineered ADAR construct. Exemplary ADAR recruitment domains include, but are not limited to, GluR-2, GluR-B(R/G), GluR-B(Q/R), GluR-6(R/G), 5HT2C, and FlnA(Q/R) domains ; See, for example, Wahlstedt, Helene, and Marie, "Site-selective versus promiscuous A-to-I editing." Wiley Interdisciplinary Reviews: RNA 2.6 (2011): 761-771, which is incorporated herein by reference in its entirety. In some embodiments, the dRNA does not contain a double-stranded portion. In some embodiments, the dRNA does not contain a hairpin structure, such as an MS2 stem-loop. In some embodiments, the dRNA contains a hairpin structure, such as an MS2 stem-loop. In some embodiments, the dRNA is single-stranded.

In some embodiments, the dRNA comprises a snoRNA sequence linked to the 5' end of the targeting RNA sequence ("5' snoRNA sequence"). In some embodiments, the dRNA comprises a snoRNA sequence linked to the 3' end of the targeting RNA sequence ("3' snoRNA sequence"). In some embodiments, the dRNA comprises a snoRNA sequence linked to the 5' end of the targeting RNA sequence ("5' snoRNA sequence") and a snoRNA sequence linked to the 3' end of the targeting RNA sequence ("3' snoRNA sequence" ). In some embodiments, the snoRNA sequence is at least about 50 nucleotides, at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides in length , at least about 100 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least about 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, at least About 160 nucleotides, at least about 170 nucleotides, at least about 180 nucleotides, at least about 190 nucleotides, or at least about 200 nucleotides. In some embodiments, the snoRNA sequence is about 50-75 nucleotides, about 75-100 nucleotides, about 100-125 nucleotides, about 125-150 nucleotides, about 150-150 nucleotides in length. 175 nucleotides, about 175-200 nucleotides, about 50-100 nucleotides, about 100-150 nucleotides, about 150-200 nucleotides, about 125-175 nucleotides Or about 100-200 nucleotides. In some embodiments, the snoRNA sequence is a C/D Box snoRNA sequence. In some embodiments, the snoRNA sequence is an H/ACA Box snoRNA sequence. In some embodiments, the snoRNA sequence is a composite C/D Box and H/ACA Box snoRNA sequence. In some embodiments, the snoRNA sequence is an orphan snoRNA sequence.

Small nucleolar RNAs (snoRNAs) are small noncoding RNA molecules that are known to direct the chemical modification of other RNAs such as ribosomal RNAs, transfer RNAs, and small nuclear RNAs. According to their specific secondary structure characteristics, there are two major categories of snoRNA: box C/D and box H/ACA. Both structural features of snoRNA enable them to bind to the corresponding RNA-binding proteins (RBPs) and accessory proteins to form functional small nucleolar ribonucleoprotein (snoRNP) complexes. Box C/D snoRNAs are thought to be related to methylation, while H/ACA box snoRNAs are thought to be related to pseudouridylation. Other snoRNA families include, for example, complex H/ACA and C/D box snoRNAs and orphan snoRNAs. The snoRNA sequences described herein may comprise naturally occurring snoRNA, portions thereof, or variants thereof.

construct

The present application provides constructs encoding dRNA and/or ADAR. In some embodiments, a construct (eg, a vector, such as a viral vector) comprising a nucleotide sequence encoding a dRNA is provided. In some embodiments, a construct (eg, a vector, such as a viral vector) comprising a nucleotide sequence encoding an ADAR is provided. In some embodiments, constructs are provided comprising a first nucleotide sequence encoding a dRNA and a second nucleotide sequence encoding an ADAR. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are operably linked to the same promoter. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are operably linked to different promoters. In some embodiments, the promoter is inducible. In some embodiments, the construct does not encode ADAR. In some embodiments, the vector further comprises nucleic acid sequences encoding ADAR3 inhibitors (eg, ADAR3 shRNA or siRNA) and/or interferon stimulators (eg, IFN-α).

As used herein, the term "construct" refers to a DNA or RNA molecule comprising a coding nucleic acid sequence that can be transcribed into RNA or expressed into a protein. In some embodiments, a construct includes one or more regulatory elements operably linked to a nucleic acid sequence encoding an RNA or protein. When the construct is introduced into a host cell, the encoding nucleic acid sequence in the construct can be transcribed or expressed under appropriate conditions.

The constructs described herein may comprise a promoter operably linked to a nucleic acid sequence encoding a dRNA such that the promoter controls the transcription or expression of the encoding nucleotide sequence. A promoter may be located 5' (upstream) of the coding nucleotide sequence under its control. The distance between a promoter and a coding sequence may be approximately the same as the distance between the promoter and the gene controlled by the promoter from the gene from which the promoter is derived. As is known in the art, changes in this distance can be accommodated without loss of promoter function. In some embodiments, the construct includes a 5' UTR and/or a 3' UTR that modulates the transcription or expression of the encoding nucleotide sequence. In some embodiments, a promoter drives the expression of two or more dRNAs.

The promoter may be a polymerase II promoter ("Pol II promoter") or a polymerase III promoter ("Pol III promoter"). In some embodiments, wherein the dRNA is a linear RNA, the construct comprises a Pol II promoter operably linked to the nucleic acid sequence encoding the dRNA. Non-limiting examples of Pol II promoters include: CMV, SV40, EF-la, CAG, and RSV. In some embodiments, the Pol II promoter is a CMV promoter.

In some embodiments, where the dRNA is a circular RNA or a linear RNA capable of forming a circular RNA, the construct includes a Pol III promoter. In some embodiments, the promoter is the U6 promoter. In some embodiments, the U6 promoter comprises gagggcctatttccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtatttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatt The nucleic acid sequence of tcgatttcttggctttatatatcttgtggaaaggacgaaacaccg.

In some embodiments, the construct is a vector encoding any of the dRNAs disclosed herein. The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules containing one or more free ends, without free ends (eg, circular); nucleic acid molecules containing DNA, RNA, or both; and Other types of polynucleotides known in the art. One type of vector is a "plasmid," which is a circular double-stranded DNA circle into which additional DNA segments can be inserted, for example, by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell and are thereby replicated together with the host genome. In addition, certain vectors are capable of directing the transcription or expression of a coding nucleotide sequence to which they are operably linked. Such vectors are referred to herein as "expression vectors."

In some embodiments, the construct is a viral vector. In some embodiments, the construct is a lentiviral vector. In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. The use of any AA serotype is considered to be within the scope of this disclosure. In some embodiments, the rAAV vector is a vector derived from an AAV serotype, including, but not limited to, the AAV ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV or mouse AAV capsid serotypes, etc. In some embodiments, the construct is flanked by one or more AAV inverted terminal repeat (ITR) sequences. In some embodiments, the construct is flanked by two AAV ITRs. In some embodiments, the AAV ITR is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype ITR. In some embodiments, the AAV ITR is an AAV2 ITR.

In some embodiments, the vector further comprises stuffer nucleic acid. In some embodiments, the filler nucleic acid is located upstream or downstream of the nucleic acid encoding the dRNA. In some embodiments, the vector is a self-complementary rAAV vector. In some embodiments, the vector comprises a first nucleic acid sequence encoding a dRNA and a second nucleic acid sequence encoding a complementary sequence to the dRNA, wherein the first nucleic acid sequence can form an intrachain base pair with the second nucleic acid sequence along most or all of its length. . In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are connected by a mutated AAV ITR, wherein the mutated AAV ITR includes a deletion of the D region and includes a mutation of the end-resolved sequence. In some embodiments, the vector is encapsulated in rAAV particles. In some embodiments, the AAV viral particles comprise AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV2/2-7m8, AAV DJ , AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV2 V708K, AAV2-HBKO, AAVDJ8, AAVPHP.B, AAVPHP.eB, AAVBR1, AAVHSC15, AAVHSC17, goat AAV, AAV1/AAV2 chimera, bovine AAV, mouse AAV or rAAV2 /HBoV1 serotype capsids.

In some embodiments, the construct further comprises a 3' twist ribozyme sequence linked to the 3' end of the nucleic acid encoding the dRNA. In some embodiments, the construct further comprises a 5' twist ribozyme sequence linked to the 5' end of the nucleic acid sequence encoding the dRNA. In some embodiments, the construct further comprises a 3' twist ribozyme sequence linked to the 3' end of the dRNA-encoding nucleic acid sequence and a 5' twist ribozyme sequence linked to the 5' end of the dRNA-encoding nucleic acid sequence. In some embodiments, the 3' twist ribozyme sequence is twisterP3 U2A and the 5' twist ribozyme sequence is twisterP1. In some embodiments, wherein the 5' twist ribozyme sequence is twisterP3 U2A and the 3' twist ribozyme sequence is twisterP1. In some embodiments, the dRNA undergoes autocatalytic cleavage. In some embodiments, the catalyzed dRNA product contains a 5'-hydroxyl group and a 2',3'-cyclic phosphate at the 3' end.

Preparation method

The dRNA described herein can be prepared using any method known in the art, including chemical synthesis and in vitro transcription. Circular dRNA can be prepared by chemical ligation of linear RNA, enzymatic ligation, or ribozyme autocatalysis. In some embodiments, circular dRNA is prepared by circularizing linear RNA in vitro.

In some embodiments, the application provides linear RNA capable of forming the circular dRNA of any of the above embodiments. In some embodiments, linear RNA can be cyclized by chemical cyclization methods using cyanogen bromide or similar condensing agents. In some embodiments, linear RNA can be cyclized by autocatalysis of a Group I intron comprising a 5' catalytic Group I intron fragment and a 3' catalytic Group I intron fragment. In some embodiments, linear RNA can be circularized by a ligase. In some embodiments, linear RNA can be circularized by T4 RNA ligase. In some embodiments, linear RNA can be circularized by DNA ligase. Suitable ligases include, but are not limited to, T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl1), and T4 RNA ligase 2 (T4 Rnl2). Circular dRNA can be purified using methods known in the art, such as by gel purification or by high performance liquid chromatography (HPLC).

In some embodiments, linear RNA can be chemically circularized to provide circular dRNA. In some chemical methods, the 5' and 3' ends of a nucleic acid (e.g., a linear or circular polyribonucleotide) include chemically reactive groups that, when brought into close proximity, can bind to the 5' and 3' ends of the molecule. A new covalent bond is formed between the 3' ends. The 5' end may contain an NHS ester reactive group and the 3' end may contain a 3'-amino-terminated nucleotide, such that in an organic solvent, the 3'-amino-terminated nucleotide at the 3' end of the linear RNA molecule will Subjects to nucleophilic attack on the 5'-NHS-ester moiety, forming a new 5'-/3'-amide bond.

In some embodiments, circular dRNA can be obtained by ribozyme autocatalytic circularization of linear RNA. In some embodiments, linear RNA is circularized in vitro. In some embodiments, autocatalytic cyclization by a ribozyme comprises (a) subjecting the linear RNA to conditions that initiate autocatalysis by the Group I intron (or 5' and 3' catalytic Group I intron fragments thereof) to provide a circularized RNA product; (b) isolating the circularized RNA product to provide circular dRNA.

In some embodiments, the method includes the step of obtaining linear RNA by first cloning a sequence encoding the linearized RNA into a plasmid vector and then linearizing the recombinant plasmid. In some embodiments, the recombinant plasmid is linearized by restriction enzyme digestion. In some embodiments, the recombinant plasmid is linearized by PCR amplification. In some embodiments, the method further includes in vitro transcription using a linearized plasmid template. In some embodiments, in vitro transcription is driven by the T7 promoter. In some embodiments, the method further includes purifying linear RNA transcripts. In some embodiments, linear RNA is purified by gel purification.

In some embodiments, the application provides a method of circularizing linear RNA (eg, purified linear RNA) by ribozyme autocatalysis of a Group I intron. During the splicing process, the 3' hydroxyl group of the guanosine nucleotide undergoes a transesterification reaction at the 5' splice site. Half of the 5' intron is excised, and the free hydroxyl group at the end of the intermediate undergoes a second transesterification at the 3' splice site, resulting in cyclization of the intermediate region and excision of the 3' intron. In some embodiments, the condition to initiate autocatalysis of the Group I intron or the 5' and 3' catalytic Group I intron fragments is the addition of GTP and Mg2 ⁺ . In some embodiments, a step is provided for circularizing linear RNA by adding GTP and Mg ²⁺ at 55°C for 15 minutes. In some embodiments, the method further includes treatment with RNase R to digest linear RNA transcripts. In some embodiments, the method further includes isolating circular dRNA. In some embodiments, the step of isolating the circular dRNA includes gel purifying the circular dRNA.

In some embodiments, circular dRNA can be obtained by circularizing linear RNA using a ligase such as RNA ligase. In some embodiments, linear RNA is circularized in vitro. In some embodiments, linear RNA can be circularized by T4 RNA ligase. In some embodiments, the linear RNA includes a 5' linker sequence located at the 5' end of the targeting RNA sequence and a 3' linker sequence located at the 3' end of the targeting RNA sequence, wherein the 5' linker sequence and the 3' linker sequence can be passed through the RNA Ligase joins each other. In non-limiting examples, linear RNA can be circularized by ligases such as T4 DNA ligase (T4 Dnl), T4 RNA ligase 1 (T4 Rnl1), and T4 RNA ligase 2 (T4 Rnl2). Linear RNA can be circularized in the presence or absence of single-stranded nucleic acid linkers (eg, splint DNA).

In some embodiments, DNA or RNA ligases can be used to enzymatically ligate a 5'-phosphorylated nucleic acid molecule (e.g., linear RNA) to the 3'-hydroxyl group of a nucleic acid (e.g., linear nucleic acid) to form a new phosphodiester key. In an example reaction, linear circular RNA was incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) for 1 hour at 37°C according to the manufacturer's protocol. Ligation reactions can occur in the presence of linear nucleic acids capable of pairing with juxtaposed 5'- and 3'-region hydroxyl groups to account for enzymatic ligation reactions. In some embodiments, the connection is a splint connection. For example, splint ligases, such as ^SPLINTR® ligase, can be used for splint ligation. For splint ligation, a single-stranded polynucleotide (splint), such as single-stranded RNA, can be designed to hybridize to both ends of a linear polyribonucleotide so that the two ends are juxtaposed when hybridized to the single-stranded splint. Thus, splint ligases catalyze the ligation of two ends of juxtaposed linear polyribonucleotides to produce circular polyribonucleotides. In some embodiments, DNA or RNA ligases can be used to synthesize circular dRNA. As non-limiting examples, the ligase may be a cyclizing ligase or a ring ligase.

IV. Treatment

The RNA editing methods and compositions described herein can be used to treat or prevent diseases or conditions in individuals, including, but not limited to, inherited genetic diseases and drug resistance.

In some embodiments, a method of editing target RNA ex vivo in cells of an individual (eg, a human individual) is provided, comprising editing the target RNA using any of the RNA editing methods described herein.

In some embodiments, a method of treating or preventing a disease or disorder in an individual (e.g., a human individual) is provided, comprising editing in a cell of the individual a gene associated with the disease or disorder using any of the RNA editing methods described herein. Target RNA. The dRNA includes a targeting RNA sequence that hybridizes to a target RNA associated with a disease or condition. In some embodiments, the method includes introducing dRNA or a construct comprising a nucleic acid encoding a dRNA into isolated cells of an individual ex vivo. In some embodiments, the method includes administering to the individual an effective amount of dRNA or a construct comprising a nucleic acid encoding a dRNA.

In some embodiments, the target RNA is associated with a disease or condition in an individual. In some embodiments, the disease or disorder is an inherited genetic disorder, or a disease or disorder associated with one or more acquired genetic mutations (eg, drug resistance). In some embodiments, the method further includes obtaining cells from the individual. In some embodiments, the ADAR is an ADAR endogenously expressed in an isolated cell. In some embodiments, the method includes introducing ADAR or a construct comprising a nucleic acid encoding ADAR into an isolated cell. In some embodiments, the methods include introducing ADAR or a construct comprising a nucleic acid encoding ADAR into cells of an individual. In some embodiments, the method further includes culturing the cells with the edited RNA. In some embodiments, the method further includes administering cells with edited RNA to the individual. In some embodiments, the disease or disorder is an inherited genetic disorder, or a disease or disorder associated with one or more acquired genetic mutations (eg, drug resistance).

Diseases and conditions suitable for treatment using the methods of the present application include diseases associated with mutations, such as G to A mutations, such as G to A mutations that result in missense mutations in RNA transcripts, early stop codons, aberrant splicing, or alternative splicing mutation. In some embodiments, the disease or condition is cancer. In some embodiments, the disease or disorder is hepatocellular carcinoma, lung cancer, pancreatic cancer, endometrial cancer, esophageal squamous cell carcinoma, or melanoma. In some embodiments, the disease or disorder is a single gene disorder. In some embodiments, the disease or disorder is a polygenic disease.

In some embodiments, a method of treating a cancer in an individual associated with a target RNA having a mutation (e.g., a G to A mutation) is provided, comprising editing in a cell of the individual using any of the RNA editing methods described herein Target RNA.

In some embodiments, a method of ameliorating symptoms of Usher syndrome in an individual is provided, comprising editing a target RNA associated with Usher syndrome in cells of the individual according to any one of the editing methods described herein. In some embodiments, a method of ameliorating symptoms of Usher syndrome in an individual is provided, comprising editing a target RNA associated with Usher syndrome in cells of the individual according to using any of the dRNAs described herein. In some embodiments, the target RNA encoding mutant Usher 2A comprises a G to A mutation relative to the target RNA encoding wild-type Usher 2A. In some embodiments, the target RNA encoding mutant Usher 2A comprises the mutation 11864G>A with reference to the target RNA encoding wild-type Usher 2A. In some embodiments, with reference to SEQ ID NO: 3, the target adenosine is located at position 101. In some embodiments, the USH2A gene mutation is NM_206933.2(USH2A)_c.11864G>A(p.Trp3955Ter).

In some embodiments of a method for ameliorating symptoms of Usher syndrome according to any one of the methods described herein, wherein the method or dRNA is used to edit a target RNA encoding mutant Usher 2A, the dRNA is introduced into the neural cell. In some embodiments, the nerve cells are sensory nerve cells. In some embodiments, the sensory nerve cells are selected from the group consisting of optic nerve cells and auditory nerve cells. In some embodiments, the optic nerve cells are cones and/or rods. In some embodiments, dRNA is introduced into host cells within or adjacent to the vitreous cavity. In some embodiments, dRNA is introduced into host cells within or adjacent to the subretinal space. In some embodiments, the host cell is a retinal epithelial cell. In some embodiments, the host cell is a retinal cell. In some embodiments of the method for improving symptoms of Usher syndrome according to any one of the methods, wherein the method or dRNA is used to edit a target RNA encoding mutant Usher 2A, the dRNA is introduced into the subretinal space and/or the vitreous cavity.

In some embodiments, the individual has Usher syndrome type I, type II, type III, or type IV. In some embodiments, the individual has Usher syndrome type II. In some embodiments, the individual is about 10 years old to about 50 years old. In some embodiments, the individual is about any of the following: 0-10 years old, 10-20 years old, 20-30 years old, 30-40 years old, or 40-50 years old. In some embodiments, the individual is about any of the following: 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, or 40 years old . In some embodiments, the individual has moderate to severe congenital deafness. In some embodiments, the individual has suffered moderate hearing loss. In some embodiments, the individual has suffered profound hearing loss. In some embodiments, the individual suffers from retinitis pigmentosa. In some embodiments, the individual has mild retinitis pigmentosa. In some embodiments, the individual has moderate retinitis pigmentosa. In some embodiments, the individual has severe retinitis pigmentosa. In some embodiments, the individual exhibits progressive loss of vision. In some embodiments, the individual does not suffer vision loss. In some embodiments, the individual has suffered mild vision loss. In some embodiments, the individual has suffered moderate vision loss. In some embodiments, the individual has suffered severe vision loss. In some embodiments, the individual exhibits progressive loss of peripheral vision and/or vision in low light. In some embodiments, the subject does not suffer a loss of peripheral vision and/or low-light vision. In some embodiments, the individual has suffered a mild loss of peripheral vision and/or vision in low light. In some embodiments, the individual has suffered a moderate loss of peripheral vision and/or vision in low light. In some embodiments, the individual has suffered a profound loss of peripheral vision and/or vision in low light. In some embodiments, the individual exhibits progressive loss of cones and/or rods. In some embodiments, the individual does not suffer a loss of cones and/or rods. In some embodiments, the individual has suffered mild loss of cones and/or rods. In some embodiments, the individual has suffered moderate loss of cones and/or rods. In some embodiments, the individual has suffered severe loss of cones and/or rods.

In some embodiments, a method of ameliorating symptoms of Usher syndrome in an individual is provided, comprising editing a target RNA associated with Usher syndrome in a cell of the individual, comprising using dRNA, wherein the dRNA is encoded in a sequence comprising SEQ ID NO: In the construct of any sequence shown in 15-293 and 317-354. In some embodiments, a method of ameliorating symptoms of Usher syndrome in an individual is provided, comprising editing a target RNA associated with Usher syndrome in a cell of the individual, comprising using a dRNA, wherein the dRNA is encoded in a sequence comprising Table A (SEQ ID NO: 15-293) or a construct of a variant of any sequence in Table B (any one of SEQ ID NO: 317-332 and SEQ ID NO: 344-354), wherein the variant is identical to the parent sequence differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide.

In some embodiments, a method of ameliorating symptoms of Usher syndrome in an individual is provided, comprising editing a target RNA associated with Usher syndrome in a cell of the individual, comprising using dRNA, wherein the dRNA comprises a gene selected from Table A (SEQ ID NO: 15-293) or any one of the cyclization sequences in Table B (any of SEQ ID NO: 317-332 and SEQ ID NO: 344-354). In some embodiments, a method of ameliorating symptoms of Usher syndrome in an individual is provided, comprising editing a target RNA associated with Usher syndrome in a cell of the individual, comprising using dRNA, wherein the dRNA comprises the expression of Table A (SEQ ID NO: 15-293) or a nucleotide encoded by a variant of any of the cyclization sequences in Table B (any of SEQ ID NO: 317-332 and SEQ ID NO: 344-354), wherein the variant is the same as Non-bold sequences differ by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide. In some embodiments, the dRNA is linear. In some embodiments, the dRNA is circular, or capable of being circularized.

In some embodiments, a method of ameliorating symptoms of Usher syndrome in an individual is provided, comprising editing a target RNA associated with Usher syndrome in a cell of the individual, comprising using a dRNA having a targeting RNA, the targeting RNA being Any one of the targeting sequences in Table A (the small capital letter sequence of any one of SEQ ID NO: 15-293) or Table B (any one of SEQ ID NO: 317-332 and SEQ ID NO: 344-354) Encoding. In some embodiments, a method of ameliorating symptoms of Usher syndrome in an individual is provided, comprising editing a target RNA associated with Usher syndrome in a cell of the individual, comprising using a dRNA having a targeting RNA, the targeting RNA being Any one of the targeting sequences in Table A (the small capital letter sequence of any one of SEQ ID NO: 15-293) or Table B (any one of SEQ ID NO: 317-332 and SEQ ID NO: 344-354) A variant encoding wherein the variant differs from the parent sequence by no more than any one of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide. In some embodiments, a method of ameliorating symptoms of Usher syndrome in an individual is provided, wherein the method comprises introducing dRNA into a cell of the individual, wherein the dRNA comprises any one of Table A (SEQ ID NO: 15-293 Sequences other than those in bold font) or nucleotides encoded by variants of any of the circularized sequences in Table B (any of SEQ ID NOs: 317-332 and SEQ ID NOs: 344-354).

In some embodiments of a method for ameliorating symptoms of Usher syndrome according to any one of the methods described herein, wherein the method or dRNA is used to edit a target RNA encoding mutant Usher 2A, compared to a corresponding individual in which the dRNA or construct is not introduced , individuals into whom dRNA or constructs encoding dRNA were introduced showed a reduction in hearing loss. In some embodiments, the introduction of a dRNA or a construct encoding a dRNA reduces hearing loss by at least 10%, 20%, 30%, 40%, 50%, 75%, compared to a corresponding individual in which the dRNA or construct is not introduced. 100%, 2x, 5x, 10x, 20x, 30x, 40x, 50x, 100x or 1000x. In some embodiments, the individual into whom the dRNA or construct encoding a dRNA is introduced does not exhibit further hearing loss. In some embodiments, the introduction of a dRNA or a construct encoding a dRNA is compared to the introduction of a corresponding individual dRNA or construct encoding a corresponding dRNA that does not include one or more mismatched regions and/or one or more linker nucleic acid sequences. Individuals exhibit reduced hearing loss. In some embodiments, the introduction of a dRNA or a construct encoding a dRNA is compared to the introduction of a corresponding individual dRNA or construct encoding a corresponding dRNA that does not include one or more mismatched regions and/or one or more linker nucleic acid sequences. Individuals who exhibit at least a 10%, 20%, 30%, 40%, 50%, 75%, 100%, 2x, 5x, 10x, 20x, 30x, 40x, 50x reduction in hearing loss . 100 times or 1000 times. In some embodiments, hearing and hearing loss are determined by audiological assessment, such as, but not limited to, auditory brainstem responses and/or otoacoustic emissions.

In some embodiments of a method for ameliorating symptoms of Usher syndrome according to any one of the methods described herein, wherein the method or dRNA is used to edit a target RNA encoding mutant Usher 2A, compared to a corresponding individual in which the dRNA or construct is not introduced , individuals into whom dRNA or constructs encoding dRNA were introduced showed reduced vision loss. In some embodiments, the introduction of a dRNA or a construct encoding a dRNA reduces vision loss by at least 10%, 20%, 30%, 40%, 50%, 75%, compared to a corresponding individual in which the dRNA or construct is not introduced. 100%, 2x, 5x, 10x, 20x, 30x, 40x, 50x, 100x or 1000x. In some embodiments, individuals into whom dRNA or a construct encoding dRNA is introduced do not exhibit further vision loss. In some embodiments, the introduction of a dRNA or a construct encoding a dRNA is compared to the introduction of a corresponding individual dRNA or construct encoding a corresponding dRNA that does not include one or more mismatched regions and/or one or more linker nucleic acid sequences. Individuals exhibit reduced vision loss. In some embodiments, the introduction of a dRNA or a construct encoding a dRNA is compared to the introduction of a corresponding individual dRNA or construct encoding a corresponding dRNA that does not include one or more mismatched regions and/or one or more linker nucleic acid sequences. Individuals exhibiting a reduction in vision loss of at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 2x, 5x, 10x, 20x, 30x, 40x, 50x . 100 times or 1000 times. In some embodiments, vision includes vision in low light. In some embodiments, vision includes peripheral vision. In some embodiments, visual acuity and vision loss are determined by an optometric evaluation, such as, but not limited to, visual field testing.

In some embodiments of a method for ameliorating symptoms of Usher syndrome according to any one of the methods described herein, wherein the method or dRNA is used to edit a target RNA encoding mutant Usher 2A, compared to a corresponding individual in which the dRNA or construct is not introduced , individuals into whom dRNA or constructs encoding dRNA were introduced showed reduced retinal cell loss. In some embodiments, introduction of a dRNA or a construct encoding a dRNA reduces retinal cell loss by at least 10%, 20%, 30%, 40%, 50%, 75% compared to a corresponding individual in which the dRNA or construct is not introduced , 100%, 2x, 5x, 10x, 20x, 30x, 40x, 50x, 100x or 1000x. In some embodiments, individuals into whom dRNA or a construct encoding dRNA is introduced do not exhibit further retinal cell loss. In some embodiments, the introduction of a dRNA or a construct encoding a dRNA is compared to the introduction of a corresponding individual dRNA or construct encoding a corresponding dRNA that does not include one or more mismatched regions and/or one or more linker nucleic acid sequences. Individuals exhibiting at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold reduction in retinal cell loss times. 100 times or 1000 times. In some embodiments, retinal cells include rods and/or cones. In some embodiments, visual cells include peripheral rods and/or cones.

In some embodiments, a method of treating an individual with mucopolysaccharidosis type I (MPS I; e.g., Hurler syndrome or Scheie syndrome) associated with a target RNA having a mutation (e.g., a G>A mutation) is provided, This includes editing target RNA in cells of an individual using any of the RNA editing methods described herein. In some embodiments, the target RNA is IDUA ^W402X (e.g., TGG>TAG mutation in exon 9).

In some embodiments, a method of treating an individual with MPS I (e.g., Hurler syndrome or Scheie syndrome) associated with a target RNA having a mutation (e.g., a G>A mutation) is provided, comprising converting a dRNA or comprising a A construct of a dRNA nucleic acid is introduced into cells isolated from an individual in vitro, wherein the dRNA contains a targeting RNA sequence that hybridizes to a target RNA associated with a disease or disorder, wherein the dRNA is capable of recruiting ADAR to detoxify a target adenosine residue in the target RNA. amino group, and wherein the dRNA is a circular RNA or is capable of forming a circular RNA.

In some embodiments, a method of treating an individual with MPS I (e.g., Hurler syndrome or Scheie syndrome) associated with a target RNA having a mutation (e.g., a G>A mutation) is provided, comprising converting a dRNA or comprising a A nucleic acid construct of dRNA is introduced into cells isolated from an individual in vitro, wherein the dRNA comprises a targeting RNA sequence that hybridizes to a target RNA associated with a disease or disorder, wherein the dRNA is encoded in any one of SEQ ID NOs: 360-374 sequence in a construct in which the dRNA is capable of recruiting ADARs to deaminate target adenosine residues in the target RNA.

In some embodiments, the ADAR is an ADAR endogenously expressed in an isolated cell. In some embodiments, the method includes introducing ADAR or a construct comprising a nucleic acid encoding ADAR into an isolated cell. In some embodiments, the target RNA is IDUA ^W402X (e.g., TGG>TAG mutation in exon 9).

In some embodiments, a method of treating or preventing MPS I (e.g., Hurler syndrome or Scheie syndrome) associated with a target RNA having a mutation (e.g., a G>A mutation) in an individual is provided, comprising administering to the individual An effective amount of dRNA or a construct comprising a nucleic acid encoding a dRNA, wherein the dRNA comprises a target RNA sequence that hybridizes to a target RNA associated with a disease or disorder, wherein the dRNA is capable of recruiting ADAR to deaminate a target adenosine residue in the target RNA , and wherein the dRNA is a circular RNA or is capable of forming a circular RNA.

In certain embodiments, the methods of treating, preventing, and/or ameliorating MPS I and/or symptoms thereof as described herein result in a significant increase in the intelligence quotient (IQ) of the treated patient, such as using Bayley in Hurler subjects. Assessed by the Infant Development Scale. In certain embodiments, the methods of treating, preventing, and/or ameliorating MPS I and/or symptoms thereof as described herein result in a significant increase in the neurocognitive IQ of the treated patient, such as the Wechsler IQ in Hurler-Scheie subjects. Measured by the simplified table (WASI). In certain embodiments, the methods of treating, preventing, and/or ameliorating MPS I and/or symptoms thereof described herein result in a significant increase in neurocognitive DQ in the treated patient, as assessed using the Bayley Scales of Infant Development.

In certain embodiments, the methods of treating, preventing, and/or ameliorating MPS I and/or symptoms thereof described herein result in significant increases in functional human IDUA levels. In certain embodiments, the methods of treating, preventing, and/or ameliorating MPS I and/or symptoms thereof described herein result in significant reductions in GAG levels, such as in serum, urine, and/or cerebrospinal fluid (CSF) samples of the patient. measured.

In some embodiments, a method of treating a disease (eg, cancer) in an individual associated with a target RNA having a mutation (eg, a G>A mutation) is provided, comprising introducing a dRNA or a construct comprising a nucleic acid encoding a dRNA. Cells isolated from an individual in vitro, wherein the dRNA contains a targeting RNA sequence that hybridizes to a target RNA associated with a disease or disorder, wherein the dRNA is capable of recruiting ADAR to deaminate a target adenosine residue in the target RNA, and wherein the dRNA is circular RNA may form circular RNA.

In some embodiments, a method of treating a disease (eg, cancer) in an individual associated with a target RNA having a mutation (eg, a G>A mutation) is provided, comprising introducing a dRNA or a construct comprising a nucleic acid encoding a dRNA. In an individual, wherein the dRNA contains a targeting RNA sequence that hybridizes to a target RNA associated with a disease or disorder, wherein the dRNA is capable of recruiting ADAR to deaminate a target adenosine residue in the target RNA, and wherein the dRNA is a circular RNA or is capable of forming Circular RNA.

In some embodiments, a method of treating a disease (eg, cancer) in an individual associated with a target RNA having a mutation (eg, a G>A mutation) is provided, comprising introducing a dRNA or a construct comprising a nucleic acid encoding a dRNA. Cells isolated in vitro or in vivo of an individual, wherein the dRNA is encoded in a construct comprising any one of the sequences set forth in SEQ ID NO: 6 and SEQ ID NO: 294-314, and wherein the dRNA is capable of recruiting ADAR to target in the target RNA Deamination of adenosine residues.

In some embodiments, the ADAR is an ADAR endogenously expressed in an isolated cell. In some embodiments, the method includes introducing ADAR or a construct comprising a nucleic acid encoding ADAR into an isolated cell. In some embodiments, the target RNA is PPIA RNA.

Generally, the dosage, schedule, and route of administration of a composition (eg, dRNA or a construct comprising a nucleic acid encoding a dRNA) can be determined based on the size and condition of the individual and in accordance with standard pharmaceutical practice. Exemplary routes of administration include intravenous, intraarterial, intraperitoneal, intrapulmonary, intravascular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, intratumoral, intraocular, or transdermal.

For the purposes of this disclosure, desirable effects of treatment include, but are not limited to, reducing the rate of disease progression, improving or alleviating disease status, and alleviating or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are alleviated or eliminated, including but not limited to reducing (or destroying) the proliferation of cancer cells, increasing cancer cell killing, and reducing symptoms caused by the disease. , prevent the spread of the disease, prevent the recurrence of the disease, improve the quality of life of patients with the disease, reduce the dosage of other drugs required to treat the disease, delay the progression of the disease, and/or extend the survival period of the individual.

The RNA editing method of the present application can be used not only for animal cells, such as mammalian cells, but also for RNA modification of plants or fungi, such as plants or fungi with endogenously expressed ADAR. The methods described herein can be used to produce genetically engineered plants and fungi with improved properties.

Also provided are any of the dRNAs, constructs, cells having edited RNA, and compositions described herein for use in any of the therapeutic methods described herein, as well as any of the dRNAs, constructs, edited cells described herein and the use of the compositions in the preparation of medicaments for the treatment of diseases or conditions.

V. Compositions, kits and articles of manufacture

Also provided herein are compositions (eg, pharmaceutical compositions) comprising any dRNA, construct, library, or host cell with edited RNA as described herein.

In some embodiments, a pharmaceutical composition is provided comprising any dRNA or construct encoding a dRNA described herein, and a pharmaceutically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences 16 ^th edition , Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations employed and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine ; Preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens such as p-hydroxybenzoate; Methyl benzoate or propyl parahydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; simple sugars, Disaccharides and other sugars, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes such as zinc - protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). In some embodiments, lyophilized formulations are provided. Pharmaceutical compositions for in vivo administration must be sterile. This is easily accomplished, for example, by filtration through sterile filtration membranes.

Also provided are kits useful in any of the RNA editing methods or therapeutic methods described herein, comprising any of the dRNA, constructs, compositions, libraries or edited host cells described herein.

In some embodiments, a kit for editing a target RNA in a host cell is provided, comprising a dRNA or a construct comprising a nucleic acid encoding a dRNA, wherein the dRNA comprises and hybridizes to a disease or disorder-associated target RNA to form an RNA duplex. A strand of targeting RNA sequence, wherein the double-stranded RNA contains one or more mismatched regions, wherein the RNA double-strand is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA. In some embodiments, the dRNA is circular. In some embodiments, the dRNA comprises a linker nucleic acid sequence flanking the termini of the targeting RNA sequence.

In some embodiments, a kit for editing a target RNA in a host cell is provided, comprising a dRNA or a construct comprising a nucleic acid encoding a dRNA, wherein the dRNA comprises hybridizes to a target RNA associated with a disease or disorder to form an RNA A double-stranded targeting RNA sequence, wherein the dRNA includes a linker nucleic acid sequence flanking the ends of the targeting RNA sequence, wherein the linker nucleic acid sequence does not substantially form any secondary structure with any portion of the dRNA, and wherein the RNA double strand is capable of recruiting RNA adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, and wherein the dRNA is a circular RNA or a linear RNA capable of forming a circular RNA.

In some embodiments, the kit further comprises an ADAR or a construct comprising a nucleic acid encoding an ADAR. In some embodiments, the kit further comprises an inhibitor of ADAR3 or a construct thereof. In some embodiments, the kit further comprises an interferon stimulator or construct thereof. In some embodiments, the kit further includes instructions for performing any of the RNA editing methods or treatments described herein.

The kit of this application is packaged appropriately. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed Mylar or plastic bags), etc. Kits may optionally provide additional components such as transfection or transduction reagents, cell culture media, buffers, and interpretive information.

Accordingly, this application also provides articles of manufacture. The article of manufacture may include a container and a label or packaging insert on or associated with the container. Suitable containers include vials (eg sealed vials), bottles, jars, flexible packaging, etc. In some embodiments, the container holds the pharmaceutical composition and may have a sterile access port (eg, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). The container holding the pharmaceutical composition may be a multi-purpose vial that allows repeated administration (eg, 2-6 administrations) of the reconstituted formulation. Package insert refers to the instructions typically included in the commercial packaging of therapeutic products that contain information regarding the indications, usage, dosage, administration, contraindications, and/or warnings for the use of such products. Additionally, the article of manufacture may include a second container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may also include other materials needed from a commercial and user perspective, including additional buffers, diluents, screening protocols, needles, and syringes.

Kits or articles of manufacture may include multiple unit doses of a pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, such as hospital pharmacies and dispensing pharmacies.

Exemplary embodiments

Embodiment 1. A method of editing a target adenosine in a target RNA in a host cell, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding a dRNA into the host cell, wherein dRNA contains a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA double strand, The RNA double strand can recruit adenosine deaminase (ADAR) that acts on RNA to deaminate the target adenosine in the target RNA. The RNA double strand contains: (a) The first mismatched region relative to the target RNA sequence, which is located between 5 nucleotides (nt) and 85 nt upstream of the target adenosine; and/or (b) a second mismatched region located 20 nt to 85 nt downstream of the target adenosine relative to the target RNA sequence; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the target RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure.

Embodiment 2. A method according to any of the preceding embodiments, wherein: (a) the RNA duplex includes a first mismatch region relative to the target RNA sequence, which is located 5 nt to 25 nt upstream of the target adenosine; and/or the RNA duplex includes a second mismatch region relative to the target RNA sequence, It is located 20nt to 45nt downstream of the target adenosine; or (b) the RNA duplex includes a first mismatch region relative to the target RNA sequence, which is located 5 nt to 15 nt upstream of the target adenosine; and/or the RNA duplex includes a second mismatch region relative to the target RNA sequence, It is located 20 nt to 45 nt downstream of the target adenosine; or (c) the RNA duplex contains a first mismatch region relative to the target RNA sequence, which is located 20nt to 40nt upstream of the target adenosine; and/or the RNA duplex contains a second mismatch region relative to the target RNA sequence, which Located 25nt to 45nt downstream of target adenosine.

Embodiment 3. A method according to any one of the preceding embodiments, wherein the first mismatch region and/or the second mismatch region comprises: (a) Target one or more non-complementary nucleotides (mismatch) in the RNA sequence; and/or (b) Deletion of one or more nucleotides in the targeted RNA sequence; and/or (c) Targeting the insertion of one or more nucleotides into an RNA sequence.

Embodiment 4. The method according to any one of the preceding embodiments, wherein the first mismatch region and/or the second mismatch region comprises: (a) Target at least one set of contiguous non-complementary nucleotides (mismatch) in the RNA sequence; and/or (b) Deletion of at least one contiguous set of nucleotides in the targeted RNA sequence; and/or (c) Insertion of at least one contiguous set of nucleotides targeting an RNA sequence.

Embodiment 5. A method according to any of the preceding embodiments, wherein: (a) the first mismatch region has a length of 1-50 nt, optionally, wherein the first mismatch region has a length of 4 nt; and/or (b) The second mismatch region has a length of 1-50 nt, optionally, wherein the second mismatch region has a length of 4 nt.

Embodiment 6. A method according to any of the preceding embodiments, wherein: (a) The length of the first mismatch region is 1-10 nt, wherein the first mismatch region contains 1-10 consecutive non-complementary nucleotides in the targeting RNA sequence or 1-10 consecutive nucleotides in the targeting RNA sequence Deletion of nucleotides; and/or (b) The length of the second mismatched region is 1-10 nt, wherein the second mismatched region contains 1-10 consecutive non-complementary nucleotides in the targeting RNA sequence or 1-10 consecutive nucleotides in the targeting RNA sequence Deletion of nucleotides.

Embodiment 7. A method according to any of the preceding embodiments, wherein: (a) The length of the first mismatched region is 4 nt, wherein the first mismatched region includes four consecutive non-complementary nucleotides in the targeting RNA sequence or four consecutive nucleotide deletions in the targeting RNA sequence; and /or (b) The length of the second mismatched region is 4 nt, wherein the second mismatched region contains four consecutive non-complementary nucleotides in the targeting RNA sequence or four consecutive nucleotide deletions in the targeting RNA sequence.

Embodiment 8. A method according to any of the preceding embodiments, wherein: (i) Targeting non-complementary nucleotides in the RNA sequence results in bubble-like structures in the RNA duplex; and/or (ii) Nucleotide deletions in the targeted RNA sequence result in bulge structures in the RNA duplex; and/or (iii) Targeted nucleotide insertion into RNA sequences results in bulge structures in the RNA duplex.

Embodiment 9. A method according to any of the preceding embodiments, wherein: (i) Targeting a set of contiguous non-complementary nucleotides in the RNA sequence results in bubble-like structures in the RNA duplex; and/or (ii) The deletion of a contiguous set of nucleotides in the targeted RNA sequence results in a bulge structure in the RNA double strand; and/or (iii) Insertion of a set of contiguous nucleotides in the targeted RNA sequence results in a bulge structure in the RNA duplex.

Embodiment 10. According to the method of any one of the preceding embodiments, the target RNA encodes a mutant Usher 2A protein; optionally, wherein the mutant Usher 2A protein comprises a missense mutation, a nonsense mutation and/or a frameshift mutation.

Embodiment 11. A method according to any one of the preceding embodiments, wherein the mutant Usher 2A protein comprises the Trp3955Ter mutation.

Embodiment 12. A method according to any one of the preceding embodiments, wherein the target RNA encoding mutant Usher 2A comprises a G to A mutation with reference to the target RNA encoding wild-type Usher 2A.

Embodiment 13. A method according to any one of the preceding embodiments, wherein the target RNA encoding mutant Usher 2A comprises the 11864G>A mutation with reference to the target RNA encoding wild-type Usher 2A.

Embodiment 14. A method according to any one of the preceding embodiments, wherein the RNA double strand further comprises a third mismatch region relative to the target RNA, wherein the third mismatch region is located relative to the first mismatch region and the second mismatch relative to the target RNA between districts.

Embodiment 15. A method according to any one of the preceding embodiments, wherein the third mismatch region comprises one or two non-complementary nucleotides in the targeting RNA sequence and/or one or two nucleotide deletions in the targeting RNA sequence.

Embodiment 16. A method according to any one of the preceding embodiments, wherein the third mismatch region relative to the target RNA sequence is located 7 nt and/or 8 nt downstream of the target adenosine; optionally, wherein the target RNA comprises a region located downstream of the target adenosine Adenosine at the 7th and/or 8th nucleotide.

Embodiment 17. A method according to any one of the preceding embodiments, wherein the target RNA comprises an "AA" sequence located 7 and 8 nucleotides downstream of the target adenosine, wherein The targeting RNA sequence contains any of the following: A, AA, U, C, CC, G, GG, or a deletion of nucleotides ("X") located 7 and 8 nucleotides downstream of the target adenosine opposite the target RNA. ).

Embodiment 18. A method according to any one of the preceding embodiments, wherein the RNA double strands comprise: (a) a first mismatched region relative to the target RNA sequence located 27 nt to 30 nt upstream of the target adenosine; and (b) A second mismatched region relative to the target RNA sequence located 31 nt to 43 nt downstream of the target adenosine.

Embodiment 19. A method according to any of the preceding embodiments, wherein: The second mismatch region relative to the target RNA sequence is located 36nt to 39nt downstream of the target adenosine, Optionally, wherein the first mismatch region is 4 nt in length, and the second mismatch region is 4 nt in length; Further optionally, wherein the first mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence.

Embodiment 20. A method according to any of the preceding embodiments, wherein: The second mismatch region relative to the target RNA sequence is located 40nt to 43nt downstream of the target adenosine, Optionally, wherein the first mismatch region is 4 nt in length, and the second mismatch region is 4 nt in length; Further optionally, wherein the first mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence.

Embodiment 21. A method according to any one of the preceding embodiments, wherein the RNA double strands comprise: (a) a first mismatched region relative to the target RNA sequence located 21 nt to 30 nt upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence located 36 nt to 39 nt downstream of the target adenosine; Optionally, wherein the first mismatch region is 10 nt in length, and the second mismatch region is 4 nt in length; Further optionally, wherein the first mismatched region comprises a deletion of ten contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence.

Embodiment 22. A method according to any one of the preceding embodiments, wherein the RNA double strands comprise: (a) a first mismatched region relative to the target RNA sequence located 21 nt to 30 nt upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence located 40 nt to 43 nt downstream of the target adenosine; Optionally, wherein the first mismatch region is 10 nt in length, and the second mismatch region is 4 nt in length; Further optionally, wherein the first mismatched region comprises a deletion of ten contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence.

Embodiment 23. A method according to any one of the preceding embodiments, wherein dRNA is: (i) Circular; or (ii) Linear and/or capable of cyclization.

Embodiment 24. A method according to any one of the preceding embodiments, wherein the dRNA further comprises one or more RNA recruitment domains, optionally, wherein the RNA recruitment domains are stem-loop structures.

Embodiment 25. A method according to any one of the preceding embodiments, wherein the linker nucleic acid sequence is from about 5 nt to about 500 nt in length.

Embodiment 26. The method according to any one of the preceding embodiments, wherein the length of the linker nucleic acid sequence is less than or equal to 70nt, optionally, the length of the linker nucleic acid sequence is 10nt-50nt, 10nt-40nt, 10nt-30nt, 10nt- Any integer between 20nt, 20nt-50nt, 20nt-40nt, 20nt-30nt, 30nt-50nt, 30nt-40nt or 40nt-50nt.

Embodiment 27. A method according to any one of the preceding embodiments, wherein the length of the linker nucleic acid sequence is from about 20nt to about 60nt; optionally, the length of the linker nucleic acid sequence is about 30nt or about 50nt.

Embodiment 28. A method according to any one of the preceding embodiments, wherein at least about 50%, 60%, 70%, 80%, 85%, 90% or 95% of any one of the linker nucleic acid sequences comprise adenosine or cytidine; any Optionally, 100% of the linker nucleic acid sequences comprise adenosine or cytidine.

Embodiment 29. A method according to any one of the preceding embodiments, wherein at least about 50% of the linker nucleic acid sequences comprise adenosine.

Embodiment 30. A method according to any one of the preceding embodiments, wherein the method increases the level of editing of the target adenosine compared to a corresponding method in which the RNA duplex does not comprise one or more mismatch regions or in which the dRNA does not comprise a linker nucleic acid sequence. .

Embodiment 31. A method according to any one of the preceding embodiments, wherein the method reduces one or more non-targets compared to a corresponding method in which the RNA duplex does not comprise one or more mismatch regions or in which the dRNA does not comprise a linker nucleic acid sequence. Adenosine's (spectator) editorial level.

Embodiment 32. A method according to any one of the preceding embodiments, wherein the non-target adenosine is within one or more mismatch regions.

Embodiment 33. A method according to any one of the preceding embodiments, wherein the non-target adenosine is outside the mismatch region.

Embodiment 34. According to the method of any one of the preceding embodiments, the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a second linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence.

Embodiment 35. According to the method of any one of the preceding embodiments, the first linker nucleic acid sequence is identical to the second linker nucleic acid sequence.

Embodiment 36. According to the method of any of the preceding embodiments, the first linker nucleic acid sequence is different from the second linker nucleic acid sequence.

Embodiment 37. A method according to any one of the preceding embodiments, wherein the dRNA is a circular RNA, and wherein the one or more linker nucleic acid sequences connect the 5' end of the targeting RNA sequence and the 3' end of the targeting RNA sequence.

Embodiment 38. A method according to any one of the preceding embodiments, wherein the dRNA is a circular RNA, wherein the dRNA further comprises a 3' exon sequence and a 5' exon sequence, the 3' exon sequence may be flanked by the targeting RNA sequence Recognized by a 3' catalytic Group I intronic fragment at the 5' end, the 5' exon sequence may be recognized by a 5' catalytic Group I intronic fragment flanking the 3' end of the targeting RNA sequence.

Embodiment 39. The method according to any one of the preceding embodiments, wherein the dRNA further comprises a 3' connecting sequence and a 5' connecting sequence.

Embodiment 40. A method according to any one of the preceding embodiments, wherein the 3' linking sequence and the 5' linking sequence are at least partially complementary to each other.

Embodiment 41. A method according to any one of the preceding embodiments, wherein the length of the 3' linking sequence and the 5' linking sequence is from about 20 nt to about 75 nt.

Embodiment 42. A method according to any one of the preceding embodiments, wherein the dRNA is circularized by RNA ligase RtcB.

Embodiment 43. A method according to any one of the preceding embodiments, wherein the dRNA is circularized by T4 RNA ligase 1 (Rnl1) or RNA ligase 2 (Rnl2).

Embodiment 44. A method according to any one of the preceding embodiments, wherein the method comprises introducing into the host cell a construct comprising a nucleic acid sequence encoding dRNA.

Embodiment 45. A method according to any one of the preceding embodiments, wherein the construct further comprises a promoter operably linked to the nucleic acid sequence encoding dRNA.

Embodiment 46. A method according to any one of the preceding embodiments, wherein the promoter is a polymerase II promoter ("Pol II promoter").

Embodiment 47. A method according to any one of the preceding embodiments, wherein the promoter is a polymerase III promoter ("Pol III promoter").

Embodiment 48. A method according to any one of the preceding embodiments, wherein the construct is a viral vector or plasmid.

Embodiment 49. A method according to any one of the preceding embodiments, wherein the construct is an adeno-associated virus (AAV) vector.

Embodiment 50. A method according to any one of the preceding embodiments, wherein the construct is a self-complementary AAV (scAAV) vector.

Embodiment 51. A method according to any one of the preceding embodiments, wherein the ADAR is endogenously expressed by the host cell.

Embodiment 52. A method according to any one of the preceding embodiments, wherein the host cell is a retinal cell.

Embodiment 53. A method according to any one of the preceding embodiments, wherein the targeting RNA sequence is more than 50 nt in length.

Embodiment 54. A method according to any one of the preceding embodiments, wherein the targeting RNA sequence is from about 100 to about 200 nt in length.

Embodiment 55. A method according to any one of the preceding embodiments, wherein the targeting RNA sequence comprises cytidine, adenosine or uridine directly opposite the target adenosine in the target RNA.

Embodiment 56. A method according to any one of the preceding embodiments, wherein the targeting RNA sequence comprises a cytidine mismatch directly opposite the target adenosine in the target RNA.

Embodiment 57. A method according to any one of the preceding embodiments, wherein the cytidine mismatch is located at least 20 nt from the 3' end of the targeting RNA sequence and at least 5 nt from the 5' end of the targeting RNA sequence.

Embodiment 58. A method according to any one of the preceding embodiments, wherein the 5' nearest neighbor of the target adenosine in the target RNA is a nucleotide selected from the group consisting of U, C, A and G, preferably U>C≈A>G, and the target The 3' nearest neighbor of the target adenosine in the RNA is a nucleotide selected from G, C, A and U, preferably G>C>A≈U.

Embodiment 59. A method according to any one of the preceding embodiments, wherein the target adenosine is in the trisaccharyl motif of UAG, and wherein the targeting RNA sequence comprises an A directly opposite to the uridine in the trisaccharyl motif, cytidine directly opposite cytidine, and cytidine, guanosine, or uridine directly opposite guanosine in the tricarboxyl motif.

Embodiment 60. A method according to any one of the preceding embodiments, wherein the target RNA is an RNA selected from the group consisting of pre-messenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long non-coding RNA and small RNA, optionally, wherein The target RNA is pre-messenger RNA.

Embodiment 61. The method according to any one of the preceding embodiments, further comprising introducing an inhibitor of ADAR3 and/or an interferon stimulator into the host cell.

Embodiment 62. A method according to any one of the preceding embodiments, comprising introducing into the host cell a plurality of dRNAs or constructs encoding dRNAs, each dRNA targeting a different target RNA.

Embodiment 63. A method according to any one of the preceding embodiments, wherein the efficiency of editing target adenosine in the target RNA is at least about 40%.

Embodiment 64. A method according to any one of the preceding embodiments, further comprising introducing ADAR into the host cell.

Embodiment 65. A method according to any one of the preceding embodiments, wherein deamination of the target adenosine in the target RNA results in a missense mutation, an early stop codon, aberrant or alternative splicing in the target RNA, or reverses a missense in the target RNA Mutations, early stop codons, abnormal splicing, or alternative splicing.

Embodiment 66. A method according to any one of the preceding embodiments, wherein deamination of the target adenosine in the target RNA results in point mutation, truncation, elongation and/or misfolding of the protein encoded by the target RNA, or by reversing the misfolding in the target RNA. Sense mutations, early stop codons, aberrant splicing, or alternative splicing result in functional, full-length, correctly folded, and/or wild-type proteins.

Embodiment 67. A method according to any one of the preceding embodiments, wherein the host cell is a eukaryotic cell, optionally, wherein the host cell is a mammalian cell.

Embodiment 68. A method according to any one of the preceding embodiments, wherein the host cell is a human cell or a mouse cell.

Embodiment 69. Edited RNA or a host cell having edited RNA produced by the method of any of the preceding embodiments.

Embodiment 70. A method of treating or preventing a disease or condition in an individual comprising editing a target RNA associated with the disease or condition in cells of the individual according to a method according to any one of the preceding embodiments.

Embodiment 71. A method according to any one of the preceding embodiments, wherein the disease or condition is an inherited genetic disease or a disease or condition associated with one or more acquired gene mutations.

Embodiment 72. A method according to any one of the preceding embodiments, wherein the disease or disorder is a single or polygenic disease or disorder.

Embodiment 73. A method of ameliorating one or more symptoms of Usher syndrome in an individual, comprising editing a target RNA associated with Usher syndrome in a cell of the individual according to the method of any one of the preceding embodiments, wherein the target RNA encodes mutation Usher 2A protein.

Embodiment 74. The method according to any one of the preceding embodiments, wherein the target RNA has a G to A mutation.

Embodiment 75. A method according to any one of the preceding embodiments, wherein the individual has Usher syndrome type II.

Embodiment 76. A method according to any one of the preceding embodiments, wherein the subject has no vision loss, or the subject has mild to moderate vision loss.

Embodiment 77. A method according to any one of the preceding embodiments, wherein the host cell is a retinal cell, optionally, wherein the host cell is a rod photoreceptor cell and/or a cone photoreceptor cell.

Embodiment 78. A method according to any one of the preceding embodiments, wherein dRNA or a construct encoding dRNA is introduced into the subretinal space and/or the vitreous cavity.

Embodiment 79. A method according to any of the preceding embodiments, wherein:

(a) Individuals into whom dRNA or a construct encoding dRNA is introduced exhibit reduced vision loss compared to corresponding individuals into whom dRNA or construct is not introduced; and/or

(b) An individual into whom a dRNA or a construct encoding a dRNA is introduced compared to a corresponding individual into whom a dRNA or a construct encoding a dRNA is introduced that does not include one or more mismatched regions and/or one or more linker nucleic acid sequences. Demonstrated reduction in vision loss.

Embodiment 80. A method according to any of the preceding embodiments, wherein: (a) Individuals into whom dRNA or a construct encoding dRNA is introduced exhibit reduced retinal cell loss compared to corresponding individuals into which dRNA or construct is not introduced; and/or (b) An individual into whom a dRNA or a construct encoding a dRNA is introduced compared to a corresponding individual into whom a dRNA or a construct encoding a dRNA is introduced that does not include one or more mismatched regions and/or one or more linker nucleic acid sequences. Demonstrated reduced retinal cell loss.

Embodiment 81. A dRNA for editing a target RNA containing a target adenosine, wherein the dRNA contains a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, The RNA double strand can recruit adenosine deaminase (ADAR) that acts on RNA to deaminate the target adenosine in the target RNA. The RNA double strand contains: (a) A first mismatched region relative to the target RNA sequence located 5 nt to 85 nt upstream of the target adenosine; and/or (b) a second mismatched region located 20 nt to 85 nt downstream of the target adenosine relative to the target RNA sequence; and wherein the dRNA comprises a linker nucleic acid sequence flanking the termini of the target RNA sequence, wherein the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure.

Embodiment 82. A dRNA according to any one of the preceding embodiments, wherein: (a) the RNA duplex includes a first mismatch region relative to the target RNA sequence, the first mismatch region is located 5 nt to 25 nt upstream of the target adenosine; and/or the RNA duplex includes a region relative to the target RNA sequence a second mismatched region located 20 nt to 45 nt downstream of the target adenosine; or (b) the RNA duplex includes a first mismatch region relative to the target RNA sequence, the first mismatch region is located 5 nt to 15 nt upstream of the target adenosine; and/or the RNA duplex includes a region relative to the target RNA sequence A second mismatched region located 20 nt to 45 nt downstream of the target adenosine; or (c) The RNA duplex includes a first mismatch region relative to the target RNA sequence, the first mismatch region is located 20 nt to 40 nt upstream of the target adenosine; and/or the RNA duplex includes a region of mismatch relative to the target RNA sequence. The second mismatch region is located 25 nt to 45 nt downstream of the target adenosine.

Embodiment 83. The dRNA according to any one of the preceding embodiments, wherein the first mismatch region and/or the second mismatch region comprises: (a) Target one or more non-complementary nucleotides (mismatch) in the RNA sequence; and/or (b) Deletion of one or more nucleotides in the targeted RNA sequence; and/or (c) Targeting the insertion of one or more nucleotides into an RNA sequence.

Embodiment 84. The dRNA according to any one of the preceding embodiments, wherein the first mismatch region and/or the second mismatch region comprises: (a) Target at least one set of contiguous non-complementary nucleotides (mismatch) in the RNA sequence; and/or (b) Deletion of at least one contiguous set of nucleotides in the targeted RNA sequence; and/or (c) Insertion of at least one contiguous set of nucleotides targeting an RNA sequence.

Embodiment 85. A dRNA according to any one of the preceding embodiments, wherein: (a) the first mismatch region has a length of 1-50 nt, optionally, wherein the first mismatch region has a length of 4 nt; and/or (b) The second mismatch region has a length of 1-50 nt, optionally, wherein the second mismatch region has a length of 4 nt.

Embodiment 86. A dRNA according to any one of the preceding embodiments, wherein: (a) The length of the first mismatch region is 1-10 nt, wherein the first mismatch region contains 1-10 consecutive non-complementary nucleotides in the targeting RNA sequence or 1-10 consecutive nucleotides in the targeting RNA sequence Deletion of nucleotides; and/or (b) The length of the second mismatched region is 1-10 nt, wherein the second mismatched region contains 1-10 consecutive non-complementary nucleotides in the targeting RNA sequence or 1-10 consecutive nucleotides in the targeting RNA sequence Deletion of nucleotides.

Embodiment 87. A dRNA according to any one of the preceding embodiments, wherein: (a) The length of the first mismatched region is 4 nt, wherein the mismatched region includes four consecutive non-complementary nucleotides in the targeting RNA sequence or four consecutive nucleotide deletions in the targeting RNA sequence; and/or (b) The length of the second mismatched region is 4 nt, wherein the mismatched region contains four consecutive non-complementary nucleotides of the targeting RNA sequence or four consecutive nucleotide deletions of the targeting RNA sequence.

Embodiment 88. A dRNA according to any one of the preceding embodiments, wherein: (i) Targeting non-complementary nucleotides in the RNA sequence results in bubble-like structures in the RNA duplex; and/or (ii) Nucleotide deletions in the targeted RNA sequence result in bulge structures in the RNA duplex; and/or (iii) Targeted nucleotide insertion into RNA sequences results in bulge structures in the RNA duplex.

Embodiment 89. A dRNA according to any one of the preceding embodiments, wherein: (i) Targeting a set of contiguous non-complementary nucleotides in the RNA sequence results in bubble-like structures in the RNA duplex; and/or (ii) The deletion of a contiguous set of nucleotides in the targeted RNA sequence results in a bulge structure in the RNA double strand; and/or (iii) Insertion of a set of contiguous nucleotides targeting an RNA sequence results in a bulge structure in the RNA duplex.

Embodiment 90. The dRNA according to any one of the preceding embodiments, wherein the mutant Usher 2A protein comprises a missense mutation, a nonsense mutation and/or a frameshift mutation.

Embodiment 91. The dRNA according to any one of the preceding embodiments, wherein the mutant Usher 2A protein comprises the Trp3955Ter mutation.

Embodiment 92. The dRNA according to any one of the preceding embodiments, wherein the target RNA encoding mutant Usher 2A comprises a G to A mutation with reference to the target RNA encoding wild-type Usher 2A.

Embodiment 93. The dRNA according to any one of the preceding embodiments, wherein the target RNA encoding mutant Usher 2A comprises the 11864G>A mutation with reference to the target RNA encoding wild-type Usher 2A.

Embodiment 94. The dRNA according to any one of the preceding embodiments, wherein the RNA double strand further comprises a third mismatch region relative to the target RNA, wherein the third mismatch region is located in the first mismatch region and the second mismatch relative to the target RNA between districts.

Embodiment 95. The dRNA according to any one of the preceding embodiments, wherein the third mismatch region comprises one or two non-complementary nucleotides in the targeting RNA sequence and/or one or two nucleotide deletions in the targeting RNA sequence.

Embodiment 96. The dRNA according to any one of the preceding embodiments, wherein the third mismatch region relative to the target RNA sequence is located 7 nt and/or 8 nt downstream of the target adenosine; optionally, wherein the target RNA comprises a region located downstream of the target adenosine Adenosine at the 7th and/or 8th nucleotide.

Embodiment 97. The dRNA according to any one of the preceding embodiments, wherein the target RNA comprises an "AA" sequence located at 7th and 8th nucleotides downstream of the target adenosine, wherein the targeting RNA sequence comprises any of the following: A, AA, U , C, CC, G, GG, or a nucleotide deletion ("X") located 7 and 8 nucleotides downstream of the target adenosine opposite the target RNA.

Embodiment 98. The dRNA according to any one of the preceding embodiments, wherein the RNA double strands comprise: (a) a first mismatched region relative to the target RNA sequence located 27 nt to 30 nt upstream of the target adenosine; and (b) A second mismatched region relative to the target RNA sequence located 31 nt to 43 nt downstream of the target adenosine.

Embodiment 99. A dRNA according to any one of the preceding embodiments, wherein: The second mismatch region relative to the target RNA sequence is located 36nt to 39nt downstream of the target adenosine, Optionally, wherein the first mismatch region is 4 nt in length, and the second mismatch region is 4 nt in length; Further optionally, wherein the first mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence.

Embodiment 100. A dRNA according to any one of the preceding embodiments, wherein: The second mismatch region relative to the target RNA sequence is located 40nt to 43nt downstream of the target adenosine, Optionally, wherein the first mismatch region is 4 nt in length, and the second mismatch region is 4 nt in length; Further optionally, wherein the first mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence.

Embodiment 101. The dRNA according to any one of the preceding embodiments, wherein the RNA double strands comprise: (a) a first mismatch region located 21 nucleotides to 30 nucleotides upstream of the target adenosine relative to the target RNA sequence; and (b) a second mismatched region relative to the target RNA sequence located 36 nucleotides to 39 nucleotides downstream of the target adenosine; Optionally, wherein the first mismatch region is 10 nt in length, and the second mismatch region is 4 nt in length; Further optionally, wherein the first mismatched region comprises a deletion of ten contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence.

Embodiment 102. The dRNA according to any one of the preceding embodiments, wherein the RNA double strands comprise: (a) a first mismatch region located 21 nucleotides to 30 nucleotides upstream of the target adenosine relative to the target RNA sequence; and (b) a second mismatch region relative to the target RNA sequence located 40 nucleotides to 43 nucleotides downstream of the target adenosine; Optionally, wherein the first mismatch region is 10 nt in length, and the second mismatch region is 4 nt in length; Further optionally, wherein the first mismatched region comprises a deletion of ten contiguous nucleotides of the targeting RNA sequence, and wherein the second mismatched region comprises a deletion of four contiguous nucleotides of the targeting RNA sequence.

Embodiment 103. The dRNA according to any one of the preceding embodiments, wherein the dRNA is: (i) Circular; or (ii) Linear and/or capable of cyclization.

Embodiment 104. The dRNA according to any one of the preceding embodiments, wherein the dRNA further comprises one or more RNA recruitment domains, optionally, wherein the RNA recruitment domains are a stem-loop structure.

Embodiment 105. The dRNA according to any one of the preceding embodiments, wherein the linker nucleic acid sequence has a length of about 5 nt to about 500 nt.

Embodiment 106. The dRNA according to any one of the preceding embodiments, wherein the length of the linker nucleic acid sequence is less than or equal to 70nt, optionally, the length of the linker nucleic acid sequence is 10nt-50nt, 10nt-40nt, 10nt-30nt, 10nt-20nt, 20nt- Any integer between 50nt, 20nt-40nt, 20nt-30nt, 30nt-50nt, 30nt-40nt or 40nt-50nt.

Embodiment 107. The dRNA according to any one of the preceding embodiments, wherein the length of the linker nucleic acid sequence is about 20nt to about 60nt; optionally, the length of the linker nucleic acid sequence is about 30nt or about 50nt.

Embodiment 108. The dRNA according to any one of the preceding embodiments, wherein at least about 50%, 60%, 70%, 80%, 85%, 90% or 95% of any one of the linker nucleic acid sequences comprise adenosine or cytidine; any Optionally, 100% of the linker nucleic acid sequences comprise adenosine or cytidine.

Embodiment 109. The dRNA according to any one of the preceding embodiments, wherein at least about 50% of the linker nucleic acid sequences comprise adenosine.

Embodiment 110. A construct comprising a nucleic acid sequence encoding the dRNA of any one of the preceding embodiments.

Embodiment 111. The construct according to any one of the preceding embodiments, wherein the construct further comprises a promoter operably linked to the nucleic acid sequence encoding dRNA, wherein the promoter is a Pol III promoter.

Embodiment 112. A host cell comprising the dRNA of any of the preceding embodiments or the construct of any of the preceding embodiments.

Embodiment 113. A kit comprising the dRNA of any of the preceding embodiments or the construct of any of the preceding embodiments, and instructions for editing a target RNA comprising a target adenosine in a host cell.

Example

The following examples are intended purely as examples of the present application and therefore should not be construed as limiting the invention in any way. The following exemplary embodiments and examples, as well as the detailed description, are provided by way of illustration and not limitation.

Usher 2A Editorial Materials and Methods

Plasmid construction

For constructs expressing linear arRNA, the sequence of arRNA was synthesized (according to Table A) and golden-gate cloned into a plasmid backbone (PackGene Biotech, LLC) (SEQ ID NO: 2). The transcription of arRNA was determined by Driven by hU6 or CMV promoter.

For the gene-encoded construct expressing circ-arRNA, we first constructed a cloning vector based on the PackGene vector, which included Twister P3 U2A, 5’ linker sequence, 3’ linker sequence and Twister P148. (See Table A). The arRNA sequence was then synthesized and Golden Gate cloned into an autocatalytic circular RNA expression vector.

For constructs expressing linear arRNA, the arRNA sequence was synthesized and Golden Gate cloned into the pLenti-sgRNA-lib 2.0 (Addgene no. 89638) backbone, and the transcription of arRNA was driven by the hU6 or CMV promoter, respectively. For the genetically encoded construct expressing circ-arRNA, we first constructed a cloning vector based on the pLenti-sgRNA-lib 2.0 vector, which included Twister P3 U2A, 5’ linker sequence, 3’ linker sequence and Twister P1. The arRNA sequence was then synthesized and Golden Gate cloned into an autocatalytic circular RNA expression vector.

To investigate whether linkers could be used to further improve editing efficiency, arRNA sequences were flanked by spacers and/or linker sequences and then Golden Gate cloned into genetically encoded vectors expressing circ-arRNA.

To reduce off-target editing, nucleotides opposite selected potential off-target adenosines are deleted and then cloned into genetically encoded vectors expressing circ-arRNA.

Specifically, the DNA sequence (NNNNNNNNN) shown in Table A was first synthesized in vitro and integrated into the vector, then digested with MiuI and KpnI, and then connected to the plasmid backbone shown in SEQ ID NO: 2. plasmid backbone acgcgtgagggcctatttccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtatttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgattt cttggctttatatatcttgtggaaaggacgaaacaccg NNNNNNNNNN tttttttggtacc (SEQ ID NO: 2)

in vitro circRNA production and purification

circRNA was produced according to methods described in: Abe, N. et al. “Preparation of Circular RNA In Vitro,” Circular RNAs . Humana Press, New York, NY, 2018. 181-192; and Chen, H. et al. . “Preferential production of RNA rings by T4 RNA ligase 2 without any splint through rational design of precursor strand,” Nucleic Acids Research 48, e54–e54 (2020). Briefly, circRNA precursors were synthesized from linearized circRNA plasmid templates by in vitro transcription (IVT) using the HISCRIBE™ T7 High Yield RNA Synthesis Kit (New England Biolabs, #E2040S). After IVT, the IVT product was treated with DNase I (New England Biolabs, #M0303S) for 30 minutes to digest the DNA template. For T4 Rnl cyclization, T4 Rnl 1 (New England Biolabs, #M0239L) or T4 Rnl 2 (New England Biolabs, #M0204L) was added to the linear circRNA precursor and incubated overnight at 37°C after DNase I digestion. . For set 1 autocatalytic cyclization, after DNase I digestion, GTP was added to the reaction at a final concentration of 2 mM, and then the reaction was incubated at 55 °C for 15 min to catalyze circRNA cyclization. Then, the circularized circ-arRNA was column purified using Monarch RNA Cleanup Kit (New England Biolabs, #T2040L). Then, the column-purified RNA was heated at 65°C for 3 minutes and cooled on ice. The reaction was treated with RNase R (Epicenter, #RNR07250) for 15 min at 37°C to enrich circRNA. RNase R-treated RNA was subjected to column purification.

To further enrich circ-arRNA, high-performance liquid chromatography (Agilent HPLC1260) was used on a 4.6 × 300 mm size exclusion column with 5 μm particle size and 2000 Å pore size (Sepax Technologies, #215980P-4630) in RNase-free RNase R-treated circ-arRNA was isolated and purified in TE buffer. circ-arRNA enriched fractions were collected and then subjected to column purification (New England Biolabs, #T2040L). To further reduce the immunogenicity of purified circ-arRNA, circ-arRNA was heated at 65°C for 3 min, cooled on ice, and then treated with fast CIP phosphatase (New England Biolabs, #M0525S). Finally, circ-arRNA was column purified and concentrated using RNA Clean & Concentrator Kit (ZYMO, #R1018).

Construction of dual fluorescent reporter gene plasmids

Dual fluorescent reporter genes were cloned by PCR amplification of DNA encoding mCherry and EGFP (the start codon ATG of EGFP was deleted), and 3×GS adapters and targeting DNA sequences were added through primers during the PCR process. The PCR products were then cleaved and ligated with type II restriction enzyme BsmB1 (Thermo) and T4 DNA ligase (NEB) and inserted into the pLenti backbone.

In order to obtain a construct expressing the USH2A gene with the pathogenic mutation (NM_206933.2(USH2A)_c.11864G>A), USH2A was amplified by mutagenesis PCR from the construct encoding the corresponding gene introducing the G>A mutation. Full-length coding sequence. The target RNA sequence (SEQ ID NO: 3) of mutant USH2A (Figure 1A) was derived from the amplification product and cloned into the above pLenti backbone (containing EGFP and cherry reporter genes) by the Gibson cloning method. This reporter gene contains an in-frame stop codon between mCherry and EGFP (Figure 1B). Fluorescence of EGFP indicates target editing efficiency on RNA. This construct is hereafter referred to as mutant USH2A dual reporter.

Lentiviral packaging and reporter gene cell line construction

The mutant USH2A dual reporter plasmid and two viral packaging plasmids, pR8.74 and pVSVG (Addgene), were co-transfected into HEK293T-WT cells through X-tremeGENE HP DNA transfection reagent. After 72 hours, supernatant virus was collected and stored at -80°C. HEK293T-WT cells were infected with lentivirus. After 72 hours, mCherry-positive cells were sorted by FACS, cultured, and clone selection was performed after limiting dilution to generate clones that stably express the dual fluorescent reporter gene system with low EGFP background. Cell lines.

To generate a stable HEK293T reporter cell line, the mutant USH2A dual reporter construct was co-transfected into HEK293T cells together with two viral packaging plasmids, pR8.74 and pVSVG. After 72 hours, supernatant virus was collected and stored at -80°C. HEK293T cells were infected with lentivirus, then mCherry-positive cells were sorted and cultured by FACS, and clone selection was performed after limiting dilution to generate a clonal cell line that stably expresses a dual fluorescent reporter gene system with no detectable EGFP background.

Mammalian cell lines and cell culture

The HEK293T cell line was from the C. Zhang laboratory (Peking University) and maintained at 37°C, 5% _CO2 in agar containing 10% fetal calf serum (Vistech SE100-011), additionally supplemented with 1% penicillin-streptomycin. Cultured in Dulbecco's modified Eagle medium (Hyclone SH30243.01).

Plasmid transfection, FACS Analysis and Next Generation Sequencing (NGS)

In order to examine the effects of circularization, target RNA length, mismatched regions and/or addition of adapters on RNA editing, the editing efficiency was measured through EGFP positivity rate and deep sequencing.

Specifically, the HEK293T reporter cell line expressing the mutated USH2A dual reporter gene was seeded in a 12-well plate (15,000 cells/well) (recorded as 0 h). 24 hours after plating, 2.5 μg of arRNA plasmid (plasmid extracted by Qiagen #12945, quantified by Nanodrop) was transfected into each well using Lipofectamine 3000 according to the manufacturer's protocol.

72 hours after plating cells (48 hours after transfection), cells from each well were dissociated with trypsin (Invitrogen 25300054), and one-sixth of the dissociated cells were used to analyze the fluorescence intensity of mCherry and GFP by flow cytometry. All remaining dissociated cells were collected using TRIzol reagent (Thermofisher 15596026) and RNA was extracted from them (Zymo Research R2052), where 1 μg of extracted RNA was reverse transcribed into cDNA (NEB E6560L). Use 5 μL of reverse transcribed cDNA as a template, and use primers ggagtgagtacggtgtgcTGAATTTATGGAATGAAGGAGACCCT (SEQ ID NO: 376) and gagttggatgctggatggACGTCACCGCATGTTAGAAGACT (SEQ ID NO: 377) for PCR amplification (final primer concentration is 0.2 μM, NEB M0492L, annealing at 63°C, 35 cycle), and sequenced the PCR products using the NGS sequencing platform of the Rice Research Institute of the Chinese Academy of Sciences (refer to the sequencing process by Liu et al. Sci China Life Sci 2019 Jan;62(1):1-7, Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems).

Use fastp (v0.19.6) to perform quality control on the raw data obtained by high-throughput sequencing, and filter out low-quality reads, reads on adapter sequences, and reads on sequences containing polyG. Subsequently, the barcode corresponding to the obtained high-quality sequencing data was split into each sample, and BWA (v0.7.17-r1188) software was used to compare it with the amplified target region sequence (see sequence below), and SAMtools (v1 .9) Format conversion to generate BAM files. Statistically compare, reorder and index the information obtained. Use REDItools (v1.2.1) software to detect all potential RNA editing sites. The software parameters are as follows: use python REDItoolDenovo.py -i -f -o, after filtering out high-frequency point mutations that appear in the control and treated samples. , using "(average mutation frequency other than A->G mutation) + 3SD" as the threshold, and the reads whose A->G mutation frequency value exceeds the threshold at the editing site point are regarded as the true frequency of the target A to G mutation.

Sequence of amplified target region:

ggagtgagtacggtgtgcTGAATTTATGGATGAAGGAGACCCTgaggcctttcacactctacgaatcgggtcagcctgtaactccaagggttcagtggagtctgtAgtcattaacacaaactctggaagctcccaagattttccagctccttgggccacgctcattcagttggaattggacaaagccaGCGGCCGCTGAGGGCAGGA AGTCTTAACATGCGGTGACGTccatccaactc ( A indicates at the site of target editing) (SEQ ID NO: 378).

Example 1. Target Usher 2A 11864 mutated ring Leaper RNA design

To show the effectiveness of the 293T reporter cell line containing the Trp3955Ter mutant Usher 2A (NM_206933.2(USH2A)_c.11864G>A), a 151nt linear arRNA (Linear-151, see Table A) and the same length of circular arRNA (Circular-151, see Table A) were transfected into the reporter cell line. Linear-151-promoted on- and off-target editing was compared to Circular-151-promoted editing using the NGS protocol described above.

As shown in Figure 2, the circularized arRNA (Circular-151) showed significantly higher editing efficiency at the target adenosine (denoted as position 0) than the corresponding linear arRNA (Linear-151).

Furthermore, linear and circularized arRNAs with targeting RNA sequences of varying lengths (specifically, Linear-51, -61, - 71, -81, -91, -101, -111, -121, -131, -141, -151 and Circular -51, -61, -71, -81, -91, -101, -111, -121 , -131, -141, -151, -161, -171, -181, -191, -201, -211, -221, as shown in Table A). Target editing efficiency was measured by the average fluorescence intensity of GFP as described above.

As shown in Figure 3, circularized arRNA containing targeting RNA sequences ranging from 121 to 151 nucleotides in length showed higher editing efficiency, as evidenced by a higher increase in the average fluorescence intensity of GFP. The gene editing efficiency of circularized arRNA can be further improved by using circularized arRNA with longer targeting RNA sequences (eg, 151 to 201 nucleotides in length).

Example 2. by targeting RNA Sequence introduction of mismatches and deletions to improve editing efficiency

As shown in Figures 2 and 3, although circularization of arRNA can increase on-target editing (at position 0), there is observable off-target editing in regions upstream and downstream of the target adenosine. Figure 4 further illustrates various non-target adenosines in USH2A mRNA.

To investigate whether off-target editing and/or on-target editing can be reduced by mismatches or deletions in the targeting RNA sequence, one or more of the regions downstream (+) or upstream (-) of the editing site opposite the target RNA were Multiple sites: +31, +35, +39, or -26, -30, -34, further modify the circular arRNA with a 171 nt targeting RNA sequence (USHER-171) as described in the table below.

Table 1: Design of mismatch regions in arRNA arRNA annotation Targeting mismatches or deletions in the RNA sequence (position relative to the target RNA) +31: +32 to +35 (32 nt to 35 nt downstream of target adenosine) +35 +36 to +39 (36 nt to 39 nt downstream of the target adenosine) +39 +40 to +43 (40 nt to 43 nt downstream of target adenosine) -26 -27 to -30 (27 to 30 nucleotides upstream of the target adenosine) -30 -31 to -34 (31 to 34 nucleotides upstream of the target adenosine) -34 -35 to -38 (35 to 38 nucleotides upstream of the target adenosine) -21x or -26x-21x -21 to -30 (21 to 30 nucleotides upstream of the target adenosine) -d21/22 -21 to -22 deletion (21 nt to 22 nt upstream of the target adenosine)

Mismatches and deletions to improve editing efficiency

Figure 5 shows an exemplary design for such a mismatch or deletion on Usher-171 (see also Table A). Specifically, modifications may include one or more of the following designs: (1) mutations in the arRNA, resulting in mismatches and the formation of bubble-like structures; (2) deletions in the arRNA, resulting in the formation of bubble-like structures in the corresponding regions of the target RNA. Mutated USH2A dual reporter cell lines were transfected with the described arRNA using Lipofectamine 3000 as described above. Target editing efficiency was measured by the average fluorescence intensity of GFP as described above. On-target and off-target editing promoted by the arRNA was determined using the NGS protocol described above.

As shown in Figure 6, mismatches or deletions in the targeting RNA sequence at one or more sites increase the editing efficiency of the target adenosine relative to the downstream (+) or upstream (-) region of the editing site, As shown by the higher increase in the average fluorescence intensity of GFP.

As shown in Figure 7, although mismatches or deletions of the targeting RNA sequence at one or more sites increased the target adenosine (at position 0) relative to the downstream (+) or upstream (-) region of the editing site ), but there was no significant increase in off-target editing compared to USHER-171.

In summary, USHER-171 arRNA with -26/+31, -26/+35 and -26+39 4bp deletions showed significantly increased editing efficiency of target adenosine, while compared with USHER-171, Produce comparable or reduced off-target editing efficiency.

Additional mismatches and deletions for improved editing efficiency

To investigate whether off-target editing could be further reduced and/or on-target editing further enhanced by targeting mismatches or deletions in the RNA sequence, the regions downstream (+) or upstream (-) of the editing site opposite the target RNA were One or more additional sites: +d7/8, or -21/22del, -21x, further modify the USHER-171 arRNA with -26/+31, -26/+35 and -26+39 4bp deletions, as follows described in the table.

Table 2: Design of mismatch regions in arRNA arRNA annotation Targeting mismatches or deletions in the RNA sequence (position relative to the target RNA) +d7/8: +7 and/or +8 deletion (7 and/or 8 nucleotides downstream of the target adenosine) -21x or -26x-21x -21 to -30 (21 to 30 nucleotides upstream of the target adenosine) -21/22del -21 to -22 deletion (21 to 22 nucleotides upstream of target adenosine)

Figure 8A shows an example of such additional mismatches or deletions (upstream of the target adenosine relative to the target RNA) on USHER-171 arRNA with -26/+31, -26/+35 and -26+39 4bp deletions. sexual design (see also Table A). Figure 8B shows an example of such additional mismatches or deletions (downstream of the target adenosine relative to the target RNA) on USHER-171 arRNA with -26/+31, -26/+35 and -26+39 4bp deletions. sexual design (see also Table A). Mutated USH2A dual reporter cell lines were transfected with the arRNA using Lipofectamine 3000 as described above. Target editing efficiency was measured by the average fluorescence intensity of GFP as described above. On-target and off-target editing promoted by the arRNA was determined using the NGS protocol described above.

As shown in Figure 9, mismatches or deletions of the targeting RNA sequence at one or more sites increased the editing efficiency of targeted adenosine relative to the downstream (+) or upstream (-) region of the editing site, As shown by the higher increase in the average fluorescence intensity of GFP. In particular, deletion of ten consecutive nucleotides (-26x-21x) in the targeted RNA sequence 21 to 30 nt upstream (-) of the editing site most significantly increased target editing (US+35x-26x- 21x and US+39x-26x-21x).

As shown in Figure 10, although additional mismatches or deletions of the targeting RNA sequence at one or more sites increased the target adenosine (in position 0), but there was no significant increase in off-target editing compared to USHER-171. In particular, deletion of ten consecutive nucleotides (-26x-21x) in the targeted RNA sequence relative to 21 to 30 nt (-) upstream of the editing site more significantly increased target editing (US+35x-26x- 21x and US+39x-26x-21x).

In summary, USHER-171 arRNA with +35x-26x-21x and +39x-26x-21x deletions exhibits significantly increased editing efficiency of target adenosine, whereas USHER-171 exhibits comparable or Reduced off-target editing efficiency.

Example 3. Reduce off-target editing with flexible linkers

To investigate whether off-target editing could be reduced by using linkers that do not hybridize to the target RNA and essentially do not form secondary structure, as shown in Figure 11, a 5' ("left" flexible linker of the targeting RNA sequence flanking the arRNA was added , or L-flexible linker) or 3' ("right" flexible linker, or R-flexible linker) flexible linker (with 10 nt, 20 nt, or 30 nt length). (See Table A for relevant dRNA sequences).

Mutated USH2A dual reporter cell lines were transfected with the described arRNA using Lipofectamine 3000 as described above. Target editing efficiency was measured by the average fluorescence intensity of GFP as described above. On-target and off-target editing promoted by the arRNA was determined using the NGS protocol described above.

Figure 12 shows that arRNA containing L-flexible linkers and R-flexible linkers ranging from 10 nt in length to 30 nt in length exhibited similar on-target editing, as indicated by a comparable increase in the mean fluorescence intensity of GFP.

As shown in Figure 13, adding an L-flexible linker flanking the 5' of the targeting RNA sequence more significantly reduced off-target editing relative to the 5' targeting RNA sequence, where the R flanking the 3' of the targeting RNA sequence - Flexible linkers more significantly reduce off-target editing relative to 3' targeted RNA sequences.

Example 4. By targeting relative to non-target adenosine RNA mismatches in and / or missing to improve editing efficiency

flexible linker sequence

In order to investigate whether the flexible linker described in Example 3 can be used to reduce off-target editing of USHER-171 arRNA with mismatched regions described in Example 2 above, by flanking the flexible linker sequence (from 10nt to 50nt) in Target the RNA sequence, or alternatively target the end of the RNA sequence to further modify the arRNA (US+35x-26x-21x and US+39x-26x-21x).

The arRNAs with mismatched regions (US+35x-26x-21x and US+39x-26x-21x) were generated either without flexible sequence (0nt) or with flexible linker sequence (10nt, 20nt, 30nt, 40nt , 50nt), and transfected into mutant USH2A dual reporter cell lines using Lipofectamine 3000 as described above. Target editing efficiency was measured by the average fluorescence intensity of GFP as described above. On-target and off-target editing promoted by the arRNA was determined using the NGS protocol described above.

As shown in Figure 15 (first and third sets of data from the left), adding flexible linker sequences slightly reduced the on-target performance of arRNAs with mismatched regions (US+35x-26x-21x and US+39x-26x-21x). Editing efficiency, and this decrease increases with the length of the flexible linker sequence, as shown by the average fluorescence intensity of GFP. However, compared to the parental USHER-171 arRNA, even with a 50nt linker sequence, on-target editing efficiency was still high (see comparison of average fluorescence intensity with Figure 6).

As shown in Figure 16, adding flexible linker sequences significantly reduced off-target editing of arRNAs with mismatched regions (US+35x-26x-21x and US+39x-26x-21x), and this reduction seemed to be beneficial for longer flexible The linker sequences were more effective, with longer flexible linker sequences (30nt, 40nt, 50nt) apparently removing almost all detectable off-target edits except those located 7 and 8 cores downstream of the target editing site (position 0) Non-target adenosine at the nucleotide.

Solving the problem of residual editing of non-target adenosine

To further address the issue of non-target editing at positions +7 and +8, the arRNAs (US+35x-26x-21x and US+39x-26x-21x) described in Example 2 were modified as shown in Figure 14A , to introduce mismatches (e.g. deletions) relative to non-target adenosines at positions +7 and +8. Such arRNAs are further modified by flanking, or replacing the termini of, the targeting RNA sequence with flexible linker sequences (from 10nt to 50nt) (as shown in Figure 14B).

Generation of the arRNAs with mismatched regions (US+35x-26x-21x+D7/8 and US+39x-26x-21x+D7/8), either without the flexible linker sequence (Ont) or with the flexible linker sequence (10nt, 20nt, 30nt, 40nt, 50nt) and transfected into mutant USH2A dual reporter cell lines using Lipofectamine 3000 as described above. Target editing efficiency was measured by the average fluorescence intensity of GFP as described above. On-target and off-target editing promoted by the arRNA was determined using the NGS protocol described above.

As shown in Figure 15, although the additional mismatch D7/8 (deletion of positions 7 and 8 relative to the target RNA) reduced arRNA (US+35x-26x-21x+D7/8 and US+39x-26x-21x +D7/8), but further addition of the flexible linker sequence did not result in a further significant reduction in target editing efficiency (see the second and fourth data sets from the left), as shown by the average fluorescence intensity of GFP. In any case, on-target editing efficiency was still high compared to the parental USHER-171 arRNA, even with the D7/8 mismatch and added linker sequence (see comparison of average fluorescence intensity with Figure 6).

As mentioned above, adding a 30nt-50nt long flexible linker sequence virtually eliminated all off-target editing of arRNAs with mismatched regions (US+35x-26x-21x and US+39x-26x-21x) except downstream of the target editing site Non-target adenosine at nucleotides 7 and 8 (Fig. 16 center panel). As shown in Figure 16 (right panel), 40nt and 50nt flexible linker sequences were used to add mismatched regions at positions +7 and +8 relative to the non-target adenosine (US+35x-26x-21x+D7/8 and US+39x-26x-21x+D7/8), virtually eliminating all off-target editing while apparently retaining a high on-target editing efficiency of over 60% (see also comparison with the parental USHER-171 arRNA in Figure 7) .

further improvement relative to target RNA of +7/8 mismatch region

To investigate which type of mismatch (non-complementarity or deletion) in the targeting RNA is most effective in reducing bystander editing of non-target adenosines at positions +7 and +8, various mismatch combinations (relative to At positions +8 and +7 of the target RNA, A, AA, U, UU, C, CC, G, GG, or “X” = no nucleotide) were used to generate additional arRNA and matched with no mismatch ( UU) and the parent USHER-171 (see Figure 17).

The above arRNA was transfected into mutant USH2A dual reporter cell lines using Lipofectamine 3000 as described above. Target editing efficiency was measured by the average fluorescence intensity of GFP as described above. On-target and off-target editing promoted by the arRNA was determined using the NGS protocol described above.

As shown in Figure 18, the mismatch of +7/8 relative to the target RNA resulted in a slight decrease in the on-target editing efficiency of US+35x-21x and US+39x-21x arRNA, but the on-target editing efficiency was roughly the same as that of the parent USHER- 171 arRNA equivalent.

As shown in Figure 19, with the exception of “CC”, all mismatch combinations of +7/8 relative to the target RNA resulted in a significant reduction or complete elimination of non-specific editing of non-target adenosines at positions +7 and +8 .

To further elucidate mismatches in the targeted RNA sequence that effectively reduce bystander editing of non-target adenosines at positions +7 and +8, but minimally reduce target editing (at position 0), all mismatch combinations (At positions +8 and +7 in the targeting RNA relative to the target RNA, AA, AU, AC, AG, UA, UU, UC, UG, CA, CU, CC, CG, GA, GU, GC, GG, A, U, C, G, or “X”) were used to generate additional arRNA and paired with arRNA that had no mismatch (UU) and missing nucleotides relative to +8 and +7 of the target RNA (“X” ”) for comparison (see Figure 20).

As shown in Figures 21, 22, and 23, although the mismatch at +7/8 relative to the target RNA resulted in US+35x-21x ( 30nt adapter) (Figure 21, upper panel), US+35x (Figure 21, lower panel), US+35x-21x (50nt adapter) (Figure 22), and 85-C-85 (Figure 23) arRNA have slight on-target editing efficiency Reduced, but the number of mismatched nucleotides in the targeting RNA sequence relative to +8 and +7 of the target RNA is reduced compared to the deletions ("X") of nucleotides +8 and +7 relative to the target RNA. Select combinations that result in higher on-target editing, as shown by the average fluorescence intensity of GFP between "UU" and "X" (between the dashed lines in Figures 21, 22 and 23). As can be seen in Figures 21, 22, and 23, in the presence of two consecutive non-target adenosines ("UU") in the target RNA facilitated by an arRNA with no mismatch relative to the non-target AA (i.e., "UU") In the case of high on-target editing but substantial bystander editing, arRNAs with "U" mismatches relative to non-target AA can promote On-target editing at AA is slightly reduced but bystander editing is significantly reduced, whereas arRNAs with nucleotide deletions ("X") relative to non-target AA promote further reduction but almost complete elimination of on-target editing at non-target AA Edited by The Spectator.

In summary, with the introduction of mismatched regions (US+35x-26x-21x, US+39x-26x-21x) and flexible linker sequences (30nt, 40nt, 50nt), the editing efficiency and specificity of arRNA can be obtained A huge improvement. Additional mismatch regions can also be introduced to account for any residual off-target editing (such as +D7/8 in the case of USHER-171 arRNA editing).

Example 5. Compare mismatched regions created by deletions and insertions

Deletions and insertions in mismatched regions

To compare the effects of deletions and insertions as mismatched regions on improving target editing, one or more sites in the downstream (+) or upstream (-) region of the editing site opposite the target RNA: -26, + 35, modified USHER-171 arRNA as described in Table 3 and shown in Figures 24 and 25.

Table 3: Design of mismatch regions in arRNA arRNA annotation Target deletions or insertions in the RNA sequence (location relative to the target RNA) -26 Missing(n) -26 -B(n) Deletions starting 27 nucleotides upstream of the target adenylate and n nucleotides further upstream +35 Missing(n) +35 -B(n) Deletions starting 36 nucleotides upstream of the target adenylate and n nucleotides further upstream -26 insert(n) Insertion of n nucleotides (A) between 26 and 27 nucleotides upstream of the target adenylate +35 insert(n) Insertion of n nucleotides (A) between 35 and 36 nucleotides downstream of the target adenylate

As shown in Figure 26, insertion or deletion of 1 to 10 nucleotides in the targeting RNA sequence at the indicated site relative to the downstream (+35) or upstream (-26) region of the editing site, The editing efficiency of target adenosine is increased, as shown by a higher increase in the average fluorescence intensity of GFP.

Figures 27 and 29 further illustrate target RNA complementarity of targeting RNA sequences with mismatched regions. As shown in Figure 27, in the case where the target RNA sequence has no deletion, 4nt deletion or 50nt deletion at position +35, the total length complementary to the target RNA is still 171nt (including the target A/C mismatch). Specifically, the 50 nt complementary region (relative to the target RNA) downstream of the target adenosine starts at position +35 (Usher +35-B0) in the targeting RNA sequence without deletion, and ends at position +35-B0 in the targeting RNA sequence with a 4 nt deletion. A position 4 nt from position +35 (Usher +35-B4), or a position 50 nt from position +35 (Usher +35-B50) in the targeting RNA sequence with a 50 nt deletion. As shown in Figure 29, in the case where the targeting RNA sequence has no deletion, 4nt deletion or 50nt deletion at position -26, the total length complementary to the targeting RNA is still 171nt (including the target A/C mismatch). Specifically, the 59nt complementary region upstream of the target adenosine (relative to the target RNA) starts at position -26 (Usher-26-B0) in the targeting RNA sequence without deletion, and ends at position -26-B0 in the targeting RNA sequence with a 4nt deletion. at a position 4 nt from -26, or 50 nt from -26 in the targeting RNA sequence with a 50 nt deletion (Usher -26-B50).

Figure 28 (left panel) shows that deletions of 4 to 20 nucleotides in the targeted RNA sequence in the indicated region downstream of the editing site (+35) increased the editing efficiency of the target adenosine, as measured by the average fluorescence of GFP. This is shown by a higher increase in light intensity. Figure 28 (right panel) further shows deletions of 4 to 50 nucleotides in the targeted RNA sequence in the region indicated downstream of the editing site (+35), and in the region indicated upstream of the editing site (-26). Deletion of a further 4 nucleotides in the targeting RNA sequence of the region significantly improved the editing efficiency of the target adenosine, as shown by a higher increase in the average fluorescence intensity of GFP.

Figure 30 (left panel) shows that deletions of 4 to 30 nucleotides in the targeted RNA sequence in the indicated region upstream of the editing site (-26) resulted in similar editing efficiencies for target adenosine, as average fluorescence of GFP. Comparable increases in light intensity are shown. Figure 30 (right) further shows deletions of 4 to 50 nucleotides in the targeted RNA sequence in the region indicated upstream of the editing site (-26), and in the region indicated downstream (+35) of the editing site. Deletion of a further 4 nucleotides in the targeting RNA sequence significantly improved the editing efficiency of the target adenosine, as shown by a higher increase in the average fluorescence intensity of GFP.

Figure 31 shows that deletion of 4 to 20 nucleotides in the targeted RNA sequence in the two indicated regions upstream (-26) and downstream (+35) of the editing site can significantly improve the editing efficiency of the target adenosine. As shown by the higher increase in the average fluorescence intensity of GFP.

Example 6. Comparing the ability of flexible linkers to reduce off-target editing

As shown in Example 3, off-target editing can be reduced by using linkers that do not hybridize to the target RNA and do not substantially form secondary structures flanking the targeting RNA sequence. To investigate the types of flexible linkers that might be more effective in reducing off-target editing, for the indicated targeting RNA sequences (+35x-21x and +35x-21x+d7/8), various flexible linkers (50 nt) were added to the target 3' to the RNA sequence (see Figure 32) and generate arRNA according to the protocol described in Materials and Methods above (see Table A for relevant dRNA sequences).

Mutated USH2A dual reporter cell lines were transfected with the described arRNA using Lipofectamine 3000 as described above. Target editing efficiency was measured by the average fluorescence intensity of GFP as described above. On-target and off-target editing promoted by the arRNA was determined using the NGS protocol described above.

As shown in Figure 33, with the exception of the AT linker, arRNA with the remaining 50 nt linkers tested showed similar on-target editing efficiencies, as shown by a comparable increase in the mean fluorescence intensity of GFP.

Example 7. Studying stem loops' ability to increase target editing

Katrekar et al. reported in Nature Biotechnology (2022) that certain stem-loop structures can improve on-target editing, for example, comparing GluR2 100.50, Alu 100.50, U6+27 100.50 with linear 100.50 (Figure 34).

To investigate whether these stem-loop structures can enhance on-target editing of various circular arRNAs or circular arRNAs with mismatched regions, 85-C-85 (USHER-171), -21x+35x-R-flexlinker30 and -21+ were generated 35x-R-flexlinker50 arRNA and corresponding arRNAs further modified by adding GluR2, Alu or U6+27 stem-loop structures (Figure 35) (see Table A for related dRNA sequences).

Mutant USH2A dual reporter cell lines were transfected with the described arRNA using Lipofectamine 3000 as described above. Target editing efficiency was measured by the average fluorescence intensity of GFP as described above. On-target and off-target editing promoted by the arRNA was determined using the NGS protocol described above.

As shown in Figure 36, compared to arRNA without a stem-loop structure (control), addition of a stem-loop structure (GluR2, Alu, or U6+27) resulted in a reduction in on-target editing, such as less mean fluorescence intensity of GFP Increase shown.

Example 8. among rhesus monkeys Usher 2A Editor

Rhesus Monkey Cell Lines and Cell Culture

Rhesus monkey kidney cell line LLC-MK2 cells (ATCC number: CCL-7) were cultured using DMEM (Hyclone SH30243.01) containing 10% FBS (Vistech SE100-011). As described above, the USH2A dual reporter gene corresponding to rhesus monkey USH2A (XM_005540847.1:c.12183A>A) was constructed and stably introduced into LLC-MK2 cells.

Plasmid transfection, FACS Analysis and Next Generation Sequencing (NGS)

To study the effects of circularization, target RNA length, mismatched regions, and/or the addition of linkers on RNA editing, editing efficiency was measured by deep sequencing.

Specifically, LLC-MK2 cells expressing mutant USH2A were seeded in 12-well plates (15,000 cells/well) (recorded as 0 h). 24 hours after plating, 2.5 μg of arRNA encoding plasmid (SEQ ID NO: 4) (plasmid extracted by Qiagen #12945 and quantified by Nanodrop) was transfected into each well using Lipofectamine 3000 according to the manufacturer's protocol.

72 hours after cell plating (48 hours after transfection), cells in each well were dissociated with trypsin (Invitrogen 25300054), and one-sixth of the dissociated cells were used to analyze the fluorescence intensity of mCherry and GFP by flow cytometry. All remaining dissociated cells were collected using TRIzol reagent (Thermofisher 15596026) and RNA was extracted from them (Zymo Research R2052), of which 1 μg of extracted RNA was reverse transcribed into cDNA (NEB E6560L). Use 5 μL of reverse transcribed cDNA as a template, and use primers ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTA (SEQ ID NO: 379) and gagttggatgctggatggCTTAATCACCATTGGTTCTCA (SEQ ID NO: 11) for PCR amplification (final primer concentration 0.2 μM, NEB M0492L, annealing at 63°C, 35 cycles ), the PCR product was sequenced using the NGS sequencing platform of the Rice Research Institute of the Chinese Academy of Sciences (refer to the sequencing process of Liu et al. Sci China Life Sci 2019 Jan;62(1):1-7, Hi-TOM: a platform for high- throughput tracking of mutations induced by CRISPR/Cas systems).

Use fastp (v0.19.6) to perform quality control on the raw data obtained by high-throughput sequencing, and filter out low-quality reads, reads on adapter sequences, and reads on sequences containing polyG. Subsequently, the barcode corresponding to the obtained high-quality sequencing data was split into each sample, and the BWA (v0.7.17-r1188) software was used to compare it with the sequence of the amplified target region (see sequence below), and SAMtools ( v1.9) format conversion to generate BAM files. Statistically compare, reorder and index the information obtained. Use REDItools (v1.2.1) software to detect all potential RNA editing sites. The parameters are as follows: use python REDItoolDenovo.py -i -f -o. After filtering out high-frequency point mutations that appear in the control and treated samples, use "(Average mutation frequency other than A->G mutation) + 3SD" is used as the threshold, and the reads whose A->G mutation frequency value exceeds the threshold at the editing site are regarded as the true frequency of the target A to G mutation.

Sequence of the amplified target region: ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTAgaaccattcacaacatatctcattggtgttgtggctgcaaaccatgcaggagaaattttaagcccctggactctgattcaaacctt A gaatcttccccaagtggactgagaaactttatagtagaacagaaagagaatggccgggcattgctactacagtggtcagag cctaTGAGAACCAATGGTGTGATTAAGccatccagcatccaactc ( A indicates the site of target editing) (SEQ ID NO: 12).

among rhesus monkeys Usher 2A in vivo editing

To study the in vivo editing of Ush2A mRNA, mutant rhesus monkeys endogenously expressing Ush2A (XM_005540847.1:c.12183A>A) were edited via Ush2A-specific arRNA.

The arRNA encoding plasmid (SEQ ID NO: 5) was packaged into adeno-associated virus of serotype AAV8 by Guangzhou Paizhen Biotechnology Co., Ltd. with a titer of 5 × 10 ¹³ genome copies/mL. AAV (packaging arRNA) or control (NaCl) was injected through the subretinal space of both eyes of rhesus monkeys. Specifically, each monkey was injected with 20 μL of 0.9% sodium chloride (NaCl), a solution containing 1 × 10 ¹¹ genome copies of AAV (low dose), and a solution containing 3 × 10 ¹¹ genome copies of AAV (medium dose). dose), or a solution containing 1 × ¹⁰¹² genome copies of AAV (high dose). One month after injection, the rhesus monkeys were euthanized, the left eye was removed, the retina was peeled off, samples were collected using TRIzol reagent (Thermofisher 15596026), and RNA was extracted from them (Zymo Research R2052), of which 1 μg of the extracted RNA was reverse transcribed into cDNA (NEB E6560L). Using 5 μL of reverse transcribed cDNA as a template, PCR amplification was performed using primers ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTA (SEQ ID NO: 379) and gagttggatgctggatggGAGCCCGTCACTGAAGATGTTGTAT (SEQ ID NO: 13). Sequencing analysis was performed as described above for the rhesus monkey cell line.

Sequence of the amplified region: ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTAgaaccattcacaacatatctcattggtgttgtggctgcaaaccatgcaggagaaattttaagcccctggactctgattcaaacctt A gaatcttccccaagtggactgagaaactttatagtagaacagaaagagaatggccgggcattgctactacagtggtcagagccta tgagaaccaatggtgtgattaagacATACAACATCTTCAGTGACGGGCTCccatccagcatccaactc (SEQ ID NO: 14) ( A indicates the site of target editing).

As shown in Figure 37, monkey kidney cells expressing the Ush2A dual reporter gene showed similar on-target editing and bystander editing patterns to in vivo monkey retinal cells endogenously expressing Ush2A after the introduction of arRNA, thus illustrating the use of the Ush2A reporter Genes are suitable to explore mechanisms that improve on-target editing efficiency and specificity.

Example 9. in monkey kidney cells PPIA Editor

Rhesus Monkey Cell Lines and Cell Culture

Rhesus monkey kidney cell line LLC-MK2 cells (ATCC number: CCL-7) were cultured using DMEM (Hyclone SH30243.01) containing 10% FBS (Vistech SE100-011). Peptidyl prolyl isomerase A (PPIA) is endogenously expressed in LLC-MK2 cells.

Plasmid transfection, FACS Analysis and Next Generation Sequencing (NGS)

In order to study the effects of circularization, target RNA length, mismatched regions and/or addition of adapters on RNA editing, deep sequencing was performed to detect editing efficiency.

To investigate whether off-target editing and/or enhance on-target editing can be reduced by mismatching or deletion of the targeting RNA sequence, one or more regions downstream (+) or upstream (-) of the editing site opposite the target RNA were sites: +5, +31, +35, +40, or -23, -28, -26, -30, -34, further modifying the circular arRNA with a 171 nt targeting RNA sequence (USHER-171), As described in the table below.

Table 4: Design of mismatch regions in arRNA arRNA annotation Deletions in the target RNA sequence (position relative to the target RNA) +5 +5 (5 nucleotides downstream of target adenosine) +31 +32 to +35 (32 nt to 35 nt downstream of target adenosine) +35 +36 to +39 (36 nt to 39 nt downstream of target adenosine) +40 +41 to +44 (41 nt to 44 nt downstream of target adenosine) -twenty three -23 (23 nucleotides upstream of target adenosine) -28 -28 (28 nucleotides upstream of target adenosine) -26 -27 to -30 (27 nucleotides to 30 nucleotides upstream of the target adenosine) -30 -31 to -34 (31 to 34 nucleotides downstream of the target adenosine) -34 -35 to -38 (35 nt to 38 nt downstream of target adenosine)

To investigate whether off-target editing could be reduced by using linkers that do not hybridize to the target RNA and form essentially no secondary structure, a 5' flexible linker (10 nt, 20 nt in length) flanking the targeting RNA sequence of the PPIA-171 arRNA was added. nt, 30 nt, 40nt or 50 nt) ("left" flex joint, or L-flex joint). (See Table A for relevant dRNA sequences).

Specifically, PP1A-expressing LLC-MK2 cells were seeded in 12-well plates (15,000 cells/well) (recorded as 0 h). 24 hours after plating, 2.5 μg of the above arRNA encoding plasmid (SEQ ID NO: 6 (85-C-85) or SEQ ID NO: 7 (at -28 -23 +5) was prepared using Lipofectamine 3000 according to the manufacturer's protocol. U deletion at )) (plasmid extracted by Qiagen #12945 and quantified by Nanodrop) was transfected into each well. Alternatively, arRNA can be packaged into AAV (1x1013 genome copies/mL) and infected with 3µL of virus per well.

Seventy-two hours after cell plating (48 hours after transfection), cells from each well were dissociated with trypsin (Invitrogen 25300054), and one-sixth of the dissociated cells were used to analyze the fluorescence intensity of mCherry and GFP by flow cytometry. All remaining dissociated cells were collected using TRIzol reagent (Thermofisher 15596026) and RNA was extracted from them (Zymo Research R2052), of which 1 μg of extracted RNA was reverse transcribed into cDNA (NEB E6560L). Use 5 μL of reverse transcribed cDNA as a template, and use primers ggagtgagtacggtgtgcAATATTGTGGAGGCCATGGAGC (SEQ ID NO: 8) and gagttggatgctggatggCCAGCTAGGCATGGGAGAACAAG (SEQ ID NO: 9) for PCR amplification (final primer concentration 0.2 μM, NEB M0492L, annealing at 63°C, 35 cycles ), the PCR product was sequenced using the NGS sequencing platform of the Rice Research Institute of the Chinese Academy of Sciences (refer to the sequencing process of Liu et al. Sci China Life Sci 2019 Jan;62(1):1-7, Hi-TOM: a platform for high- throughput tracking of mutations induced by CRISPR/Cas systems).

Use fastp (v0.19.6) to perform quality control on the raw data obtained by high-throughput sequencing, and filter out low-quality reads, reads on adapter sequences, and reads on sequences containing polyG. Subsequently, the barcode corresponding to the obtained high-quality sequencing data was split into each sample, and the BWA (v0.7.17-r1188) software was used to compare it with the sequence of the amplified target region (see sequence below), and SAMtools ( v1.9) format conversion to generate BAM files. Statistically compare, reorder and index the information obtained. Use REDItools (v1.2.1) software to detect all potential RNA editing sites. The parameters are as follows: use python REDItoolDenovo.py -i -f -o. After filtering out high-frequency point mutations that appear in the control and treated samples, use "(Average mutation frequency other than A->G mutation) + 3SD" is used as the threshold, and the reads whose A->G mutation frequency value exceeds the threshold at the editing site are regarded as the true frequency of the target A to G mutation.

Sequence of the amplified target region: ggagtgagtacggtgtgcAATATTGTGGAGGCCATGGAGCgctttgggtccaggaatggcaagaccagcaagaagatcaccattgctgactgtggacaactcgaataagtttgacttgtgttttatcttaaccaccagaccattccttctgt A gctcaggagagcacccctccaccccatttgctcgcagtatcct agaatctttgtgctctcgctgcagttccctttgggttccatgttttcCTTGTTCTCTCCCATGCCTAGCTGGccatccagcatccaactc (SEQ ID NO: 10) ( A indicates the site of target editing).

among rhesus monkeys PPIA in vivo editing

To study in vivo editing of PPIA, rhesus monkeys were edited via PPIA-specific arRNA.

The arRNA plasmid containing the U deletion at -28-23+5 (SEQ. ID NO: 7) was packaged into adeno-associated virus of serotype AAV8 by Paizhen Biotechnology Co., Ltd. with a titer of 6 × 10 ¹³ genomes copies/mL. Combine AAV (packaging arRNA) with 1 × 10 ¹³ genome copies of AAV/kg body weight (low dose), 3 × 10 ¹¹ genome copies of AAV solution (medium dose), or 1 × 10 ¹² genome copies of AAV solution (high dose) intravenously. Two weeks after injection, lung tissue samples were obtained via biopsy. One month after injection, the rhesus monkeys were euthanized and their livers were removed. For each time point, samples were collected using TRIzol reagent (Thermofisher 15596026), from which RNA was extracted (Zymo Research R2052), where 1 μg of extracted RNA was reverse transcribed into cDNA (NEB E6560L). Use 5 μL of reverse-transcribed cDNA as a template, and perform PCR amplification according to the above method. Sequencing analysis was the same as that for the rhesus cell line described above.

As shown in the upper left panel of Figure 38, compared with PPIA-171 (same as 85-c-85), the downstream (+5) or upstream (-28, -23) region relative to the editing site, at the selected position Deletions in the targeting RNA sequence at the metasite increased the editing efficiency of the target adenosine (at position 0) with no apparent changes in off-target editing. As shown in Figure 38, monkey kidney cells showed similar on-target and bystander editing patterns to PPIA as monkey liver cells in vivo after the introduction of arRNA (with U deletion), thus illustrating the use of monkey kidney cells to explore modifications to improve Suitable for target editing efficiency and specificity. Further mismatched regions of the targeted RNA sequence were then tested in monkey kidney cells.

As shown in Figure 39, compared to PPIA-171, although deletion of the targeting RNA sequence at selected position sites relative to the downstream (+) or upstream (-) region of the editing site increased the targeting of adenosine ( Editing efficiency at position 0), but no significant increase in off-target editing. In particular, deletions in the targeted RNA sequence 31 and 40 nt (+31, +40) downstream of the editing site increased target editing more significantly.

As shown in Figure 40, the addition of flexible linker sequences (L-10, L-20, L-30, L-40, L-50) flanking the 5' of the targeting RNA sequence significantly reduced the interaction with PPIA-171 arRNA. The 5' targeting RNA sequence is relatively off-target editing, while the addition of flexible linker sequences (R-10, R-20, R-40, R-50) flanking the 3' targeting RNA sequence significantly reduces interaction with PPIA-171 The 3' targeting RNA sequence of arRNA is relatively off-target edited, and this reduction appears to be more effective for longer flexible linker sequences, where longer flexible linker sequences (40nt, 50nt) apparently eliminate almost all detectable off-target editing, except for the non-target adenosine +11 nucleotides downstream and -29 nucleotides upstream of the target editing site (position 0).

In summary, by introducing mismatch regions and flexible linker sequences into arRNA, the editing efficiency and specificity of arRNA for endogenously expressed RNA can be greatly improved.

Example 10 : Reduction of mutations in rhesus monkeys Usher 2A RNA Off-target editing in editing

This example demonstrates that bystander (off-target) editing can be reduced by engineering a circ-arRNA (SEQ ID NO: 315) targeting mutant Usher 2A in rhesus monkeys. Rhesus monkey kidney cell line LLC-MK2 cells (ATCC number: CCL-7) were cultured as described above.

The first method is to introduce deletions at different sites opposite the target RNA in the downstream (+) or upstream (-) region of the editing site. The DNA sequence (NNNNNNNNN) shown in Table B was first synthesized in vitro and integrated into the vector, followed by digestion with MiuI and KpnI, and then connected to the plasmid backbone shown in SEQ ID NO: 316. To further test the effectiveness of introduced deletions in reducing off-target editing, deletions at two different sites were combined.

As shown in Figure 41, compared with the circ-arRNA before optimization (SEQ ID NO: 317), at -26, -30, -34, +31, +35 or +39 (SEQ ID NO: 318-323) The 4 bp deletion reduces off-target editing. With the exception of a 4 bp deletion at -34 and a 4 bp deletion at +39, these modifications did not reduce editing efficiency at target (position 0). Off-target editing was further reduced by introducing two deletions at two sites (SEQ ID NO: 324-332) (Figure 42).

Second, combine the double deletions at -26 and +35 and the left and right flex joints. As shown in Figure 43, compared with the circ-arRNA before optimization (SEQ ID NOs: 344), the circ-arRNA with 4bp deletions at -26 and +35 has a 20nt right linker and a 20-50nt left linker ( SEQ ID NO: 345-348) significantly reduces off-target editing while retaining high on-target editing efficiency.

As further demonstrated in Figure 44, introducing deletions at -26 and +35 and adding a 20nt right linker and a 30nt left linker significantly reduced off-target editing. There are still off-target edits at non-target adenines at -7, -6, -5, +2, +3, +12 and +13.

To eliminate residual off-target editing at these sites, different deletions were performed on the circ-arRNA (with 4 bp at -26 and +35), as well as the 20 nt right linker and 30 nt left linker (SEQ ID NO: 351-354). Uracil at positions including -5, +3 and/or +13. As shown in Figure 45, deletion of three uracils at -5, +3 and +13 eliminated all residual editing at non-target adenosines.

Example 11 : by in IDUA RNA Introducing mismatches in editing to reduce off-target editing

This example demonstrates that off-target editing can be reduced by introducing mismatches into the targeting RNA targeting IDUA mRNA.

circ-arRNA Design and plasmid construction:

To construct a plasmid expressing circ-arRNA to target mutant IDUA mRNA encoding the W402X mutation in IDUA protein, the scAAV-U6-CAG-GFP vector (PackGene Biotech, LLC) was modified, in which GFP was replaced by blue fluorescent protein (BFP) replace. Briefly, the vector was digested using AgeI and EcoRI enzymes, and the digested fragments were recovered by gel extraction. BFP was amplified by PCR using the primers shown in SEQ ID NO: 355-356, and then the amplified BFP product was Golden Gate cloned into the vector. In addition, a CMV enhancer (CMVE) sequence (SEQ ID NO: 357) was inserted before the U6 promoter. The circularization sequence T (SEQ ID NO: 358) was inserted after the U6 promoter, which included 2 BsmbI restriction sites for subsequent introduction of the arRNA coding sequence. The final plasmid construct is scAAV-CMVE-U6-T-CAG-BFP.

ArRNA (101 nt long) was designed to include a 30 nt linker at the 3' end containing only adenine and cytosine (SEQ ID NO: 359), and in the region downstream (+) or upstream (-) of the editing site Various mismatches at one or more sites relative to the target RNA (SEQ ID NOs: 360-374). The "M" in the name of arRNA stands for "mismatch", and the number after the "M" indicates the number of mismatched nucleotides. The "D" in the arRNA name stands for "deletion," and the number after the "D" indicates the number of deleted nucleotides. For example, +30M2 and -6M2 refer to two mismatches in the target RNA sequence starting from a position corresponding to 30 nt downstream of the editing site, and two mismatches starting from a position corresponding to 6 nt upstream of the editing site.

IDUA Reporter gene cell line construction:

HEK293T cells were cultured as described above. The mutant IDUA gene (NM_000203.4 (IDUA)-c.1205G>A (p.Trp402Ter)) and the target RNA sequence about 100nt upstream and downstream of the target site were cloned into the pLenti backbone (containing mCherry and GFP) using the Gibson cloning method described above. reporter gene). This reporter contains an in-frame stop codon between mCherry and GFP. This plasmid was then co-transfected into HEK293T cells, mCherry-positive cells were sorted by FACS and cultured, followed by clonal selection as described above. The fluorescence of GFP indicates the efficiency of targeted editing on RNA.

Contains mismatches circ-arRNA target RNA On-target and off-target editing:

IDUA reporter cells expressing mutant IDUA genes were seeded in 24-well plates (15,000 cells/well) (recorded as 0 h). As described above, 24 hours after plating, cells were transfected with 1.0 μg of one of the plasmids expressing circ-arRNA (plasmids extracted by Qiagen #12945 and quantified by Nanodrop) in 3.0 μl of transfection reagent (Roche, X-tremeGENE HP DNA Transf.Reag). 48 hours after transfection, cells were sorted by FACS to detect BFP expression and GFP expression. Target editing efficiency was measured by the average fluorescence intensity of GFP as described above. On-target and off-target editing promoted by the arRNA was determined using an NGS protocol as described above.

As shown in Figure 46, the mismatch affected on-target editing efficiency as indicated by the percentage of BFP-positive cells expressing GFP compared to the control arRNA85-C-15-3'AC30. BL5 showed an increase in on-target editing efficiency, BL6 and BL14 inhibited on-target editing efficiency similar to the control, while BL7, BL8, BL15, BL16, BL18 and BL19 had a decrease in on-target editing efficiency.

As shown in Figure 47, off-target editing was significantly reduced in arRNAs such as BL5, BL6, BL14, BL16 and BL18. This example further demonstrates that modifying arRNA such as adding mismatches can reduce off-target editing while maintaining on-target editing efficiency. sequence listSEQ ID NO: 1 (PackGene plasmid backbone) ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagaggggagtgtagccatgctctaggaagatcaattcaattcacgcgtgagggcctatttcccatgattcct tcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttGTGGAAAGGACGAAAC gAAGCTTgaattcGGTACCcgcgTcgacattgattattgactagctctggtcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtggagtatttacggtaaactgcccacttggcagtacatcaagt gtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctactcgaggccacgttctgcttcactctccccatctcccccccctccccaccccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggcgg gggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagcgggatcagccaccgcggtgg cggcctagagtcgacgaggaactgaaaaaccagaaagttaactggtaagtttagtctttttgtcttttatttcaggtcccggatccggtggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttctgctctaaaagctgcggaattgtacccgcggccga tccaccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtga ccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgactt caaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagt ccgccctgagcaaagacccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaatcgaattcGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTG TCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAGGCCGCAGGAACCCCTAGTGATGGAGTTGGccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagc gcgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcatta agcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttccttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaactt gattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaa atttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccctttt ttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcgg tattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgca caacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggtaaagttgcaggaccacttctgcgctcggccctt ccggctggctggttttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatact ttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagtttttcgtccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacca ccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgt cttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgc acgaggggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctg cgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggttcccgactggaaagc gggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggccttaattagg Plasmid backbone including targeting RNA sequence ( NNNNNNNNNN ) acgcgtgagggcctatttccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtatttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgattt cttggctttatatatcttgtggaaaggacgaaacaccg NNNNNNNNNN tttttttggtacc (SEQ ID NO: 2) SEQ ID NO: 3 – Mutated Ush2A target RNA ( Arepresents target adenosine) gcccttgaatttatggatgaaggagacaccctgaggcctttcacactctacgaatatcgggtcagagcctgtaactccaagggttcagtggagagtctgt Agtcattaacacaaactctggaagctccacctcaagattttccagctccttgggctcaagccacgagtgctcattcagttctgttgaattggacaaagcca SEQ ID NO: 4 – Plasmid encoding arRNA targeting mutated Ush2A SEQ ID NO: 5 – Plasmid encoding arRNA targeting mutated Ush2A SEQ ID NO: 6 – Plasmid encoding 171 nt circular arRNA targeting PPIA Plasmid SEQ ID NO : 7 - Plasmid encoding a 171nt circular arRNA (with U deletion at -28 -23 +5) targeting PPIA SEQ ID NO: 8 - Primers for PCR amplification of reverse transcribed cDNA of PPIA ggagtgagtacggtgtgcAATATTGTGGAGGCCATGGAGC SEQ ID NO: 9 - Primers for PCR amplification of the reverse transcribed cDNA of PPIA gagttggatgctggatggCCAGCTAGGCATGGGAGAGAACAAG SEQ ID NO: 10 - Sequence of the target region of the amplified PPIA ggagtgagtacggtgtgcAATATTGTGGAGGCCATGGAGCgctttgggtccaggaatggcaagaccagcaagaagatcaccattgctgactgtggacaactcgaataagtttgacttgt gttttatcttaaccaccagaccattccttctgtAgctcaggagagcacccctccaccccatttgctcgcagtatcctagaatctttgtgctctcgctgcagttccctttgggttccatgttttcCTTGTTCTCTCCCATGCCTAGCTGGccatccagcatccaactc SEQ ID NO: 11 – Primers for PCR amplification of reverse transcribed cDNA of USHER 2A gagttggatgctggatggCTTAATCACACCATTGGTTCTCA SEQ ID NO: 12 - Sequence of amplified target region of USHER 2A ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTAgaaccattcacaacatatctcattggtgttgtggctgcaaaccatgcaggagaaattttaagcccctggactctgattcaaacctt AgaatcttccccaagtggactgagaaactttatagtagaacagaaagagaatggccgggcattgctactacagtggtcagagcctaTGAGAACCAATGGTGTGATTAAGccatccagcatccaactc SEQ ID NO: 13 – Primers for PCR amplification of reverse transcribed cDNA of USHER 2A gagttggatgctggatggGAGCCCGTCACTGAAGATGTTGTAT SEQ ID NO: 14 - Sequence of amplified target region of USHER 2A ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTAgaaccattcacaacatatctcattggtgttgtggctgcaaaccatgcaggagaaattttaagcccctggactctgattcaaacctt AgaatcttccccaagtggactgagaaactttatagtagaacagaaagagaatggccgggcattgctactacagtggtcagagcctatgagaaccaatggtgtgattaagacATACAACATCTTCAGTGACGGGCTCccatccagcatccaactc surface A :Sequences containing linear RNA (hereinafter referred to as "linear") and linear RNA capable of being circularized (hereinafter referred to as "circularized") targeting RNA sequences *Uncapitalized sequence = targeting RNA sequence ("targeting sequence"). *Sequences not in bold = part of the circularized arRNA ("circularized sequence"). Figure label In vitro synthetic sequence (corresponding to NNNNNNNNNNNNNNNNNNNNNN in SEQ ID NO:2). Figure 2 Linear-151 gaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgt (SEQ ID NO: 15) Circular-151 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 16) Figure 3 Linear-51 agcttccagagtttgtgttaatgaccacagactctccactgaacccttgga (SEQ ID NO: 17) Linear-61 ggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttac (SEQ ID NO: 18) Linear-71 cttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggct (SEQ ID NO: 19) Linear-81 aaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgac (SEQ ID NO: 20) Linear-91 gctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgat (SEQ ID NO: 21) Linear-101 aaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcg (SEQ ID NO: 22) Linear-111 agcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagag (SEQ ID NO: 23) Linear-121 gcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtga (SEQ ID NO: 24) Linear-131 tcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggc (SEQ ID NO: 25) Linear-141 agcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcag (SEQ ID NO: 26) Linear-151 gaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgt (SEQ ID NO: 27) Circular-51 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGagcttccagagtttgtgttaatgaccacagactctccactgaacccttggaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 28) Circular-61 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAG ggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 29) Circular-71 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 30) Circular-81 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 31) Circular-91 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 32) Circular-101 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 33) Circular-111 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 34) Circular-121 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 35) Circular-131 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 36) Circular-141 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 37) Circular-151 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 38) Circular-161 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctcctCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 39) Circular-171 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 40) Circular-181 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataaCTGCCATCAGTCGGCGTGGACT GTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 41) Circular-191 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGttgtccaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataaattcaCTGCCAT CAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 42) Circular-201 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtggctttgtccaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataaattca agggcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 43) Circular-211 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGGCCGCtggctttgtccaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccata aattcaagggcGCTAGCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 44) Circular-221 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGCAGCGGCCGCtggctttgtccaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcat ccataaattcaagggcGCTAGCAGGACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 45) Figure 6 Figure 7 USHER-171 (4bp deletion and 4bp mismatch share the same sequence) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 46) non-target GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgctgtccaggacggtcccggcctgcgacacttcggcccagctgctcctcatctgcggggcgggggggggccgtcgccgcgtggggtcgttgcccagccgcccaggacggacccagggccgggccCTGCCAT CAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 47) US-26 4bp mismatch GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagAATGaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 48) US-26 4bp deletion GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 49) US-30 4bp mismatch GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacTCCGtctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 50) US-30 4bp deletion GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttactctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 51) US-34 4bp mismatch GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggcAGACacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 52) US-34 4bp deletion GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggcacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 53) US+31 4bp mismatch GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatGAACaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 54) US+31 4bp deletion GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaataggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 55) US+35 4bp mismatch GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaTTTActtgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 56) US+35 4bp deletion GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 57) US+39 4bp mismatch GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcACCTaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 58) US+39 4bp deletion GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 59) US-26+31 4bp mismatch GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatGAACaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagAATGaggctctgaccccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 60) US-26+31 4bp deletion GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaataggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 61) US-26+35 4bp mismatch GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaTTTActtgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagAATGaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 62) US-26+35 4bp deletion GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggaaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 63) US-26+39 4bp mismatch GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcACCTaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagAATGaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 64) US-26+39 4bp deletion GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 65) US-30+39 4bp mismatch GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcACCTaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacTCCGtctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 66) US-30+39 4bp deletion GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttactctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 67) US-34+39 4bp mismatch GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcACCTaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggcAGACacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 68) US-34+39 4bp deletion GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggcacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 69) Figure 9 Figure 10 USHER-171 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 70) non-target GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgctgtccaggacggtcccggcctgcgacacttcggcccagctgctcctcatctgcggggcgggggggggccgtcgccgcgtggggtcgttgcccagccgcccaccgtccgagggccgggccCTGCCAT CAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 71) US+35X-26X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 72) US+35X-26X-21X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 73) US+35X-26X&21/22 Del GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 74) US+35X-26X Del 78 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 75) US+35X-26X Del 78-21X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 76) US+35X-26X Del 78&21/22 Del GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 77) US+39X-26X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 78) US+39X-26X-21X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 79) US+39X-26X&21/22 Del GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 80) US+39X-26X Del 78 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 81) US+39X-26X Del 78-21X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 82) US+39X-26X Del 78&21/22 Del GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 83) Figure 12 Figure 13 L-10 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGAAAAACAAAAgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 84) L-20 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGAAAAAAAAAAAAAAAAAAtcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 85) L-30 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGAAAAACAAAAAAAAAAAAAAAAAAAAAagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 86) R-10 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 87) R-20 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 88) R-30 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 89) Figure 15 Figure 16 US+35X-26X-21X (0nt) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 90) US+35X-26X-21X 10nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 91) US+35X-26X-21X 20nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 92) US+35X-26X-21X 30nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 93) US+35X-26X-21X 40nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 94) US+35X-26X-21X 50nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 95) US+35X-26X-21X-D78 (0nt) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 96) US+35X-26X-21X-D78 10nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 97) US+35X-26X-21X-D78 20nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 98) US+35X-26X-21X-D78 30nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 99) US+35X-26X-21X-D78 40nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 100) US+35X-26X-21X-D78 50nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 101) US+39X-26X-21X (0nt) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 102) US+39X-26X-21X 10nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 103) US+39X-26X-21X 20nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 104) US+39X-26X-21X 30nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 105) US+39X-26X-21X 40nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 106) US+39X-26X-21X 50nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 107) US+39X-26X-21X-D78 (0nt) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 108) US+39X-26X-21X-D78 10nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 109) US+39X-26X-21X-D78 20nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 110) US+39X-26X-21X-D78 30nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 111) US+39X-26X-21X-D78 40nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 112) US+39X-26X-21X-D78 50nt GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 113) Figure 18 Figure 19 USHER-171 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 114) non-target GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgctgtccaggacggtcccggcctgcgacacttcggcccagctgctcctcatctgcggggcgggggggggccgtcgccgcgtggggtcgttgcccagccgcccaccgtccgagggccgggccCTGCCAT CAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 115) 35X-21X-A GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 116) 35X-21X-AA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAAaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 117) 35X-21X-U GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 118) 35X-21X-UU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTTaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 119) 35X-21X-C GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 120) 35X-21X-CC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCCaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 121) 35X-21X-G GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 122) 35X-21X-GG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGGaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 123) 35X-21X-X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 124) 35X-21X-A GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgAaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 125) 35X-21X-AA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgAAaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 126) 35X-21X-U GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgUaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 127) 35X-21X-UU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgUUaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 128) 35X-21X-C GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgCaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 129) 35X-21X-CC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgCCaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 130) 35X-21X-G GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgGaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 131) 35X-21X-GG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgGGaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 132) 35X-21X-X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagcaaatcttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 133) Figure 21 35X-21X-AC30-AA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAAaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 134) 35X-21X-AC30-AU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgATaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 135) 35X-21X-AC30-AC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgACaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 136) 35X-21X-AC30-AG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAGaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 137) 35X-21X-AC30-UA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTAaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 138) 35X-21X-AC30-UU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTTaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 139) 35X-21X-AC30-UC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTCaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 140) 35X-21X-AC30-UG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTGaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 141) 35X-21X-AC30-CA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCAaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 142) 35X-21X-AC30-CU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCTaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 143) 35X-21X-AC30-CC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCCaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 144) 35X-21X-AC30-CG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCGaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 145) 35X-21X-AC30-GA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGAaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 146) 35X-21X-AC30-GU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGTaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 147) 35X-21X-AC30-GC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGCaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 148) 35X-21X-AC30-GG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGGaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 149) 35X-21X-AC30-A GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 150) 35X-21X-AC30-U GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 151) 35X-21X-AC30-C GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 152) 35X-21X-AC30-G GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 153) 35X-21X-AC30-X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 154) 35X-AA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAAaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 155) 35X-AU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgATaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 156) 35X-AC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgACaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 157) 35X-AG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAGaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 158) 35X-UA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTAaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 159) 35X-UU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTTaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 160) 35X-UC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTCaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 161) 35X-UG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTGaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 162) 35X-CA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCAaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 163) 35X-CU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCTaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 164) 35X-CC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCCaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 165) 35X-CG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCGaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 166) 35X-GA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGAaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 167) 35X-GU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGTaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 168) 35X-GC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGCaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 169) 35X-GG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGGaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 170) 35X-A GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 171) 35X-U GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 172) 35X-C GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 173) 35X-G GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 174) 35X-X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 175) Figure 22 35X-21X-AC50-AA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAAaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 176) 35X-21X-AC50-AU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgATaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 177) 35X-21X-AC50-AC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgACaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 178) 35X-21X-AC50-AG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAGaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 179) 35X-21X-AC50-UA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTAaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 180) 35X-21X-AC50-UU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTTaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 181) 35X-21X-AC50-UC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTCaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 182) 35X-21X-AC50-UG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTGaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 183) 35X-21X-AC50-CA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCAaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 184) 35X-21X-AC50-CU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCTaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 185) 35X-21X-AC50-CC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCCaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 186) 35X-21X-AC50-CG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCGaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 187) 35X-21X-AC50-GA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGAaatgaccacagactctccactgaacccaggctAAAAAACAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 188) 35X-21X-AC50-GU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGTaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 189) 35X-21X-AC50-GC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGCaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 190) 35X-21X-AC50-GG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 191) 35X-21X-AC50-A GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAaatgaccacagactctccactgaacccaggctAAAAAAAAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 192) 35X-21X-AC50-U GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgTaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 193) 35X-21X-AC50-C GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgCaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 194) 35X-21X-AC50-G GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgGaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 195) 35X-21X-AC50-X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 196) Figure 23 85-C-85-AA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgAAaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 197) 85-C-85-AU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgATaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 198) 85-C-85-AC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgACaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 199) 85-C-85-AG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgAGaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 200) 85-C-85-UA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgTAaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 201) 85-C-85-UU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgTTaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 202) 85-C-85-UC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgTCaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 203) 85-C-85-UG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgTGaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 204) 85-C-85-CA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgCAaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 205) 85-C-85-CU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgCTaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 206) 85-C-85-CC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgCCaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 207) 85-C-85-CG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgCGaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 208) 85-C-85-GA GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgGAaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 209) 85-C-85-GU GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgGTaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 210) 85-C-85-GC GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgGCaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 211) 85-C-85-GG GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgGGaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 212) 85-C-85-A GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgAaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 213) 85-C-85-U GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgTaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 214) 85-C-85-C GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgCaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 215) 85-C-85-G GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgGaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 216) 85-C-85-X GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 217) Figure 26 Deletion-0 or insertion-0 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 218) Missing-1 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagtacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 219) Missing-2 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 220) Missing-3 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagcaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 221) Missing-4 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 222) Missing-7 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 223) Missing-10 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagtgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 224) Insert -1 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCACATGCGACGACTGCAGCAACACACAACGAATGATGACTCGGGGGCCCCCCAAGGGGAAAATAATTGGGGGGGGGGGGGGGTTCCCCCTGAACCTGACCCTTT GGAGAGAGGGCTGACCCCCGATTCGTGTGTGCCTCTCTCTCTCTCTCCCAGCCAGCGCGGGGGGGGGGGGGGGGGGGGGGGGGGGCTGGGGGGGGGACTGCGCGCGCGCCCCTGTGTGCCCCCTGT AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 225) insert-2 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCACATGCGACGACTGGCAGCAACACACAACGAATGAGAGAGCTCGGGGGCCCCCCCAGGGAAACTGGGGGGGGGGGGGGGGGAgATCACCTGAACCCCCT TGGAGAATTACAGGGCTGACCCCCGATTCGTGTGCCTCTCTCTCTCTCTCTCCCAGCAGCGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTGGGGGGGGGTGCGGGGGGCGCGCGCATCCTCTGCCCTGCCCCCTCGAT AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 226) insert-3 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCGACGACTGGCAGCAACACAACGAATGATGACTCTCGGGGCCCCCAAGGGGGGGGGGGGGGGGGGGGGGGGGGGGAgACACCTCCTGACTGAACCCCCC TTGGAGAAAAAAAATACGCTCTGACCCCCCGATTCGTGTGTGCCTCTCTCTCTCTCTCCCAGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTGTGTGTGTGTGGGCGCGCGCGCGCCTCCTCCCCCCCCCCCCCCCCCCCTCCCTCCCCCTC AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 227) insert-4 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatAAAActtgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagAAAAttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACT GTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 228) insert-7 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatAAAAAAActtgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagAAAAAAAttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGT GGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 229) Insert -10 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatAAAAAAAAAActtgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagAAAAAAAAAAttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCG GCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 230) Figure 28 85-C-85 &+35-B0 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 231) -26X & +35-B0 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 232) 85-C-85 &+35-B4 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 233) -26X & +35-B4 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 234) 85-C-85 &+35-B10 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGttgtccaattcaacagaactgaatgagcactcgtggcttgagcccaaggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 235) -26X & +35-B10 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGttgtccaattcaacagaactgaatgagcactcgtggcttgagcccaaggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 236) 85-C-85 &+35-B20 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgccgctggctttgtccaattcaacagaactgaatgagcactcgtggcttgcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 237) -26X & +35-B20 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgccgctggctttgtccaattcaacagaactgaatgagcactcgtggcttgcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 238) 85-C-85 &+35-B30 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgccctcagcggccgctggctttgtccaattcaacagaactgaatgagcaccttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 239) -26X & +35-B30 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgccctcagcggccgctggctttgtccaattcaacagaactgaatgagcaccttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 240) 85-C-85 &+35-B40 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgacttcctctgccctcagcggccgctggctttgtccaattcaacagaactcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 241) -26X & +35-B40 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgacttcctctgccctcagcggccgctggctttgtccaattcaacagaactcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 242) 85-C-85 &+35-B50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcatgttagaagacttcctctgccctcagcggccgctggctttgtccaattcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 243) -26X & +35-B50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcatgttagaagacttcctctgccctcagcggccgctggctttgtccaattcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 244) Figure 30 85-C-85 &-26-B0 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 245) +35X & -26-B0 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 246) 85-C-85 &-26-B4 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 247) +35X & -26-B4 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 248) 85-C-85 &-26-B10 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagtgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataaattcaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 249) +35X & -26-B10 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagtgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataaattcaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 250) 85-C-85 &-26-B20 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttcgtagagtgtgaaaggcctcagggtgtctccttcatccataaattcaagggcgctagCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 251) +35X & -26-B20 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttcgtagagtgtgaaaggcctcagggtgtctccttcatccataaattcaagggcgctagCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 252) 85-C-85 &-26-B30 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggaggtgaaaggcctcagggtgtctccttcatccataaattcaagggcgctagcaggaccgggCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 253) +35X & -26-B30 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggaggtgaaaggcctcagggtgtctccttcatccataaattcaagggcgctagcaggaccgggCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 254) 85-C-85 &-26-B40 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagtcagggtgtctccttcatccataaattcaagggcgctagcaggaccggggttttcttccCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 255) +35X & -26-B40 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagtcagggtgtctccttcatccataaattcaagggcgctagcaggaccggggtttcttccCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 256) 85-C-85 &-26-B50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagtccttcatccataaattcaagggcgctagcaggaccggggttttcttccacgtctcctgCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 257) +35X & -26-B50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagtccttcatccataaattcaagggcgctagcaggaccggggttttcttccacgtctcctgCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 258) Figure 31 -26-B0+35-B0 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 259) -26-B4+35-B4 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 260) -26-B0+35-B10 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGttgtccaattcaacagaactgaatgagcactcgtggcttgagcccaaggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagtgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataaattcaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 261) -26-B0+35-B20 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCACATGCGACGACTGGCAGCCCGCTGGGCTTGTCAACAACAACAATGAGAGAGAGCTCTCTGTGTGTGTGGGGGGGGGGGGGGGGGGAGGGAGAGAGAGACCTGACTGACCCCTTTG gagtttcgtgggtgaaaggcctcggggtcccccccattcaagGCGCTGCCCAGCAGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGACTCCCTCAAAT AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 262) -26-B0+35-B30 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgccctcagcggccgctggctttgtccaattcaacagaactgaatgagcaccttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggaggtgaaaggcctcagggtgtctccttcatccataaattcaagggcgctagcaggaccgggCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 263) -26-B0+35-B40 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgacttcctctgccctcagcggccgctggctttgtccaattcaacagaactcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagtcagggtgtctccttcatccataaattcaagggcgctagcaggaccggggttttcttccCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 264) -26-B0+35-B50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcatgttagaagacttcctctgccctcagcggccgctggctttgtccaattcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagtccttcatccataaattcaagggcgctagcaggaccggggttttcttccacgtctcctgCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 265) Figure 33 USHER-171 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 266) non-target GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgctgtccaggacggtcccggcctgcgacacttcggcccagctgctcctcatctgcggggcgggggggggccgtcgccgcgtggggtcgttgcccagccgcccaccgtccgagggccgggccCTGCCAT CAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 267) +35X-21X-AC50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 268) +35X-21X+d78-AC50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 269) +35X-21X-AG50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctAAAAAGAAAAAAGAAAAAAAAGAAAAAAAAAAGGAAAAAAAGAAAAGAGACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 270) +35X-21X+d78-AG50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctAAAAAGAAAAAAGAAAAAAAAGAAAAAAAAAAGGAAAAAAAGAAAAGAGACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 271) +35X-21X-AT50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctAAAAATAAAAAATAAAAAAAATAAAAAAAAAATTAAAAAAATAAAATATACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 272) +35X-21X+d78-AT50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctAAAAATAAAAAATAAAAAAAATAAAAAAAAAATTAAAAAAATAAAATATACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 273) +35X-21X-CA50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctCCCCCACCCCCCACCCCCCCCACCCCCCCCCAACCCCCCCACCCACCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 274) +35X-21X+d78-CA50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctCCCCCACCCCCCACCCCCCCCACCCCCCCCCAACCCCCCCACCCACCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 275) +35X-21X-CT50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctCCCCCCTCCCCTCCCCCCCCCCCCCCCCCTTCCCCCCCTCCCCTCCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 276) +35X-21X+d78-CT50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctCCCCCTCCCCTCCCCCCCCCCCCCCCCCTTCCCCCCCTCCCCTCCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 277) +35X-21X-GT50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctGGGTTGGTGTTGGTGGGTTGGTGTTGGTGGGTTGGTGGGTTGTGGTTGCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 278) +35X-21X+d78-GT50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctGGGTTGGTGTTGGTGGGTTGGTGTTGGTGGGTTGGTGGGTTGTGGTTGCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 279) +35X-21X-ACGT50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAGAAAACAAAAAAAAAACCAAATAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 280) +35X-21X+d78-ACGT50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgaatgaccacagactctccactgaacccaggctAAAAACAAAAAACAAAGAAAACAAAAAAAAAACCAAATAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 281) Figure 36 85-C-85-Control GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 282) 85-C-85+GluR2 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgtggaatagtataacaatatgctaaatgttgttatagtatcccaccaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaagg cctcagggtgtctccttcatcgtggaatagtataacaatatgctaaatgttgttatagtatcccacCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 283) 85-C-85+U6+27 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgtgctcgcttcggcagcacatatactagtcgaccaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtg tctccttcatctctagagcggacttcggtccgcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 284) 85-C-85+Alu GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGggccgggcgcggtggctcacgcctgtaatcccagcactttggggggccgaggcggggagaattgcttgagcccaggagttcgagaccagcctgggcaacatagcgagaccccgtctcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttg tgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcagccgggcgtggtggcgcgcgcctgtagtcccagctactcgggaggctgaggcaggaggatcgcttgagcccaggagttcgaggctgcagtgagctatgatcgcgccact gcactccagcctgggcgacagagcgagaccctgtctcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 285) -21X+35X-RAC30-Control GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 286) -21X+35X-RAC30+GluR2 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgtggaatagtataacaatatgctaaatgttgttatagtatcccaccaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagacaaaaaaaaaccaaaaaa acaaaacacagtggaatagtataacaatatgctaaatgttgttatagtatcccacCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 287) -21X+35X-RAC30+U6+27 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgtgctcgcttcggcagcacatatactagtcgaccaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctctgacccgatattcgtagagacaaaaaaaaaccaaaaaacaaaacacat ctagagcggacttcggtccgcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 288) -21X+35X-RAC30+Alu GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGggccgggcgcggtggctcacgcctgtaatcccagcactttggggggccgaggcggggagattgcttgagcccaggagttcgagaccagcctgggcaacatagcgagaccccgtctccaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgtgtta atgaccacagactctccactgaacccaggctctgacccgatattcgtagagacaaaaaaaaaaccaaaaaacaaaacacaagccgggcgtggtggcgcgcgcctgtagtcccagctactcgggaggctgaggcaggaggatcgcttgagcccaggagttcgaggctgcagtgagctatgatcgcgccactgcactccagcc tgggcgacagagcgagaccctgtctcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 289) -21X+35X-RAC50-Control GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctaaaaacaaaaaacaaaaaaaacaaaaaaaaaccaaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 290) -21X+35X-RAC50+GluR2 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgtggaatagtataacaatatgctaaatgttgttatagtatcccaccaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctaaaaacaaaaaacaaaaaaaaaaaaaaa aaccaaaaaaacaaaacacagtggaatagtataacaatatgctaaatgttgttatagtatcccac CTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 291) -21X+35X-RAC50+U6+27 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgtgctcgcttcggcagcacatatactagtcgaccaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctaaaaacaaaaaacaaaaaaaccaaaaaaaaaaccaaaaaa aacaaaacacatctagagcggacttcggtccgcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 292) -21X+35X-RAC50+Alu GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGggccgggcgcggtggctcacgcctgtaatcccagcactttggggggccgaggcggggagaattgcttgagcccaggagttcgagaccagcctgggcaacatagcgagaccccgtctccaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgtgtta atgaccacagactctccactgaacccaggctaaaaacaaaaaacaaaaaaaacaaaaaaaaaccaaaaaaaaacacaagccgggcgtggtggcgcgcgcctgtagtcccagctactcgggaggctgaggcaggaggatcgcttgagcccaggagttcgaggctgcagtgagctatgatcgcg ccactgcactccagcctgggcgacagagcgagaccctgtctcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 293) Figure 39 PPIA-171 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacaaacattgacacttcctgggactggaaagtaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtttcagaagtaacccattactgcttaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 294) PPIA+31-26 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacaaacacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaatgtttcagaagtaacccattactgcttaaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 295) PPIA+31-30 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacaaacacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaagtgttcagaagtaacccattactgcttaaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 296) PPIA+31-34 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacaaacacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtgaagtaacccattactgcttaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 297) PPIA+35-26 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacattgacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaatgtttcagaagtaacccattactgcttaaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 298) PPIA+35-30 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacattgacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaagtgttcagaagtaacccattactgcttaaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 299) PPIA+35-34 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacattgacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtgaagtaacccattactgcttaaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 300) PPIA+40-26 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactaaacattgacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaatgtttcagaagtaacccattactgcttaaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 301) PPIA+40-30 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactaaacattgacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaagtgttcagaagtaacccattactgcttaaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 302) PPIA+40-34 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactaaacattgacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtgaagtaacccattactgcttaaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 303) Figure 40 PPIA-171 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacaaacattgacacttcctgggactggaaagtaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtttcagaagtaacccattactgcttaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 304) L-10 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGAAAAACAAAAcagttgctgcctacactttcaatattccactcaacaaacattgacacttcctgggactggaaagtaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtttcagaagtaacccattactgcttaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 305) L-20 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGAAAAACAAAAAACAAAAAAActacactttcaatattccactcaacaaacattgacacttcctgggactggaaagtaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtttcagaagtaacccattactgcttaaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 306) L-30 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGAAAAACAAAAAAAAAAAAAAAAAAAAAaatattccactcaacaaacattgacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtttcagaagtaacccattactgcttaaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 307) L-40 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGAAAAAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAtcaacaaacattgacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtttcagaagtaacccattactgcttaaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 308) L-50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGAAAAAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACAttgacacttcctgggactggaaagtaaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtttcagaagtaacccattactgcttaaaaaaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 309) Figure 40 PPIA-171 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacaaacattgacacttcctgggactggaaagtaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtttcagaagtaacccattactgcttaaaataaagcctacatcaacactcttaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 310) R-10 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacaaacattgacacttcctgggactggaaagtaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtttcagaagtaacccattactgcttaaaataaagcctacatcACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 311) R-20 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacaaacattgacacttcctgggactggaaagtaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtttcagaagtaacccattactgcttaaaataaAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 312) R-40 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacaaacattgacacttcctgggactggaaagtaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgtttcagaagtaaAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 313) R-50 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGccaccatgcccagttgctgcctacactttcaatattccactcaacaaacattgacacttcctgggactggaaagtaaaaaaatccaagtactgtgtagaattaagcaaacaagtgatgttAAAAACAAAAAACAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO: 314) SEQ ID NO: 315 – Mutant mf-Ush2A target RNA ( Arefers to target adenosine) ggaacaagccatcaagcccacctgttcggattagaaccattcacaacatatctcattggtgttgtggctgcaaaccatgcaggagaaattttaagcccctggactctgattcaaacctt Agaatcttccccaagtggactgagaaactttatagtagaacagaaagagaatggccgggcattgctactacagtggtcagagcctatgagaaccaatggtgtgattaag SEQ ID NO: 316 – Reporter plasmid sequence acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttta cggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattgg ttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccggggagctgcatg tgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct gataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatcctt gagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataac catgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactact tactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggacccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggat gaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatca aaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaact ctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaa agcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacg ccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtcctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcc tgcggccattcggtacaattcacgcgtgagggcctatttccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtatttgcagtttttaaaattatgttttaaaattatgttttaaaatggactatcatatgcttaccgtaactt gaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccg NNNNNNNNNNtttttttttggtaccaggtcttgaaaggagtgggcgcgtgtcgacattgattattgactagctctggtcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatt tacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctactcgaggccacgttctgcttcactctccccatctcccccccctccccaccccccaattttgtatttatttattttt ttaattattttgtgcagcgatggggggcggggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcgg cgggcgggagcgggatcagccaccgcggtggcggccctagagtcgacgaggaactgaaaaaccagaaagttaactggtaagtttagtctttttgtcttttatttcaggtcccggatccggtggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttct gctctaaaagctgcggaattgtacccgcggccgatccaccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcacca ccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcga caccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccccccatcggcgacggccccg tgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaggaacaagccatcaagcccacctgttcggattagaaccattcacaacatatctcattggtgttgtggctgca aaccatgcaggagaaattttaagcccctggactctgattcaaaccttagaatcttccccaagtggactgagaaactttatagtagaacagaaagagaatggccgggcattgctactacagtggtcagagcctatgagaaccaatggtgtgattaaggaattccgctcgagataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctcctt ttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttagttcttgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgtttatttgtgaaatt tgtgatgctattgctttatttgtaaccatctagctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaattgcattcattttatgtttcaggttcaggggggagatgtgggaggttttttaaagcggccgcaggaacccctagtgatggagttggccactccctctct gcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagt Table B: Sequences containing targeting RNAs capable of being circularized. *uncapitalized sequence = targeting RNA sequence *Sequences not in bold = part of arRNA after circularization *Capital sequence in bold = part of arRNA that will be cut off after circularization Figure label In vitro synthetic sequence (corresponding to NNNNNNNNNN in SEQ ID NO:316) Figure 41 Before optimization (SEQ ID NO: 317) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatggtttgcagccacaacacca atgagatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4 bp deletion at -26 (SEQ ID NO: 318) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggctatttctcctgcatggtttgcagccacaacaccaatgaga tatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4 bp deletion at -30 (SEQ ID NO: 319) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaactcctgcatggtttgcagccacaacaccaatgaga tatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4 bp deletion at -34 (SEQ ID NO: 320) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaattttgcatggtttgcagccacaacaccaatg agatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4 bp deletion at +31 (SEQ ID NO: 321) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttcttaaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatggtttgcagccacaacaccaat gagatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4 bp deletion at +35 (SEQ ID NO: 322) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaaatttctcctgcatggtttgcagccacaacaccaatgaga tatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4 bp deletion at +39 (SEQ ID NO: 323) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctcttttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaaatttctcctgcatggtttgcagccacaacaccaatgaga tatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC Figure 42 4bp deletion at -26+31 (SEQ ID NO: 324) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttcttaaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggctatttctcctgcatggtttgcagccacaacaccaatgagata tgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -30+31 (SEQ ID NO: 325) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttcttaaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaactcctgcatggtttgcagccacaacaccaatgagata tgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -34+31 (SEQ ID NO: 326) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttcttaaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaattttgcatggtttgcagccacaacaccaatgaga tatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26+35 (SEQ ID NO: 327) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggctatttctcctgcatggtttgcagccacaacaccaatgagatatg ttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -30+35 (SEQ ID NO: 328) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaactcctgcatggtttgcagccacaacaccaatgagatatg ttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -34+35 (SEQ ID NO: 329) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaattttgcatggtttgcagccacaacaccaatgagatat gttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26+39 (SEQ ID NO: 330) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctcttttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggctatttctcctgcatggtttgcagccaacaccaatgagatatg ttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -30+39 (SEQ ID NO: 331) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctcttttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaactcctgcatggtttgcagccaacaccaatgagatatg ttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -34+39 (SEQ ID NO: 332) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctcttttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaattttgcatggtttgcagccacaacaccaatgagatat gttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC Figure 43 Before optimization (SEQ ID NO: 344) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatggtttgcagccacaacacca atgagatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26& +35 AC linker at R20 & L20 (SEQ ID NO: 345) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaagcaatgcccggccattctctttctgactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggctatttctcctgcatggtttgcagccaca acaccaataaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4 bp deletion at -26 & +35 AC linker at R20 & L30 (SEQ ID NO: 346) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaaaaaaaaaggccattctctttctgactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggctatttctcctgcatggtttgcag ccacaacaccaataaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26& +35 AC linker at R20 & L40 (SEQ ID NO: 347) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaacaaaaaaaaaccaaaaaatttctgactataaagtttctcagtccacttggggaagatcCaaggttttgaatcagagtccaggggctatttctcctgcatggtttg cagccacaacaccaataaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26& +35 AC linker at R20 & L50 (SEQ ID NO: 348) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaacaaaaaaaaaccaaaaaaaaaaaccaaaaaaacaaaacacaactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggctatttctcctgcatgg tttgcagccacaacaccaataaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC Figure 45 Before optimization (SEQ ID NO: 349) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatggtttgcagccacaacacca atgagatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26& +35 AC linker at R20 & L30 (SEQ ID NO: 350) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaaaaaaaaaggccattctctttctgactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggctatttctcctgcatggtttgcag ccacaacaccaataaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26& +35 AC linker at R20 & L30 U deletion at -5 +3 (SEQ ID NO: 351) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaaaaaaaaaggccattctctttctgactataaagtttctcagtccacttggggaagatcCaaggttgaatcagagtccaggggctatttctcctgcatggtttgcagcc acaacaccaataaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26& +35 AC linker at R20 & L30 U deletion at -5 +13 (SEQ ID NO: 352) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaacaaaaaaaaggccattctctttctgactataaagtttctcagtccactggggaagattcCaaggttgaatcagagtccaggggctatttctcctgcatggtttgcagccac aacaccaataaccaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26& +35 AC linker at R20 & L30 U deletion at +3 +13 (SEQ ID NO: 353) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaaaaaaaaaggccattctctttctgactataaagtttctcagtccactggggaagatcCaaggtttgaatcagagtccaggggctatttctcctgcatggtttgcagcc acaacaccaataaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26& +35 AC linker at R20 & L30 U deletion at -5 +3+13 (SEQ ID NO: 354) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaacaaaaaaaaggccattctctttctgactataaagtttctcagtccactggggaagatcCaaggttgaatcagagtccaggggctatttctcctgcatggtttgcagccac aacaccaataaccaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC SEQ ID NO: 355 – BFP forward primer GCACCATGAGCGAGCTG SEQ ID NO: 356 – BFP reverse primer TTAATTGAGTTTGTGCCC SEQ ID NO: 357 – CMV enhancer (CMVE) tacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccg cctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctactcgaggccacgttctgctt SEQ ID NO: 358 – Cyclization sequence GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCTAACCATGCCGACTGATGGCAGGAGACGGGATCCACGTCTCTCTGCCATCAGTCGGCGTGGACTGTAGAACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC SEQ ID NO: 359 – AC30 Connector Acacacacacacacacacacacacacac label In vitro synthetic sequences targeting RNA BL0 (SEQ ID NO: 360) cgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgctgtccaggacggtcccggcctgcgacacttcggcccagagctgctcctcatACACACACACACACACACACACACAC BL1 (+30M2, -6M2) (SEQ ID NO: 361) cgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgctgtaaaggacggtcccggcctgcgacacttcggcccagagctcgtcctcatACACACACACACACACACACACACACAC BL2 (+30M3, -6M3) (SEQ ID NO: 362) cgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgctggaaaggacggtcccggcctgcgacacttcggcccagagctcgacctcatACACACACACACACACACACACACACAC BL3 (+30M4, -6M4) (SEQ ID NO: 363) cgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgctagaaaggacggtcccggcctgcgacacttcggcccagagctcgagctcatACACACACACACACACACACACACACAC BL4 (+30M5, -6M5) (SEQ ID NO: 364) cgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgcgagaaaggacggtcccggcctgcgacacttcggcccagagctcgaggtcatACACACACACACACACACACACACACAC BL5 (+60M2, +30M2, -6M2) (SEQ ID NO: 365) cgggccctgggggcggtgggcgccagccaggacgcccaccgtgtggttgctgtaaaggacggtcccggcctgcgacacttcggcccagagctcgtcctcatACACACACACACACACACACACACACAC BL6 (+60M3, +30M3, -6M3) (SEQ ID NO: 366) cgggccctgggggcggtgggcggacgccaggacgcccaccgtgtggttgctggaaaggacggtcccggcctgcgacacttcggcccagagctcgacctcatACACACACACACACACACACACACACAC BL7 (+60M4, +30M4, -6M4) (SEQ ID NO: 367) cgggccctgggggcggtgggccgacgccaggacgcccaccgtgtggttgctagaaaggacggtcccggcctgcgacacttcggcccagagctcgagctcatACACACACACACACACACACACACAC BL8 (+60M5, +30M5, -6M5) (SEQ ID NO: 368) cgggccctgggggcggtggggcgacgccaggacgcccaccgtgtggttgcgagaaaggacggtcccggcctgcgacacttcggcccagagctcgaggtcatACACACACACACACACACACACACACAC BL9 (+41M3) (SEQ ID NO: 369) cgggccctgggggcggtgggcgctggccaggacgcccaccgacaggttgctgtccaggacggtcccggcctgcgacacttcggcccagagctgctcctcatACACACACACACACACACACACACACAC BL10 (+40M5) (SEQ ID NO: 370) cgggccctgggggcggtgggcgctggccaggacgcccaccaacaagttgctgtccaggacggtcccggcctgcgacacttcggcccagagctgctcctcatACACACACACACACACACACACACACAC BL111 (+40M5, -6M2) (SEQ ID NO: 371) cgggccctgggggcggtgggcgctggccaggacgcccaccaacaagttgctgtccaggacggtcccggcctgcgacacttcggcccagagctcgtcctcatACACACACACACACACACACACACAC BL12 (+40M5, -6M3) (SEQ ID NO: 372) cgggccctgggggcggtgggcgctggccaggacgcccaccaacaagttgctgtccaggacggtcccggcctgcgacacttcggcccagagctcgacctcatACACACACACACACACACACACACAC BL13 (+40M5, -6M5) (SEQ ID NO: 373) cgggccctgggggcggtgggcgctggccaggacgcccaccaacaagttgctgtccaggacggtcccggcctgcgacacttcggcccagagctcgaggtcatACACACACACACACACACACACACACAC BL14 (+41M3, -6M2) (SEQ ID NO: 374) cgggccctgggggcggtgggcgctggccaggacgcccaccgacaggttgctgtccaggacggtcccggcctgcgacacttcggcccagagctcgtcctcatACACACACACACACACACACACACACAC SEQ ID NO: 375 - 3×GS Connector GGGGS SEQ ID NO: 376 – Primers for PCR amplification of reverse transcribed cDNA of USHER 2A ggagtgagtacggtgtgcTGAATTTATGGATGAAGGAGACACCCT SEQ ID NO: 377 – Primers for PCR amplification of reverse transcribed cDNA of USHER 2A gagttggatgctggatggACGTCACCGCATGTTAGAAGACT SEQ ID NO: 378 - Sequence of amplified target region of USHER 2A ggagtgagtacggtgtgcTGAATTTATGGAATGAAGGACACCCTgaggcctttcacactctacgaatatcgggtcagagcctgtaactccaagggttcagtggagagtctgt AgtcattaacacaaactctggaagctccacctcaagattttccagctccttgggctcaagccacgagtgctcattcagttctgttgaattggacaaagccaGCGGCCGCTGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTccatccagcatccaactc SEQ ID NO: 379 – Primers for PCR amplification of reverse transcribed cDNA of USHER 2A ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTA

without

Figure 1A shows the sequence of the PCR amplification product of the USH2A full-length coding sequence introducing G>A mutation by mutagenesis PCR. Figure 1B shows a schematic diagram of genetically encoded arRNA from U6 or CMV promoters. A 151-nt arRNA targeting fluorescent reporter gene 1 was expressed under the human U6 or CMV promoter. For reporter gene 1, the mCherry and EGFP genes were connected by a sequence containing the 3×GGGGS (SEQ ID NO: 375) coding region and an in-frame UAG stop codon. Reporter-expressing cells produce only mCherry protein, and targeted editing of the UAG stop codon of the reporter transcript converts UAG to UIG, allowing downstream EGFP expression. Figure 2 shows the target RNA in USH2A reporter cell lines after the introduction of a linear arRNA with a 151-nt targeting RNA sequence (Linear-151) or a circular arRNA with a 151-nt (Circular-151) targeting RNA sequence. The percentage of each adenosine in the sequence that is edited, as measured by sequencing. Position 0 represents the target adenosine, "-" represents the upstream position of the target RNA, and "+" represents the downstream position of the target RNA. Figure 3 shows on-target editing efficiencies in USH2A reporter cell lines after introduction of linear or circular arRNA with targeting RNA sequence lengths as indicated on the x-axis compared to untreated (UT) or mock transfection, As measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 4 shows non-target adenosines in USH2A that are either upstream (upper panel) or downstream of the editing site (lower panel). Figure 5 shows a schematic diagram of the use of non-complementary base pairs ("4bp mismatch") and deletion of the targeted RNA sequence ("4bp deletion") to generate mismatched regions in arRNA design. Figure 6 shows the on-target editing efficiency in the USH2A reporter cell line after the introduction of USHER-171 arRNA, non-targeting arRNA, or arRNA with mismatched regions as described in Table 1, as measured by the average fluorescence of the GFP reporter. Light intensity (MFI) measurement. Figure 7 shows the percentage of each adenosine edited in the target RNA sequence in the USH2A reporter cell line after the introduction of USHER-171 arRNA, non-targeting arRNA, or arRNA with mismatched regions as described in Table 1, as Measured by sequencing. Position 0 represents the target adenosine, "-" represents the upstream position of the target RNA, and "+" represents the downstream position of the target RNA. Figure 8A shows a schematic representation of the use of deletions of targeted RNA sequences at positions upstream of the target adenosine (relative to the target RNA) to generate mismatched regions in arRNA design. Figure 8B shows a schematic representation of the use of deletions of targeted RNA sequences to generate mismatched regions in arRNA designs located downstream of the target adenosine (relative to the target RNA). Figure 9 shows the on-target editing efficiency in the USH2A reporter cell line after the introduction of USHER-171 arRNA, non-targeting arRNA, or arRNA with mismatched regions as described in Table 2, as measured by the average fluorescence of the GFP reporter Strength (MFI) measured. Figure 10 shows the percentage of each adenosine edited in the target RNA sequence in the USH2A reporter cell line after the introduction of USHER-171 arRNA, non-targeting arRNA, or arRNA with mismatched regions as described in Table 2, as determined by Measured by sequencing. Position 0 represents the target adenosine, "-" represents the upstream position of the target RNA, and "+" represents the downstream position of the target RNA. Figure 11 is a schematic depicting the use of a flexible linker flanking the 5'("left" flexible linker, or L-flexible linker) of the arRNA's targeting RNA sequence (top panel), or the 3' of the arRNA's targeting RNA sequence. '("right" flex joint, or R-flex joint) (picture below). Figure 12 shows arRNA containing an L-flexible linker (L) or an R-flexible linker (R) with the linker lengths indicated in Figure 11 as indicated on the x-axis (10-nt, 20-nt, 30-nt). Finally, on-target editing efficiency in the USH2A reporter cell line, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 13 shows the results in USH2A reporter cells after the introduction of arRNA containing L-flexible linker (L) or R-flexible linker (R) of the indicated linker lengths (10-nt, 20-nt, 30-nt). The percentage of each adenosine in the line that is edited, as measured by sequencing. Position 0 represents the target adenosine, "-" represents the upstream position of the target RNA, and "+" represents the downstream position of the target RNA. Figure 14A is a schematic depicting a circular arRNA with a 10 bp deletion upstream of the target adenosine (-26x-21x) and a 4 bp deletion downstream of the target adenosine (+35x or +39x) in the targeting RNA sequence. There is also a nucleotide deletion downstream of the target adenosine (+d7/8) relative to the non-target adenosine (all positions relative to the target RNA). Figure 14B is a schematic depicting a circular arRNA with a 10 bp deletion upstream of the target adenosine (-26x-21x) and a 4 bp deletion downstream of the target adenosine (+35x or +39x) in the targeting RNA sequence. There is also a nucleotide deletion downstream of the target adenosine (+d7/8) relative to the non-target adenosine (all positions relative to the target RNA), where the targeting RNA sequence is also surrounded by a 3' flexible linker ("right" flexible linker, or R-flexible joint) side connection. Figure 15 shows the results of introducing USHER-171 arRNA, non-targeting arRNA, or arRNA with mismatched regions with or without AC linkers as described in Figure 14 (at 10-nt, 20-nt, 30-nt). nt, 40-nt, 50-nt), on-target editing efficiency in the USH2A reporter cell line, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 16 shows the results of introducing USHER-171 arRNA, non-targeting arRNA, or arRNA with mismatched regions with or without AC linkers as described in Figure 14 (at 10-nt, 20-nt, 30-nt). nt, 40-nt, 50-nt), as measured by sequencing, the percentage of each adenosine edited in the USH2A reporter cell line. Position 0 represents the target adenosine, "-" represents the upstream position of the target RNA, and "+" represents the downstream position of the target RNA. Figure 17 is a schematic depicting further refinement of the mismatch of the hydroxyl group (originally "UU") of the non-target adenosine relative to the target adenosine downstream (+8 and +7). Figure 18 shows the results of introducing USHER-171 arRNA, non-targeting arRNA or having the mismatched region (+39x-21x or +35x-21x, see Figure 14) and downstream of the target adenosine to the non-target adenosine (+8 and +7) After the arRNA changes the base group (originally "UU") to the base group indicated on the x-axis (A, AA, U, UU, C, CC, G, GG, or "X"), USH2A On-target editing efficiency in reporter cell lines, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 19 shows that when USHER-171 arRNA is introduced, or has the mismatched region (+39x-21x or +35x-21x, see Figure 14) and is downstream of the target adenosine, non-target adenosine (+8 and +7) USH2A reporter cell line after arRNA changing the relative base (originally "UU") to the base indicated on the x-axis (A, AA, U, UU, C, CC, G, GG or "X") The percentage of target adenosine (0) and non-target adenosine (+7, +8) in the target RNA sequence that is edited, as measured by sequencing. Position 0 represents the target adenosine, "-" represents the upstream position of the target RNA, and "+" represents the downstream position of the target RNA. Figure 20 is a schematic depicting further refinement of the mismatch of the hydroxyl group (originally "UU") of the non-target adenosine relative to the target adenosine downstream (+d7/8). Figure 21 shows the introduction of base groups (originally "UU") with the mismatched region (+35x-21x-FlexLinker30 or +35x) and opposite the non-target adenosine (+8 and +7) downstream of the target adenosine. ) on-target editing efficiency in the USH2A reporter cell line after changing arRNA to the base indicated on the x-axis, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 22 shows that upon introduction of the base group (originally "UU") with the mismatched region (+35x-21x-FlexLinker50) and opposite the non-target adenosine (+8 and +7) downstream of the target adenosine, it is changed to The x-axis indicates on-target editing efficiency in the USH2A reporter cell line after base-based arRNA, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 23 shows that upon introduction of the base group (originally "UU") with the mismatched region (+35x-21x-FlexLinker50) and opposite the non-target adenosine (+8 and +7) downstream of the target adenosine, it is changed to The x-axis indicates on-target editing efficiency in the USH2A reporter cell line after base-based arRNA, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 24 is a schematic depicting the deletion of a base group of a targeting RNA sequence located in the upstream (-26) or downstream (+35) region of the target adenosine relative to the target RNA. Figure 25 is a schematic depicting the insertion of alkyl groups into a targeting RNA sequence located in a region upstream (-26) or downstream (+35) of a target adenosine relative to the target RNA. Figure 26 shows that after the introduction of arRNA with the deletion or insertion (1, 2, 3, 4, 7, 10 nt in length) in the upstream or downstream region relative to the target RNA (-26+35), On-target editing efficiency in the USH2A reporter cell line, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 27 is a schematic depicting the deletion of alkyl groups in the target RNA in the region downstream (+35) of the target adenosine relative to the target RNA. Figure 28 shows with solid bars (left) the introduction of arRNA with the deletion (0, 4, 10, 20, 30, 40 or 50 nt in length) in the downstream region relative to the target RNA (+35) Finally, on-target editing efficiency in the USH2A reporter cell line, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 28 also shows with open bars (right) the introduction of the deletion (0, 4, 10, 20, 30, 40 or 50 nt in length) in the downstream region relative to the target RNA (+35), and On-target editing efficiency of the USH2A reporter cell line as measured by the mean fluorescence intensity (MFI) of the GFP reporter after having a further 4 nt deletion of arRNA in the downstream region relative to the target RNA (-26). Figure 29 is a schematic depicting the deletion of an alkyl group of a targeting RNA located in the region (-26) upstream of the target adenosine relative to the target RNA. Figure 30 shows with solid bars (left) the introduction of arRNA with the deletion (0, 4, 10, 20, 30, 40 or 50 nt in length) in the upstream region relative to the target RNA (-26) Finally, on-target editing efficiency in the USH2A reporter cell line, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 30 also shows with open bars (right) the introduction of the deletion (0, 4, 10, 20, 30, 40 or 50 nt in length) in the upstream region relative to the target RNA (-26), and On-target editing efficiency of the USH2A reporter cell line as measured by the mean fluorescence intensity (MFI) of the GFP reporter after having a further 4 nt deletion of arRNA in the downstream region relative to the target RNA (+35). Figure 31 shows the introduction of deletions (lengths 0, 4, 10, 20, 30) in the upstream region relative to the target RNA (-26), and in the downstream region relative to the target RNA (+35). , 40 or 50 nt) arRNA, on-target editing efficiency of USH2A reporter cell lines, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 32 shows the sequence and length of flexible linkers used to flank targeting RNA sequences. Figure 33 shows that in the USH2A reporter cell line after the introduction of arRNA with the mismatched regions described (+35x-21X, +35x-21X+d78 or control arRNA) flanked by the flexible linkers described in Figure 32 The on-target editing efficiency, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 34 shows previously reported on-target editing efficiencies of arRNAs with different lengths of targeting RNA sequences and containing illustrated stem-loops and/or circularizations. Figure 35 is a schematic depicting the addition of stem loops (GluR, U6+27, Alu) to arRNA. Figure 36 shows the introduction of USHER-171 arRNAs (85-c-85) with or without further modifications to the stem loop shown in Figure 34 (GluR, U6+27, Alu) or with the mismatched region and On-target editing efficiency in the USH2A reporter cell line after USHER-171 of linkers (-21x+35x-R-flexlinker30 and -21+35x-R-flexlinker50), as measured by the average fluorescence intensity of the GFP reporter ( MFI) measured. Figure 37 shows the editing pattern in Rhesus monkey kidney cells after transfection of Ush2A targeting arRNA compared with the editing pattern in the eyes of Rhesus monkeys after subretinal injection of AAV packaged with Ush2A targeting arRNA using the Ush2A reporter gene. On-target and bystander editing modes (below). Figure 38 shows the editing pattern in the liver of rhesus monkeys after intravenous injection of AAV packaged with PPIA-targeting arRNA (targeting RNA sequence of 171 nt in length, 85-C-85) compared to On-target and bystander editing patterns of peptidyl prolyl isomerase A (PPIA) in rhesus monkey kidney cells following the corresponding arRNA with mismatched region (U deletion at -28-23+5) (pictured left). Figure 39 shows the introduction of target adenosine downstream mismatched regions (+31, +35, +40) and target adenosine upstream mismatched regions (-26, -30, -34) relative to the target RNA with the indicated On-target and bystander editing patterns of PPIA in rhesus monkey kidney cells after combining PPIA-specific arRNA (targeting RNA sequence of 171 nt in length). Figure 40 shows the results of introducing PPIA-specific arRNA (PPIA-171), either at 5' (L-10, L-20, L-30, L-40, L-50) or 3' (R-10, R -20, R-40, R-50) flanked by the corresponding arRNA (PPIA-171) with flexible linkers of the indicated lengths (10, 20, 30, 40, 50 nt) of the targeting RNA sequence, rhesus monkeys On-target and bystander editing patterns of PPIA in kidney cells. Figure 41 shows the on-target and bystander editing patterns of USH2A in rhesus monkey kidney cells after the introduction of USH2A-specific arRNA with the deletion (length 4) in the region upstream or downstream of the target adenosine. Figure 42 shows the on-target and bystander editing patterns of USH2A in rhesus monkey kidney cells after the introduction of USH2A-specific arRNA with 2 deletions (length 4) in the region upstream or downstream of the target adenosine. Figure 43 shows that after introduction of USH2A-specific arRNA with the indicated combinations of target adenosine downstream and upstream mismatch regions relative to the target RNA and flexible linkers at 5' and 3', in rhesus monkey kidney cells On-target and bystander editing modes of USH2A. Figure 44 shows before optimization, after optimization by first deleting 4 bp in the upstream region (-26) relative to the target RNA and in the downstream region (+35) relative to the target RNA, and additionally including in the 3' On-target and bystander editing of USH2A in rhesus monkey kidney cells after the introduction of USH2A-specific arRNA following optimization of a 20 bp adapter flanked by the targeting RNA sequence at the 5' end and a 30 bp adapter flanked by the targeting RNA sequence at the 5' end model. Figure 45 shows the introduction of USH2A-specific arRNA with the indicated combinations of mismatched regions downstream and upstream of the target adenosine relative to the target RNA, flexible linkers at 5' and 3', and one or more uracil deletions. Later, on-target and bystander editing patterns of USH2A in rhesus monkey kidney cells. Figure 46 shows the on-target editing efficiency in the IDUA reporter cell line after the introduction of the IDUA targeting arRNA (85-c-15) with the mismatched region and 3' linker, as measured by the average fluorescence of the GFP reporter Strength (MFI) measured. Figure 47 shows on-target and bystander editing patterns of IDUA targeting arRNA with the mismatched region and 3' linker.

TW202339775A_112103066_SEQL.xml

Claims (50)

A method of editing a target adenosine in a target RNA in a host cell, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding said dRNA into the host cell, wherein said dRNA comprises a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein the RNA double strand is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, Wherein the RNA double strand contains: (a) a first mismatch region relative to the target RNA sequence, which is located 5 nucleotides (nt) to 85 nt upstream of the target adenosine; and/or (b) a second mismatched region relative to the target RNA sequence located 20 nt to 85 nt downstream of the target adenosine; and wherein said dRNA comprises a linker nucleic acid sequence flanking the termini of said targeting RNA sequence, wherein said linker nucleic acid sequence does not hybridize to said target RNA and does not substantially form secondary structure. A method as described in request item 1, wherein: (a) the RNA duplex includes a first mismatch region relative to the target RNA sequence, the first mismatch region is located 5 nt to 25 nt upstream of the target adenosine; and/or the RNA duplex The strand comprises a second mismatch region relative to the target RNA sequence, the second mismatch region being located 20 nt to 45 nt downstream of the target adenosine; or (b) the RNA duplex includes a first mismatch region relative to the target RNA sequence, the first mismatch region is located 5 nt to 15 nt upstream of the target adenosine; and/or the RNA duplex The strand comprises a second mismatched region relative to the target RNA sequence, the second mismatched region being located 20 nt to 45 nt downstream of the target adenosine; or (c) the RNA double strands comprise a first mismatch region relative to the target RNA sequence, the first mismatch region is located 20nt to 40nt upstream of the target adenosine; and/or the RNA double strands A second mismatch region relative to the target RNA sequence is included, the second mismatch region being located 25 nt to 45 nt downstream of the target adenosine. The method according to claim 1 or 2, wherein the first mismatch region and/or the second mismatch region includes: (a) One or more non-complementary nucleotides (mismatch) in the targeting RNA sequence; and/or (b) one or more nucleotides of the targeting RNA sequence are deleted; and/or (c) Insertion of one or more nucleotides into the targeting RNA sequence. The method according to any one of claims 1-3, wherein the first mismatch region and/or the second mismatch region includes: (a) at least one set of contiguous non-complementary nucleotides (mismatch) in the targeting RNA sequence; and/or (b) Deletion of at least one group of contiguous nucleotides in the targeting RNA sequence; and/or (c) Insertion of at least one set of contiguous nucleotides of the targeting RNA sequence. A method as described in any of requests 1-4, wherein: (a) The length of the first mismatch region is 1-50nt, optionally, wherein the length of the first mismatch region is 4nt; and/or (b) The second mismatch region has a length of 1-50 nt, optionally, wherein the second mismatch region has a length of 4 nt. A method as described in any of claims 1-5, wherein: (a) The length of the first mismatch region is 1-10 nt, wherein the first mismatch region includes 1-10 consecutive non-complementary nucleotides in the targeting RNA sequence or the targeting Deletion of 1-10 consecutive nucleotides in the RNA sequence; and/or (b) The length of the second mismatch region is 1-10 nt, wherein the second mismatch region includes 1-10 consecutive non-complementary nucleotides in the targeting RNA sequence or the targeting Deletion of 1-10 consecutive nucleotides in the RNA sequence. A method as described in claim 5 or 6, wherein: (a) The length of the first mismatch region is 4 nt, wherein the first mismatch region includes four consecutive non-complementary nucleotides in the targeting RNA sequence or four nucleotides in the targeting RNA sequence. consecutive nucleotide deletions; and/or (b) The length of the second mismatch region is 4 nt, wherein the second mismatch region includes four consecutive non-complementary nucleotides in the targeting RNA sequence or four nucleotides in the targeting RNA sequence. Consecutive nucleotides are missing. A method as described in any of requests 1-7, wherein: (i) Non-complementary nucleotides in the targeting RNA sequence lead to bubble-like structures in the RNA double strands; and/or (ii) The deletion of nucleotides in the targeting RNA sequence results in a bulge structure in the RNA double strand; and/or (iii) Nucleotide insertion in the targeting RNA sequence results in a bulge structure in the RNA duplex. A method as described in any of requests 1-8, wherein: (i) A set of contiguous non-complementary nucleotides in the targeting RNA sequence results in a bubble-like structure in the RNA duplex; and/or (ii) Deletion of a set of contiguous nucleotides in the targeting RNA sequence results in a bulge structure in the RNA double strand; and/or (iii) Insertion of a set of contiguous nucleotides in the targeting RNA sequence results in a bulge structure in the RNA duplex. The method of any one of claims 1-9, wherein the target RNA encodes a PPIA protein. The method of claim 10, wherein the target RNA encoding the PPIA protein contains a target adenosine in an untranslated region (UTR). The method of claim 10 or 11, wherein the target RNA encodes wild-type PPIA. The method of any one of claims 1-12, wherein the RNA double-stranded further comprises a third mismatch region relative to the target RNA. The method of claim 13, wherein the third mismatch region includes one or two non-complementary nucleotides in the targeting RNA sequence and/or one or two cores of the targeting RNA sequence. The nucleotide is missing. The method of claim 13 or 14, wherein the third mismatch region relative to the target RNA sequence is located 5 nt, 7 nt and/or 8 nt downstream of the target adenosine; optionally, wherein the The target RNA includes an adenosine located 5, 7 and/or 8 nucleotides downstream of the target adenosine. The method according to any one of claims 10-15, wherein the RNA double strands comprise: (a) a first mismatch region relative to the target RNA sequence located 27 nt to 38 nt upstream of the target adenosine; and (b) A second mismatch region relative to the target RNA sequence located 32 nt to 35 nt downstream of the target adenosine. A method as described in request item 16, wherein: The first mismatch region relative to the target RNA sequence is located 27 nt to 30 nt upstream of the target adenosine, Optionally, wherein the first mismatch region is 4 nt in length, and the second mismatch region is 4 nt in length; Further optionally, wherein the first mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence, and wherein the second mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence. Deletion of nucleotides. A method as described in request item 16, wherein: The first mismatch region relative to the target RNA sequence is located 31 nt to 34 nt upstream of the target adenosine, Optionally, wherein the first mismatch region is 4 nt in length, and the second mismatch region is 4 nt in length. Further optionally, wherein the first mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence, and wherein the second mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence. Deletion of nucleotides. A method as described in request item 16, wherein: The first mismatch region relative to the target RNA sequence is located 35nt to 38nt upstream of the target adenosine, Optionally, wherein the first mismatch region is 4 nt in length, and the second mismatch region is 4 nt in length; Further optionally, wherein the first mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence, and wherein the second mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence. Deletion of nucleotides. The method according to any one of claims 10-15, wherein the RNA double strands comprise: (a) a first mismatch region relative to the target RNA sequence located 27 nt to 38 nt upstream of the target adenosine; and (b) A second mismatch region relative to the target RNA sequence located 41 nt to 44 nt downstream of the target adenosine. A method as described in request item 20, wherein: The first mismatch region relative to the target RNA sequence is located 27nt to 30nt upstream of the target adenosine, Optionally, wherein the first mismatch region is 4 nt in length, and the second mismatch region is 4 nt in length. Further optionally, wherein the first mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence, and wherein the second mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence. Deletion of nucleotides. A method as described in request item 20, wherein: The first mismatch region relative to the target RNA sequence is located 31 nt to 34 nt upstream of the target adenosine, Optionally, wherein the first mismatch region is 4 nt in length, and the second mismatch region is 4 nt in length; Further optionally, wherein the first mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence, and wherein the second mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence. Deletion of nucleotides. A method as described in request item 20, wherein: The first mismatch region relative to the target RNA sequence is located 35nt to 38nt upstream of the target adenosine, Optionally, wherein the first mismatch region is 4 nt in length, and the second mismatch region is 4 nt in length. Further optionally, wherein the first mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence, and wherein the second mismatch region includes a deletion of four consecutive nucleotides of the targeting RNA sequence. Deletion of nucleotides. The method according to any one of claims 1-23, wherein the dRNA is: (i) Circular; or (ii) Linear and/or capable of cyclization. The method of any one of claims 1-24, wherein the dRNA further comprises one or more RNA recruitment domains, optionally, wherein the RNA recruitment domains are stem-loop structures. The method of any one of claims 1-25, wherein the length of the linker nucleic acid sequence is from about 5 nt to about 500 nt. The method of any one of claims 1-26, wherein at least about 50%, 60%, 70%, 80%, 85%, 90% or 95% of any one of the linker nucleic acid sequences comprises adenosine or cytidine; optionally, wherein 100% of said linker nucleic acid sequences comprise adenosine or cytidine. The method of any one of claims 1-27, wherein at least about 50% of the linker nucleic acid sequences comprise adenosine. The method of any one of claims 1-28, wherein compared to a corresponding method in which the RNA double strand does not comprise one or more mismatch regions or wherein the dRNA does not comprise a linker nucleic acid sequence, the The method improves the editing level of the target adenosine. The method of any one of claims 1-29, wherein compared to a corresponding method in which the RNA double strand does not comprise one or more mismatch regions or wherein the dRNA does not comprise a linker nucleic acid sequence, the Methods reduce (bystander) editing levels of one or more non-target adenosines. The method of any one of claims 1-30, wherein the dRNA comprises a first linker nucleic acid sequence flanking the 5' end of the targeting RNA sequence and a first linker nucleic acid sequence flanking the 3' end of the targeting RNA sequence. Second linker nucleic acid sequence. The method of any one of claims 1-31, wherein the dRNA is a circular RNA, and wherein the one or more linker nucleic acid sequences connect the 5' end of the targeting RNA sequence and the targeting 3' end of the RNA sequence. The method according to any one of claims 1-32, wherein the dRNA is a circular RNA, wherein the dRNA further includes a 3' exon sequence and a 5' exon sequence, the 3' exon The sequence is recognized by a 3' catalytic Group I intronic fragment flanking the 5' end of the targeting RNA sequence, and the 5' exon sequence is recognized by a 3' end of the targeting RNA sequence. Identification of 5' catalytic group I intronic fragments. The method according to any one of claims 1-33, wherein the dRNA further comprises a 3' connecting sequence and a 5' connecting sequence. The method of any one of claims 1-34, wherein the method includes introducing into a host cell a construct comprising a nucleic acid sequence encoding the dRNA. The method of claim 35, wherein the construct further comprises a promoter operably linked to the nucleic acid sequence encoding the dRNA. The method of any one of claims 1-36, wherein the targeting RNA sequence comprises cytidine, adenosine or uridine directly opposite the target adenosine in the target RNA. The method of any one of claims 1-37, wherein the targeting RNA sequence contains a cytidine mismatch directly opposite a target adenosine in the target RNA. The method according to any one of claims 1-38, wherein the 5' nearest neighbor of the target adenosine in the target RNA is a nucleotide selected from U, C, A and G, preferably U>C≈ A>G, and the 3' nearest neighbor of the target adenosine in the target RNA is a nucleotide selected from G, C, A and U, preferably G>C>A≈U. The method of any one of claims 1-39, wherein the target adenosine is in the trisaccharyl motif of UAG, and wherein the targeting RNA sequence comprises the same as in the trisaccharyl motif. A directly opposite uridine, cytidine directly opposite the target adenosine, and cytidine, guanosine or uridine directly opposite guanosine in the trisaccharyl motif. The method of any one of claims 1-40, wherein the target RNA is an RNA selected from the group consisting of pre-messenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long non-coding RNA and small RNA. , optionally, wherein the target RNA is pre-messenger RNA. The method of any one of claims 1-41, wherein the efficiency of editing target adenosine in the target RNA is at least about 40%. The method of any one of claims 1-42, further comprising introducing ADAR into the host cell. An edited RNA produced by the method of any one of claims 1-43 or a host cell having edited RNA. A method of ameliorating one or more symptoms in an individual of: acute and chronic inflammatory diseases, cerebral hypoxia-ischemia, cancers such as hepatocellular carcinoma, lung cancer, pancreatic cancer, endometrial cancer, esophageal squamous cell carcinoma, and Melanoma, viral diseases such as AIDS, hepatitis C, measles and influenza A, the method comprising editing a target associated with PPIA in cells of the individual according to the method of any one of claims 1-43 RNA, wherein the target RNA encodes a PPIA protein. A dRNA for editing a target RNA comprising a target adenosine, wherein the dRNA comprises a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA double strand, wherein the RNA double strand is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in the target RNA, Wherein the RNA double strand contains: (a) a first mismatch region relative to the target RNA sequence located 5 nt to 85 nt upstream of the target adenosine; and/or (b) a second mismatched region relative to the target RNA sequence located 20 nt to 85 nt downstream of the target adenosine; and wherein said dRNA comprises a linker nucleic acid sequence flanking the termini of said targeting RNA sequence, wherein said linker nucleic acid sequence does not hybridize to said target RNA and does not substantially form secondary structure. The dRNA of claim 46, wherein the target RNA encodes a PPIA protein. A construct comprising a nucleic acid sequence encoding a dRNA as described in claim 46 or 47. A host cell comprising a dRNA as described in claim 46 or 47 or a construct as described in claim 48. A kit comprising a dRNA as described in claim 46 or 47 or a construct as described in claim 48, and instructions for editing a target RNA containing a target adenosine in a host cell.