Improved RNA editing method

An editing and targeting technology, applied in the fields of RNA editing and gene editing, can solve the problems of reduced editing efficiency and inability to edit

Pending Publication Date: 2022-01-07
EDIGENE THERAPEUTICS (BEIJING) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

When the 5' upstream residue is G, the editing efficiency is significantly reduced, even close to zero, and the difference between the two is more than 10 times
This sho

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0163] Example 1: Construction of a three-base motif reporter system and corresponding arRNA

[0164] First, we constructed a reporter system containing 16 three-base motifs. Since in the LEAPER literature, the difference in editing efficiency when the three-base motif is UAG has been tested (Qu et al., 2019), in this example, in order to maintain the consistency of the control, arRNA can complement other than the editing site The paired parts all use the same sequence design as in the LEAPER literature, such as Figure 5 shown. The original plasmid Reporter1 was donated by Professor Wei Wensheng, School of Life Sciences, Peking University. The plasmid map is as follows: Figure 14 As shown, the plasmid contains the sequences shown in Table 4. Synthesize 16 kinds of three-base motif-related primers as shown in Table 1, and according to the methods well known to researchers in the field in "J. Sambrook, M.R. Green, Molecular Cloning Experiment Guide (Fourth Edition), 2017" ...

Embodiment 2

[0166] Example 2: Comparing the editing efficiency of different arRNAs to the UAG three-base motif through the positive ratio of GFP

[0167] Such as Figure 5 Shown are 16 kinds of target RNAs described in Example 1, which all have the GFP green fluorescent protein nucleic acid sequence at the 3' end of the target sequence. This sequence normally translates correctly and fluoresces green. But when the three-base motif is UAG, because UAG is a stop codon, translation will stop there, and then it cannot be translated into GFP. In this example, the A in the UAG three-base motif was edited by the LEAPER system. If the editing is successful, UAG will be converted into UIG, and UIG will be recognized as UGG during the translation process, so that the translation will not be terminated, so that the downstream GFP can be translated normally. Therefore, we can roughly judge the level of editing efficiency of different arRNAs through the size of the positive ratio of GFP.

[0168] ...

Embodiment 3

[0177] Embodiment 3: The RNA editing efficiency determination of GAN tribasic motif

[0178] In this example, 16 kinds of arRNA were transfected to the reporter system cells respectively containing the three base motifs of UAG, GAA, GAU, GAC, and GAG, and the transfection steps were the same as in Example 2.

[0179] After 72h (48h after transfection), the sample was collected by TRIZOL and RNA was extracted (TRIzol Reagent, ambientREF15596026), and 1 μg of RNA was reverse-transcribed, and the reverse transcription system was 20 μL ( One-Step gDNARemoval and cDNA Synthesis SuperMix, full gold AT311-02), take 1 μL of the reverse transcription product and perform PCR with the following pair of primers: ggagtgagtacggtgtgcGACGAGCTGTACAAGCTGCAGGG (SEQ ID NO: 1), gagttggatgctggatggTGGTGCAGATGAACTTCAGGGTCAG (lowercase The letters indicate the primer adapters required by the Hi-Tom kit) for PCR amplification and library construction with the Hi-Tom kit (Novogene, REF PT045).

[0180...

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Abstract

The invention relates to a method of editing a target RNA at a position of a target residue in a host cell. The method comprises the steps: introducing a deaminase recruitment RNA (arRNA) or a construct coding the arRNA into the host cell, wherein the arRNA comprises a complementary RNA sequence that hybridizes to the target RNA, wherein the target residue is located in a tribase motif, the tribase motif comprises a 5'-nearest neighbor residue of the target residue in the target RNA (upstream residue), the target residue, and a 3'-nearest neighbor residue of the target residue in the target RNA (downstream residue), wherein the tribase motif is not UAG, and wherein the complementary RNA sequence comprises a mismatch directly opposed to the upstream or downstream residue of the target RNA. The invention also relates to the arRNA used in the method, RNA obtained through the RNA editing method, the host cell containing the RNA and application of the RNA editing method in treatment of diseases.

Description

technical field [0001] The invention belongs to the field of gene editing, in particular to the field of RNA editing, comprising introducing deaminase recruiting RNA (dRNA, also called arRNA) or a construct encoding the arRNA into host cells, and editing target RNA at the target residue position of the host cell . Background technique [0002] CRISPR technology [0003] In recent years, genome editing technology led by CRISPR (Clustered regularly interspaced short palindromicrepeats, WO2014018423A3) is developing rapidly, and has had a profound impact on many fields of biology and medicine. Many scientific researchers and biotechnology companies are also working to bring this technology to the clinic. In September 2019, Professor Deng Hongkui of Peking University and his collaborators published an article, reporting for the first time the results of clinical trials using CRISPR technology to edit stem cells and reinfuse them into patients to treat their AIDS and leukemia....

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/867C12N5/10
CPCC12N15/113C12N15/86C12N2310/10C12N2740/15043
Inventor 袁鹏飞易泽轩刘能银
Owner EDIGENE THERAPEUTICS (BEIJING) INC
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