TW202346596A - Engineered adar-recruiting rnas and methods of use for usher syndrome - 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 encoding a mutant Usher 2A protein. 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 use in USHER syndrome

[ Cross-references to related applications ]

This application claims the right of priority to International Application No. PCT/CN2022/085144 filed on April 2, 2022, the entire content of which is incorporated herein by reference. [ Reference to electronic sequence listing ]

The entire contents of the electronic sequence listing (792642002640SEQLIST-traditional CN.xml; size: 412KB; creation date: May 10, 2023) are incorporated herein by reference.

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

Genome editing is a powerful tool for biomedical research and development of treatments for disease. Editing technologies using engineered nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Cas proteins of the CRISPR system, have been applied to manipulate the genomes of a large number of organisms. Recently, new RNA editing tools have been developed taking advantage of deaminase proteins, such as adenosine deaminase (ADAR) acting on RNA. 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 of 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, ADAR proteins or their catalytic domains are fused to λN peptides, SNAP tags, or Cas proteins (dCas13b), and guide RNAs are designed to recruit chimeric ADAR proteins to the target site. Overexpression of ADAR1 or ADAR2 proteins and guide RNA containing R/G motifs has also been reported to 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 therapy is via viral vectors, but the limited loading size of highly desirable adeno-associated virus (AAV) vectors (approximately 4.5 kb) makes it challenging to accommodate both protein and guide RNA. Furthermore, it has recently been reported that overexpression of ADAR1 is oncogenic in multiple myeloma due to aberrant hyperediting on RNA and generates a large number of global off-target edits. In addition, ectopic expression of proteins or their domains of non-human origin carries the potential risk of causing immunogenicity. Furthermore, 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 result 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 early onset, severe congenital deafness, early visual impairment, and a small window period for intervention; while types III and IV are relatively rare and comprehensive Symptoms are mild. People with Usher syndrome type II begin to gradually experience vision loss between the ages of 10 and 20. The initial stage of the disease is night blindness, which eventually progresses to vision loss. This allows us to have a longer treatment window and hopefully halt the gradual 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 sequenced USH2A gene mutations, 13% were NM_206933.2 (USH2A)_ c.11864 position G>A (p.Trp3955Ter), which is the most common single point 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 load. Typically, lentivirus can be used to deliver payloads of no more than 10,000 base pairs, while adeno-associated viruses 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 functions of Usherin because the patient still lacks 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 RNA splicing, 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 the splicing process. This method is similar to the previous method - because the mutated exons are skipped, full-length Usherin is still not obtained in the end.

In 2020, Boya Jiyin Company 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 editing of target RNA. Adenosine use. 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 the arRNA is capable of recruiting an adenosine deaminase (ADAR) to target in the target RNA. Adenosine deamination. This can safely and effectively perform in vivo base editing from "A" to "I" bases on RNA, repairing the pathogenic mutation site, and achieving the purpose of treating diseases such as Usher syndrome.

While there are different gene or RNA editing methods for specific diseases (such as those caused by Usher2A mutations), the search for a method that can truly repair the disease-causing 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 incorporated by reference in their entirety.

The present application provides methods for editing a target adenosine in a target RNA encoding the mutant Usher2A protein and comprising a target adenosine in a host cell, the method comprising converting a deaminase recruiting RNA (dRNA ) or a construct comprising a nucleic acid encoding said dRNA is introduced into said host cell, wherein said dRNA comprises a targeting RNA sequence capable of hybridizing to a target RNA to form an RNA duplex, wherein said RNA duplex is capable of recruiting An adenosine deaminase (ADAR) on RNA to deaminate a target adenosine in a target RNA, wherein the RNA duplex includes one or more mismatch regions, and wherein the dRNA includes a linker nucleic acid sequence, which flanking the end of the targeting RNA sequence. Further provided herein are methods for 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 RNA comprising a target adenosine (e.g., a target RNA encoding said mutant Usher2A protein) in a host cell, comprising converting a deaminase recruiting RNA (dRNA) or comprising encoding said A construct of a dRNA nucleic acid is introduced into the host cell, wherein the dRNA includes the targeting RNA sequence, the sequence is capable of hybridizing with the target RNA to form an RNA duplex, and wherein the RNA duplex is capable of recruiting RNA an adenosine deaminase (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 in the 5 nucleotides to 85 nucleotides upstream of the target adenosine; and/or (b) 20 nucleotides downstream of the target adenosine relative to the second mismatch region of the target RNA sequence to 85 nucleotides, 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 hybridize to the target RNA Form secondary structure. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence located 5 nucleotides to 25 nucleotides upstream of the target adenosine; and/ Or wherein the RNA duplex comprises a second mismatch region relative to the target RNA sequence, located 20 nucleotides to 45 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence located 5 nucleotides to 15 nucleotides upstream of the target adenosine; and/ Or wherein the RNA duplex comprises a second mismatch region relative to the target RNA sequence, located 20 nucleotides to 45 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence located 20 nucleotides to 40 nucleotides upstream of the target adenosine; and/ Or wherein the RNA duplex comprises a second mismatch region relative to the target RNA sequence, located 25 nucleotides to 45 nucleotides downstream of the target adenosine. 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 is a dRNA for editing a target RNA (e.g., a target RNA encoding the mutant Usher2A protein), comprising the targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, Wherein the dRNA includes the targeting RNA sequence, the sequence can hybridize with the target RNA to form an RNA duplex, wherein the RNA duplex can recruit adenosine deaminase (ADAR) that acts on RNA to make 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 nucleotides upstream of the target adenosine nucleotide; and/or (b) located 20 nucleotides to 85 nucleotides downstream of the target adenosine relative to the second mismatch region of the target RNA sequence, and wherein said The dRNA contains linker nucleic acid sequences flanking the ends of the targeting RNA sequences, wherein the linker nucleic acid sequences do not hybridize to the target RNA and do not substantially form secondary structure. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence located 5 nucleotides to 25 nucleotides upstream of the target adenosine; and/ Or wherein the RNA duplex comprises a second mismatch region relative to the target RNA sequence, located 20 nucleotides to 45 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence located 5 nucleotides to 15 nucleotides upstream of the target adenosine; and/ Or wherein the RNA duplex comprises a second mismatch region relative to the target RNA sequence, located 20 nucleotides to 45 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence located 20 nucleotides to 40 nucleotides upstream of the target adenosine; and/ Or wherein the RNA duplex comprises a second mismatch region relative to the target RNA sequence, located 25 nucleotides to 45 nucleotides downstream of the target adenosine. 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 a deletion of one or more nucleotides in the targeting RNA. In some embodiments, the first mismatch region and/or the second mismatch region comprises an insertion of one or more nucleotides in the targeting RNA. In some embodiments, the first mismatch region and/or the second mismatch region comprise at least one contiguous set of non-complementary nucleotides (mismatches) in the targeting RNA. In some embodiments, the first mismatched region and/or the second mismatched region comprises a deletion of at least one contiguous set of nucleotides in 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 in the targeting RNA. In some embodiments, the first mismatch region is 1-50 nucleotides in length. In some embodiments, the second mismatch 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 consecutive non-complementary nucleotides in the targeting RNA or the target Deletions of 1-10 consecutive nucleotides into RNA. In some embodiments, the second mismatch region is 1-10 nucleotides in length, wherein the second mismatch region includes 1-10 consecutive non-complementary nucleotides in the targeting RNA or the target Deletions of 1-10 consecutive nucleotides into RNA. In some embodiments, the first mismatch region is 4 nucleotides in length. In some embodiments, the second mismatch region is 4 nucleotides in length. In some embodiments, the first mismatch region is 4 nucleotides in length, wherein the first mismatch region comprises 4 consecutive non-complementary nucleotides in the targeting RNA or in the targeting RNA. Deletion of 4 consecutive nucleotides. In some embodiments, the second mismatch region is 4 nucleotides in length, wherein the second mismatch region comprises 4 consecutive non-complementary nucleotides in the targeting RNA or in the targeting RNA. Deletion of 4 consecutive nucleotides. In some embodiments, non-complementary nucleotides in the targeting RNA cause bubbling in the RNA duplex. In some embodiments, deletions of nucleotides in the targeting RNA result in bulges in the RNA duplex. In some embodiments, insertion of nucleotides in the targeting RNA results in a bulge in the RNA duplex. In some embodiments, a contiguous set of non-complementary nucleotides in the targeting RNA results in bubbling in the RNA duplex. In some embodiments, deletion of a contiguous set of nucleotides in the targeting RNA results in a bulge in the RNA duplex. In some embodiments, insertion of a contiguous set of nucleotides in the targeting RNA results in a bulge in the RNA duplex.

In some embodiments, mutant Usher2A proteins comprise missense mutations, nonsense mutations, and/or frameshift mutations. In some embodiments, the mutant Usher2A protein comprises the Trp3955Ter mutation. In some embodiments, the target RNA encoding the mutant Usher2A protein contains a G to A mutation compared to the target RNA encoding the wild-type Usher2A protein. In some embodiments, the target RNA encoding the mutant Usher2A protein includes a G>A mutation at position 11864 compared to the target RNA encoding the wild-type Usher2A protein.

In some embodiments according to any one of the above methods or dRNA, wherein the RNA duplex further comprises a third mismatch region relative to the target RNA, wherein the third mismatch region relative to the target RNA The alignment region is located between the first mismatch region and the second mismatch region. In some embodiments, the third mismatch region comprises 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; alternatively, wherein the target RNA is located Adenosine is included at the 7th and/or 8th nucleotide downstream of the target adenosine. In some embodiments, the targeting RNA comprises an "AA" sequence 7 and 8 nucleotides downstream of the target adenosine, wherein the targeting RNA sequence comprises any one selected from: A , AA, U, C, CC, G, GG or a nucleotide deletion ("X") opposite the 7th and 8th nucleotides downstream of the target adenosine in the target RNA.

In some embodiments according to any of the above methods or dRNA, wherein the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence, located upstream of the target adenosine 27 nucleotides to 30 nucleotides; and (b) 31 nucleotides to 43 nucleotides downstream of the target adenosine relative to the second mismatch region of the target RNA sequence at. In some embodiments, the second mismatch region is located 36 nucleotides to 39 nucleotides downstream of the target adenosine relative to the target RNA sequence; alternatively, wherein the first mismatch The length of the region is 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 in the targeting RNA sequence, wherein the second mismatch region includes a deletion of 4 contiguous nucleotides in the targeting RNA sequence. missing.

In some embodiments according to any of the above methods or dRNA, wherein said RNA duplex comprises: (a) a first mismatch region relative to said target RNA sequence, located on said target adenosine 21 nucleotides to 30 nucleotides downstream, and (b) 36 nucleotides to 39 nucleotides downstream of the target adenosine relative to the second mismatch region of the target RNA sequence acid; optionally, the length of the first mismatch region is 10 nucleotides, and the length of the second mismatch region is 4 nucleotides. In some embodiments, the first mismatch region includes a deletion of 10 contiguous nucleotides in the targeting RNA sequence, and wherein the second mismatch region includes 4 contiguous nucleotides in the targeting RNA sequence. Lack of acid.

In some embodiments according to any of the above methods or dRNA, wherein said RNA duplex comprises: (a) a first mismatch region relative to said target RNA sequence, located on said target adenosine 21 nucleotides to 30 nucleotides downstream of the target adenosine, and (b) 40 nucleotides to 43 nucleotides downstream of the target adenosine relative to the second mismatched region of the target RNA sequence. nucleotide; optionally, the length of the first mismatch region is 10 nucleotides, and the length of the second mismatch region is 4 nucleotides. In some embodiments, the first mismatch region includes a deletion of 10 contiguous nucleotides in the targeting RNA sequence, and wherein the second mismatch region includes 4 contiguous nucleotides in the targeting RNA sequence. Lack of acid.

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 can be circularized. 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, the length of the linker nucleic acid sequence is less than or equal to 70nt, optionally, wherein 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 any integer between 40nt-50nt. In some embodiments, the linker nucleic acid sequence is from about 20 nt to about 60 nt in length; optionally, wherein the linker nucleic acid sequence is about 30 nt in length, or is about 50 nt in length. In some embodiments, the linker nucleic acid sequence contains adenosine or cytidine at least about any of: 50%, 60%, 70%, 80%, 85%, 90%, or 95%. In some embodiments, at least 50% of the linker nucleic acid sequences comprise adenosine.

In some embodiments according to any of the above methods, the method has an improvement compared to a corresponding method in which the RNA duplex does not comprise one or more mismatch regions or the dRNA does not comprise a linker nucleic acid sequence. Editing levels of target adenosine. In some embodiments, the method reduces one or more non-target adenosine compared to a corresponding method in which the RNA duplex does not comprise one or more mismatch regions or the dRNA does not comprise a linker nucleic acid sequence. (bystander) editing level. In some embodiments, non-target adenosine is located in one or more mismatch regions. In some embodiments, the non-target adenosine is located outside the mismatch region.

In some embodiments according to any of the above methods or dRNA, 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. Two linker nucleic acid sequences. 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 is a circular RNA, wherein the 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 that is recognized by a 3' catalytic Type I intronic fragment flanking the 5' end of the targeting RNA sequence, and a 5' exon sequence that is recognized by a 5' catalytic type I intronic fragment flanking the 3' end of the targeting RNA sequence. In some embodiments, the dRNA further comprises 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 from about 20 to about 75 nucleotides in length. In some embodiments, dRNA is circularized by RNA ligase RtcB. In some embodiments, dRNA is circularized by T4 RNA ligase 1 (Rnl1) or RNA ligase 2 (Rnl2).

In some embodiments according to any of the above methods or dRNA, the method comprises 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 promoter is a polymerase II promoter ("Pol III promoter"). In some embodiments, the promoter is a polymerase III promoter ("Pol III promoter"). In some embodiments, the construct is a viral vector or plasmid. In some embodiments, the construct is an adeno-associated virus (AAV) vector. In some embodiments, the construct is a self-complementary AAV (scAAV) vector. In some embodiments, ADARs are endogenously expressed by the host cell. In some embodiments, the host cell is a retinal cell.

In some embodiments according to any of the above methods or dRNA, the targeting RNA sequence is greater than 50 nt in length. In some embodiments, the targeting RNA sequence is about 100 to about 200 nt in length. In some embodiments, the targeting RNA sequence comprises cytidine, adenosine, or uridine directly opposite the 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, with a priority of 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, with a priority of G>C>A≈U. In some embodiments, the target adenosine is located in a UAG trihydroxyl motif, and wherein the targeting RNA contains an A directly opposite a uridine in the trihydroxyl motif and an A directly opposite the target adenosine. the opposite cytidine, and a cytidine, guanosine, or uridine directly opposite the guanosine in the tricarboxylic motif. In some embodiments, the target RNA is an RNA selected from the group consisting of precursor messenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long non-coding RNA, and small RNA, optionally, wherein the target RNA is the precursor messenger RNA.

In some embodiments according to any of the above methods, the method further comprises: introducing an ADAR3 inhibitor and/or an interferon stimulator into the host cell. In some embodiments, the methods comprise introducing multiple dRNAs or constructs into the host cell, each construct directed against a different target RNA. In some embodiments, the efficiency of editing the target RNA is at least 40%. In some embodiments, the method further comprises introducing ADAR into the host cell. In some embodiments, deamination of a target adenosine in the target RNA results in a missense mutation, premature stop codon, aberrant or alternative splicing in the target RNA, or a missense mutation, premature stop codon in the target RNA Stop codon, aberrant splicing, or reversal of alternative splicing. In some embodiments, target adenosine deamination in the target RNA results in point mutations, truncation, elongation and/or misfolding of the protein encoded by the target RNA, or through missense mutations, premature termination in the target RNA Codons, aberrant splicing, or reversal of alternative splicing to produce functional, full-length, correctly folded, and/or wild-type proteins. In some embodiments, the host cell is a eukaryotic cell, optionally, wherein the host cell is a mammalian cell. In some embodiments, the host cell is a human or mouse cell. Edited RNA and host cells having edited RNA produced by any of the methods described herein are also provided.

In one aspect, a method for treating or preventing a disease or disorder in an individual is provided, comprising editing and encoding the mutant Usher2A protein in a cell of the individual according to any of the above editing methods, or according to any of the above uses of dRNA. The target RNA of the protein contains the target adenosine associated with the disease or disorder. 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. In some embodiments, the disease or disorder is a single or polygenic disease or disorder.

In one aspect, a method for alleviating the symptoms of Usher syndrome in an individual is provided, comprising editing in a cell of the individual a cell containing adenosine, a target associated with Usher syndrome, according to any one of the editing methods described above, or according to the use of any one of the dRNAs described above. Target RNA. In some embodiments, the target RNA contains a G to A mutation. In some embodiments, the individual comprises Usher syndrome type II. In some embodiments, the individual has no vision loss, or wherein the individual has mild to moderate vision loss. In some embodiments, the host cells are retinal cells, optionally, wherein the host cells are rods and/or cones. In some embodiments, dRNA or a construct encoding the dRNA is introduced into the subretinal space and/or vitreous cavity. In some embodiments, an individual into whom the dRNA or a construct encoding the dRNA is introduced exhibits reduced vision loss compared to a corresponding individual into whom the dRNA or construct encoding the dRNA is not introduced. In some embodiments, the dRNA is introduced or a construct encoding a corresponding dRNA is introduced compared to an individual in which a corresponding dRNA or construct encoding a corresponding dRNA is introduced that does not include one or more mismatch regions and/or one or more linker nucleic acid sequences. Individuals with the dRNA construct showed reduced vision loss. In some embodiments, an individual into which the dRNA or a construct encoding the dRNA is introduced exhibits reduced loss of retinal cells compared to a corresponding individual into which the dRNA or construct encoding the dRNA is not introduced. In some embodiments, use of a corresponding dRNA or a construct encoding a corresponding dRNA that does not include one or more mismatch regions and/or one or more linker nucleic acid sequences is compared to a corresponding individual using the dRNA or construct encoding the corresponding dRNA. Individuals with the dRNA constructs showed reduced retinal cell loss.

In some embodiments, host cells comprising any of the constructs or dRNA described above are provided. In some embodiments, a kit comprising any of the above constructs or dRNA is provided, 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 various embodiments described herein may be combined with other embodiments of the present 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 RNA ("dRNA") or ADAR recruiting RNA ("arRNA"), or constructs containing nucleic acids encoding these arRNAs, It is used to edit target RNA containing target adenosine in host cells. In one aspect, the present application discloses an improved Leaper-based editing method that exhibits significantly higher editing efficiency and specificity and provides a solution to the bystander off-target problem through its optimization.

"LEAPER" (Leveraging Endogenous ADAR for Programmable Editing on RNA) has been previously developed by the inventor of the present application, 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 native or exogenously introduced ADAR1 or ADAR2 to convert adenosine to inosine at specific sites on the target RNA. Therefore, in some systems RNA editing can be achieved 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 the targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex contains one or more mismatch regions, the mismatch The region is located upstream and/or downstream of the target adenosine; and wherein the dRNA includes a linker nucleic acid sequence that, in some embodiments, does not hybridize to the target RNA and does not substantially form secondary structure . The mismatch region may comprise one or more non-complementary hydroxyl groups or a deletion of one or more hydroxyl groups in the targeting RNA. The method described here has been successfully used to correct pathogenic point mutations, such as USH2A mutations. The improved LEAPER method may provide broad applicability for therapeutics and biomedical research.

Accordingly, one aspect of the present application provides a method of editing a target adenosine in a target RNA encoding the mutant Usher2A protein in a host cell, comprising incorporating a deaminase recruiting RNA (dRNA) or comprising a nucleic acid encoding the dRNA. The construct is introduced into the host cell, wherein the dRNA includes the targeting RNA sequence, the sequence is capable of hybridizing with the target RNA to form an RNA duplex, and wherein the RNA duplex is capable of recruiting adenoids that act on the RNA. An aldehyde deaminase (ADAR) deaminates a target adenosine in a target RNA, wherein the RNA duplex comprises (a) a first mismatch region relative to the target RNA sequence located on the target adenosine 20 nucleotides to 40 nucleotides downstream; and/or (b) 25 nucleotides to 45 nucleotides downstream of the target adenosine relative to the second mismatch region of the target RNA sequence. at a nucleotide, 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 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 the dRNA into the host cell, Wherein the dRNA includes the targeting RNA sequence, the sequence can hybridize with the target RNA to form an RNA duplex, wherein the RNA duplex can recruit adenosine deaminase (ADAR) that acts on RNA to make the target RNA Deamination of a target adenosine in wherein the RNA duplex comprises a first mismatch region relative to the target RNA sequence, 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 the 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 an adenosine deaminase (ADAR) enzyme acting on RNA to render the target in the target RNA Adenosine deamination, wherein the RNA duplex 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 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. 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 the dRNA into the host cell, Wherein the dRNA includes the targeting RNA sequence, the sequence can hybridize with the target RNA to form an RNA duplex, wherein the RNA duplex can recruit adenosine deaminase (ADAR) that acts on RNA to make 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 20 nucleotides to 40 nucleotides upstream of the target adenosine nucleotide; and (b) located 25 nucleotides to 45 nucleotides downstream of the target adenosine relative to a second mismatch region of the target RNA sequence, 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 secondary structure.

Another aspect of the present application provides a method of editing a target RNA associated with Usher syndrome (eg, a target RNA encoding the mutant Usher2A protein), wherein the method comprises converting a deaminase recruiting RNA (dRNA) or comprising encoding A construct of the nucleic acid of said dRNA is introduced into a host cell of an individual, wherein said dRNA comprises said targeting RNA sequence, said sequence is capable of hybridizing to the target RNA to form an RNA duplex, wherein said RNA duplex is capable of recruiting An RNA-based adenosine deaminase (ADAR) deaminates a target adenosine in a target RNA, wherein the RNA duplex comprises: (a) a first mismatch region comprising, relative to the target RNA sequence, the a deletion of about 4 to about 10 consecutive nucleotides located 21 nucleotides to 37 nucleotides upstream of the target adenosine in the targeting RNA; and optionally (b) a second mismatch A region comprising a deletion of about 4 to about 10 contiguous nucleotides in the target RNA located 31 nucleotides to 43 nucleotides downstream of the target adenosine relative to the target RNA sequence. ; and optionally (c) a third mismatch region relative to the target RNA, located between the first mismatch region and the second mismatch region, wherein the targeting RNA sequence of the third mismatch region includes a sequence selected from Any of the following: A, AA, U, C, CC, G, GG or a nucleotide deletion ("X") located 3rd, 7th and 8th downstream of the target adenosine, or at the 13th nucleotide, or at the 5th nucleotide upstream, and optionally, wherein the dRNA includes a linker nucleic acid sequence flanking the end of the targeting RNA sequence, wherein the The linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure, and wherein the target RNA sequence in the dRNA is about 150 to about 220 nt in length. In some embodiments, the target RNA encodes a protein comprising a Trp3955Ter mutant Usher2A. 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 definitions incorporated by reference.

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

As used herein, the term "bulge" refers to an asymmetric bubbling region in a nucleic acid duplex formed by one or more unpaired nucleotides (eg, non-target adenosine) of the nucleic acid duplex. The bulges described herein may have a completely unpaired region on one strand without any corresponding complementary region on the opposite strand. Additionally, the bulges described herein may be formed from two non-complementary regions (one in each strand) having different numbers of nucleotides, possibly further containing unpaired nucleotides, so The nucleotides described do not form Watson-Crick base pairs. The longer of the two non-complementary regions contains 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 side chain, i.e., the opposite side chain contains Nucleic acid sequences that are complementary to the nucleic acid sequences flanking the bulge, but the opposite side chain does not contain at least one nucleotide opposite the nucleotide in the bulge (eg, non-target adenosine). As used herein, "bulge" does not include a region of completely mismatched nucleotides located within one strand of a nucleic acid duplex, i.e., the opposite side strand contains a nucleotide that is not complementary to every nucleotide in the bulge , which results in symmetric bubbling in the nucleic acid double strands. In some examples, the bulge includes 1, 2, 3, 4, 5, or more than 5 nucleotides on the strand with unpaired nucleotides.

When the first nucleic acid strand and the second nucleic acid strand form a double-stranded nucleic acid region, base pairing of the first nucleotide in the first nucleic acid strand with the second nucleotide in the second nucleic acid strand is described herein. are "opposite" to each other, or "correspond" to each other, that is, the first nucleotide is opposite to the second nucleotide, and the second nucleotide is opposite to the first nucleotide.

The terms "polynucleotide", "nucleic acid", "nucleotide sequence" and "nucleic acid sequence" are used interchangeably. They refer to polymeric forms of nucleotides of any length, whether deoxynucleotides or ribonucleotides, or analogs thereof.

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

The terms "Group I intron" and "Group I catalytic intron" are used interchangeably to refer to self-splicing ribozymes capable of catalyzing their own excision from RNA precursors . The type I intron consists of two fragments, a 5' catalytic type I intron fragment and a 3' catalytic type I intron fragment, which retain their folding and catalytic functions (i.e., self-splicing activity). In its native environment, the 5' catalytic type I intronic fragment contains a 5' exon flanking its 5' terminus, which contains a 5' exon that is recognized by the 5' catalytic type I intronic fragment subsequence; whereas the 3' catalytic type I intron fragment contains a 3' exon flanking its 3' end, which contains a 3' exon sequence recognized by the 3' catalytic type I intron fragment . The terms "5' exon sequence" and "3' exon sequence" as used herein are annotated according to the order of the exons relative to the type I introns in their native environment.

As used herein, the terms "adenine", "guanine", "cytosine", "thymine" and "hypoxanthine" refer to the nucleobase itself. The terms "adenosine," "guanosine," "cytidine," "thymidine," "uridine," and "inosine" refer to the nucleobase attached to a ribose or deoxyribose molecule. The term "nucleoside" refers to a nucleoside attached to ribose or deoxyribose. The term "nucleotide" refers to the respective nucleo-ribosyl-phosphate or nucleo-deoxyribosyl-phosphate salt. 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 ("U"), thymine and thymidine (abbreviated as "T"), inosine and hypoxanthine (abbreviated as "I"), can be used interchangeably to refer to the corresponding nucleobase, nucleoside or nucleotides. The terms nucleoside, nucleoside and nucleotide are sometimes 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. The disease or disorder may be caused in whole or in part by changes, such as mutations, in the wild-type, naturally occurring protein corresponding to the functional protein.

As used herein, expression of a region "located x to y nucleotides upstream of the target adenosine" means that a region may be expressed from "x to y nucleotides upstream of the target adenosine" Start with any nucleotide. For example, when the mismatch 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 mismatch region can range from Either starts with: 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 upstream relative to the adenosine of the target RNA , or 30nt to 34nt. As used herein, the expression that a region is "located x to y nucleotides downstream of the target adenosine" means that a region may be located "x to y nucleotides downstream of the target adenosine" Start with any nucleotide. For example, when the mismatch 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 mismatch region can range from Either starts with: 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, 42nt to 51nt, or 43nt to 52nt.

The present disclosure provides several types of compositions based on polynucleotides or polypeptides, including variants and derivatives. These include, for example, substitutional, insertional, deletional and covalent variants and derivatives. The term "derivative" is synonymous with the term "variant" and generally refers to a molecule that is 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 vector constructs comprising one or more vector constructs described herein, one or more transcripts thereof, to a host cell. As a basic platform, the present invention can realize targeted editing of RNA, such as precursor messenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long non-coding RNA and small RNA (such as miRNA). The methods of the present application may employ 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 the host cells. Traditional viral-based and non-viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. This method 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 with episomal or integrated genomes for delivery to host cells.

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

As used herein, "operably linked", when referring to a first nucleic acid sequence operably linked to a second nucleic acid sequence, refers to a situation where 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 it 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. Generally, operably linked nucleic acid sequences are contiguous and the open reading frames are aligned when required to bind two protein coding regions.

As used herein, "ligated" refers to joining nucleic acid sequences directly or indirectly, for example, through an intervening nucleic acid sequence.

As used herein, "complementarity" refers to the ability of one 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 are capable of forming hydrogen bonds (i.e., Watson-Crick base pairs) with a second nucleic acid (e.g., approximately 5, 6, 7, 8, 9, 10 out of 10, respectively). are approximately 50%, 60%, 70%, 80%, 90% and 100% complementary). "Perfectly complementary" means that all contiguous residues of one nucleic acid sequence form hydrogen bonds with the same number of contiguous residues of a second nucleic acid sequence. As used herein, "substantially complementary" means a degree of complementarity of at least about 70%, 75%, 80% over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides. Any 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 usually sequence-dependent and vary based on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence will specifically hybridize to the 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" or "hybridize" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonds between nucleotide residues. Hydrogen bonding can occur through Watson Crick base pairing, Hoogstein binding, or any other sequence-specific manner. A sequence that hybridizes to a given sequence is called a "complement" of the given sequence.

"Subject", "patient" or "individual" includes mammals, such as humans or other animals, and is typically humans. In some embodiments, the subject (eg, 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 of any appropriate age, including infants, teenagers, 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 in the context of clinical pathology intended to produce a beneficial and desirable effect on the natural processes of the individual or cell being treated. 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, if one or more symptoms associated with a progressive disease (e.g., Usher syndrome) are alleviated or eliminated, including but not limited to reduced host cell loss (e.g., reduced rod photoreceptor cell loss), reduced host cell loss, dependence, a person is successfully "treated". Reduce 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 patients with the disease, reduce the dosage of other drugs needed to treat the disease, delay or prevent the progression of the disease (e.g., delay or prevent the loss of vision). loss), and/or prolong the survival of the individual.

As used herein, the term "effective amount" or "therapeutically effective amount" of a substance is at least the lowest concentration required to produce measurable improvement or prevention of a particular disease. The effective amount here 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 one in which any toxic or harmful effects of the treatment are outweighed by the beneficial effects of the treatment. For progressive diseases (e.g., diseases characterized by progressive loss of function), an effective amount includes an amount sufficient to prevent or delay the 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 retinal cell loss). 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 shrink tumors and/or reduce the rate of tumor growth (eg, inhibit tumor growth) or prevent or delay other unnecessary 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 in one or more times. The 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 down to a certain extent, and preferably stop the infiltration of cancer cells into surrounding organs ; (iv) inhibit (i.e. slow down, preferably stop to a certain extent) the metastasis of tumors; (v) inhibit the growth of tumors; (vi) prevent or delay the occurrence and/or recurrence of tumors; (vii) reduce the risk of tumors recurrence rate, and/or (viii) some degree of alleviation of one or more symptoms associated with the cancer. An effective amount can be administered in one or more times. For the purposes of this statement, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to accomplish, directly or indirectly, prevention or treatment. As is clinically understood, an effective amount of one drug, compound or pharmaceutical composition may or may not be achieved together with another drug, compound or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and a single dose of an agent may be considered to be effective given the quantity.

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 characteristic found in nature, as distinguished from mutant or variant forms.

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

"Recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector containing one or more heterologous sequences (i.e., nucleic acid sequences of non-AAV origin) flanked by at least one, and in embodiments two, AAV reverse terminal repeats (ITR). Such rAAV vectors, when present in a host cell that has been infected with an appropriate helper virus (or is expressing an appropriate helper function) and is expressing the AAV rep and cap gene products (i.e., the AAV Rep and Cap proteins), can be replicated and Packaged into infectious virus particles. When an rAAV vector is integrated into a larger polynucleotide (such as in a chromosome or into another vector or plasmid used for cloning or transfection), then the rAAV vector can be called a "proto-vector". Can be "rescued" by copying and wrapping in the presence of AAV wrapper functionality and appropriate helper functions. rAAV vectors can be in any of a variety of forms, including but not limited to plasmids, linear artificial chromosomes, complexed with lipids, packaged within liposomes, and packaged in viral particles, especially AAV particles. The rAAV vector can be packaged into the outer shell of the AAV virus to produce "recombinant adeno-associated virus particles (rAAV particles)".

The "AAV inverted terminal repeat (ITR)" sequence, a term well known in the art, is a sequence of approximately 145 nucleotides that is present at both ends of the native single-stranded AAV genome. The most distal 125 nucleotides of the ITR can exist in two different orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome. The most distal 125 nucleotides also contain several shorter self-complementary regions (termed A, A', B, B', C, C', and D regions), allowing intrastrand base pairing to occur within the ITR This part happens.

Accordingly, polynucleotides encoding peptides or polypeptides containing 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, sequence tags or amino acids, such as one or more lysines, can be added to the polypeptide sequence (eg, at the N- or C-terminus). Sequence tags can be used for detection, purification, or localization of peptides. Lysine can be used to increase the solubility of the peptide or allow biotinylation. Additionally, amino acid residues located in the carboxyl and amino-terminal regions of the amino acid sequence of a polypeptide or protein can be selectively deleted to provide a truncated sequence. Certain amino acids (e.g., C-terminal residues or N-terminal residues) can be selectively deleted depending on the use of the sequence, e.g., expression of the sequence as part of a soluble larger sequence, or attachment to a solid support .

The terms "non-naturally occurring" or "engineered" are used interchangeably and mean artificial involvement. These terms, when referring to a nucleic acid molecule or polypeptide, mean that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it is naturally associated and found in nature.

As used herein, "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and/or the subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. The transcript and encoded polypeptide may collectively be referred to as the "gene product". If the polynucleotide is from genomic DNA, expression may involve splicing of the mRNA in a eukaryotic cell.

As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient, or stabilizer that is non-toxic to the cells or mammals to which it is exposed at the doses and concentrations employed. Typically physiologically acceptable carriers are pH buffered aqueous solutions. Non-limiting examples of physiologically acceptable carriers include buffers, such as phosphoric acid, citric acid, and other organic acids; antioxidants, including ascorbic acid; 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, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, 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 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 drug for treating a disease or condition). In some embodiments, the article or kit is marketed, distributed, or sold as a unit 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 "consisting of" and/or "consisting essentially of" aspects and embodiments.

It will be understood that, whether or not the term "about" is expressly used, each quantity given herein means the actual given value and also means an approximation of that given value that can be reasonably inferred in accordance with ordinary skill in the art , contains equivalents and approximations resulting from experimental and/or measurement conditions for that given value. Reference here to "about" a value or parameter includes (and describes) changes to the value or parameter itself. For example, a description that mentions "about X" includes a description of "X".

As used herein, reference to "other than" a value or parameter generally means and describes a value or parameter "other than". For example, the method is not used to treat type X disease means that the method is used to treat a type of disease other than 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 is further noted that the scope of the application may be drafted to exclude any optional elements. Therefore, this statement is intended to serve as a prerequisite for the use of exclusive terms such as "only" and "only" in connection with the recitation of elements of the claimed patent scope or the use of "negative" limitations.

As used herein, the term "and/or" such as the phrase "A and/or B" is intended to include A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used herein, such as "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. Methods of RNA editing

Provided herein are methods of editing 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 the dRNA, wherein the dRNA contains a compound capable of hybridizing to the target RNA. A targeting RNA sequence to form an RNA duplex, 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 is The strand comprises one or more mismatch regions relative to the target RNA, and 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 essentially does not form secondary structure. The dRNA can be any dRNA described in Section 3 ("dRNA, Constructs and Libraries") below. 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 method uses a construct comprising a nucleic acid sequence encoding the dRNA. The construct may be any of the constructs described in Section 3 below.

In some embodiments, methods are provided for editing a target adenosine in a target RNA (eg, a target RNA encoding the mutant Usher2A protein) in a host cell, comprising converting a deaminase recruiting RNA (dRNA) or comprising encoding the mutant Usher2A protein. The construct of the nucleic acid of the dRNA is introduced into the host cell, wherein the dRNA includes the targeting RNA sequence, and the sequence is capable of hybridizing with the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA adenosine deaminase (ADAR) deaminates a target adenosine in a target RNA, wherein the RNA duplex includes (a) a first mismatch region relative to the target RNA sequence, located in the target 5 nucleotides to 85 nucleotides upstream of adenosine; and/or (b) 20 nucleotides downstream of the target adenosine relative to the second mismatch region of the target RNA sequence to 85 nucleotides, wherein the dRNA includes a linker nucleic acid sequence flanking the terminus of the targeting RNA sequence, and 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 mismatch region being: 5 nucleotides to 25 nucleotides upstream of the target adenosine at, or 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 at a position: 20 nucleotides to 65 nucleotides downstream of the target adenosine, or 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 mismatch region is 1-50 nucleotides in length; and/or the second mismatch 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%, compared to methods using dRNA that does not include a linker nucleic acid sequence. 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x or more.

In some embodiments, methods are provided for editing a target adenosine in a target RNA (eg, a target RNA encoding the mutant Usher2A protein) in a host cell, comprising converting a deaminase recruiting RNA (dRNA) or comprising a deaminase recruiting RNA (dRNA) encoding the mutant Usher2A protein. The construct of the nucleic acid of the dRNA is introduced into the host cell, wherein the dRNA includes the targeting RNA sequence, and the sequence is capable of hybridizing with the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting RNA adenosine deaminase (ADAR) to deaminate a target adenosine in a target RNA, wherein the RNA duplex includes (a) a first mismatch region relative to the target RNA sequence, located in the 20 nucleotides to 40 nucleotides upstream of the target adenosine; and/or (b) 25 nucleotides downstream of the target adenosine relative to the second mismatch region of the target RNA sequence to 45 nucleotides, wherein the dRNA includes a linker nucleic acid sequence flanking the end of the targeting RNA sequence, and the linker nucleic acid sequence does not hybridize to the target RNA and does not substantially form secondary structure. In some embodiments, the first mismatch region is 1-50 nucleotides in length; and/or the second mismatch 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 compared to methods using dRNA that does not include a linker nucleic acid sequence. Times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.

In some embodiments, provided are methods of reducing non-target adenosine editing (also referred to herein as "bystander editing") in a target RNA (e.g., a target RNA encoding the mutant Usher2A protein) in a host cell, comprising: A deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA is introduced into the host cell, wherein the dRNA comprises a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the The RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate a target adenosine in a target RNA, wherein the RNA duplex comprises: (a) a sequence relative to the target RNA The first mismatch region is located 20 nucleotides to 40 nucleotides upstream of the target adenosine; and/or (b) the second mismatch region relative to the target RNA sequence is located at the 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, and the linker nucleic acid sequence does not hybridize to the target RNA , and basically does not form secondary structure. In some embodiments, the first mismatch region is 1-50 nucleotides in length; and/or the second mismatch 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 (such as a target RNA encoding the mutant Usher2A protein) in a host cell is provided, comprising converting a deaminase recruiting RNA (dRNA) or comprising encoding A construct of the nucleic acid of the dRNA is introduced 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 adenoids that act on the RNA. Adenosine deaminase (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 in the target adenosine 20 nucleotides to 40 nucleotides upstream of the target adenosine; and/or (b) 25 nucleotides to 25 nucleotides downstream of the target adenosine relative to the second mismatch region of the target RNA sequence. 45 nucleotides, wherein the dRNA includes a linker nucleic acid sequence flanking the terminus of the targeting RNA sequence, and 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 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 comprises a mismatch region relative to the target RNA sequence, located 26 nucleotides upstream of the target adenosine. In some embodiments, the RNA duplex comprises a mismatch region relative to the target RNA sequence, located 30 nucleotides upstream of the target adenosine. In some embodiments, the RNA duplex comprises a mismatch region relative to the target RNA sequence, located 34 nucleotides upstream of the target adenosine. In some embodiments, the RNA duplex comprises a mismatch region relative to the target RNA sequence 25 nucleotides to 45 nucleotides downstream of a target adenosine. In some embodiments, the RNA duplex comprises a mismatch region relative to the target RNA sequence, located 31 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex comprises a mismatch region relative to the target RNA sequence, located 35 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex comprises a mismatch region relative to the target RNA sequence, 39 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex comprises a mismatch region relative to the target RNA sequence, located 40 nucleotides downstream of the target adenosine. In some embodiments, the RNA duplex comprises a mismatch region relative to the target RNA sequence, located 41 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region is 1-50 nucleotides in length; and/or the second mismatch 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%, compared to methods using dRNA that does not include a linker nucleic acid sequence. 1x, 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 in the targeting RNA (mismatch); and/or (b) one or more nucleosides in the targeting RNA Deletion of acid; and/or (c) insertion of one or more nucleotides in the targeted RNA. In some embodiments, the second mismatch region comprises: (a) one or more non-complementary nucleotides in the targeting RNA (mismatch); and/or (b) one or more nucleosides in the targeting RNA Deletion of acid; and/or (c) insertion of one or more nucleotides in the targeted RNA. In some embodiments, the first mismatch region comprises: (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 targeted RNA. In some embodiments, the second mismatch region comprises: (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 targeted RNA. In some embodiments, non-complementary nucleotides in the targeting RNA cause bubbling in the RNA duplex. In some embodiments, deletions of nucleotides in the targeting RNA result in bulges in the RNA duplex. In some embodiments, insertion of nucleotides in the targeting RNA results in a bulge in the RNA duplex. In some embodiments, a contiguous set of non-complementary nucleotides in the targeting RNA causes bubbling in the RNA duplex. In some embodiments, deletion of a contiguous set of nucleotides in the targeting RNA results in a bulge in the RNA duplex. In some embodiments, insertion of a contiguous set of nucleotides in the targeting RNA results in a bulge in the RNA duplex.

In some embodiments, the first mismatch region is 1-50 nucleotides in length. In some embodiments, the second mismatch region is 1-50 nucleotides in length. In some embodiments, the first mismatch region is any of about 1-10, 10-20, 20-30, 30-40, or 40-50 nucleotides in length. In some embodiments, the first mismatch region is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 in length , any of 19 or 20 nucleotides. In some embodiments, the second mismatch region is any of about 1-10, 10-20, 20-30, 30-40, or 40-50 nucleotides in length. In some embodiments, the length of the second mismatch 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 mismatch region is 1-10 nucleotides in length; and/or the second mismatch region is 1-10 nucleotides in length. In some embodiments, the first mismatch region includes 1-10 consecutive non-complementary nucleotides in the targeting RNA. In some embodiments, the first mismatch region comprises a deletion of 1-10 contiguous nucleotides in the targeting RNA. In some embodiments, the second mismatch region contains 1-10 consecutive non-complementary nucleotides in the targeting RNA. In some embodiments, the second mismatch region comprises a deletion of 1-10 contiguous nucleotides in the targeting RNA.

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

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 the mutant Usher2A protein according to any of the above methods for editing a target adenosine in a target RNA in a host cell. In some embodiments, mutant Usher2A proteins comprise missense mutations, nonsense mutations, and/or frameshift mutations. In some embodiments, the mutant Usher2A protein is a truncated Usher2A protein. In some embodiments, the target RNA encodes a mutant Usher2A protein comprising the Trp3955Ter mutation. In some embodiments, the target RNA encoding the mutant Usher2A contains a G to A mutation compared to the target RNA encoding the wild-type Usher2A protein. In some embodiments, the target RNA encoding the mutant Usher2A protein includes a G>A mutation at position 11864 compared to the target RNA encoding the wild-type Usher2A protein. In some embodiments the target RNA is endogenously expressed.

In some embodiments, a method of editing a target RNA encoding the mutant Usher2A protein in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA into the A host cell, wherein the dRNA comprises the targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting adenosine deaminase (ADAR) acting on RNA Deaminating a target adenosine in a target RNA, wherein the RNA duplex comprises: (a) a first mismatch region 20 nucleotides upstream of the target adenosine relative to the target RNA sequence to 40 nucleotides; and/or (b) 25 nucleotides to 45 nucleotides downstream of the target adenosine relative to a second mismatch region of the target RNA sequence, wherein , the dRNA includes a linker nucleic acid sequence flanking the end of the targeting RNA sequence, and 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 protein comprising a Trp3955Ter mutant Usher2A. In some embodiments, the target RNA encoding the mutant Usher2A protein comprises a G to A mutation compared to the target RNA encoding the wild-type Usher2A protein. In some embodiments, the target RNA encoding the mutant Usher2A protein includes a G>A mutation at position 11864 compared to the target RNA encoding the wild-type Usher2A protein. In some embodiments, the target adenosine is located at position 101 compared to SEQ ID NO. 3.

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 the mutant Usher2A protein, the RNA duplex further comprises in the third mismatch region of the target RNA. In some embodiments, a third mismatch region is located between the first mismatch region and the second mismatch region relative to the target RNA. In some embodiments, the third mismatch region comprises 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 comprises 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 one selected from: A, AA, U , C, CC, G, GG or a nucleotide deletion ("X") opposite the 7th and 8th nucleotides downstream of the target adenosine in the target RNA.

In some embodiments, according to any of the above methods of editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes the mutant Usher2A protein, the RNA duplex comprises: (a ) relative to the first mismatch region of the target RNA sequence, located 27 nucleotides to 30 nucleotides (e.g., 27 nucleotides) upstream of the target adenosine; and (b) relative to The second mismatch region of the target RNA sequence is located 31 nucleotides to 43 nucleotides downstream of the target adenosine. In some embodiments, the second mismatched region is located 32 nucleotides to 35 nucleotides (eg, 32 nucleotides) downstream of the target adenosine relative to the target RNA sequence. In some embodiments, the second mismatch region is located 36 nucleotides to 39 nucleotides (eg, 36 nucleotides) downstream of the target adenosine relative to the target RNA sequence. 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 mismatch region is 4 nucleotides in length. In some embodiments, the first mismatch 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 mismatch region comprises a deletion of four consecutive nucleotides in the targeting RNA.

In some embodiments, according to any of the above methods of editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes the mutant Usher2A protein, the RNA duplex comprises: (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 The mismatch region is located 36 to 43 nucleotides downstream of the target adenosine. In some embodiments, the second mismatch region is located 36 nucleotides to 39 nucleotides downstream of the target adenosine relative to the target RNA sequence. In some embodiments, the second mismatch region is located 40 nucleotides to 43 nucleotides downstream of the target adenosine relative to the target RNA sequence. In some embodiments, the first mismatch region is 10 nucleotides in length. In some embodiments, the first mismatch region comprises a deletion of ten consecutive nucleotides in the targeting RNA. In some embodiments, the second mismatch region is 4 nucleotides in length. In some embodiments, the second mismatch region comprises a deletion of four consecutive nucleotides in the targeting RNA.

In some embodiments, according to any of the above methods of editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes the mutant Usher2A protein, the RNA duplex comprises: (a ) a first mismatch region located 26 nucleotides to 35 nucleotides upstream of the target adenosine relative to the target RNA sequence; and (b) a second mismatch region relative to the target RNA sequence The mismatch region is located 35 to 44 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region is 1 nucleotide in length and the second mismatch region is 1 nucleotide in length. In some embodiments, the first mismatch region is 2 nucleotides in length and the second mismatch region is 2 nucleotides in length. In some embodiments, the first mismatch region is 3 nucleotides in length and the second mismatch region is 3 nucleotides in length. In some embodiments, the first mismatch region is 4 nucleotides in length and the second mismatch region is 4 nucleotides in length. In some embodiments, the first mismatch region is 7 nucleotides in length and the second mismatch region is 7 nucleotides in length. In some embodiments, the first mismatch region is 10 nucleotides in length and the second mismatch region is 10 nucleotides in length. In some embodiments, the first mismatch region includes an insertion of one nucleotide in the targeting RNA and the second mismatch region includes an insertion of one nucleotide in the targeting RNA. In some embodiments, a first mismatch region includes an insertion of two contiguous nucleotides in the targeting RNA and a second mismatch region includes an insertion of two contiguous nucleotides in the targeting RNA. In some embodiments, the first mismatch region includes an insertion of three contiguous nucleotides in the targeting RNA and the second mismatch region includes an insertion of three contiguous nucleotides in the targeting RNA. In some embodiments, the first mismatch region includes an insertion of four contiguous nucleotides in the targeting RNA and the second mismatch region includes an insertion of four contiguous nucleotides in the targeting RNA. In some embodiments, the first mismatch region includes an insertion of seven contiguous nucleotides in the targeting RNA and the second mismatch region includes an insertion of seven contiguous nucleotides in the targeting RNA. In some embodiments, the first mismatch region includes an insertion of ten contiguous nucleotides in the targeting RNA and the second mismatch region includes an insertion of ten contiguous nucleotides in 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 the mutant Usher2A protein, the RNA duplex comprises: relative to The mismatch region of the target RNA sequence is located at 26 nucleotides to 37 nucleotides upstream of the target adenosine. In some embodiments, the mismatch region is located 26 nucleotides to 29 nucleotides downstream of the target adenosine relative to the target RNA sequence. In some embodiments, the mismatch region is located 30 nucleotides to 33 nucleotides downstream of the target adenosine relative to the target RNA sequence. In some embodiments, the mismatch region is located 34 nucleotides to 37 nucleotides downstream of the target adenosine relative to the target RNA sequence. In some embodiments, the mismatch region is 4 nucleotides in length. In some embodiments, the mismatch region comprises a deletion of four consecutive nucleotides in the targeting RNA.

In some embodiments, according to any of the above methods of editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes the mutant Usher2A protein, the RNA duplex comprises: (a ) a first mismatch region located 26 nucleotides to 37 nucleotides upstream of the target adenosine relative to the target RNA sequence; and (b) a second mismatch region relative to the target RNA sequence The mismatch region is located 31 to 39 nucleotides downstream of the target adenosine. 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 mismatch region relative to the target RNA sequence is located 31 nucleotides to 34 nucleotides downstream of the target adenosine. In some embodiments, the second 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 mismatch region relative to the target RNA sequence is located 39 nucleotides to 42 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region is 4 nucleotides in length. In some embodiments, the first mismatch 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 mismatch region comprises a deletion of four consecutive nucleotides in the targeting RNA.

In some embodiments, according to any of the above methods of editing a target adenosine in a target RNA in a host cell, wherein the target RNA encodes the mutant Usher2A protein, the RNA duplex comprises: (a ) a first mismatch region located 26 nucleotides to 29 nucleotides upstream of the target adenosine relative to the target RNA sequence; and (b) a second mismatch region relative to the target RNA sequence The mismatch region is located 35 to 38 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region is 4 nucleotides in length. In some embodiments, the first mismatch 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 mismatch region comprises a deletion of four consecutive nucleotides in the targeting RNA. In some embodiments, the RNA duplex further comprises a third mismatch region relative to the target RNA sequence, located 5 nucleotides upstream of the target adenosine, and relative to the target RNA The fourth mismatch region of the sequence is located 3 nucleotides downstream of the target adenosine. In some embodiments, the third mismatch region contains a deletion of a uracil in the targeting RNA. In some embodiments, the fourth mismatch region contains a deletion of one uracil in the targeting RNA. In some embodiments, the RNA duplex further comprises a third mismatch region relative to the target RNA sequence, located 5 nucleotides upstream of the target adenosine and relative to the target RNA sequence The fourth mismatch region is located 13 nucleotides downstream of the target adenosine. In some embodiments, the third mismatch region contains a deletion of a uracil in the targeting RNA. In some embodiments, the fourth mismatch region contains a deletion of one uracil in the targeting RNA. In some embodiments, the RNA duplex further comprises a third mismatch region relative to the target RNA sequence, located 3 nucleotides downstream of the target adenosine and relative to the target RNA sequence. The fourth mismatch region is located 13 nucleotides downstream of the target adenosine. In some embodiments, the third mismatch region contains a deletion of a uracil in the targeting RNA. In some embodiments, the fourth mismatch region contains a deletion of one uracil in the targeting RNA. In some embodiments, the RNA duplex further comprises a third mismatch region relative to the target RNA sequence, located 5 nucleotides upstream of the target adenosine, relative to the target RNA sequence a fourth mismatch region located 3 nucleotides downstream of the target adenosine, and a fifth mismatch region relative to the target RNA sequence located 13 nucleotides downstream of the target adenosine at. In some embodiments, the third mismatch region contains a deletion of a uracil in the targeting RNA. In some embodiments, the fourth mismatch region contains a deletion of one uracil in the targeting RNA. In some embodiments, the fifth mismatch region contains a deletion of one uracil in the targeting RNA.

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 in length , any of 300, 350, 400, 450 or 500nt, 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 Any integer between 40nt-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 any one of 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the linker nucleic acid sequences comprise adenosine or cytidine. In some embodiments, about 50% to 60%, 60% to 70%, 70% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% of the linker Either of the 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 any one of 50%, 60%, 70%, 80%, 85%, or 90% 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% From % to 95% of the adapter nucleic acid sequences contain adenosine.

In some embodiments, 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 and/or the dRNA does not comprise a linker nucleic acid sequence. . In some embodiments, the method shows a level of editing of a target adenosine compared to a corresponding method wherein the RNA duplex does not comprise one or more mismatch regions and/or wherein the dRNA does not comprise a linker nucleic acid sequence. An increase of at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2x, 5x, 10x, 20x, 50x, or 100% More than times. In some embodiments, the method reduces one or more non-target adenosines compared to a corresponding method wherein the RNA duplex does not comprise one or more mismatch regions and/or the dRNA does not comprise a linker nucleic acid sequence. (spectator) editing level. In some embodiments, the method exhibits one or more non- The editing level of the target adenosine is reduced by at least approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20 times, 50 times or more than 100 times. In some embodiments, the non-target adenosine is located within one or more mismatch regions. In some embodiments, the non-target adenosine is located 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 that is recognized by a 3' catalytic type I intronic fragment targeting the 5' end of the RNA sequence, and a 3' exon sequence that is recognized by a 5' end of the targeting RNA sequence. '5' exon sequence recognized by the catalytic type I intronic fragment. In some embodiments, the dRNA further comprises a 3' linker sequence and a 5' linker sequence. In some embodiments, the double-stranded RNA contains a bulge at each non-target adenosine in a target RNA, such as a target RNA encoding the mutant Usher2A protein.

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 targeting RNA sequence in the dRNA is about 150 to about 220 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 targeting RNA sequence in the dRNA is about 170 nt (eg, 171 nt) in length. 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, a method of editing a target RNA encoding the mutant Usher2A protein in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA into the A host cell, wherein the dRNA comprises the targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting adenosine deaminase (ADAR) acting on RNA Deaminating a target adenosine in a target RNA, wherein the RNA duplex comprises: (a) a first mismatch region comprising 27 nucleotides to 30 nucleotides upstream of the target adenosine, relative to A deletion of four consecutive nucleotides in the target RNA sequence; and (b) a second mismatch region comprised from 32 nucleotides to 35 nucleotides downstream of the target adenosine, or 36 nuclei A deletion of four consecutive nucleotides relative to the target RNA sequence from nucleotide 39 to nucleotide 39, or from nucleotide 40 to nucleotide 43. and (c) a third mismatched region relative to the target RNA, located between the first mismatched region and the second mismatched region, wherein the targeting RNA sequence of the third mismatched region includes any one selected from the following Item: A, AA, U, C, CC, G, GG or a nucleotide deletion ("X") opposite the 7th and 8th nucleotides downstream of the target adenosine in the target RNA, and wherein the dRNA comprises a linker nucleic acid sequence flanking the ends 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 Length approximately 150 to approximately 220nt. 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 can be circularized. In some embodiments, the targeting RNA encodes a Trp3955 Ter mutant Usher2A protein.

In some embodiments, a method of editing a target RNA encoding the mutant Usher2A protein in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA into the A host cell, wherein the dRNA comprises the targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting adenosine deaminase (ADAR) acting on RNA Deaminating a target adenosine in a target RNA, wherein the RNA duplex comprises: (a) a first mismatch region comprising 21 nucleotides to 30 nucleotides upstream of the target adenosine, relative to a deletion of 10 consecutive nucleotides in the target RNA sequence in the target RNA; and (b) a second mismatch region comprised from 36 nucleotides to 39 nucleotides downstream of the target adenosine, or 40 nucleotides to 43 nucleotides, a deletion of 4 consecutive nucleotides in the target RNA relative to the target RNA sequence; and (c) a third mismatch relative to the target RNA Region, located between the first mismatch region and the second mismatch region, wherein the targeting RNA sequence of the third mismatch region includes any one selected from the following: A, AA, U, C, CC, G, GG or a nucleotide deletion ("X") opposite the 7th and 8th nucleotides downstream of a target adenosine in the target RNA, and wherein the dRNA comprises an adapter nucleic acid flanking the terminus of the target RNA sequence 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 about 150 to about 220 nt in length. 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 can be circularized. In some embodiments, the targeting RNA encodes a Trp3955Ter mutant Usher2A protein.

In some embodiments, a method of editing a target RNA encoding the mutant Usher2A protein in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA into the A host cell, wherein the dRNA comprises the targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting adenosine deaminase (ADAR) acting on RNA Deaminating a target adenosine in a target RNA, wherein the RNA duplex comprises: (a) a first mismatch region comprising 26 nucleotides to 29 nucleotides upstream of the target adenosine, at 30 nucleotides to 33 nucleotides upstream of the target adenosine, or 34 nucleotides to 37 nucleotides upstream of the target adenosine, four consecutive nucleotides relative to the target RNA sequence deletion of acid; and (b) a second mismatched region comprised from 31 nucleotides to 34 nucleotides downstream of the target adenosine, or from 35 nucleotides to 38 nucleotides downstream of the target adenosine a deletion of four consecutive nucleotides at the nucleotide, or 39 nucleotides to 42 nucleotides downstream of the target adenosine relative to the target RNA sequence, and wherein the dRNA comprises a linker nucleic acid sequence, The linker nucleic acid sequence flanks 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 a secondary structure, and wherein the length of the targeting RNA sequence in the dRNA is about 150 to approximately 220nt. 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 can be circularized. In some embodiments, the target RNA encodes a protein comprising a Trp3955Ter mutant Usher2A.

In some embodiments, a method of editing a target RNA encoding the mutant Usher2A protein in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA into the A host cell, wherein the dRNA comprises the targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting adenosine deaminase (ADAR) acting on RNA Deaminating a target adenosine in a target RNA, wherein the RNA duplex includes a mismatch region comprised between 26 nucleotides and 29 nucleotides upstream of the target adenosine, or between 26 and 29 nucleotides upstream of the target adenosine. A deletion of four consecutive nucleotides 30 nucleotides to 33 nucleotides upstream of the target adenosine, 34 nucleotides to 37 nucleotides upstream of the target adenosine, relative to the target RNA sequence, And wherein the length of the targeting RNA sequence in the dRNA is about 150 to about 220 nt. In some embodiments, the dRNA is circular or can be circularized. In some embodiments, the target RNA encodes a protein comprising a Trp3955Ter mutant Usher2A.

In some embodiments, a method of editing a target RNA encoding the mutant Usher2A protein in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA into the A host cell, wherein the dRNA comprises the targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting adenosine deaminase (ADAR) acting on RNA Deaminating a target adenosine in a target RNA, wherein the RNA duplex includes a mismatch region comprised 31 nucleotides to 34 nucleotides downstream of the target adenosine, or between 31 and 34 nucleotides downstream of the target adenosine. Deletion of four consecutive nucleotides 35 to 38 nucleotides downstream, or 39 to 42 nucleotides downstream of the target adenosine relative to the target RNA sequence , and wherein the length of the targeting RNA sequence in the dRNA is about 150 to about 220 nt. In some embodiments, the dRNA is circular or can be circularized. In some embodiments, the target RNA encodes a protein comprising a Trp3955Ter mutant Usher2A.

In some embodiments, a method of editing a target RNA encoding the mutant Usher2A protein in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA into the A host cell, wherein the dRNA comprises the targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting adenosine deaminase (ADAR) acting on RNA Deamination of target adenosine in target RNA. 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 can be circularized. In some embodiments, the target RNA encodes a protein comprising a Trp3955Ter mutant Usher2A.

In some embodiments, a method of editing a target RNA encoding the mutant Usher2A protein in a host cell is provided, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA into the A host cell, wherein the dRNA comprises the targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex is capable of recruiting adenosine deaminase (ADAR) acting on RNA Deaminating a target adenosine in a target RNA, wherein the RNA duplex comprises: (a) a first mismatch region comprising 26 nucleotides to 29 nucleotides upstream of the target adenosine, relative to Deletion of four consecutive nucleotides in the target RNA sequence; (b) a second mismatch region comprising 35 nucleotides to 38 nucleotides downstream of the target adenosine, relative to the target Deletion of four consecutive nucleotides of the RNA sequence, and 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, and 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 can be circularized. In some embodiments, the target RNA encodes a protein comprising a Trp3955Ter mutant Usher2A.

In some embodiments, the targeting RNA sequence comprises a cytidine, adenosine, or uridine directly opposite a target adenosine residue in a target RNA, such as a target RNA encoding the mutant Usher2A protein. In some embodiments, the targeting RNA sequence contains a cytidine mismatch directly opposite a target adenosine residue in the target RNA. In some embodiments, the cytidine mismatch is located at least 5 nucleotides, eg, 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, with a priority of U>C≈A>G, and the target adenosine residue The 3' nearest neighbor of a residue is a nucleotide selected from G, C, A, and U, with a priority of 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 a G. In some embodiments, the 3' nearest neighbor of the target adenosine residue is C.

In some embodiments, the target adenosine residue is a three-alpha base motif in a target RNA (eg, a target RNA encoding the mutant Usher2A protein) selected from the group consisting of: UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA and GAU. In certain 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 trigonyl motif The G in is directly opposite 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 of editing a plurality of target RNAs (e.g., at least about 2, 3, 4, 5, 10, 20, 50, 100, 1000, or more) in a host cell, the method being A plurality of the dRNA or one or more constructs encoding the dRNA is introduced into the host cell.

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 post-mitotic cell. In some embodiments, the host cell is a cell of the central nervous system (CNS), such as a brain cell, eg, a cerebellar cell. In some embodiments, the cells are neural cells. In some embodiments, the nerve cell is a sensory nerve cell. 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 within 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 cells are located in retinal epithelial cells. In some embodiments, the host cell is a retinal cell.

In some embodiments, the ADAR is 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 endogenously expressed by the host cell. In some embodiments, the ADAR is an ADAR endogenously encoded by the host cell, wherein the introduction of the ADAR comprises 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, the ADAR is ADAR1 and/or ADAR2. In some embodiments, the ADAR is one or more ADARs selected from the group consisting of hADAR1, hADAR2, mouse ADAR1, and ADAR2. In some embodiments, the ADAR is ADAR1, such as the p110 isomer of ADAR1 ("ADAR1 ^p110 ") and/or the p150 isomer of ADAR1 ("ADAR1 ^p150 "). In some embodiments, the ADAR is ADAR2. In some embodiments, the ADAR is ADAR2 expressed by a host cell, e.g., ADAR2 expressed by cerebellar cells.

In some embodiments, the ADAR 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 a fusion protein that does not include 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 the MS2 hairpin fused to the complementary RNA sequence in the dRNA.

In some embodiments, the host cell expresses ADAR1 (eg, ADAR1 ^p110 and/or ADAR1 ^p150 ) at high levels, e.g., at least about 10%, 20%, 50%, 100% relative to the protein expression level of beta-tubulin. , any of 2-fold, 3-fold, 5-fold or more. In some embodiments, the host cell expresses ADAR2 at a high level, e.g., at least about 10%, 20%, 50%, 100%, 2-fold, 3-fold, 5-fold, or more relative to beta-tubulin expression levels. any of. In some embodiments, the host cell has a lower ADAR3 expression level, e.g., no more than 5-fold, 3-fold, 2-fold, 100%, 50%, 20%, or less relative to the beta-tubulin expression level. any of.

In certain embodiments, the methods do not induce an immune response, such as an innate immune response. In certain embodiments, the methods do not induce interferon and/or interleukin expression in the host cell. In some embodiments, the methods do not induce expression of IFN-β and/or IL-6 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, biolistic methods, viral particles, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, electroporation, nanoparticles, exocytosis bodies, cellular microvesicles, or gene guns, naked DNA, and artificial virus particles.

Delivery of nucleic acids using RNA- or DNA-based viral systems is highly efficient in targeting the virus to specific cells and transporting the viral payload into the nucleus. In certain embodiments, the method comprises introducing a viral vector (eg, AAV, such as scAAV, or a lentiviral vector) encoding the dRNA into the host cell. For example, the constructs described herein can be any of the viral vectors described in Section 3 "dRNA, Constructs and Libraries" below.

In some embodiments, the method comprises introducing a plasmid encoding the dRNA into the host cell. In some embodiments, the methods comprise electroporation of dRNA (eg, synthetic dRNA) into the host cell. In some embodiments, the method includes transfecting dRNA into a host cell.

In certain embodiments, the editing efficiency of the target RNA (eg, the target RNA encoding the mutant Usher2A protein) is 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, the efficiency of editing is determined by Sanger sequencing. In some embodiments, the efficiency of editing is determined by next-generation sequencing. In some embodiments, the efficiency of editing is determined by assessing the expression of a reporter gene, such as a fluorescent reporter gene (eg, EGFP).

In certain embodiments, the methods have low off-target editing rates. In certain embodiments, the method has an editing efficiency of less than about 1% (e.g., no more than about 0.5%, 0.1%) of non-target A in the target RNA (eg, the target RNA encoding the mutant Usher2A protein) , 0.05%, 0.01%, 0.001% or any of lower). In some embodiments, the method does not edit non-target A in the target RNA. In some embodiments, the method has an editing efficiency of A in a non-target RNA of less than about 0.1% (e.g., no more than any of about 0.05%, 0.01%, 0.005%, 0.001%, 0.0001%, or less). a).

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 may lead to changes in the amino acid sequence encoded by the mRNA. For example, a point mutation can be introduced into the mRNA due to the conversion of "A" to "I", and innate or acquired point mutations in the mRNA can be reversed to produce a wild-type gene product. Amino acid sequencing using methods known in the art can be used to detect any changes in the amino acid residues in the encoded protein. Stop codon modifications can be determined by assessing whether a functional, extended, truncated, full-length and/or wild-type protein is present. 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) may produce a read-through mutation and/or Elongated proteins, or truncated proteins encoded by the target RNA, may be reversed to produce a functional, full-length and/or wild-type protein. 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 splicing encoded in the target RNA that may Alternative splice sites can be reversed to create a functional, correctly folded, full-length and/or wild-type protein. In some embodiments, the present application contemplates editing of innate and acquired genetic changes, for example, missense mutations, premature stop codons, aberrant splicing, or target RNAs encoded by alternative splice sites. Using known methods to assess the function of the protein encoded by the target RNA can reveal whether the RNA editing has the desired effect. Because deamination of adenosine (A) to inosine (I) can correct mutant A at the target position in the mutant RNA encoding the protein, identification of deamination to inosine can provide an assessment of whether a functional protein is present, or is caused by mutant adenosine The presence of disease- or resistance-associated RNA is reversed or partially reversed. Likewise, since deamination of adenosine (A) to inosine (I) may introduce point mutations in the resulting protein, identification of deamination to inosine may provide functional indications for identifying the cause of disease or factors associated with disease.

Effects of target adenosine deamination include, for example, point mutations, premature 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 relevant to disease, whether they are inherited or caused by acquired genetic mutations, or may induce changes in RNA and/or proteins that are involved in the development of drug resistance. Structural and functional changes. Therefore, the dRNA, the construct encoding the dRNA and the RNA editing method of the present application can be used to prevent or treat inherited genetic diseases or disorders or to be related to disease-related diseases by changing the structure and/or function of RNA and/or proteins. Diseases or conditions associated with acquired genetic mutations.

In some embodiments, the target RNA is precursor 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 (such as miRNA, pri-miRNA, pre-miRNA, piRNA, siRNA, snoRNA, snRNA, exRNA or scaRNA) . Deamination of target adenosine involves, 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 function. Enhance. In some embodiments, deamination of a target adenosine in the target RNA changes the expression level of one or more downstream molecules of the target RNA (eg, protein, RNA, and/or metabolites). A change in the expression level of a downstream molecule can be an increase or decrease in the expression level.

Some embodiments of the present application involve multiplex editing of target RNA in a host cell, which is useful for editing different variants or different genes of the target gene in the host cell. In some embodiments, wherein the method comprises 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 modifications in multiple (eg, at least 2, 3, 5, 10, 50, 100, 1000 or more) target RNAs in the host cell. In some embodiments, the methods comprise editing multiple target RNAs in multiple host cell populations. In some embodiments, each host cell population receives a dRNA or dRNAs with a different target RNA than the other host cell populations.

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 an inosine. In some embodiments, the host cell contains a target RNA (eg, a target RNA encoding the mutant Usher2A protein) that has a missense mutation, a premature 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 methods restore the function of the target RNA. III . dRNA , constructs and libraries

The application further provides dRNAs, constructs encoding the dRNAs, and libraries comprising a plurality of dRNAs or constructs thereof, which may be used in any of the RNA editing methods or therapeutic methods described herein. This is intended to mean that any features and parameters of the dRNAs or constructs described herein may be combined with one another as if each combination were described individually.

In one aspect, the application provides a dRNA for editing a target RNA, 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 adenosine deamination that acts on the RNA. enzyme (ADAR) to deaminate a target adenosine in a target RNA, wherein the RNA duplex includes one or more mismatch regions relative to the target RNA, and wherein the dRNA includes a linker nucleic acid sequence located at Flanks the terminus of a targeting 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 dRNA described in Section 3 ("dRNA, Constructs and Libraries") below. 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 method uses a construct comprising a nucleic acid sequence encoding the dRNA. The construct may be any of the constructs described in Section 3 below.

In some embodiments, dRNA for editing a target RNA (eg, a target RNA encoding the mutant Usher2A protein) is provided, comprising a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein said An RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in a target RNA, wherein the RNA duplex comprises: (a) relative to the target RNA sequence a first mismatch region 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 at the 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, and wherein the linker nucleic acid sequence does not hybridize to the target RNA , and basically does not form secondary structure. In some embodiments, the RNA duplex comprises a first mismatch region relative to the target RNA sequence located: 5 nucleotides to 25 nucleotides upstream of the target adenosine, or 5 nucleotides to 15 nucleotides upstream of 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 located: 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 mismatch region is 1-50 nucleotides in length; and/or the second mismatch 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, dRNA for editing a target RNA (eg, a target RNA encoding the mutant Usher2A protein) is provided, comprising a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein said An RNA duplex is capable of recruiting RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in a target RNA, wherein the RNA duplex comprises: (a) relative to the target RNA sequence a first mismatch region 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 at the 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, the first mismatch region is 1-50 nucleotides in length; and/or the second mismatch 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 in the targeting RNA (mismatch); and/or (b) one or more nucleosides in the targeting RNA Deletion of acid; and/or (c) insertion of one or more nucleotides in the targeted RNA. In some embodiments, the second mismatch region comprises: (a) one or more non-complementary nucleotides in the targeting RNA (mismatch); and/or (b) one or more nucleosides in the targeting RNA Deletion of acid; and/or (c) insertion of one or more nucleotides in the targeted RNA. In some embodiments, the first mismatch region comprises: (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 targeted RNA. In some embodiments, the second mismatch region comprises: (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 targeted RNA. In some embodiments, non-complementary nucleotides in the targeting RNA cause bubbling in the RNA duplex. In some embodiments, deletions of nucleotides in the targeting RNA result in bulges in the RNA duplex. In some embodiments, insertion of nucleotides in the targeting RNA results in a bulge in the RNA duplex. In some embodiments, a contiguous set of non-complementary nucleotides in the targeting RNA causes bubbling in the RNA duplex. In some embodiments, deletion of a contiguous set of nucleotides in the targeting RNA results in a bulge in the RNA duplex. In some embodiments, insertion of a contiguous set of nucleotides in the targeting RNA results in a bulge in the RNA duplex.

In some embodiments, the first mismatch region is 1-50 nucleotides in length. In some embodiments, the second mismatch region is 1-50 nucleotides in length. In some embodiments, the first mismatch region is any of about 1-10, 10-20, 20-30, 30-40, or 40-50 nucleotides in length. In some embodiments, the length of the first mismatch 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 mismatch region is any of about 1-10, 10-20, 20-30, 30-40, or 40-50 nucleotides in length. In some embodiments, the length of the second mismatch region is approximately 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 mismatch region is 1-10 nucleotides in length; and/or the second mismatch region is 1-10 nucleotides in length. In some embodiments, the first mismatch region includes 1-10 consecutive non-complementary nucleotides in the targeting RNA. In some embodiments, the first mismatch region comprises a deletion of 1-10 contiguous nucleotides in the targeting RNA. In some embodiments, the second mismatch region contains 1-10 consecutive non-complementary nucleotides in the targeting RNA. In some embodiments, the second mismatch region comprises a deletion of 1-10 contiguous nucleotides in the targeting RNA.

In some embodiments, the first mismatch region is 4 nucleotides in length; and/or the second mismatch region is 4 nucleotides in length. In some embodiments, the first mismatch region includes 4 consecutive non-complementary nucleotides in the targeting RNA. In some embodiments, the first mismatch region comprises a deletion of 4 consecutive nucleotides in the targeting RNA. In some embodiments, the second mismatch region includes 4 consecutive non-complementary nucleotides in the targeting RNA. In some embodiments, the second mismatch region comprises a deletion of 4 consecutive nucleotides in 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, the target RNA encodes a mutant Usher2A protein according to any of the dRNAs described herein. In some embodiments, mutant Usher2A proteins comprise missense mutations, nonsense mutations, and/or frameshift mutations. In some embodiments, the mutant Usher2A protein is a truncated Usher2A protein. In some embodiments, the target RNA encodes a mutant Usher2A protein comprising the Trp3955Ter mutation. In some embodiments, the target RNA encoding the mutant Usher2A contains a G to A mutation compared to the target RNA encoding the wild-type Usher2A protein. In some embodiments, the target RNA encoding the mutant Usher2A protein includes a G>A mutation at position 11864 compared to the target RNA encoding the wild-type Usher2A protein.

In some embodiments, 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 adenosine deamination that acts on the RNA. enzyme (ADAR) to deaminate a target adenosine in a target RNA, wherein the target RNA encodes the mutant Usher2A protein, wherein the dRNA comprises a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein the RNA duplex comprises: (a) a first mismatch region located 20 nucleotides to 40 nucleotides upstream of the target adenosine relative to the target RNA sequence; and/or (b) a second mismatch region located 25 nucleotides to 45 nucleotides downstream of the target adenosine relative to the target RNA sequence, and wherein the dRNA comprises a linker nucleic acid sequence, said Linker nucleic acid sequences flank the ends of the targeting RNA sequence, wherein the linker nucleic acid sequences do not hybridize to the target RNA and do not substantially form secondary structure. In some embodiments, the target RNA encodes a protein comprising a Trp3955Ter mutant Usher2A. In some embodiments, the target RNA encoding the mutant Usher2A protein comprises a G to A mutation compared to the target RNA encoding the wild-type Usher2A protein. In some embodiments, the target RNA encoding the mutant Usher2A protein includes a G>A mutation at position 11864 compared to the target RNA encoding the wild-type Usher2A protein. In some embodiments, the target adenosine is located at position 101 compared to SEQ ID NO. 3.

In some embodiments of the dRNA according to any one of the invention, wherein the target RNA encodes the mutant Usher2A protein, the RNA duplex further comprises a third mismatch region relative to the target RNA. In some embodiments, a third mismatch region is located between the first mismatch region and the second mismatch region relative to the target RNA. In some embodiments, the third mismatch region comprises 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 comprises 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 one selected from: A, AA, U , C, CC, G, GG or a nucleotide deletion ("X") opposite the 7th and 8th nucleotides downstream of the target adenosine in the target RNA.

In some embodiments of the dRNA according to any one of the invention, wherein the target RNA encodes the mutant Usher2A protein, the RNA duplex comprises: (a) a first sequence relative to the target RNA a mismatch region located 27 nucleotides to 30 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence located below the target adenosine Swim 31 nucleotides to 43 nucleotides. In some embodiments, the second mismatch region is located 32 nucleotides to 35 nucleotides downstream of the target adenosine relative to the target RNA sequence. 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 mismatch region relative to the target RNA sequence is located 40 nucleotides to 43 nucleotides downstream of the target adenosine. In some embodiments, the first mismatch region is 4 nucleotides in length. In some embodiments, the first mismatch 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 mismatch region comprises a deletion of four consecutive nucleotides in the targeting RNA.

In some embodiments of the dRNA according to any one of the invention, wherein the target RNA encodes the mutant Usher2A protein, the RNA duplex comprises: (a) a first sequence relative to the target RNA a mismatch region located 21 nucleotides to 30 nucleotides upstream of the target adenosine; and (b) a second mismatch region relative to the target RNA sequence located below the target adenosine Swim 36 nucleotides to 43 nucleotides. In some embodiments, the second mismatch region is located 36 nucleotides to 39 nucleotides downstream of the target adenosine relative to the target RNA sequence. In some embodiments, the second mismatch region is located 40 nucleotides to 43 nucleotides downstream of the target adenosine relative to the target RNA sequence. In some embodiments, the first mismatch region is 10 nucleotides in length. In some embodiments, the first mismatch region comprises a deletion of ten consecutive nucleotides in the targeting RNA. In some embodiments, the second mismatch region is 4 nucleotides in length. In some embodiments, the second mismatch region comprises a deletion of four consecutive nucleotides in the targeting RNA.

In some embodiments, the dRNA is circular. In some embodiments, the 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 can be cyclized, the cyclized dRNA comprises any one of the cyclization sequences in Table A (non-bold font of any of SEQ ID NOs. 15-314 sequence) encoding nucleotides. In some embodiments, wherein the dRNA is circular or can be circularized, the circularized dRNA comprises a non-font sequence consisting of any one of the circularization sequences in Table A (any one of SEQ ID NOs. 15-314 A nucleotide encoded by a variant of the bold sequence), wherein the variant differs from the non-bold sequence by no more than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide Any of the acids. In some embodiments, the targeting RNA sequence is encoded by any of the targeting sequences in Table A (the lowercase letter sequence 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 lowercase letter sequence of any of SEQ ID NO. 15-314), wherein the variant is identical to the parent The sequences differ by no more than any of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide.

In some embodiments according to any 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 , any of 300, 350, 400, 450 or 500nt, 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 Any integer between 40nt-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 any of: 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the linker nucleic acid sequences comprise adenosine or cytidine. In some embodiments, about 50% to 60%, 60% to 70%, 70% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 99% of the linker Either of the 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, the linker nucleic acid sequence contains adenosine at least about any of: 50%, 60%, 70%, 80%, 85%, or 90%. In some embodiments, the linker nucleic acid sequence comprises adenosine at about any of: 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% % to 85%, 85% to 90% or 90% to 95%.

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 that is recognized by a 3' catalytic type I intronic fragment targeting the 5' end of the RNA sequence, and a 3' exon sequence that is recognized by a 5' end of the targeting RNA sequence. '5' exon sequence recognized by the catalytic type I intronic fragment. In some embodiments, the dRNA further comprises a 3' linker sequence and a 5' linker sequence. In some embodiments, the double-stranded RNA contains a bulge at 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 about 100 to about 200 nt in length. In some embodiments, the targeting RNA sequence in the dRNA is about 150 to about 220 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 targeting RNA sequence in the dRNA is about 170 nt (eg, 171 nt) in length. 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, 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 adenosine deamination that acts on the RNA. enzyme (ADAR) to deaminate a target adenosine in a target RNA, wherein the target RNA encodes the mutant Usher2A protein, wherein the dRNA comprises a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein said RNA duplex comprises: (a) a first mismatch region comprised between 21 nucleotides and 30 nucleotides upstream of a target adenosine, relative to said target RNA sequence in the targeting RNA Deletion of 10 consecutive nucleotides; and (b) a second mismatched region comprised from 36 nucleotides to 39 nucleotides downstream of the target adenosine, or 40 nucleotides to 43 nucleotides downstream At acid, the deletion of 4 consecutive nucleotides in the target RNA relative to the target RNA sequence. and (c) a third mismatched region relative to the target RNA, located between the first mismatched region and the second mismatched region, wherein the targeting RNA sequence of the third mismatched region includes any one selected from the following Item: A, AA, U, C, CC, G, GG or a nucleotide deletion ("X") opposite the 7th and 8th nucleotides downstream of the target adenosine in the target RNA, 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 secondary structure, and wherein the targeting RNA sequence in the dRNA Length approximately 150 to approximately 220nt. 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 can be circularized. In some embodiments, the targeting RNA encodes a Trp3955Ter mutant Usher2A protein.

In some embodiments, 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 adenosine deamination that acts on the RNA. enzyme (ADAR) to deaminate a target adenosine in a target RNA, wherein the target RNA encodes the mutant Usher2A protein, wherein the dRNA comprises a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, wherein said RNA duplex comprises: (a) a first mismatch region comprised between 21 nucleotides and 30 nucleotides upstream of a target adenosine, relative to said target RNA sequence in the targeting RNA Deletion of 10 consecutive nucleotides; and (b) a second mismatched region comprised from 36 nucleotides to 39 nucleotides downstream of the target adenosine, or 40 nucleotides to 43 nucleotides downstream At acid, the deletion of 4 consecutive nucleotides in the target RNA relative to the target RNA sequence. and (c) a third mismatched region relative to the target RNA, located between the first mismatched region and the second mismatched region, wherein the targeting RNA sequence of the third mismatched region includes any one selected from the following Item: A, AA, U, C, CC, G, GG or a nucleotide deletion ("X") opposite the 7th and 8th nucleotides downstream of the target adenosine in the target RNA, 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 secondary structure, and wherein the targeting RNA sequence in the dRNA Length approximately 150 to approximately 220nt. 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 can be circularized. In some embodiments, the targeting RNA encodes a Trp3955Ter mutant Usher2A protein.

In one aspect, the application provides a dRNA for editing a target RNA (such as a target RNA encoding the mutant Usher2A protein), comprising a targeting RNA sequence capable of hybridizing with the target RNA to form a double-stranded RNA, wherein the double-stranded RNA The RNA contains bulges made of non-target adenosine in the target RNA. In some embodiments, the targeting RNA sequence has one or more uridine residues opposite one or more non-target adenosines deleted in the sequence complementary to the targeting RNA. In some embodiments, the dRNA is linear RNA. In some embodiments, the dRNA is a 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 about 5 nt to about 500 nt in length, such as about 50 nt to 200 nt in length. In some embodiments, the linker nucleic acid sequence comprises a polyadenosine (polyA), polyguanosine (polyG), or polycytidine (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 a 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 dRNA or a plurality of constructs described herein.

In one aspect, the application provides a composition or host cell comprising any of the deaminase recruiting RNAs or 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 such that the targeting RNA sequence hybridizes to the target RNA. In some embodiments, the targeting RNA sequence is at least about any one of 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, or 99%, or more Complementarity is to any of at least about 20, 40, 60, 80, 100, 150, 200, or more contiguous nucleotides in the target RNA. In some embodiments, the dsRNA formed by hybridization between the targeting RNA sequence and the target RNA has one or more (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) Non-Watson-Crick base pair (i.e. mismatch).

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

Unpaired nucleotides in dsRNA create bulges. In some embodiments, the dsRNA formed by hybridizing the target RNA to the dRNA contains protuberances containing non-target adenosine in the target RNA. The bulges in the dsRNA formed when the dRNA hybridizes to the target RNA contain non-target adenosines from the target RNA. The bulge may be a mononucleotide bulge, i.e., containing an unpaired non-target adenosine, or a polynucleotide bulge, i.e., containing additional unpaired or mismatched nucleotides, located on an unpaired, non-target adenosine. Glycoside flanking. In some embodiments, the bulge may comprise more than one (e.g., 2, 3, 4, 5 or more) unpaired nucleotides in the target RNA, i.e., the bulge is composed of unpaired nucleotides, directly Located on the 5' and/or 3' flank of non-target adenosine residues. In some embodiments, the bulge may comprise one or more (e.g., 2, 3, 4, 5, or more) unpaired nucleotides located directly 5' and/or 3' of the non-target adenosine residue. flank. In some embodiments, the bulge includes unpaired non-target adenosine, one or more unpaired nucleotides flanking the 5' and/or 3' of the non-target adenosine residue, and one or more unpaired nucleotides. The paired nucleotides are located on the 5' and/or 3' flank of the non-target adenosine residue. In some embodiments, the protrusions are 1nt, 2nt, 3nt or longer.

In some embodiments, the double-stranded RNA comprises two or more bulges, such as any of 2, 3, 4, 5, 6 or more bulges, wherein each bulge comprises a target RNA of non-target adenosine. In some embodiments, the double-stranded RNA contains a bulge 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 mismatches, e.g., any one of 1, 2, 3, 4, 5, 6, 7 (e.g., the same type or different types of mismatch). In some embodiments, the dsRNA formed by hybridization of dRNA and target RNA contains one or more mismatches, such as 1, 2, 3, 4, 5, 6, 7 mismatches selected from the group consisting of: G-A, C-A, U-C, A-A, G-G, C-C and U-U.

In some embodiments, in addition to the mismatch region, the targeting RNA sequence may further comprise one or more guanosines, such as 1, 2, 3, 4, 5, 6 or more Gs, each of which is directly associated with The non-target adenosine in the target RNA is opposite. In some embodiments, in addition to the mismatch region, the targeting RNA sequence may further comprise 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 comprises a single linker nucleic acid sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence located at the 5' end of the targeting RNA sequence. In some embodiments, the dRNA comprises a linker nucleic acid sequence located at the 3' end of the targeting RNA sequence. In some embodiments, the dRNA includes a first linker nucleic acid sequence located at the 5' end of the targeting RNA sequence, and a second linker nucleic acid sequence located 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' end and the 3' end 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 linker nucleic acid sequence (comprising a first linker nucleic acid sequence and a second linker nucleic acid sequence) is at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 in length , any of 150, 200, 250, 300, 350, 400, 450 or 500nt. In some embodiments, the linker nucleic acid sequence (comprising the first linker nucleic acid sequence and the second linker nucleic acid sequence) is no more than about 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80 in length , any of 70, 60, 50, 40, 30, 20, 10 or 5nt. In some embodiments, the linker nucleic acid sequence (comprising a first linker nucleic acid sequence and a 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, Any of 50-400 or 50-500nt. In some embodiments, the linker nucleic acid sequence (comprising 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 (comprising 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 known in the art, including, for example, RNAfold, can predict the secondary structure of RNA. 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 approximately more than any one of 3, 4, 5, 6, or more bases in length. In some embodiments, the linker nucleic acid sequence does not comprise a complementary region having a length of more than 3, 4, 5, or 6 nucleotides. In some embodiments, the first linker nucleic acid sequence has no region of complementarity relative to the second linker nucleic acid sequence that is more than 3, 4, 5, or 6 nucleotides in length.

The linker nucleic acid sequence (comprising a first linker nucleic acid sequence and a second linker nucleic acid sequence) may be a mono- or di-nucleotide repeating sequence, or a random sequence. In some embodiments, the linker nucleic acid sequence comprises a polyadenosine (polyA), polyguanosine (polyG), or polycytidine (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' end and the 3' end of the circular dRNA sequence.

ADARs (eg, human ADAR enzymes) edit double-stranded RNA (dsRNA) structures with varying specificities, depending on different factors. An important factor is the degree of complementarity of the two strands that make up the dsRNA sequence. Complete complementarity between dRNA and target RNA typically causes the catalytic domain of ADAR to deaminate adenosine in an indiscriminate manner. The specificity and efficiency of ADAR can be altered by introducing mismatches in the dsRNA region. For example, the use of A-C mismatches is best recommended to increase the specificity and efficiency of deamination of adenosine to be edited. The formation of dsRNA between dRNA and its target RNA does not necessarily require complete complementarity, only basic complementarity in the hybridization and formation of dsRNA between dRNA and target RNA. In some embodiments, the dRNA sequence or single-stranded RNA region thereof has at least any one of about 70%, 80%, 85%, 90%, or 95% sequence complementarity to the target RNA when optimally aligned. Optimal alignment can be determined by using any suitable sequence alignment algorithm, of which non-limiting examples include the Smith-Waterman algorithm, the Needleman-Wimsch algorithm, algorithms based on the Burrows-Wheeler transform (eg, Burrows Wheeler Aligner).

The nucleotides adjacent to the target adenosine also affect the specificity and efficiency of deamination. For example, for the purpose of specificity and efficiency of adenosine deamination, the 5' nearest neighbor of the target adenosine to be edited in the target RNA sequence has a priority of U>C≈A>G. In the target RNA sequence to be edited, The 3' nearest neighbor of the target adenosine has a priority of G>C>A≈U. In some embodiments, when the target adenosine in the target RNA can be located in any one of the following three alkyl motifs: UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA and GAU, the specificity and efficiency of the adenosine deamination is higher than that of adenosine with other trisaccharyl motifs. In some embodiments, if the target adenosine to be edited is located in the three-aminoyl motif UAG, UAC, UAA, UAU, CAG, CAC, AAG, AAC or AAA, the deamination efficiency of the adenosine is much higher than that of other Adenosine in the motif. For the same trisaccharide motif, different dRNA designs can also lead to different deamination efficiencies. Taking the three-alkyl motif UAG as an example, in some embodiments, when the dRNA contains 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 using other dRNA sequences. In some embodiments, when the dRNA includes ACC, ACG, or ACU opposite the UAG of the target RNA, the editing efficiency of 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, the target RNA may have one or more adenosines (referred to here as "non-target A") that are not suitable for editing. Regarding these adenosines, it is best to reduce their editing efficiency as much as possible. In some embodiments, provided herein is the introduction of a first mismatch region in the dRNA relative to the target RNA sequence, located 20 nucleotides to 40 nucleotides upstream of the target adenosine. ; and/or a second mismatched region located 25 nucleotides to 45 nucleotides downstream of the target adenosine relative to the target RNA sequence, which results in a bulge in the mismatched region and/or Bubbles, where editing of the target adenosine can be increased and off-target editing of non-target adenosine can be reduced. The dRNA may further comprise one or more unpaired nucleotides and/or one or more mismatched nucleotides directly 5' or 3' flanking the non-target adenosine. As used herein, the term "mismatch" refers to the unpairing of one nucleotide in the first strand of a double-stranded nucleic acid with any nucleotide 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 have a deletion of one or more nucleotides (e.g., U) opposite the first non-target adenosine, and/or to a second directly edited portion of the target RNA. Non-target adenosine is the direct opposite of guanosine.

The level of specificity required and the efficiency with which target RNA sequences are edited may depend on the application. According to the description of this patent application, those skilled in the art will be able to design dRNA that is complementary or substantially complementary to the target RNA sequence according to their needs, and through a series of trial and error methods, obtain the results they want. As used herein, the term "mismatch" may refer to opposing nucleotides in double-stranded RNA (dsRNA) that do not form a complete 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 pairing as an example, a target adenosine residue is to be edited in the target RNA, and the dRNA is designed to contain a C opposite to the A to be edited, resulting in an A-C error in the dsRNA formed by hybridization between the target RNA and dRNA. match. Here, the term "mismatch" may also refer to the deletion of a nucleotide from one strand of a double-stranded RNA (dsRNA), thereby resulting in no nucleotide pairing on the strand opposite 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 targeting RNA sequence in the dRNA comprises a length of at least about 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 110, 120, 130, Any of 140, 150, 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 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- Any one of 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, 105-140, or 105-155 nucleotides. In some embodiments, the targeting RNA sequence in the dRNA is 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 targeting RNA sequence in the dRNA is about 170 nt (eg, 171 nt) in length. 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 a cytidine, adenosine, or uridine directly opposite a target adenosine residue in a target RNA, such as a target RNA encoding the mutant Usher2A protein. In some embodiments, the targeting RNA sequence contains a cytidine mismatch directly opposite a target adenosine residue in the target RNA. In some embodiments, the cytidine mismatch is located at least 5 nucleotides, eg, 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 (eg, 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 (eg, at least 25, 30, 35, or more nucleotides) from the 3' end of the targeting RNA sequence and 5 nucleotides from the 3' end of the targeting RNA sequence 'At least 5 nucleotides (such as at least 10, 15, 20, 25, 30 or more nucleotides) from the end. 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 20 nucleotides (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1) of the center of the targeting sequence in the dRNA. nucleotides).

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

In some embodiments, the target adenosine residue is located in a three-alpha base motif in a target RNA, such as a target RNA encoding the mutant Usher2A protein, selected from the group consisting of: UAG, UAC, UAA, UAU, CAG, CAC, CAA, CAU, AAG, AAC, AAA, AAU, GAG, GAC, GAA and GAU. In certain 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 trigonyl motif The G in is directly opposite 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. dRNA may be completely single-stranded, or may have one or more (such as 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 any of 200, 150, 100, 50, 40, 30, 20, 10, or fewer base pairs. 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) discloses oligonucleotides for editing RNA that include double-stranded regions, and/or stem-loop regions, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the dRNA does not contain a stem loop or double-stranded region. In some embodiments, the dRNA comprises an ADAR recruitment domain. In some embodiments, the dRNA does not comprise an ADAR recruitment domain.

The dRNA may 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 to 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. Chemical modifications can in some embodiments increase the stability and effectiveness of dRNA-facilitated editing. 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 are hereby incorporated in their 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 comprise nucleotides with phosphorothioate linkages. In some embodiments, the dRNA does not comprise any one selected from: 2'-fluoronucleotides, 2'-O-methyl nucleotides, or nucleotides with phosphorothioate linkages. In some embodiments, the dRNA contains one or more chemical modifications. In some embodiments, the dRNA contains one or more chemically modified nucleotides. In some embodiments, the dRNA comprises one or more 2'-fluoronucleotides. In some embodiments, the dRNA contains one or more 2'-O-methyl nucleotides. In some embodiments, the dRNA comprises one or more nucleotides with phosphorothioate linkages. In some embodiments, the dRNA contains 2'-O-methyl and phosphorothioate linkage modifications on only the first three and last three residues. In some embodiments, the dRNA is not an antisense oligonucleotide (ASO).

The dRNA may further comprise one or more other expression elements to promote expression and/or circularization of the dRNA.

In some embodiments, the dRNA further comprises a 3' exon sequence that is recognized by a 3' catalytic type I intronic fragment at the 5' end of the targeting RNA sequence, and a 3' exon sequence that is recognized by the 3' end of the targeting RNA sequence. The 5' catalytic type I intron fragment recognizes the 5' exon sequence. In some embodiments, the type I catalytic intron of the T4 phage Td gene is bisected in a manner to preserve structural elements critical for nuclease folding. Exon fragment 2 is then ligated upstream of exon fragment 1 and the targeting RNA sequence is inserted between the exon-exon junctions (optionally, the 5' and/or 3' ends are flanked by linker nucleic acid sequence).

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

In some embodiments, the dRNA comprises the targeting RNA sequence flanked (directly or indirectly) by 5' and/or 3' linker sequences. In some embodiments, the dRNA comprises a 3' linker sequence. In some embodiments, the dRNA comprises 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% are complementary to each other. In some embodiments, the 3' linker sequence and the 5' linker sequence are completely complementary. In some embodiments, the 5' and/or 3' linker sequences are further flanked by 5'-Twister nuclease and/or 3'-Twister nuclease, respectively.

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 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 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 CTGCCATCAGTCGGCGTGG ACTGTAG. In some embodiments, the 5' linker sequence comprises the sequence AACCATGCCGACT GATGGCAG.

In some embodiments, the dRNA is a circular RNA comprising a linker sequence that directly or indirectly connects the 5' end and the 3' end of the targeting RNA sequence. In some embodiments, the linker sequence includes a 5' linker sequence and a 3' linker sequence, which are linked to each other by a ligase, such as a T4 RNA ligase, such as Rnl1 or Rnl2.

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

In some embodiments, the 3' linker sequence and the 5' linker sequence are each at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, 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 nuclei glycosides. In some embodiments, the 3' linker sequence and the 5' linker sequence are each about 20-30 nucleotides, about 30-40 nucleotides, about 40-50 nucleotides, about 50-60 nucleosides Acid, 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, dRNA is circularized before being introduced into the host cell. In some embodiments, the 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 an ADAR with high affinity, or a nucleotide sequence that binds to an ADAR fused binding partner 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) Structural 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, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the dRNA does not contain a double-stranded portion. In some embodiments, the dRNA does not contain a hairpin, such as an MS2 stem-loop. In some embodiments, the dRNA comprises a hairpin, 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 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 ribonucleic acids (snoRNAs) are small non-coding RNA molecules known to direct the chemical modification of other RNAs, such as ribosomal RNAs, transfer RNAs and snoRNAs. According to their specific secondary structure characteristics, there are two major categories of snoRNA: box C/D and box H/ACA. These two structural features of snoRNA enable them to bind to corresponding RNA-binding proteins (RBPs) and accessory proteins to form functional small ribonucleic acid protein (snoRNP) complexes. Box C/D snoRNA is thought to be related to methylation, while H/A CA box snoRNA is thought to be related to pseudouridylation. Other families of snoRNAs 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 application provides constructs encoding said dRNA and/or ADAR. In some embodiments, a construct (eg, a vector, such as a viral vector) comprising a nucleotide sequence encoding the 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, a construct is provided comprising a first nucleotide sequence encoding the 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 an ADAR. In some embodiments, the vector further comprises a nucleic acid sequence encoding an ADAR3 inhibitor (eg, ADAR3 shRNA or siRNA) and/or an interferon stimulator (eg, IFN-α).

The term "construct" as used herein 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, the 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 the 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 the nucleic acid sequence encoding the dRNA, such that the promoter controls the transcription or expression of the encoding nucleotide sequence. A promoter can be positioned 5' (upstream) of the coding nucleotide sequence it controls. The distance between a promoter and the coding sequence may be approximately the same as the distance between the promoter and the gene it controls in the gene from which the promoter is derived. As is well known in the art, variations in this distance can be tolerated without loss of promoter function. In some embodiments, the construct comprises a 5'UTR and/or 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 a 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, wherein 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 a U6 promoter. In some embodiments. The U6 promoter contains the following nucleic acid sequence gagggcctatttccatgattccttcatatttgcatatacgatacaaggctgttagagataattagaatttgactgtaaacac aaagatattagtacaaaatgtgacgtagaaagataatttcttgggtatttgagtttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttccttggctttatatt gtggaaaggacgaaacaccg.

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, no free ends (such as circular); nucleic acid molecules composed of DNA, RNA or both, and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to 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 cell into which they are introduced (eg, bacterial vectors of bacterial origin of replication and mammalian episomal vectors). Other vectors, such as non-episomal mammalian vectors, are integrated into the host cell's genome when introduced into the host cell and are therefore 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 AAV 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 or analogs. 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 ITR of mouse AAV serotypes. In some embodiments, the AAV ITR is an AAV2 ITR.

In some embodiments, the vector further comprises a 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 the dRNA and a second nucleic acid sequence encoding a complementary sequence to the dRNA, wherein the first nucleic acid sequence may coexist with the second nucleic acid sequence along most or all of its length. Form an intrachain base pair. 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 a mutation that includes a terminal cleavage sequence. In some embodiments, the vector is packaged 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 or Capsids of rAAV2/HboV1 serotypes.

In some embodiments, the construct further comprises a 3' twister nuclease sequence linked to the 3' end of the nucleic acid encoding the dRNA. In some embodiments, the construct further comprises a 5' twister nuclease sequence linked to the 5' end of the nucleic acid sequence encoding the dRNA. In some embodiments, the construct further comprises a 3' twister ribozyme sequence linked to the 3' end of the nucleic acid sequence encoding the dRNA and a 5' twister core linked to the 5' end of the nucleic acid encoding the dRNA. enzyme sequence. In some embodiments, the 3' twister sequence is twisterP3 U2A and the 5' twister sequence is twister P1. In some embodiments, the 5' twister sequence is twister P3 U2A and the 3' twister sequence is twister P1. In some embodiments, the dRNA undergoes autocatalytic cleavage. In some embodiments, the catalyzed dRNA product contains a 5'-hydroxyl group and a 3'-terminal 2',3'-cyclic phosphate. Preparation method

The dRNA described herein can be prepared by any method known in the art, including chemical synthesis and in vitro transcription. Circular dRNA can be prepared by chemical ligation, enzymatic ligation, or nuclease autocatalysis of linear RNA. 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, the linear RNA can be cyclized by chemical cyclization using cyanogen bromide or similar condensing agents. In some embodiments, linear RNA can be cyclized by autocatalysis of a Type I intron consisting of a 5' catalytic Type I intron fragment and a 3' catalytic Type 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 circular polynucleotide) contain chemically reactive groups that, when brought into close proximity, can form between the 5'- and 3'-ends of the molecule. new covalent connections are formed. The 5'-end can contain an NHS ester reactive group, and the 3'-end can contain a 3'-amino-terminal nucleotide, so that in organic solvents, the 3'-end of the linear RNA molecule has a 3'-amino-terminal nucleotide. The nucleotide will perform a nucleophilic attack on the 5'-NHS-ester molecule to form a new 5'-/3'-amide bond.

In some embodiments, circular dRNA can be obtained by nuclease autocatalytic circularization of linear RNA. In some embodiments, linear RNA is circularized in vitro. In some embodiments, cyclization by nuclease autocatalysis comprises (a) autocatalyzing a linear RNA into an activating Type I intron (or 5' and 3' catalytic Type I intron fragments thereof) under conditions to provide a circularized RNA product, and (b) isolating the circularized RNA product to provide circular dRNA.

In some embodiments, the method includes the steps of obtaining linear RNA by first cloning a sequence encoding a linear RNA into a plasmid vector and then linearizing the recombinant plasmid. In some embodiments, recombinant plasmids are linearized by restriction endonuclease digestion. In some embodiments, the recombinant plasmid is linearized by PCR amplification. In some embodiments, the method further comprises 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 comprises purifying the linear RNA transcript. In some embodiments, the linear RNA is purified by gel purification.

In some embodiments, the application provides methods for circularizing linear RNA (eg, purified linear RNA) by nuclease autocatalysis of type I introns. During the splicing process, the 3' hydroxyl group of the guanosine nucleotide participates in the esterification reaction at the 5' splice site. The half with the 5' intron is excised, and the released hydroxyl group at the end of the intermediate participates in a second esterification reaction at the 3' splice site, resulting in cyclization of the middle region and excision of the 3' intron. In some embodiments, the condition to activate autocatalysis of the Type I intron or the 5' and 3' catalytic Type I intron fragments is the addition of GTP and Mg2 ⁺ . In some embodiments, a step is provided to circularize linear RNA by adding GTP and Mg2 ⁺ at 55°C for 15 minutes. In some embodiments, the method further comprises treating with ribonuclease R to digest the linear RNA transcript. In some embodiments, the method further comprises isolating circular dRNA. In some embodiments, the step of isolating the circular dRNA comprises gel purifying the circular dRNA.

In some embodiments, linear RNA can be circularized by using a ligase, such as RNA ligase, to obtain circular dRNA. 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 RNA ligase connects 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 aptamers (eg, splint DNA).

In some embodiments, a DNA or RNA ligase can be used to enzymatically ligate a 5'-phosphorylated nucleic acid molecule (e.g., linear RNA) to the 3'-hydroxyl group of the nucleic acid (e.g., linear nucleic acid) to form a new phosphate diester linkage. In an example reaction, linearized circular RNA was incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, Mass.) at 37°C for 1 hour according to the manufacturer's protocol. Ligation reactions can be performed in the presence of linear nucleic acids capable of simultaneous base pairing with juxtaposed 5' and 3' regions to assist the enzymatic ligation reaction. 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 polynucleotide so that the two ends can be juxtaposed when hybridized to the single-stranded splint. Splint ligases can therefore catalyze the ligation of two juxtaposed ends of a linear polynucleotide to generate a circular polynucleotide. In some embodiments, DNA or RNA ligases can be used for the synthesis of circular dRNA. As a non-limiting example, the ligase may be a ring ligase or a ring ligase. IV. Treatment methods

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, methods are provided for editing a target RNA (e.g., a target RNA encoding the mutant Usher2A protein) in cells of an individual (e.g., a human individual) in vitro, comprising using any of the RNA editing methods described herein. An editing target RNA.

In some embodiments, methods are provided for treating or preventing a disease or disorder in an individual, such as a human individual, comprising editing a target in a cell of the individual that is associated with the disease or disorder using any of the RNA editing methods described herein. RNA, wherein the dRNA comprises a targeting RNA sequence that hybridizes to a target RNA associated with the disease or condition. In some embodiments, the method comprises introducing a dRNA or a construct comprising a nucleic acid encoding the dRNA into an isolated cell outside the body of an individual. In some embodiments, the methods comprise administering to an individual an effective amount of a dRNA or a construct comprising a nucleic acid encoding the dRNA.

In some embodiments, the target RNA is associated with a disease or condition in 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). In some embodiments, the method further comprises obtaining said cells from said individual. In some embodiments, the ADAR is an ADAR endogenously expressed in an isolated cell. In some embodiments, the methods comprise introducing ADAR or a construct comprising a nucleic acid encoding ADAR into an isolated cell. In some embodiments, the methods comprise introducing an ADAR or a construct comprising a nucleic acid encoding an ADAR into a cell of an individual. In some embodiments, the method further comprises culturing the cells with edited RNA. In some embodiments, the method further comprises 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 those associated with mutations (e.g., G to A mutations, e.g., resulting in missense mutations in RNA transcripts, premature stop codons, aberrant splicing, or alternatively spliced G to A mutations) related diseases. 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 disease. In some embodiments, the disease or disorder is a polygenic disease.

In some embodiments, methods of treating cancer associated with a target RNA having a mutation (eg, a G to A mutation) in an individual (eg, a target RNA encoding the mutant Usher2A protein) are provided, comprising using the method described herein. Any of the RNA editing methods edits the target RNA in the cells of an individual.

In some embodiments, a method of alleviating symptoms of Usher syndrome in an individual is provided, comprising editing a target RNA associated with Usher syndrome in a cell of the individual according to any of the editing methods described herein. In some embodiments, methods are provided for alleviating symptoms of Usher syndrome in an individual, comprising editing a target RNA associated with Usher syndrome in a cell of the individual according to use of any dRNA described herein. In some embodiments, the target RNA encoding the mutant Usher2A protein comprises a G to A mutation relative to the target RNA encoding the wild-type Usher2A protein. In some embodiments, the target RNA encoding the mutant Usher2A protein includes a G>A mutation at position 11864 compared to the target RNA encoding the wild-type Usher2A protein. In some embodiments, the target adenosine is located at position 101 compared to SEQ ID NO. 3. In some embodiments, the USH2A gene mutation is NM_206933.2 (USH2A)_c.11864 G>A (p.Trp3955Ter).

In some embodiments according to any of the methods for ameliorating Usher syndrome symptoms described herein, wherein the method or dRNA is used to edit a target RNA encoding the mutant Usher2A protein, the dRNA is introduced into the nerve cells. In some embodiments, the nerve cell is a sensory nerve cell. 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 the vitreous cavity or into adjacent host cells. In some embodiments, dRNA is introduced into host cells located in or adjacent to the subretinal space. In some embodiments, the host cells are located in retinal epithelial cells. In some embodiments, the host cell is a retinal cell. In some embodiments according to any of the methods for ameliorating Usher syndrome symptoms, wherein said method or dRNA is used to edit a target RNA encoding said mutant Usher2A protein, said dRNA is introduced into the subretinal space and/or 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 approximately any one selected from: 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 one of: 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 has 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 low-light vision. In some embodiments, the individual does not suffer from loss of peripheral vision and/or low-light vision. In some embodiments, the individual suffers from mild peripheral vision loss and/or low-light vision loss. In some embodiments, the individual suffers moderate loss in peripheral vision and/or low-light vision. In some embodiments, the individual suffers from severe peripheral vision and/or low-light vision loss. In some embodiments, the individual exhibits progressive loss of cones and/or rods. In some embodiments, the individual does not suffer from cone and/or rod loss. In some embodiments, the individual suffers mild loss of cones and/or rods. In some embodiments, the individual suffers moderate loss of cones and/or rods. In some embodiments, the individual suffers from severe loss of cones and/or rods.

In some embodiments, methods are provided for alleviating symptoms of Usher Syndrome in an individual, 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 gene selected from the group consisting of SEQ ID In the construct of any sequence listed in NO. 15-293 and 317-354. In some embodiments, methods are provided for alleviating symptoms of Usher syndrome in an individual, comprising editing a target RNA associated with Usher syndrome in cells of the individual (comprising using dRNA, wherein the dRNA is selected from the group consisting of Table A ( SEQ ID NO.15-293) or a variant encoding of the sequence of any one of Table B (SEQ ID NO.317-354), wherein the variant does not differ from the parent sequence by more than 10, 9, 8, Any of 7, 6, 5, 4, 3, 2 or 1 nucleotide.

In some embodiments, methods are provided for alleviating symptoms of Usher syndrome in an individual, comprising editing (comprising using dRNA) a target RNA associated with Usher syndrome in a cell of the individual, wherein the dRNA comprises the composition of Table A (SEQ ID NO. 15-293) or the nucleotide encoded by the cyclization sequence of any one of Table B (SEQ ID NO. 317-354). In some embodiments, methods are provided for alleviating symptoms of Usher syndrome in an individual, comprising editing (comprising using dRNA) a target RNA associated with Usher syndrome in a cell of the individual, wherein the dRNA comprises the composition of Table A (SEQ ID NO. 15-293) or a nucleotide encoding a variant of any one of Table B (SEQ ID NO. 317-354), wherein the variant is identical to the sequence in non-bold The difference does not exceed any of 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 can be circularized.

In some embodiments, a method of alleviating 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 polypeptide having the composition of Table A (any one of SEQ ID NO. 15-293 dRNA encoded by the targeting sequence of any one of Table B (SEQ ID NO. 317-354). In some embodiments, methods are provided for alleviating symptoms of Usher syndrome in an individual, comprising editing a target RNA associated with Usher syndrome in a cell of the individual (comprising using a dRNA having a targeting RNA sequence, the sequence being represented by Table A (the lower case sequence of any one of SEQ ID NOs. 15-293) or a variant encoding a targeting sequence of any one of Table B, wherein the variant differs from the parental sequence by no more than 10, 9, Any of 8, 7, 6, 5, 4, 3, 2 or 1 nucleotide. In some embodiments, a method of alleviating symptoms of Usher Syndrome in an individual is provided, wherein the method comprises introducing a dRNA into a cell of the individual, wherein the dRNA comprises any one of Table A (SEQ ID NO. 15-293 Nucleotides encoded by the cyclization sequences in any of Table B (Sequences not in bold) or Table B (SEQ ID NO. 317-354).

In some embodiments according to any of the methods for improving Usher syndrome symptoms described herein, wherein the method or dRNA is used to edit a target RNA encoding the mutant Usher2A protein, and the dRNA is not introduced or Individuals into which the dRNA was introduced showed a reduction in hearing loss compared to corresponding individuals into which the construct encoding the dRNA was introduced. In some embodiments, the hearing loss in which the dRNA or construct encoding the dRNA is introduced is reduced by at least 10%, 20% compared to a corresponding individual in which the dRNA or construct encoding the dRNA is not introduced. , 30%, 40%, 50%, 75%, 100%, 2x, 5x, 10x, 20x, 30x, 40x, 50x, 100x or 1000x. In some embodiments, the individual into whom the dRNA or construct encoding the dRNA was introduced does not exhibit further hearing loss. In some embodiments, the introduction of a corresponding dRNA or a construct encoding a corresponding dRNA that does not include one or more mismatch regions and/or one or more linker nucleic acid sequences is compared to a corresponding individual in which the dRNA is introduced or a construct encoding the corresponding dRNA is introduced. Individuals with the dRNA constructs showed reduced hearing loss. In some embodiments, the individual into whom the dRNA or construct encoding the dRNA is introduced exhibits a reduction in hearing loss of at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 2-fold , 5 times, 10 times, 20 times, 30 times, 40 times, 50 times. 100-fold or 1000-fold compared to the corresponding individual in which the corresponding dRNA or construct encoding the corresponding dRNA is introduced, said construct does not contain one or more mismatch regions and/or one or more linker nucleic acid sequences. 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 according to any of the methods for improving Usher syndrome symptoms described herein, wherein the method or dRNA is used to edit a target RNA encoding the mutant Usher2A protein, and the dRNA is not introduced or Individuals into which the dRNA was introduced showed reduced vision loss compared to corresponding individuals encoding the construct encoding the dRNA. In some embodiments, an individual into which the dRNA or a construct encoding the dRNA is introduced exhibits at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 2x, 5x, 10x, 20x, 30x, 40x, 50x, 100x or 1000x reduction in vision loss. In some embodiments, the individual into whom the dRNA or construct encoding the dRNA has been introduced exhibits no further vision loss. In some embodiments, the introduction of a corresponding dRNA or a construct encoding a corresponding dRNA that does not include one or more mismatch regions and/or one or more linker nucleic acid sequences is compared to a corresponding individual in which the dRNA is introduced or a construct encoding the corresponding dRNA is introduced. Individuals with the dRNA construct showed reduced vision loss. In some embodiments, an individual introduced with the dRNA or a construct encoding the dRNA exhibits reduced vision loss by at least 10%, 20%, 30%, 40%, 50%, 75 %, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold or 1000-fold compared to the corresponding individual in which the corresponding dRNA or a construct encoding the corresponding dRNA was introduced, The construct does not contain one or more mismatch regions and/or one or more linker nucleic acid sequences. In some embodiments, the vision includes low-light vision. In some embodiments, the vision includes peripheral vision. In some embodiments, the visual acuity and visual acuity loss are determined by an optometric evaluation, such as, but not limited to, visual field testing.

In some embodiments according to any of the methods for improving Usher syndrome symptoms described herein, wherein the method or dRNA is used to edit a target RNA encoding the mutant Usher2A protein, and the dRNA is not introduced or Individuals into which the dRNA was introduced showed reduced retinal cell loss compared to corresponding individuals with the construct encoding the dRNA. In some embodiments, retinal cell loss to which the dRNA or construct encoding the dRNA is introduced is reduced by at least 10%, 20% compared to a corresponding individual in which the dRNA or construct encoding the dRNA is not introduced. , 30%, 40%, 50%, 75%, 100%, 2x, 5x, 10x, 20x, 30x, 40x, 50x, 100x or 1000x. In some embodiments, an individual into whom the dRNA or construct encoding the dRNA is introduced does not exhibit further loss of retinal cells. In some embodiments, an individual introduced with the dRNA or a construct encoding the dRNA exhibits a reduction in retinal cell loss of at least 10%, 20%, 30%, 40%, 50%, 75%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold or 1000-fold, compared to the corresponding individual in which the corresponding dRNA or the construct encoding the corresponding dRNA is introduced, the construct is not Contains one or more mismatch regions and/or one or more linker nucleic acid sequences. In some embodiments, the retinal cells comprise rods and/or cones. In some embodiments the vision includes peripheral rods and/or cones.

Generally, the dosage, timing, and route of administration of a composition (eg, dRNA or a construct comprising a nucleic acid encoding the 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, intravenous, 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, ameliorating or alleviating disease status, and alleviating or improving prognosis. For example, an individual is successfully "treated" if one or more of the symptoms associated with cancer are alleviated or eliminated, including but not limited to reducing the proliferation (or destruction) of cancer cells, increasing the killing of cancer cells, mitigating the effects of the disease symptoms, 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 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 modifying RNA of plants or fungi, such as for plants or fungi with endogenously expressed ADAR. The methods described herein can be used to produce genetically engineered plants and fungi with improved properties.

Further provided are any dRNA, constructs, cells with edited RNA and compositions described herein for use in any of the treatment methods described herein, as well as any dRNA, constructs, edited RNAs described herein The cells and compositions are used to make drugs to treat diseases or conditions. V. Compositions, Kits and Articles of Manufacture

Also provided herein are compositions (eg, pharmaceutical compositions) comprising any of the dRNA, constructs, libraries, or host cells comprising edited RNA described herein.

In some embodiments, pharmaceutical compositions are provided comprising any dRNA described herein or a construct encoding the dRNA, and a pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th Edition , Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citric acid and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethylammonium chloride; benzalkonium chloride, benzalkonium chloride; phenol, butyl or benzyl alcohol; alkyl benzoates such as methyl or propyl benzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin Proteins, gelatin or immunoglobulins; hydrophilic polymers such as vinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides and disaccharides and other carbohydrates, 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 Zn - 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 achieved easily by, for example, filtration through a sterile membrane.

Further 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 (eg, a target RNA encoding the mutant Usher2A protein) in a host cell is provided, comprising a dRNA or a construct comprising a nucleic acid encoding the dRNA, wherein the The dRNA comprises the targeting RNA sequence that hybridizes to a targeting RNA associated with the disease or disorder to form an RNA duplex, wherein the double-stranded RNA includes one or more mismatch regions, wherein the RNA duplex Able to recruit RNA-acting adenosine deaminase (ADAR) to deaminate target adenosine in target RNA. In some embodiments, the dRNA is circular. In some embodiments, the dRNA comprises linker nucleic acid sequences flanking the termini of the targeting RNA sequence.

In some embodiments, a kit for editing a target RNA (eg, a target RNA encoding the mutant Usher2A protein) in a host cell is provided, comprising a dRNA or a construct comprising a nucleic acid encoding the dRNA, wherein the The dRNA includes the targeting RNA sequence that hybridizes to a target RNA associated with the disease or disorder to form an RNA duplex, wherein the dRNA includes a linker nucleic acid sequence located at the end of the targeting RNA sequence, wherein the The linker nucleic acid sequence does not substantially form any secondary structure with any portion of the dRNA, wherein the RNA duplex is capable of recruiting an RNA-acting adenosine deaminase (ADAR) to deaminate the 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 a stimulator of interferon or a construct thereof. In some embodiments, the kit further comprises instructions for performing any of the RNA editing methods or treatment methods 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), and the like. Kits optionally provide additional components such as transfection or transduction reagents, cell culture media, buffers, and instructions.

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 (such as 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 IV bag or a vial with a stopper pierceable by a hypodermic needle). The container holding the pharmaceutical composition may be a reusable vial, allowing reconstitution of the formulation for repeated administration (eg, from 2 to 6 administrations). Package insert refers to the instructions usually included in the commercial packaging of therapeutic products that contain information about the indications, usage, dosage, administration, contraindications, and/or warnings of such products. Additionally, the article of manufacture may further comprise 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 further contain other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

Kits or articles of manufacture may contain 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. Methods of editing target adenosine in a target RNA encoding a mutant Usher2A protein, a construct comprising a deaminase recruiting RNA (dRNA) or a nucleic acid encoding said dRNA in a host cell introduce into the host cell; Wherein, the dRNA includes the targeting RNA sequence, and the sequence is capable of hybridizing with the target RNA to form an RNA duplex; Wherein, the RNA duplex can recruit adenosine deaminase (ADAR) that acts on RNA to deaminate target adenosine in the target RNA; Wherein, the RNA duplex contains: (a) The first mismatch region relative to the target RNA sequence is located 5 nucleotides (nt) to 85 nt upstream of the target adenosine; and/or (b) a second mismatch region relative to the target RNA sequence located 20 nt to 85 nt downstream of the target adenosine, and Wherein, the dRNA includes a linker nucleic acid sequence flanking the end of the target RNA sequence, wherein the linker nucleic acid sequence does not hybridize with the target RNA and does not substantially form a secondary structure.

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

Embodiment 3. The 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 in 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) Targeting at least one set of contiguous non-complementary nucleotides (mismatches) in the RNA sequence; and/or (b) Deletion of at least one contiguous set of nucleotides in the targeted RNA sequence; and/or (c) Targeting the insertion of at least one contiguous set of nucleotides in an RNA sequence.

Embodiment 5. The method according to any one of the preceding embodiments, 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 length of the second mismatch region is 1-50 nt, optionally, the length of the second mismatch region is 4 nt.

Embodiment 6. The method 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 includes 1-10 consecutive non-complementary nucleotides in the targeting RNA sequence or 1 in the targeting RNA sequence. -Deletion of 10 consecutive nucleotides; 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 1 in the targeting RNA sequence. -Deletion of 10 consecutive nucleotides.

Embodiment 7. The method according to any one of the preceding embodiments, wherein: (a) The length of the first mismatch region is 4 nt, wherein the first mismatch region includes 4 consecutive non-complementary nucleotides in the targeting RNA sequence or 4 consecutive nuclei in the targeting RNA sequence Deletion of nucleotides; and/or (b) The length of the second mismatch region is 4 nt, wherein the second mismatch region includes 4 consecutive non-complementary nucleotides in the targeting RNA sequence or 4 consecutive nucleotides in the targeting RNA sequence Lack of acid.

Embodiment 8. The method according to any one of the preceding embodiments, wherein: (i) Targeting non-complementary nucleotides in an RNA sequence results in bubbling in the RNA duplex; and/or (ii) targeted deletions of nucleotides in the RNA sequence that result in bulges in the RNA duplex; and/or (iii) Insertion of nucleotides in the targeted RNA sequence results in the appearance of bulges in the RNA duplex.

Embodiment 9. The method according to any one of the preceding embodiments, wherein: (i) Targeting a contiguous set of non-complementary nucleotides in an RNA sequence results in bubbling in the RNA duplex; and/or (ii) The deletion of a contiguous set of nucleotides in the targeted RNA sequence results in the appearance of a bulge in the RNA duplex; and/or (iii) Insertion of a contiguous set of nucleotides in the targeting RNA sequence results in a bulge in the RNA duplex.

Embodiment 10. The method according to any one of the preceding embodiments, wherein the mutant Usher2A protein comprises a missense mutation, a nonsense mutation and/or a frameshift mutation.

Embodiment 11. The method according to any one of the preceding embodiments, wherein the mutant Usher2A protein comprises the Trp3955Ter mutation.

Embodiment 12. The method of any one of the preceding embodiments, wherein the target RNA encoding the mutant Usher2A protein comprises a G to A mutation compared to the target RNA encoding the wild-type Usher2A protein.

Embodiment 13. The method according to any one of the preceding embodiments, wherein the target RNA encoding the mutant Usher2A protein comprises a G>A mutation at position 11864, which mutation is compared to the target RNA encoding the wild-type Usher2A protein. .

Embodiment 14. The method of any one of embodiments 1-13, wherein the RNA duplex further comprises a third mismatch region relative to the target RNA, wherein relative to the target RNA, the The third mismatch region is located between the first mismatch region and the second mismatch region.

Embodiment 15. The 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 the targeting RNA sequence Deletion of one or two nucleotides.

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

Embodiment 17. The method of any one of the preceding embodiments, wherein the targeting RNA comprises an "AA" sequence at the 7th and 8th nucleotides downstream of the target adenosine, in The targeting RNA sequence includes any one selected from the group consisting of: A, AA, U, C, CC, G, GG or a nucleotide deletion ("X") that is the same as the target adenosine downstream of the target RNA. The 7th and 8th nucleotides are opposite.

Embodiment 18. The method according to any one of the preceding embodiments, wherein the RNA duplex comprises (a) a first mismatch region located 27nt to 30nt upstream of the target adenosine relative to the target RNA sequence, and (b) The second mismatch region relative to the target RNA sequence is located 31 nt to 43 nt downstream of the target adenosine.

Embodiment 19. The method 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 length of the first mismatch region is 4nt and the length of the second mismatch region is 4nt; Further optionally, wherein the first mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence, and wherein the second mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence. Lack of acid.

Embodiment 20. The method 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 length of the first mismatch region is 4nt and the length of the second mismatch region is 4nt; Further optionally, wherein the first mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence, and wherein the second mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence. Lack of acid.

Embodiment 21. The method according to any one of the preceding embodiments, wherein the RNA duplex comprises: (a) a first mismatch region located 21 nt to 30 nt upstream of the target adenosine relative to the target RNA sequence; and (b) a second mismatch region relative to the target RNA sequence located 36nt to 39nt downstream of the target adenosine; Optionally, the length of the first mismatching region is 10nt, and the length of the second mismatching region is 4nt; Further optionally, wherein the first mismatch region includes a deletion of 10 consecutive nucleotides in the targeting RNA sequence, and wherein the second mismatch region includes 4 consecutive nucleotides in the targeting RNA sequence Lack of acid.

Embodiment 22. The method according to any one of the preceding embodiments, wherein the RNA duplex comprises: (a) a first mismatch region located 21 nt to 30 nt upstream of the target adenosine relative to the target RNA sequence; and (b) a second mismatch region relative to the target RNA sequence, located 40nt to 43nt downstream of the target adenosine; Optionally, wherein the length of the first mismatch region is 10nt and the length of the second mismatch region is 4nt; Further optionally, wherein the first mismatch region includes a deletion of 10 consecutive nucleotides in the targeting RNA sequence, and wherein the second mismatch region includes 4 consecutive nucleotides in the targeting RNA sequence Lack of acid.

Embodiment 23. The method according to any one of the preceding embodiments, wherein the dRNA is: (i) cyclic; or (ii) Linear and/or capable of being cyclized.

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

Embodiment 25. The 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, wherein 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 any integer between 40nt-50nt.

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

Embodiment 28. The method of any one of the preceding embodiments, wherein at least about any one of: 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the linker nucleic acid sequence Contains adenosine or cytidine; optionally, wherein 100% of the linker nucleic acid sequences comprise adenosine or cytidine.

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

Embodiment 30. The method according to any one of the preceding embodiments, wherein said RNA duplex does not comprise one or more mismatch regions or the dRNA does not comprise a linker nucleic acid sequence compared to a corresponding method. Methods have improved editing levels of target adenosine.

Embodiment 31. The method of any one of the preceding embodiments, wherein the method has a reduced level of one or more non-target adenosine (bystander) edits compared to a corresponding method, wherein the RNA double The strand does not contain one or more mismatch regions, or the dRNA does not contain the linker nucleic acid sequence.

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

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

Embodiment 34. The method of any one of the preceding embodiments, 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.

Embodiment 35. The method of any one of the preceding embodiments, wherein the first linker nucleic acid sequence and the second linker nucleic acid sequence are identical.

Embodiment 36. The method of any one of the preceding embodiments, wherein the first linker nucleic acid sequence and the second linker nucleic acid sequence are different.

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

Embodiment 38. The 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, which can be flanked at the 5' end of the targeting RNA sequence. Recognition of the 3' catalytic type I intron fragment, and 5' exon sequences, which are recognized by the 5' catalytic type I intron 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' linker sequence and a 5' linker sequence.

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

Embodiment 41. The method of any one of the preceding embodiments, wherein the 3' linker sequence and the 5' linker sequence are from about 20 nt to about 75 nt in length.

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

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

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

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

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

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

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

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

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

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

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

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

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

Embodiment 55. The method of any one of the preceding embodiments, wherein the targeting RNA sequence comprises a cytidine, adenosine, or uridine in the targeting RNA that is directly opposite a target adenosine.

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

Embodiment 57. The method of 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 at.

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

Embodiment 59. The method of any one of the preceding embodiments, wherein the target adenosine is located in a UAG trihydroxyl motif, and wherein the targeting RNA sequence comprises a sequence directly associated with A against uridine, cytidine directly against the target adenosine, and cytidine, guanosine, or uridine directly against guanosine in the trisacryl motif.

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

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

Embodiment 62. The method of any one of the preceding embodiments, comprising introducing into the host cell a plurality of dRNAs each directed to a different target RNA or a construct encoding the dRNA.

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

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

Embodiment 65. The method of any one of the preceding embodiments, wherein deamination of a target adenosine in the target RNA results in a missense mutation, a premature stop codon, aberrant splicing, or variable splicing in the target RNA. Splicing, or missense mutations in the target RNA, premature stop codons, aberrant splicing, or reversal of alternative splicing.

Embodiment 66. The method of any one of the preceding embodiments, wherein deamination of a 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 missense mutations, premature stop codons, aberrant splicing or reversal of alternative splicing in the target RNA to produce functional, full-length, correctly folded and/or wild-type proteins.

Embodiment 67. The method of 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. The method of any one of the preceding embodiments, wherein the host cell is a human cell or a mouse cell.

Embodiment 69. Edited RNA produced by a method according to any one of the preceding embodiments or a host cell comprising edited RNA.

Embodiment 70. A method for 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 according to the method of any one of the preceding embodiments.

Embodiment 71. The method of any one of the preceding embodiments, wherein the disease or disorder is an inherited genetic disease or a disease or disorder associated with one or more acquired genetic mutations.

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

Embodiment 73. A method of alleviating one or more symptoms of Usher syndrome in an individual, comprising editing a target RNA associated with Usher syndrome in cells of the individual according to the method of any one of the preceding embodiments.

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

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

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

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

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

Embodiment 79. The method of any one of the preceding embodiments, wherein: (a) an individual into whom said dRNA or a construct encoding said dRNA is introduced exhibits reduced vision loss compared to a corresponding individual into whom said dRNA or construct encoding said dRNA is not introduced; and/or (b) Introducing said dRNA or encoding said dRNA compared to a corresponding individual in which a corresponding dRNA or a construct encoding a corresponding dRNA is introduced which does not comprise one or more mismatch regions and/or one or more linker nucleic acid sequences. Individuals with the construct exhibit reduced vision loss.

Embodiment 80. The method of any one of the preceding embodiments, wherein: (a) an individual into whom the dRNA or construct encoding the dRNA has been introduced exhibits reduced loss of retinal cells compared to a corresponding individual into whom the dRNA or construct encoding the dRNA has not been introduced; and/or (b) Introducing said dRNA or encoding said dRNA compared to a corresponding individual in which a corresponding dRNA or a construct encoding a corresponding dRNA is introduced which does not comprise one or more mismatch regions and/or one or more linker nucleic acid sequences. Individuals with the construct showed reduced loss of retinal cells.

Embodiment 81. dRNA for editing a target RNA encoding the mutant Usher2A protein and comprising a target adenosine, wherein the dRNA comprises a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex, Wherein, the RNA duplex can recruit adenosine deaminase (ADAR) that acts on RNA to deaminate the target adenosine in the target RNA, Wherein, the RNA duplex contains (a) The first mismatch region relative to the target RNA sequence is located 5nt to 85nt upstream of the target adenosine; and/or (b) a second mismatch region relative to the target RNA sequence is located 20 nt to 85 nt downstream of the target adenosine, and Wherein, the dRNA includes a linker nucleic acid sequence flanking the end of the target RNA sequence, wherein the linker nucleic acid sequence does not hybridize with the target RNA and does not substantially form a secondary structure.

Embodiment 82. The dRNA according to any one of the preceding embodiments, wherein: (a) the RNA duplex contains a first mismatch region relative to the target RNA sequence, located 5nt to 25nt upstream of the target adenosine; and/or the RNA duplex contains a region relative to the target RNA sequence; The second mismatch region of the target RNA sequence is located 20nt to 45nt downstream of the target adenosine; or (b) the RNA duplex contains a first mismatch region relative to the target RNA sequence, located 5nt to 15nt upstream of the target adenosine; and/or the RNA duplex contains a region relative to the target RNA sequence. The second mismatch region of the target RNA sequence is located 20nt to 45nt downstream of the target adenosine; or (c) the RNA duplex comprises a first mismatch region relative to the target RNA sequence, located 20 nucleotides to 40 nucleotides upstream of the target adenosine; and/or the RNA duplex The body includes a second mismatch region relative to the target RNA sequence, located 25 nucleotides to 45 nucleotides 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 include: (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 in 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 include (a) Targeting at least one contiguous set of non-complementary nucleotides (mismatches) in the RNA sequence; and/or (b) Deletion of at least one contiguous set of nucleotides in the targeted RNA sequence; and/or (c) Targeting the insertion of at least one contiguous set of nucleotides in an RNA sequence.

Embodiment 85. The dRNA according to any one of the preceding embodiments, 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 length of the second mismatch region is 1-50 nt, optionally, the length of the second mismatch region is 4 nt.

Embodiment 86. The 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 includes 1-10 consecutive non-complementary nucleotides in the targeting RNA sequence or 1 in the targeting RNA sequence. -Deletion of 10 consecutive nucleotides; and/or (b) The length of the second mismatch region is any one of embodiments 81-84, wherein the second mismatch region includes 1-10 consecutive non-complementary nucleotides in the targeting RNA sequence or the Deletion of 1-10 consecutive nucleotides in the target RNA sequence.

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

Embodiment 88. The dRNA according to any one of the preceding embodiments, wherein: (i) Targeting non-complementary nucleotides in an RNA sequence results in bubbling in the RNA duplex; and/or (ii) The deletion of nucleotides in the targeted RNA sequence results in the appearance of bulges in the RNA duplex; and/or (iii) Insertion of nucleotides in the targeted RNA sequence results in bulges in the RNA duplex.

Embodiment 89. The dRNA according to any one of the preceding embodiments, wherein: (i) Targeting a contiguous set of non-complementary nucleotides in an RNA sequence results in bubbling in the RNA duplex; and/or (ii) deletion of a contiguous set of nucleotides in the targeted RNA sequence resulting in a bulge in the RNA duplex; and/or (iii) Insertion of a contiguous set of nucleotides in a targeted RNA sequence results in the appearance of a bulge in the RNA duplex. Embodiment 90. The dRNA of any one of the preceding embodiments, wherein the mutant Usher2A protein comprises a missense mutation, a nonsense mutation and/or a frameshift mutation.

Embodiment 91. The dRNA of any one of the preceding embodiments, wherein the mutant Usher2A protein comprises the Trp3955Ter mutation.

Embodiment 92. The dRNA of any one of the preceding embodiments, wherein the target RNA encoding the mutant Usher2A protein comprises a G to A mutation compared to the target RNA encoding a wild-type Usher2A protein.

Embodiment 93. The dRNA of any one of the preceding embodiments, wherein the target RNA encoding the mutant Usher2A protein comprises a G>A mutation at position 11864 that is compared to the target RNA encoding the wild-type Usher2A protein. .

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

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 the targeting RNA sequence Deletion of one or two nucleotides.

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 7nt and/or 8nt downstream of the target adenosine; optionally , wherein the target RNA contains adenosine at the 7th and/or 8th nucleotide downstream of the target adenosine.

Embodiment 97. The dRNA of any one of the preceding embodiments, wherein the targeting RNA comprises an "AA" sequence 7 and 8 nucleotides downstream of the target adenosine, wherein The targeting RNA sequence includes any one selected from the group consisting of: A, AA, U, C, CC, G, GG or a nucleotide deletion ("X") that is the same as the target adenosine downstream of the target RNA. The 7th and 8th nucleotides are opposite.

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

Embodiment 99. The 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 length of the first mismatch region is 4nt and the length of the second mismatch region is 4nt; Further optionally, wherein the first mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence, and wherein the second mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence. Lack of acid.

Embodiment 100. The 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, Alternatively, wherein the length of the first mismatch region is 4nt and the length of the second mismatch region is 4nt, Further optionally, the first mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence, and the second mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence. Missing.

Embodiment 101. The dRNA of any one of the preceding embodiments, wherein the RNA duplex comprises: (a) located 21 nt to 30 nt upstream of the target adenosine relative to the first mismatch region of the target RNA sequence; and (b) a second mismatch region relative to the target RNA sequence, located 36nt to 39nt downstream of the target adenosine, Optionally, wherein the length of the first mismatch region is 10nt and the length of the second mismatch region is 4nt; Further optionally, wherein the first mismatch region includes a deletion of 10 consecutive nucleotides in the targeting RNA sequence, and wherein the second mismatch region includes 4 consecutive nucleotides in the targeting RNA sequence Lack of acid.

Embodiment 102. The dRNA of any one of the preceding embodiments, wherein the RNA duplex comprises: (a) a first mismatch region located 21 nt to 30 nt upstream of the target adenosine relative to the target RNA sequence; and (b) a second mismatch region relative to the target RNA sequence, located 40nt to 43nt downstream of the target adenosine; Optionally, wherein the length of the first mismatch region is 10nt and the length of the second mismatch region is 4nt; Further optionally, wherein the first mismatch region includes a deletion of 10 consecutive nucleotides in the targeting RNA sequence, and wherein the second mismatch region includes 4 consecutive nucleotides in the targeting RNA sequence Lack of acid.

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

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 stem-loop structures.

Embodiment 105. The dRNA of any one of the preceding embodiments, wherein the linker nucleic acid sequence is from about 5 nt to about 500 nt in length.

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, wherein 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 any integer between 40nt-50nt.

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

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

Embodiment 109. The dRNA of any one of the preceding embodiments, wherein at least about 50% of the linker nucleic acid sequence comprises adenosine.

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

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

Embodiment 112. A host cell comprising a dRNA according to any one of the preceding embodiments or a construct according to any one of the preceding embodiments.

Embodiment 113. A kit comprising a dRNA according to any one of the preceding embodiments or a construct according to any one of the preceding embodiments, and for editing in a host cell a mutation encoding a target adenosine. Specification of target RNA for Usher2A protein.

Example

The following examples are purely for illustrative purposes and should not be construed as limiting this application in any way. The following exemplary embodiments and examples, as well as the detailed description, are provided by way of illustration and not by way of limitation.

used for Usher2A Editorial Materials and Methods

Plasmid construction

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

For the construct of genetically encoded expression circular arRNA, we first constructed a cloning vector based on the PackGene vector, which contained Twister P3 U2A, 5' linker sequence, 3' linker sequence, and Twister P148. (See Table A). The sequence of arRNA was then synthesized and cloned into an autocatalytic circular RNA expression vector via the Golden Gate method.

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

To investigate whether adapters could be used to further improve editing efficiency, spacer sequences and/or adapter sequences were flanked by the arRNA sequence and then cloned into genetically encoded vectors expressing circular arRNA using the Golden Gate method.

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

Specifically, the DNA sequence (NNNNNNNNN) shown in Table A was first synthesized in vitro and incorporated into the vector, followed by MiuI and KpnI digestion, and then ligated to the plasmid backbone shown in SEQ ID NO.2. plasmid backbone acgcgtgagggcctatttccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtatttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgattt cttggctttatatatcttgtggaaaggacgaaacaccg NNNNNNNNNN tttttttggtacc (SEQ ID NO.2)

ring RNA In vitro production and purification

According to 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," is described in Nucleic Acids Research 48, e54-e54 (2020) to produce circular RNA. Briefly, circular RNA precursors were synthesized by in vitro transcription (IVT) from linearized circular RNA plasmid templates 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 circularization, add T4 Rnl 1 (New England Biolabs, #M0239L) or T4 Rnl 2 (New England Biolabs, #M0204L) to the linear circular RNA precursor and incubate overnight at 37°C after DNase I digestion. For type 1 autocatalytic cyclization, after DNase I digestion, GTP at a final concentration of 2 mM was added to the reaction, and the reaction was incubated at 55°C for 15 min to catalyze cyclization of circRNA. Then, the circularized circular RNA 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 minutes at 37°C to enrich for circular RNA. RNA treated with RNase R was column purified.

To further enrich circRNA, purified RNase R-treated circRNA was purified in RNase-free TE buffer using high performance liquid chromatography (Agilent HPLC1260) 4.6 × 300 mm exclusion column (Sepax Technologies, #215980P-4630, Particle size 5 μm, pore size 2000 Å) was analyzed. Circular RNA-enriched fractions were collected and then subjected to column purification (New England Biolabs, #T2040L). To further reduce the immunogenicity of purified circRNA, circRNA was heated at 65°C for 3 min, cooled on ice, and then treated with Fast CIP Phosphatase (New England Biolabs, #M0525S). Finally, the circular RNA was column purified and concentrated using an RNA cleaning and concentration kit (ZYMO, #R1018).

Construction of dual fluorescent reporter plasmids

The DNA encoding mCherry and EGFP (the first codon ATG of EGFP was deleted) was amplified by PCR, the dual fluorescent reporter was cloned, and the 3×GS adapter and targeting DNA sequence were added through primers during the PCR process. The PCR product was then cleaved and ligated with type II restriction enzyme BsmB1 (Thermo) and T4 DNA ligase (NEB), and then inserted into the pLenti backbone.

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

Lentiviral packaging and reporter cell line construction

The mutated 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, and 72 hours later, mCherry-positive cells were sorted by FACS, cultured, and then subjected to clonal selection after limiting dilution to generate a clonal cell line stably expressing the dual fluorescent reporter system with an EGFP background Low.

To generate a stable HEK293T reporter cell line, the mutated USH2A dual reporter construct was co-transfected into HEK293T cells with two viral packaging plasmids, pR8.74 and pVSVG. After 72 hours, the 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 clonal selection was performed after limiting dilution to generate a clonal cell line stably expressing the dual-fluorescent reporter system that has no detectable EGFP background.

Mammalian cell lines and cell culture

The HEK293T cell line was obtained from C. Zhang's laboratory (Peking University) and cultured in Dulbecco's Modified Eagle Medium (Hyclone SH30243.01) and 10% fetal calf serum (Vistech SE100-011) supplemented with 1% penicillin-streptomycin , using 5% CO ₂ , 37℃.

Plasmid transfection, FACS Analysis and next-generation sequencing (NGS)

To test 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 was seeded in a 12-well plate (15,000 cells/well) (recorded as 0 hours). 24 hours after seeding, 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 seeding the cells (48 hours after transfection), cells in each well were separated with trypsin (Invitrogen 25300054), and one-sixth of the separated cells were used for flow cytometry analysis of the fluorescence intensity of mCherry and GFP. All remaining isolated 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 ggagtgagtacggtgtgcTGAATTTATGGAATGAAGGAGACCCT (SEQ ID NO. 376) and gagttggatgctggatggACGTCACCGCATGTTAGAAGACT (SEQ ID NO. 377) for PCR amplification (final primer concentration 0.2 μM, NEB M0492L, annealing at 63°C, 35 cycles) , the PCR products were sent for sequencing, using the NGS sequencing platform of the Rice Research Institute of the Chinese Academy of Sciences (for the sequencing process, please refer to 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 aptamer sequences, and reads on sequences containing polyG. Subsequently, the barcode corresponding to the obtained high-quality sequencing data was divided into each sample, and the BWA (v0.7.17-r1188) software was used to compare it with the amplified target region sequence (see below for the sequence), and the barcode was analyzed through SAM tools ( v1.9) Format conversion to generate BAM files. The information obtained is statistically compared, reordered and indexed. 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 both the control group and the treatment group samples , using "(average mutation frequency other than A->G mutations) + 3SD" as the threshold, the read length whose A->G mutation frequency value exceeds the threshold at the editing site is regarded as the true frequency of the target A to G mutation.

Sequence of amplified target region:

ggagtgagtacggtgtgcTGAATTTATGGATGAAGGAGACCCTgaggcctttcacactctacgaatcgggtcagcctgtaactccaagggttcagtggagtctgtAgtcattaacacaaactctggaagctcaagattttccagctccttgggctcaagccacggctcattcagttgattggacaaagccaGCGGCCGCTGAGGGCAGG AAGTCTAACATGCGGTGACGTccatagcatccaactc ( A indicates on-target editing) (SEQ ID NO. 378).

Example 1. Target Usher2A 11864 mutated ring Leaper RNA design

To show the effectiveness of the 293T reporter cell line containing the Trp3955Ter mutant Usher2A (NM_206933.2(USH2A)_c. G>A at position 11864), this reporter cell line was assayed for 151 nt linear arRNA using Lipofectamine 3000 as described above. (Linear-151, see Table A) and circular arRNA of the same length (circular-151, see Table A) were transfected. Using the NGS protocol described above, on- and off-target editing facilitated by linear-151 was compared to that using cyclic-151.

As shown in Figure 2, the editing efficiency of the circular arRNA (circular-151) on the target adenosine (denoted as position 0) is significantly higher than that of the corresponding linear arRNA (linear-151).

Furthermore, linear and circular arRNAs with targeting RNA sequences of different lengths (specifically, linear -51, -61, -71, -81, -91, -101, -111, -121, -131, - 141, -151 and cyclization -51, -61, -71, -81, -91, -101, -111, -121, -131, -141, -151, -161, -171, -181, - 191, -201, -211, -221, as shown in Table A) were transfected with the mutated USH2A dual reporter 293T cell line using Lipofectamine 3000 as described above. As described above, the efficiency of on-target editing was measured by the average fluorescence intensity of GFP.

As shown in Figure 3, circular arRNA composed of targeting RNA sequences with a length of 121 to 151 nucleotides showed higher editing efficiency, which can be obtained from the higher increase in the average fluorescence intensity of GFP Prove. The gene editing efficiency of circular arRNA can be further improved by using circular arRNAs with longer targeting RNA sequences (e.g., 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 are significant off-target editing in the upstream and downstream regions of the target adenosine. Figure 4 further depicts various non-target adenosines in USH2A mRNA.

To investigate whether on-target editing can be reduced and/or increased by mismatching or deletion of the targeting RNA sequence, a circular arRNA (USHER-171) with a 171 nt targeting RNA sequence downstream of the editing site (+) or One or more sites in the upstream (-) region opposite the targeting RNA (+31, +35, +39, or -26, -30, -34 as described in the table below) are further modified.

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

Mismatches and deletions for improved editing efficiency

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

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

As shown in Figure 7, although relative to the downstream (+) or upstream (-) region of the editing site, the mismatch or deletion of one or more sites of the targeting RNA sequence increases the target adenosine ( 0 bit) editing efficiency, but there is no significant increase in off-target editing compared with USHER-171.

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

Additional mismatches and deletions for editing efficiency

To investigate whether off-target editing could be further reduced and/or whether on-target editing could be further enhanced by targeting mismatches or deletions in the RNA sequence, a pair of genes with -26/+31, -26/+35 and -26+39 4bp The deleted USHER-171 arRNA has one or more additional sites in the downstream (+) or upstream (-) region of the editing site opposite the targeting RNA (+d7/8, or - as described in the table below) 21/22del, -21x) for further modification.

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

Figure 8A shows additional mismatches or deletions on USHER-171 arRNA with -26/+31, -26/+35 and -26+39 4bp deletions (upstream relative to the target adenosine of the target RNA) An exemplary design (see also Table A). Figure 8B shows 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. Exemplary design (see also Table A). Transfection of the arRNA with the mutant USH2A dual reporter cell line was performed 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 facilitated by the arRNA was determined using the NGS protocol described above.

As shown in Figure 9, relative to the downstream (+) or upstream (-) region of the editing site, the mismatch or deletion of one or more sites in the targeting RNA sequence enhances the editing of the target adenosine. efficiency, manifested by a higher increase in the average fluorescence intensity of GFP. In particular, deletion of 10 consecutive nucleotides (-26x-21x) in the target RNA sequence relative to 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 one or more sites in the targeting RNA sequence increase the target adenosine relative to the downstream (+) or upstream (-) region of the editing site (0 bit) editing efficiency, but there is no significant increase in off-target editing compared to USHER-171. In particular, deletion of 10 consecutive nucleotides (-26x-21x) in the target 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 exhibited significantly increased editing efficiency of target adenosine while exhibiting comparable or decreased editing efficiency compared to USHER-171 off-target editing efficiency.

Example 3. Use flexible linkers to reduce off-target editing

To investigate whether off-target editing could be reduced by using linkers that do not hybridize to the target RNA and do not form secondary structures, arRNAs were placed 5' ("left" flexible linker, or L-flexible linker) or 3' of the targeting RNA sequence. ("Right" flexlinker, or R-flexlinker) flank the flexlinker (10-nt, 20-nt, or 30-nt length) as shown in Figure 11. (Related dRNA sequences are shown in Table A).

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

Figure 12 shows that arRNA consisting of a 10-nt length L-flexible linker and a 30-nt length R-flexible linker exhibited similar on-target editing, as reflected by a comparable increase in the average fluorescence intensity of GFP.

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

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

flexible linker sequence

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, for arRNA (US+35x-26x-21x and US+39x-26x -21x), further modified by adding flanking flexible linker sequences (from 10nt to 50nt) to the targeting RNA sequence or by replacing the ends of the targeting RNA sequence with flanking flexible linker sequences (from 10nt to 50nt).

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

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

As shown in Figure 16, after adding the flexible linker sequence, the off-target editing of arRNA with mismatched regions (US+35x-26x-21x and US+39x-26x-21x) is significantly reduced, and it has a longer flexible linker sequence. Yes, this reduction effect seems to be more obvious, with longer flexible linker sequences (30nt, 40nt, 50nt), which can be significantly removed [except for the 7th and 8th nucleotides downstream of the target editing site (position 0) of non-target adenosine] nearly all detectable off-target edits.

Solving the problem of residual off-target adenosine editing

To further address the problem of off-target editing at positions +7 and +8, the arRNA described in Example 2 (US+35x- 26x-21x and US+39x-26x-21x), as shown in Figure 14A. The arRNA is further modified by adding flanking flexible linker sequences (from 10nt to 50nt) to the targeting RNA sequence (as shown in Figure 14B), or by replacing the ends of the targeting RNA sequence with flanking flexible linker sequences.

The arRNA with mismatch region (US+35x-26x-21x+D7/8 and US+39x-26x-21x+D7/8) does not contain a flexible linker sequence (Ont) or contains a flexible linker. Sequences (10nt, 20nt, 30nt, 40nt, 50nt) were transfected into mutant USH2A dual reporter cell lines using Lipofectamine 3000 as described above. The efficiency of target editing was measured by the average fluorescence intensity of GFP as described above. On-target and off-target editing facilitated by the arRNA was measured using the NGS protocol described above.

As shown in Figure 15, although the additional mismatch D7/8 (relative to the deletion of positions 7 and 8 of the target RNA) reduced the arRNA (US+35x-26x-21x+D7/8 and US+39x -26x-21x+D7/8), but further addition of flexible linker sequences did not lead to a further significant decrease in on-target editing efficiency (see the second and fourth sets of data from the left), such as the average fluorescence intensity of GFP shown. 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 mean fluorescence intensity comparison with Figure 6).

As mentioned above, adding a 30nt-50nt long flexible linker sequence almost eliminated (except for the non-target adenosine at the 7th and 8th nucleotides downstream of the target editing site (Fig. 16 middle panel)) all cases with mismatches Off-target editing of arRNA regions (US+35x-26x-21x and US+39x-26x-21x). As shown in Figure 16 (right panel), increasing non-target adenosine relative to positions +7 and +8 (US+35x-26x-21x+D7/8 and US+39x-26x-21x+D7/8) The mismatch region, as well as the addition of 40nt and 50nt flexible linker sequences, virtually eliminated 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 the target RNA of +7/8 mismatch region

To investigate which type of mismatch (non-complementary or deletion) in the targeting RNA is most effective in reducing bystander editing of non-target adenosines at positions +7 and +8, various mismatches were used when generating additional arRNA combinations (A, AA, U, UU, C, CC, G, GG or "X" = nucleotide deletions located at positions +8 and +7 relative to the target RNA in the target RNA) and combined with Compare the unmismatched (UU) and parental USHER-171 (see Figure 17).

The above arRNA was transfected into the mutant USH2A dual reporter cell line using Lipofectamine 3000 as described above. Target editing efficiency was measured by GFP mean fluorescence intensity as described above. On-target and off-target editing facilitated by the arRNA was measured 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 average. Comparable to parental USHER-171 arRNA.

As shown in Figure 19, with the exception of "CC", all mismatch combinations relative to +7/8 of the target RNA resulted in significantly reduced or complete non-specific editing of non-target adenosines at positions +7 and +8 eliminate.

To further elucidate the mismatches in the targeted RNA sequence that effectively reduce non-target adenosine bystander editing at positions +7 and +8, but minimally reduce on-target editing (position 0), all mismatch combinations (AA, AU, AC, AG, UA, UU, UC, UG, CA, CU, CC, CG, GA, GU, GC, GG, A, U, C, G, or "X", located in the target RNA relative to Positions +8 and +7 of the target RNA) were used to generate additional arRNAs with arRNAs without mismatches (UU) and with deletions of nucleotides relative to +8 and +7 of the target RNA. arRNA("X") for comparison (see Figure 20).

As shown in Figures 21, 22, and 23, although a mismatch of +7/8 relative to the target RNA resulted in an arRNA without any mismatch ("UU") at +7/8: US+35x-21x (30nt linker) (top panel in Figure 21), US+35x (bottom panel in Figure 21), US+35x-21x (50nt linker) (Figure 22) and 85-C-85 (Figure 23), the target editing efficiency decreased slightly, Selecting mismatched nucleotide combinations at +7 and +8 relative to the target RNA in the target RNA sequence can result in higher on-target editing efficiencies compared to Relative to the nucleotide deletion ("X") at +7 and +8 of the target RNA, as shown by the average fluorescence intensity of GFP between "UU" and "X" (Figures 21, 22 and between the dotted lines of 23). It can be understood from Figures 21, 22, and 23, in which two consecutive non-target adenosines ("non-target AA") in the target RNA are facilitated by the arRNA with no mismatch relative to the non-target AA (i.e. "UU"). ") (like +7/8 in this example) high on-target editing but substantial bystander editing; slight reduction in non-target AA contributed by arRNA with "U" mismatch relative to non-target AA on-target editing, but significantly reduced bystander editing; whereas further reduced on-target editing but almost complete elimination of on-target editing on non-target AA was facilitated by arRNA with a nucleotide deletion ("X") relative to the non-target AA Edited by The Spectator.

In summary, with the introduction of mismatch 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 editing of USHER-171 arRNA).

Example 5. Compare mismatched regions created by deletions and insertions

Deletions and insertions located in mismatched regions

To compare the effects of deletions and insertions as mismatched regions to improve target editing, USHER-171arRNA was compared with the target RNA in the downstream (+) or upstream (-) region of the editing site (-26, +35). One or more sites are modified. As described in Table 3 and shown in Figures 24 and 25.

Table 3: Design of mismatch regions in arRNA arRNA annotation Deletions or insertions in the target RNA sequence (location relative to the target RNA) -26 missing(n)-26-B(n) Starting 27 nucleotides upstream of the target adenosine, delete n nucleotides further upstream. +35Missing(n)+35-B(n) Starting 36 nucleotides downstream of the target adenosine, delete n nucleotides further downstream. -26 insert(n) Insert n nucleotides (A) between 26 and 27 nucleotides upstream of the target adenosine. +35 insert(n) Insert n nucleotides between 35 and 36 nucleotides downstream of the target adenosine (A)

As shown in Figure 26, relative to the downstream ( +35 ) or upstream (-26) region of the editing site, 1 to 10 nucleotides are inserted or deleted in the targeted RNA sequence of the site, increasing The editing efficiency of target adenosine is improved, which is reflected by a higher increase in the average fluorescence intensity of GFP.

Figures 27 and 29 further illustrate targeting RNA complementarity of targeting RNA sequences with mismatched regions. As shown in Figure 27, in the case where the targeting RNA sequence has no deletion at position +35, contains a 4nt deletion, or a 50nt deletion, the total complementary length to the targeting RNA is still 171nt (including the target error in A/C match). Specifically, the 50nt complementary region of the target adenosine starts from the +35 position of the target RNA sequence (no deletion, Usher +35-B0) from the furthest downstream of the target adenosine (relative to the target RNA), a distance of 4 nt from position +35 of the targeting RNA sequence (with a 4 nt deletion, Usher +35-B4), or 50-nt from position +35 of the targeting RNA sequence (with a 50 nt deletion, Usher +35-B50). As shown in Figure 29, in the case where there is no deletion, a 4nt deletion, or a 50nt deletion at position -26 of the targeting RNA sequence, the total length complementary to the targeting RNA is still 171nt (including the targeting A/C mismatch). Specifically, the 59nt complementary region of the target adenosine starts from the -26 position (no deletion, Usher -26-B0) of the target adenosine (relative to the target RNA) upstream of the target adenosine, and is 59 nt from the target adenosine. 4 nt toward position -26 of the RNA sequence (with a 4 nt deletion, Usher -26-B4), or 50 -nt from position -26 of 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 region indicated downstream of the editing site (+35) increased the editing efficiency of the target adenosine, as indicated by GFP A higher increase in mean fluorescence intensity. Figure 28 (right) further shows deletions of 4 to 50 nucleotides in the targeted RNA sequence in the region indicated downstream of the editing site (+35), as well as in the region indicated upstream of the editing site (-26). Further deletion of 4 nucleotides in the target RNA sequence in the region shown can significantly improve the editing efficiency of the target adenosine, which is reflected in 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 designated region upstream of the editing site (-26) resulted in similar editing efficiencies of the target adenosine, as demonstrated by the Considerable increase in mean fluorescence intensity. Figure 30 (right panel) further shows deletions of 4 to 50 nucleotides in the targeted RNA sequence in the designated region upstream (-26) of the editing site, and in the designated region downstream (+35) of the editing site. Further deletion of 4 nucleotides in the targeting RNA sequence can significantly improve the editing efficiency of the target adenosine, which is reflected by a higher increase in the average fluorescence intensity of GFP.

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

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

As shown in Example 3, off-target editing can be reduced by using linkers flanking the target RNA that do not hybridize to the target RNA and do not substantially form secondary structures. To investigate which type of flexible linker is more effective at reducing off-target editing, various flexible linkers (50-nt) were added 3' to the targeting RNA sequence (see Figure 32), using the specified targeting RNA sequence (+ 35x-21x and +35x-21x+d7/8), and arRNA was generated according to the protocol described in Materials and Methods above (relevant dRNA sequences are shown in 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 facilitated by the arRNA was measured using the NGS protocol described above.

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

Example 7. Studying stem loops to increase on-target editing capabilities

Katrekar et al. reported in Nature Biotechnology (2022) that certain stem-loop structures can improve 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 improve on-target editing of various circular arRNAs or circular arRNAs with mismatched regions, we generated 85-C-85 (USHER-171), -21x+35x-R-flexible Linker 30 and -21+35x-R-flexible linker 50 arRNAs as well as the corresponding arRNAs further modified by the addition of GluR2, Alu, or U6+27 stem-loop structures (Figure 35) (see Table A for relevant dRNA sequences).

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 facilitated by the arRNA was measured using the NGS protocol described above.

As shown in Figure 36, the addition of a stem-loop structure (GluR2, Alu, or U6+27) resulted in a decrease in on-target editing, as evidenced by a smaller increase in the mean fluorescence intensity of GFP compared to arRNA without a stem-loop structure (control). Show.

Example 8. mutants in rhesus monkeys Usher2A Protein editing

Rhesus Monkey Cell Lines and Cell Culture

Rhesus monkey kidney cell line LLC-MK2 cells (ATCC number: CCL-7) were cultured in DMEM (Hyclone SH30243.01) containing 10% FBS (Vistech SE100-011). Mutated USH2A dual reporters were stably introduced into LLC-MK2 cells.

Plasmid transfection, FACS Analysis and next-generation sequencing (NGS)

To study the effects of circularization, length of targeting RNA, addition of mismatch regions and/or linkers on RNA editing, editing efficiency was determined by deep sequencing.

Specifically, LLC-MK2 cells expressing mutated USH2A were seeded in 12-well plates (15,000 cells/well) (recorded as 0 hours). 24 hours after seeding, 2.5 μg of arRNA encoding plasmid (SEQ ID NO. 4) (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 seeding the cells (48 hours after transfection), cells in each well were separated with trypsin (Invitrogen 25300054), and one-sixth of the separated cells were used for flow cytometry analysis of the fluorescence intensity of mCherry and GFP. All remaining isolated cells were collected using TRIzol reagent (Thermofisher 15596026) and RNA was extracted (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 (for the sequencing process, please refer to 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 aptamer 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 amplified target region sequence (see below for the sequence), and the barcode was analyzed through SAM tools ( v1.9) format conversion to generate BAM files. The information obtained is statistically compared, reordered and indexed. 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 treatment group samples, Using "(average mutation frequency other than A->G mutations) + 3SD" as the threshold, read lengths with A->G mutation frequency values higher than the threshold at the editing site are regarded as the true frequency of target A to G mutations.

Sequence of the amplified target region: ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTAgaaccattcacaacatctcattggtgttgtggctgcaaaccatgcaggagaaattttaagcccctggactgattcaaaccttAgaatcttccaagtggactgagaaactttatagtagaaagatggccgcattgctactacagtggtcagcctaTGAGAACCAAT GGTGATTAAGccatcatccaactc ( A represents the target editing position) (SEQ ID NO. 12).

In vivo editing in rhesus monkeys Usher2A situation

To study in vivo editing of USH2A mRNA with G->A mutations, mutated USH2A-specific arRNA editing was performed in rhesus monkeys endogenously expressing G->A mutated USH2A.

The arRNA encoding plasmid (SEQ ID NO.5) was packaged into an adeno-associated virus of AAV8 serotype by Guangzhou PackGene Biotechnology Co., Ltd. with a titer of 5×10 ¹³ genome copies/ml. AAV (packaged with arRNA) or control (NaCl) was injected into the subretinal space of both eyes of rhesus monkeys. Specifically, each monkey was injected with 20 μL of % 0.9 NaCl (NaCl), a solution containing 1 × ¹⁰ genomic copies of AAV (low dose), a solution containing 3 × ¹⁰ genomic copies of AAV (medium dose), or a solution containing 1 ×10 solution of ¹² genome copies of AAV (high dose). One month after the injection, the rhesus monkey was 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 ggagtgagtacggtgtgcCAT CAAGCCCACCTGTTCGGATTA (SEQ ID NO. 379) and gagttggatgctggatggGAG CCCGTCACTGAAGATGTTGTAT (SEQ ID NO. 13). Sequencing analysis was performed as described above for the rhesus monkey cell line.

Sequence of the amplified region: ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTAgaaccattcacaacatctcattggtgttgtggctgcaaaccatgcaggagaaattttaagcccctggactgattcaaaccttAgaatcttccaagtggactgagaaactttatagtagaacagaggtgcattgctactacagtggtcagcctatgagaacatggtgtg attaagacATACAACATCTTCAGTGACGGGCTCccatagccaactc (SEQ ID NO. 14) ( A represents the target editing site).

As shown in Figure 37, monkey kidney cells expressing mutated USH2A dual reporters showed similar on-target editing and bystander editing patterns to monkey retinal cells endogenously expressing mutated USH2A in vivo after introducing arRNA, thus illustrating the use of Ush2A. Reporters are suitable to explore mechanisms that improve the efficiency and specificity of on-target editing.

Example 10 : Reducing off-target editing in RNA editing of rhesus monkey mutant Usher2A

This example shows that by engineering the circRNA-targeting mutant Usher2A protein (SEQ ID NO. 315) of rhesus monkeys, bystander (off-target) editing can be reduced. 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 incorporated into the vector, followed by MiuI and KpnI digestion, and then ligated to the plasmid backbone shown in SEQ ID NO. 316. To further test the effect of adding deletions on reducing off-target editing, deletions at two different sites were combined.

As shown in Figure 38, compared with the circRNA before optimization (SEQ ID NO. 317), at -26, -30, -34, +31, +35 or +39 (SEQ ID NO. 318 - 323) 4bp deletion reduces off-target editing. Except for the 4 bp deletion at -34 and the 4 bp deletion at +39, these modifications did not reduce the on-target (position 0) editing efficiency. Off-target editing was further reduced by adding two deletions (SEQ ID NO. 324-332) at two sites (Figure 39).

The second approach is to add flexible linkers of varying lengths flanking the 5' ("left" flexible linker, or LAC) or 3' ("right" flexible linker, or RAC) of the circRNA's targeting RNA sequence as described above. . The circular RNA with adapters (SEQ ID NO. 334-343) was compared with the circular RNA before optimization (SEQ ID NO. 333). As shown in Figure 40, adding a LAC adapter to the 5' flank of the targeting RNA sequence more significantly reduces off-target editing relative to the 5' targeting RNA sequence, where a RAC adapter is added to the 3' flank of the targeting RNA sequence, Off-target editing relative to 3' targeted RNA sequences is more significantly reduced.

Finally, combine the double misses at -26 and +35 with the left and right flex joints. As shown in Figure 41, compared with the pre-optimized circular RNA (SEQ ID NO. 344), there are 4 bp deletions at -26 and +35 with a 20nt right linker and a 20-50nt left linker (SEQ ID NO. .345-348) of circRNA, off-target editing is significantly reduced while maintaining high off-target editing efficiency.

As shown in Figure 42, deletions were added at -26 and +35, and a 20nt right linker and a 30nt left linker were added, greatly reducing 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 circRNAs were made based on 4 bp deletions at -26 and +35, as well as a 20 nt right linker and a 30 nt left linker (SEQ ID NO. 351-354). Deletion of uracil at positions including -5, +3 and/or +13. As shown in Figure 43, deletion of the three uracils at -5, +3, and +13 eliminated editing of all remaining non-target adenosines. sequence listSEQ ID NO.1 (PackGene plasmid backbone) ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagaggggagtgtagccatgctctaggaagatcaattcaattcacgcgtgagggcctatttcccatgattcctt catatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttGTGGAAAGGACGAAACACCg AAGCTTgaattcGGTACCcgcgTcgacattgattattgactagctctggtcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtg tatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctactcgaggccacgttctgcttcactctccccatctcccccccctccccaccccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcgggg gggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcgggagcgggatcagccaccgcggtggc ggcctagagtcgacgaggaactgaaaaaccagaaagttaactggtaagtttagtctttttgtcttttatttcaggtcccggatccggtggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttctgctctaaaagctgcggaattgtacccgcggccgat ccaccggtcgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgacc accctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcacccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttca aggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtcc gccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaatcgaattcGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGTGCATTC TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGAGGCCGCAGGAACCCCTAGTGATGGAGTTGGccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcg cgcagccttaattaacctaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaag cgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttga ttagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaaatt taacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttt tgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggt attatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgca acatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggtaaagttgcaggaccacttctgcgctcggcccttcc ggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactt tagattgatttaaaacttcattttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagtttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaccacc gctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtc ttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcac gagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggccttttacggttcctggccttttgctggccttttgctcacatgttctttcctgc gttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggttcccgactggaaagcgg gcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccagatttaattaaggccttaattagg Plasmid backbone containing the targeting RNA sequence ( NNNNNNNNNN ) acgcgtgagggcctatttccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtatttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgattt cttggctttatatatcttgtggaaaggacgaaacaccg NNNNNNNNNN tttttttggtacc (SEQ ID NO.2) SEQ ID NO.3 – Target RNA of mutant Ush2A ( Astands for target adenosine) gcccttgaatttatggatgaaggagacaccctgaggcctttcacactctacgaatatcgggtcagagcctgtaactccaagggttcagtggagagtctgt Agtcattaacacaaactctggaagctccacctcaagattttccagctccttgggctcaagccacgagtgctcattcagttctgttgaattggacaaagcca SEQ ID NO.4 – Plasmid encoding arRNA targeting mutant Ush2A acgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgcttta cggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattgg ttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccggggagctgcatg tgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccct gataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatcctt gagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataac catgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactact tactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggacccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggat gaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatca aaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaact ctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaa agcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacg ccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtcctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcc tgcggccattcggtacaattcacgcgtgagggcctatttccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtatttgcagtttttaaaattatgttttaaaattatgttttaaaatggactatcatatgcttaccgtaactt gaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgGCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCTaaccatgccgactgatggcagtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggttt gaatcagagtccaggggcttaaaatttctcctgcatggtttgcagccacaacaccaatgagatatgttgtgaatggttctgccatcagtcggcgtggactgtagaacactgccaatgccggtcccaagcccggtaaaagtggagggtacagtccacgctttttttttggtaccaggtcttgaaaggagtgggcgc gtgtcgacattgattattgactagctctggtcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtac gccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctactcgaggccacgttctgcttcactctccccatctcccccccctccccaccccccaattttgtatttatttttttaattattttgtgcagcgatgggggcggggggggggggggggggcgc gcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcgggcggggagcgggatcagccaccgcggtggcggccctagagtcg acgaggaactgaaaaaccagaaagttaactggtaagtttagtctttttgtcttttatttcaggtcccggatccggtggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttctgctctaaaagctgcggaattgtacccgcggccgatccaccggtcg ccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctac ggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaa catcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaa agaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaggaacaagccatcaagcccacctgttcggattagaaccattcacaacatatctcattggtgttgtggctgcaaaccatgcaggagaaattttaagcccctggactctgattcaaaccttagaatcttccc caagtggactgagaaactttatagtagaacagaaagagaatggccgggcattgctactacagtggtcagagcctatgagaaccaatggtgtgattaaggaattccgctcgagataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgct tcccgtatggctttcattttctcctccttgtataaatcctggttagttcttgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgtttatttgtgaaatttgtgatgctattgctttatttgtaaccatctagctttatttgtgaaatt tgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaacaacaattgcattcattttatgtttcaggttcaggggggagatgtgggaggttttttaaagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgc ccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagt SEQ ID NO.5 – Plasmid encoding arRNA targeting mutant Ush2A aacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgtcctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactagg ggttcctgcggccattcggtacaattcacgcgtgagggcctatttccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtatttgcagtttttaaaattatgttttaaaatggactatcatatgcttacc gtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccgGCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCTaaccatgccgactgatggcagtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaa ggtttgaatcagagtccaggggcttaaaatttctcctgcatggtttgcagccacaacaccaatgagatatgttgtgaatggttctgccatcagtcggcgtggactgtagaacactgccaatgccggtcccaagcccggtaaaagtggagggtacagtccacgctttttttcttattggcgctggtgaacggacttcctct gagggcctatttccatgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtatttgcagtttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatata tcttgtggaaaggacgaaacaccgGCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCTaaccatgccgactgatggcagtaggctctgaccactgtagtagcaatgcccggccattctctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttct cctgcatggtttgcagccacaacaccaatgagatatgttgtgaatggttctgccatcagtcggcgtggactgtagaacactgccaatgccggtcccaagcccggataaaagtggagggtacagtccacgcttttttttggtaccaggtcttgaaaggagtgggcgcgtgtcgacattgattattgactagctctggtc gttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcc cgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctactcgaggccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttatttttaattattttgtgcagcgatggggggcggggggggggggggggggggcgcgcgccaggcggggcggggcggggcgagg cggggcggggcgaggcggagaggtgcggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcgcgggcgggagcgggatcagccaccgcggtggcggccctagagtcgacgaggaactgaaaaaccagaaagttaactggtaag tttagtctttttgtcttttatttcaggtcccggatccggtggtggtgcaaatcaaagaactgctcctcagtggatgttgcctttacttctaggcctgtacggaagtgttacttctgctctaaaagctgcggaattgtacccgcggccgatccaccggtcgccaccatggtgagcaagggcgaggagctgttca ccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccacggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctacccc gaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagcc acaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacaccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcct gctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaatcgaattccgctcgagataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcatttt ctcctccttgtataaatcctggttagttcttgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgtttatttgtgaaatttgtgatgctattgctttatttgtaaccatctagctttatttgtgaaatttgtgatgctattgctttattt gtaaccattataagctgcaataaaacaagttaacaacaacaattgcattcattttatgtttcaggttcaggggggagatgtgggaggtttttaaagcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgccc gggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcg ccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcggggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgcc ctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctg ctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcat gataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatt ttgccttcctgttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgt attgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggat catgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggt ttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaa aacttcatttttaatttaaaaggatctaggtgaagatccttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttctgcgcgtaatctgctgcttgcaaacaaaaaaccaccgctaccagc ggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggtt ggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagctt ccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaa SEQ ID NO.11 – Primers for PCR amplification of Usher2A reverse transcribed cDNA gagttggatgctggatggCTTAATCACACCATTGGTTCTCA SEQ ID NO.12 - Amplified Usher2A target region sequence ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTAgaaccattcacaacatatctcattggtgttgtggctgcaaaccatgcaggagaaattttaagcccctggactctgattcaaaccttAgaatcttccccaagtggactgagaaactttatagtagaacagaaagagaatggccgggcattgctactacagtggtcagagcctaTGAGAACCAATGG TGTGATTAAGccatccagcatccaactc SEQ ID NO.13 – Primers for PCR amplification of Usher2A reverse transcribed cDNA gagttggatgctggatggGAGCCCGTCACTGAAGATGTTGTAT SEQ ID NO.14 - Amplified Usher2A target region sequence ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTAgaaccattcacaacatatctcattggtgttgtggctgcaaaccatgcaggagaaattttaagcccctggactctgattcaaacctt AgaatcttccccaagtggactgagaaactttatagtagaacagaaagagaatggccgggcattgctactacagtggtcagagcctatgagaaccaatggtgtgattaagacATACAACATCTTCAGTGACGGGCTCccatccagcatccaactc surface A:Sequences, including linear RNA (hereinafter referred to as "linear") and linear RNA that can be circularized (hereinafter referred to as "circular"), the sequence of the targeting RNA *Uncapitalized sequence = targeting RNA sequence ("targeting sequence"). *Non-bold sequence = part of the arRNA after circularization ("circularization sequence"). Figure label In vitro synthesized sequence (corresponding to SEQ ID NO.2 NNNNNNNNNN ) Figure 2 Linear-151 gaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgt (SEQ ID NO. 15) ring-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) ring-51 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGagcttccagagtttgtgttaatgaccacagactctccactgaacccttggaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.28) ring-61 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAG ggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.29) Ring-71 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.30) Ring-81 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.31) ring-91 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.32) Ring-101 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.33) ring-111 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.34) ring-121 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.35) ring-131 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.36) ring-141 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.37) ring-151 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.38) ring-161 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGgaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctcctCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.39) ring-171 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.40) ring-181 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGcaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataaCTGCCATCAGTCGGCGTGGACT GTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.41) ring-191 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGttgtccaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataaattcaCTGCCAT CAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.42) Ring-201 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtggctttgtccaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccataaattca agggcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.43) Ring-211 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGGCCGCtggctttgtccaattcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcctcagggtgtctccttcatccata aattcaagggcGCTAGCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.44) Ring-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 AACCATGCCGACTGATGGCAGcgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgctgtccaggacggtcccggcctgcgacacttcggcccagctgctcctcatctgcggggcgggggggggccgtcgccgcgtggggtcgttgcccagccgcccaccgtccgagggccgggccCTGCCAT 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 GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCTAACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggaaaatcttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccttggagttacaggctctgacccgatattcgtagagtgtgaaaggcc tcagggtgtctccttcatcCTGCCATCAGTCGGCGTGGACTGTAGAACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.114) non-target GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCTAACCATGCCGACTGATGGCAGcgggccctggggggcggtgggcgctggccaggacgcccaccgtgtggttgctgtccaggacggtcccggcctgcgacacttcggcccagctgctcctcatctgcggggcgggggggccgtcgccgcgtgg ggtcgttgcccagccgccccccgacccagggccgggccCTGCCATCAGTCGGCGTGGACTGTAGAACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (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 AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgAAaatgaccacagactctccactgaacccaggctAAAAAACAAAAAAAAAAAACAAAAAAAAAACCAAAAAAACAAAACACACTGCCATCAGTCGGCGTGGACTGTAG 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 AACCATGCCGACTGATGGCAGcgggccctgggggcggtgggcgctggccaggacgcccaccgtgtggttgctgtccaggacggtcccggcctgcgacacttcggcccagctgctcctcatctgcggggcgggggggggccgtcgccgcgtggggtcgttgcccagccgcccaccgtcccagggccgggccCTGCCAT 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 AACCATGCCGACTGATGGCAGcaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctCCCCCTCCCCTCCCCCCCCCCCCCCCCCTTCCCCCCCTCCCCTCCTGCCATCAGTCGGCGTGGACTGTAG 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 AACCATGCCGACTGATGGCAGgtgctcgcttcggcagcacatatactagtcgaccaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgttaatgaccacagactctccactgaacccaggctaaaaacaaaaaacaaaaaaaccaaaaaaaaaaccaaaaa aacaaaacacatctagagcggacttcggtccgcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.292) -21X+35X-RAC50+Alu GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGggccgggcgcggtggctcacgcctgtaatcccagcactttggggggccgaggcggggagattgcttgagcccaggagttcgagaccagcctgggcaacatagcgagaccccgtctccaacagaactgaatgagcactcgtggcttgagcccaaggagctggacttgaggtggagcttccagagtttgtgtgtta atgaccacagactctccactgaacccaggctaaaaacaaaaaacaaaaaaaacaaaaaaaaaccaaaaaaaaacacaagccgggcgtggtggcgcgcgcctgtagtcccagctactcgggaggctgaggcaggaggatcgcttgagcccaggagttcgaggctgcagtgagctatgatcgcg ccactgcactccagcctgggcgacagagcgagaccctgtctcCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC (SEQ ID NO.293) SEQ ID NO.315 – Mutant mf-Ush2A target RNA ( Astands for 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 circularizable targeting RNAs *Uncapitalized sequence = targeting RNA sequence *Sequence in capital letters other than bold = part of arRNA after circularization *Capital sequence with bold font = part of arRNA that is cleaved 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 4bp deletion at -26 (SEQ ID NO.318) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggctatttctcctgcatggtttgcagccacaacaccaatgaga tatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -30 (SEQ ID NO.319) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaactcctgcatggtttgcagccacaacaccaatgaga tatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -34 (SEQ ID NO.320) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaattttgcatggtttgcagccacaacaccaatg agatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at +31 (SEQ ID NO.321) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttcttaaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatggtttgcagccacaacaccaat gagatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at +35 (SEQ ID NO.322) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaaatttctcctgcatggtttgcagccacaacaccaatgaga tatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp 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.333) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatggtttgcagccacaacacca atgagatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC LAC-10 (SEQ ID NO.334) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaccactgtagtagcaatgcccggccattctctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaaatttctcctgcatggtttgcagccac aacaccaatgagatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC LAC-20 (SEQ ID NO.335) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatggttt gcagccacaacaccaatgagatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC LAC-30 (SEQ ID NO.336) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaacaaaaaaaaggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatgg tttgcagccacaacaccaatgagatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC LAC-40 (SEQ ID NO.337) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaacaaaaaaaaaccaaaaaatttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctg catggtttgcagccacaacaccaatgagatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC LAC-50 (SEQ ID NO.338) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaaaaaaaaaaccaaaaaaacaaaaccaactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcct gcatggtttgcagccacaacaccaatgagatatgttgtgaatggttCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC RAC-10 (SEQ ID NO.339) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatggtttgcagccacaacacca atgagatatgttacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC RAC-20 (SEQ ID NO.340) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatggtttgcagccacaacacca ataaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC RAC-30 (SEQ ID NO.341) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatggtttgcagccaacaaa aaaaaaaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC RAC-40 (SEQ ID NO.342) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttctcctgcatggaacaaaaaaaaaa aaaaaaaaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC RAC-50 (SEQ ID NO.343) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGtaggctctgaccactgtagtagcaatgcccggccattctctttctgttctactataaagtttctcagtccacttggggaagattcCaaggtttgaatcagagtccaggggcttaaaatttcaaaaacaaaaacaaaaaaa acaaaaaaaaaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC Figure 44 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 4bp 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 46 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 -5 U deletion at +3 (SEQ ID NO.351) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaaaaaaaaaggccattctctttctgactataaagtttctcagtccacttggggaagatcCaaggttgaatcagagtccaggggctatttctcctgcatggtttgcagcc acaacaccaataaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26& +35 AC linker at R20 & L30 -5 U deletion at +13 (SEQ ID NO.352) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaacaaaaaaaaggccattctctttctgactataaagtttctcagtccactggggaagattcCaaggttgaatcagagtccaggggctatttctcctgcatggtttgcagccac aacaccaataaccaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26& +35 AC linker at R20 & L30 +3 U deletion at +13 (SEQ ID NO.353) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaaaaaaaaaggccattctctttctgactataaagtttctcagtccactggggaagatcCaaggtttgaatcagagtccaggggctatttctcctgcatggtttgcagcc acaacaccaataaccaaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 4bp deletion at -26& +35 AC linker at R20 & L30 -5 +3 U deletion at +13 (SEQ ID NO.354) GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT AACCATGCCGACTGATGGCAGaaaaacaaaaaacaaaaaaaacaaaaaaaaggccattctctttctgactataaagtttctcagtccactggggaagatcCaaggttgaatcagagtccaggggctatttctcctgcatggtttgcagccac aacaccaataaccaaaaaacaaaacacaCTGCCATCAGTCGGCGTGGACTGTAG AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC SEQ ID NO.375 - 3×GS Connector GGGGS SEQ ID NO.376 – Primers for PCR amplification of Usher2A reverse transcribed cDNA ggagtgagtacggtgtgcTGAATTTATGGATGAAGGAGACACCCT SEQ ID NO.377 – Primers for PCR amplification of Usher2A reverse transcribed cDNA gagttggatgctggatggACGTCACCGCATGTTAGAAGACT SEQ ID NO.378 - Amplified Usher2A target region sequence ggagtgagtacggtgtgcTGAATTTATGGAATGAAGGACAACCCTgaggcctttcacactctacgaatatcgggtcagagcctgtaactccaagggttcagtggagagtctgtAgtcattaacacaaactctggaagctccacctcaagattttccagctccttgggctcaagccacgagtgctcattcagttctgttgaattggac aaagccaGCGGCCGCTGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTccatccagcatccaactc SEQ ID NO.379 – Primers for PCR amplification of Usher2A reverse transcribed cDNA ggagtgagtacggtgtgcCATCAAGCCCACCTGTTCGGATTA

without

Figure 1A shows the PCR amplification product sequence of the USH2A full-length coding sequence introducing G>A mutation by mutagenesis PCR. Figure 1B shows a schematic diagram of genetic encoding of arRNA via U6 or CMV promoters. A 151 nt arRNA targeting fluorescent reporter-1 was expressed under the human U6 or CMV promoter. For reporter 1, the mCherry and EGFP genes were connected by a sequence containing the 3×GGGS (SEQ ID NO. 375) coding region and an in-frame UAG stop codon. Cells expressing the reporter only produce 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 USH2A reporter cells measured by sequencing after the introduction of linear arRNA with a 151-nt targeting RNA sequence (linear-151) or circular arRNA with a 151-nt targeting RNA sequence (circular-151). The percentage of each adenosine edited in the target RNA sequence in the 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 3 shows the on-target editing efficiency in the USH2A reporter cell line after the introduction of linear or circular arRNA compared with untreated (UT) or blank control (mock) transfection, as measured by the average fluorescence intensity of the GFP reporter ( MFI) measured. Figure 4 shows non-target adenosine in USH2A located 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 deletions in the targeting 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 mean fluorescence intensity of the GFP reporter (MFI) measured. Figure 7 shows the percentage of each adenosine in the target RNA sequence in the USH2A reporter cell line after the introduction of USHER-171 arRNA, non-targeting arRNA, or arRNA with mismatch 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 diagram 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 diagram of the use of deletions of targeted RNA sequences downstream of the target adenosine (relative to the target RNA) to generate mismatched regions in arRNA design. 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 mean fluorescence intensity of the GFP reporter (MFI) measured. Figure 10 shows the percentage of each adenosine in the target RNA sequence in the USH2A reporter cell line after the introduction of USHER-171 arRNA, non-targeting arRNA, or arRNA with mismatch regions as described in Table 2, 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 11 is a diagram depicting the 5' flanking ("left" flexible linker, or L-flexible linker) of an arRNA targeting RNA sequence (top panel) or the 3' flanking ("right" flexible linker,) of an arRNA targeting RNA sequence. or R-flexible joint) (below) Schematic diagram of using a flexible joint. Figure 12 shows the on-target editing efficiency in the USH2A reporter cell line after the introduction of arRNA containing the L-flexible linker (L) or R-flexible linker (R) described in Figure 11, with the x-axis indicating linker length (10 -nt, 20-nt, 30-nt), which was measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 13 shows that after the introduction of arRNA containing L-flexible linker (L) or R-flexible linker (R) of specific linker lengths (10-nt, 20-nt, 30-nt), the expression of arRNA in the USH2A reporter cell line was The percentage of each adenosine 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 in the targeting RNA sequence (all positions relative to the target RNA) upstream of the target adenosine (-26x-21x) and There is a 4 bp deletion downstream (+35x or +39x) and a further deletion of the nucleotide opposite the non-target adenosine downstream of the target adenosine (+d7/8). 14B is a schematic depicting a circular arRNA with a 10 bp deletion in the targeting RNA sequence (all positions relative to the target RNA) upstream of the target adenosine (-26x-21x) and downstream of the target adenosine ( +35x or +39x) with a 4 bp deletion and a further deletion of nucleotides opposite the non-target adenosine downstream of the target adenosine (+d7/8), where the targeting RNA sequence further has 3 bp flanking it 'Flexible Joint ("Right" Flexible Joint, or R-Flexible Joint). Figure 15 shows the results of introducing USHER-171 arRNA, non-targeting arRNA, or with or without (at 10-nt, 20-nt, 30-nt, 40-nt, 50-nt) as described in Figure 14 On-target editing efficiency in USH2A reporter cell lines after arRNA with the mismatched region of the AC linker, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 16 shows that when introducing USHER-171 arRNA, non-targeting arRNA, or with or without (at 10-nt, 20-nt, 30-nt, 40-nt, 50-nt) as described in Figure 14 Percentage of each adenosine edited in the USH2A reporter cell line after arRNA in the mismatched region of the AC linker, 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 17 is a further detailed schematic depicting the mismatch of the alkyl group (originally "UU") relative to the non-target adenosine downstream of the target adenosine (+8 and +7). Figure 18 shows the introduction of USHER-171 arRNA, non-targeting arRNA or having the mismatch region (+39x--21x or +35x-21x, see Figure 14) and the non-target adenosine downstream of the target adenosine ( +8 and +7) after the corresponding base (originally "UU") becomes the arRNA of the base shown on the X-axis (A, AA, U, UU, C, CC, G, GG or "X"), On-target editing efficiency of the USH2A reporter cell line, measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 19 shows that when introducing USHER-171 arRNA or having the mismatched region (+39x-21x or +35x-21x, see Figure 14) and non-target adenosine downstream of the target adenosine (+7/8) After the arRNA whose base group is changed to the base group shown on the x-axis (A, AA, U, UU, C, CC, G, GG or "X"), the edited target adenosine (0 ) and the percentage of non-target adenosine (+7, +8) 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 further detailed schematic depicting the mismatch of the alkyl group (formerly "UU") relative to the non-target adenosine downstream of the target adenosine (+d7/8). Figure 21 shows the introduction of alkyl groups (original groups) with the mismatch region (+35x-21x-flexible linker 30 or +35x) and will be opposite to the non-target adenosine (+8 and +7) downstream of the target adenosine. On-target editing efficiency in the USH2A reporter cell line after making changes to arRNA as shown on the X-axis for "UU"), as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 22 shows the introduction of an alkyl group (originally "UU") that has the mismatch region (+35x-21x-flexible linker 50) and will be opposite the non-target adenosine (+8 and +7) downstream of the target adenosine. ") On-target editing efficiency in the USH2A reporter cell line after making changes to arRNA as shown on the x-axis, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 23 shows the introduction of alkyl groups (originally "UU") that have the mismatch region (+35x-21x-flexible linker 50) and will be opposite the non-target adenosine (+8 and +7) downstream of the target adenosine. ") On-target editing efficiency in the USH2A reporter cell line after making changes to arRNA as shown on the x-axis, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 24 is a schematic depicting deletion of alkyl groups of a targeted RNA sequence relative to the upstream (-26) or downstream (+35) region of the target adenosine. Figure 25 is a schematic depicting the insertion of alkyl groups into targeted RNA sequences relative to the upstream (-26) or downstream (+35) region of the target adenosine. Figure 26 shows that after introducing arRNA with the deletion or insertion (length 1, 2, 3, 4, 7, 10 nt) relative to the upstream or downstream region of the target RNA (-26+35), On-target editing efficiency in USH2A reporter cell lines, as measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 27 is a schematic diagram depicting the deletion of alkyl groups in the downstream (+35) region of the target RNA 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) relative to the downstream region (+35) of the target RNA. Finally, the on-target editing efficiency of the USH2A reporter cell line was measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 28 further shows with open bars (right) the introduction of the deletion (0, 4, 10, 20, 30, 40 or 50 nt in length) relative to the downstream region (+35) of the target RNA and On-target editing efficiency of the USH2A reporter cell line, measured by the mean fluorescence intensity (MFI) of the GFP reporter, after further having a 4nt deletion of arRNA relative to the upstream region (-26) of the target RNA. Figure 29 is a schematic diagram depicting the deletion of alkyl groups in the upstream region (-26) of the target RNA 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) relative to the upstream region (-26) of the target RNA. Finally, the on-target editing efficiency of the USH2A reporter cell line was measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 30 further shows with open bars (right panel) the introduction of the upstream region (-26) relative to the target RNA with the deletion (0, 4, 10, 20, 30, 40 or 50 nt in length) On-target editing efficiency of the USH2A reporter cell line as measured by the mean fluorescence intensity (MFI) of the GFP reporter after further having a 4nt deletion of arRNA relative to the downstream region (+35) of the target RNA . Figure 31 shows the introduction of deletions (lengths 0, 4, 10, 20, 30, 40) in the upstream region (-26) and the downstream region (+35) relative to the target RNA. or 50nt) arRNA, the on-target editing efficiency of the USH2A reporter cell line, which was 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 after the introduction of arRNA with the mismatch region (+35x-21X, +35x-21X+d78 or control arRNA) and flanked by the flexible linkers described in Figure 32, the USH2A reporter cell line On-target editing efficiency, measured by the mean fluorescence intensity (MFI) of the GFP reporter. Figure 34 shows the on-target editing efficiencies of previously reported arRNAs targeting RNAs of different lengths and containing illustrated stem-loops and/or circularizations. Figure 35 is a schematic diagram describing the addition of stem loops (GluR, U6+27, Alu) to arRNA. Figure 36 shows that after introducing USHER-171 arRNA (85-c-85), or having the mismatch region and linker (-21x+35x-R-flexible linker 30 and -21+35x-R-flexible linker 50), And the on-target editing efficiency of the USH2A reporter cell line with or without further stem-loop modified USHER-171 arRNA as described in Figure 34 (GluR, U6+27, Alu), as measured by the average fluorescence intensity of the GFP reporter ( MFI) measured. Figure 37 shows the editing pattern in rhesus monkey kidney cells with Ush2A reporter (bottom panel) after transfection of Ush2A targeting arRNA compared to the editing pattern in rhesus monkey eyes after transfection with AAV packaged with Ush2A targeting arRNA. Target and spectator editing modes. Figure 38 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 upstream or downstream region of the target adenosine. Figure 39 shows on-target and bystander editing patterns of USH2A in rhesus monkey kidney cells after the introduction of USH2A-specific arRNA with two deletions (length 4) in the upstream or downstream region of the target adenosine. . Figure 40 shows that when introducing USH2A-specific arRNA or targeting RNA sequences 5' (L-10, L-20, L-30, L-40, L-50) or 3' (R-10, R-20, R-40, R-50) on-target and bystander editing modes of USH2A in rhesus monkey kidney cells following the corresponding arRNA flanked by flexible linkers of specified lengths (10, 20, 30, 40, 50 nt). Figure 41 shows on-target and bystander editing patterns of USH2A in rhesus monkey kidney cells following the introduction of USH2A-specific arRNA with shown downstream and downstream of target adenosine relative to the target RNA. The combination of the mismatch region upstream and having flexible linkers at 5' and 3'. Figure 42 shows the on-target and bystander editing patterns of USH2A in rhesus monkey kidney cells after the introduction of USH2A-specific arRNA before and after optimization, wherein the optimization was performed by first modifying the arRNA relative to the target RNA. The upstream region (-26) and the downstream region (+35) relative to the target RNA are deleted by 4 bp and additionally contain a 20 bp linker flanking the 3' end of the targeting RNA sequence and 5 bp at the 3' end of the targeting RNA sequence. The 'ends are flanked by 30bp adapters. Figure 43 shows the on-target and bystander editing patterns of USH2A in rhesus monkey kidney cells after the introduction of USH2A-specific arRNA with the shown arRNA downstream and upstream of the target adenosine relative to the target RNA. A combination of mismatched regions and the insertion of one or more uridines outside the flexible linker at the 5' and 3' ends.

TW202346596A_112103033_SEQL.xml

Claims (50)

A method of editing a target adenosine in a target RNA encoding a mutant Usher2A protein in a host cell, comprising introducing a deaminase recruiting RNA (dRNA) or a construct comprising a nucleic acid encoding the dRNA into the host cell. Said host cell, Wherein, the dRNA includes a targeting RNA sequence that is capable of hybridizing with the target RNA to form an RNA duplex, Wherein, the RNA duplex can recruit adenosine deaminase (ADAR) that acts on RNA to deaminate target adenosine in the target RNA; Wherein, the RNA duplex contains: (a) The first mismatch region relative to the target RNA sequence is located 5 nucleotides (nt) to 85 nt upstream of the target adenosine; and/or (b) a second mismatch region located 20 nt to 85 nt downstream of the target adenosine relative to the target RNA sequence; and Wherein, the dRNA includes a linker nucleic acid sequence located at the end of the target RNA sequence, wherein the linker nucleic acid sequence does not hybridize with the target RNA and basically does not form a secondary structure. The method as described in request item 1, wherein (a) the RNA duplex contains a first mismatch region relative to the target RNA sequence, which is located 5nt to 25nt upstream of the target adenosine; and/or the RNA duplex contains a region relative to the target RNA sequence; A second mismatch region of the target RNA sequence located 20nt to 45nt downstream of the target adenosine; or (b) the RNA duplex contains a first mismatch region relative to the target RNA sequence, which is located 5nt to 15nt upstream of the target adenosine; and/or the RNA duplex contains a region relative to the target RNA sequence; A second mismatch region of the target RNA sequence located 20nt to 45nt 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 region relative to the target RNA sequence; The second mismatch region of the target RNA sequence is located 25nt to 45nt downstream of the target adenosine. The method as claimed in 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) Deletion of one or more nucleotides in the targeting RNA sequence; and/or (c) Insertion of one or more nucleotides in 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 include: (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 contiguous set of nucleotides in the targeting RNA sequence. A method as described in any one 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 length of the second mismatch region is 1-50 nt, optionally, the length of the second mismatch region is 4 nt. A method as described in any of requests 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 1-10 consecutive non-complementary nucleotides in the targeting RNA sequence. Deletion of 1-10 consecutive nucleotides; 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 1-10 consecutive non-complementary nucleotides in the targeting RNA sequence. Deletion of 1-10 consecutive nucleotides. 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 4 consecutive non-complementary nucleotides in the targeting RNA sequence or 4 consecutive nuclei in the targeting RNA sequence Deletion of nucleotides; and/or (b) The length of the second mismatch region is 4 nt, wherein the second mismatch region includes 4 consecutive non-complementary nucleotides in the targeting RNA sequence or 4 consecutive nuclei in the targeting RNA sequence Deletion of nucleotides. A method as described in any of requests 1-7, wherein (i) Targeting non-complementary nucleotides in an RNA sequence results in bubbles in the RNA duplex; and/or (ii) Deletion of nucleotides in the targeted RNA sequence results in a bulge in the RNA duplex; and/or (iii) Insertion of nucleotides in the targeted RNA sequence results in the appearance of bulges in the RNA duplex. A method as described in any of requests 1-8, wherein (i) Targeting a contiguous set of non-complementary nucleotides in an RNA sequence results in bubbling in said RNA duplex; and/or (ii) The deletion of a contiguous set of nucleotides in the targeted RNA sequence results in the appearance of a bulge in the RNA duplex; and/or (iii) Insertion of a contiguous set of nucleotides in a targeted RNA sequence results in the appearance of a bulge in the RNA duplex. The method according to any one of claims 1-9, wherein the mutant Usher2A protein contains missense mutations, nonsense mutations and/or frameshift mutations. The method of any one of claims 1-10, wherein the mutant Usher2A protein comprises the Trp3955Ter mutation. The method of any one of claims 1-11, wherein the target RNA encoding the mutant Usher2A protein comprises a G to A mutation compared to the target RNA encoding the wild-type Usher2A protein. The method of any one of claims 1-12, wherein the target RNA encoding the mutant Usher2A protein contains a G>A mutation at position 11864, which mutation is compared to the target RNA encoding the wild-type Usher2A protein. The method of any one of claims 1-13, wherein the RNA duplex further comprises a third mismatch region relative to the target RNA, wherein the third mismatch region relative to the target RNA Located between the first mismatch region and the second mismatch region. The method of claim 14, wherein the third mismatch region comprises one or two non-complementary nucleotides in the targeting RNA sequence and/or one or two in the targeting RNA sequence Deletion of nucleotides. The method of claim 14 or 15, wherein the third mismatch region relative to the target RNA sequence is located 7nt and/or 8nt downstream of the target adenosine; optionally, wherein the target RNA Adenosine is included at the 7th and/or 8th nucleotide downstream of the target adenosine. The method of any one of claims 14-16, wherein the target RNA includes "AA" located at the 7th and 8th nucleotides downstream of the target adenosine, wherein the target The RNA sequence includes any one selected from: A, AA, U, C, CC, G, GG or a nucleotide deletion ("X") downstream of the target adenosine 7 and 8 target RNA at nucleotides relative to each other. The method of any one of claims 10-17, wherein the RNA duplex comprises: (a) a first mismatch region relative to the target RNA sequence located 27nt to 30nt upstream of the target adenosine; and (b) A second mismatch region relative to the target RNA sequence located 31 nt to 43 nt downstream of the target adenosine. A method as described in request 18, wherein The second mismatch region relative to the target RNA sequence is located 36nt to 39nt downstream of the target adenosine; Optionally, wherein the length of the first mismatch region is 4nt and the length of the second mismatch region is 4nt; Further optionally, wherein the first mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence, and the second mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence. missing. A method as described in request 18, wherein The second mismatch region relative to the target RNA sequence is located 40nt to 43nt downstream of the target adenosine; Optionally, wherein the length of the first mismatch region is 4nt and the length of the second mismatch region is 4nt; Further optionally, the first mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence, and the second mismatch region includes a deletion of 4 consecutive nucleotides in the targeting RNA sequence. Missing. The method of any one of claims 10-17, wherein the RNA duplex comprises: (a) a first mismatch region located 21 nt to 30 nt upstream of the target adenosine relative to the target RNA sequence; and (b) a second mismatch region relative to the target RNA sequence located 36nt to 39nt downstream of the target adenosine; Optionally, wherein the length of the first mismatch region is 10nt and the length of the second mismatch region is 4nt; Further optionally, wherein the first mismatch region includes a deletion of 10 consecutive nucleotides in the targeting RNA sequence, and wherein the second mismatch region includes 4 consecutive nucleotides in the targeting RNA sequence. Lack of acid. The method of any one of claims 10-17, wherein the RNA duplex comprises: (a) a first mismatch region located 21 nt to 30 nt upstream of the target adenosine relative to the target RNA sequence; and (b) a second mismatch region relative to the target RNA sequence, located 40nt to 43nt downstream of the target adenosine; Optionally, wherein the length of the first mismatch region is 10nt and the length of the second mismatch region is 4nt; Further optionally, wherein the first mismatch region includes a deletion of 10 consecutive nucleotides in the targeting RNA sequence, and wherein the second mismatch region includes 4 consecutive nucleotides in the targeting RNA sequence. Lack of acid. The method according to any one of claims 1-22, wherein the dRNA is: (i) cyclic; or (ii) Linear and/or capable of cyclization. The method according to any one of claims 1-23, 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 1-24, wherein the length of the linker nucleic acid sequence is from about 5nt to about 500nt. The method of any one of claims 1-25, wherein at least about any of the following, the linker nucleic acid sequence contains adenosine or cytidine: 50%, 60%, 70%, 80%, 85%, 90% or 95%; optionally, wherein 100% of the linker nucleic acid sequences contain adenosine or cytidine. The method of any one of claims 1-26, wherein, compared to a corresponding method in which the RNA duplex does not comprise one or more mismatch regions or wherein the dRNA does not comprise a linker nucleic acid sequence, The method has enhanced editing levels for the target adenosine. The method of any one of claims 1-27, wherein, compared to a corresponding method in which the RNA duplex does not comprise one or more mismatch regions or wherein the dRNA does not comprise a linker nucleic acid sequence, The method has reduced (bystander) editing levels for one or more non-target adenosines. The method of claim 28, wherein the non-target adenosine is located within one or more mismatch regions. The method of claim 28, wherein the non-target adenosine is located outside the mismatch region. 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 located at the 3' end of the targeting RNA sequence. flanking second linker nucleic acid sequence. The method of any one of claims 1-31, wherein the dRNA is a circular RNA, and wherein 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 end. The method of any one of claims 1-32, wherein the method comprises introducing a construct comprising a nucleic acid sequence encoding the dRNA into the host cell. The method of claim 33, wherein the construct further comprises a promoter operably linked to the nucleic acid sequence encoding the dRNA. The method of any one of claims 1-34, 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 1-35, wherein the 5' nearest neighbor of the target adenosine in the target RNA is a nucleotide selected from U, C, A and G, with a priority of 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, and the priority is G>C>A≈U. The method of any one of claims 1-36, wherein the target adenosine is located in a UAG trihydroxyl motif, and wherein the targeting RNA sequence comprises a direct link to a uridine in the trihydroxyl motif. Opposite A, cytidine directly opposite the target adenosine, and cytidine, guanosine, or uridine directly opposite the guanosine in the trisacryl motif. The method of any one of claims 1-37, wherein the target RNA is an RNA selected from the group consisting of precursor messenger RNA, messenger RNA, ribosomal RNA, transfer RNA, long non-coding RNA and small RNA, optionally, wherein the target RNA is a precursor messenger RNA. The method of any one of claims 1-38, wherein the efficiency of editing target adenosine in the target RNA is at least about 40%. The method of any one of claims 1-39, further comprising introducing ADAR into the host cell. The method of any one of claims 1-40, wherein deamination of target adenosine in the target RNA results in missense mutations, premature stop codons, aberrant splicing or alternative splicing in the target RNA, Or missense mutations, premature stop codons, abnormal splicing or reversal of alternative splicing in the target RNA. An edited RNA produced by the method of any one of claims 1-41 or a host cell comprising edited RNA. A method of alleviating one or more symptoms of Usher syndrome in an individual, comprising editing a target RNA associated with Usher syndrome in cells of the individual according to the method of any one of claims 1-41. The method of claim 43, wherein the target RNA contains a G to A mutation. A dRNA for editing a target RNA encoding a mutant Usher2A protein and comprising a target adenosine, wherein the dRNA comprises a targeting RNA sequence capable of hybridizing to the target RNA to form an RNA duplex; Wherein, the RNA duplex can recruit adenosine deaminase (ADAR) that acts on RNA to deaminate target adenosine in the target RNA; Wherein, the RNA duplex contains: (a) a first mismatch region relative to the target RNA sequence located 5nt to 85nt upstream of the target adenosine; and/or (b) a second mismatch region relative to the target RNA sequence located 20 nt to 85 nt downstream of the target adenosine, and Wherein, the dRNA includes a linker nucleic acid sequence located at the end of the target RNA sequence, wherein the linker nucleic acid sequence does not hybridize with the target RNA and does not substantially form a secondary structure. A dRNA as described in claim 45, wherein: (i) Targeting a contiguous set of non-complementary nucleotides in an RNA sequence results in bubbling in said RNA duplex; and/or (ii) The deletion of a contiguous set of nucleotides in the targeted RNA sequence results in the appearance of a bulge in the RNA duplex; and/or (iii) Insertion of a contiguous set of nucleotides in a targeted RNA sequence results in the appearance of a bulge in the RNA duplex. The dRNA of claim 45 or 46, wherein the targeting RNA encodes a mutant Usher2A protein; optionally, wherein the mutant Usher2A protein contains a missense mutation, a nonsense mutation and/or a frameshift mutation. A construct comprising a nucleic acid sequence encoding the dRNA of any one of claims 45-47. A host cell comprising the dRNA of any one of claims 45-47 or the construct of claim 48. A kit comprising the dRNA as described in any one of claims 45-47 or the construct as described in claim 48, and instructions for editing target RNA containing target adenosine in a host cell, said The target RNA encodes the mutant Usher2A protein.

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