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RNA fixed-point editing by artificially constructing RNA editing enzyme and related application

An editing enzyme and editing technology, applied in the field of RNA-directed editing and related applications, can solve the problems of difficult transfer of genomic loci, low-efficiency gene introduction, and long-term safety concerns of genomic DNA editing.

Inactive Publication Date: 2020-10-20
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) The current CRISPR-mediated base editing system is not accurate enough, and the efficiency of single base editing is low. There is generally an editing window, and additional mutations will be introduced when editing the target site
[0005] (2) The CRISPR system is very large, and the current gene transfer technology is difficult to wrap the entire CRISPR system in the same system at one time for delivery, so there are inefficient gene transfer and low editing efficiency during the treatment process
[0006] (3) Limited by the size of the transgene, some genomic sites are difficult to transfer
[0009] Not least, long-term safety concerns with genomic DNA editing have been a major concern since genomic DNA changes are present throughout the cell's lifetime

Method used

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  • RNA fixed-point editing by artificially constructing RNA editing enzyme and related application
  • RNA fixed-point editing by artificially constructing RNA editing enzyme and related application
  • RNA fixed-point editing by artificially constructing RNA editing enzyme and related application

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Experimental program
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Effect test

Embodiment 1

[0226] ADAR is an adenosine editing enzyme that can catalyze the deamination of RNA adenosine to form inosine (adenosine-to-inosine, A-to-I). Three ADAR catalytic domains from different sources were cloned, and the linker and RNA binding protein PUF fusion, developed three different artificial RNA editing enzymes (RNA editase), named the system as artificial P UF- A DAR R NA s sequence e ditors(PARSE-d,PARSE1,PARSE2)( figure 1 )

[0227] Detect the editing activity of the three RNA editing enzymes PARSE-d, PARSE1 and PARSE2 at the cellular level, transfer the PARSE RNA editing enzyme and the GFP reporter gene plasmid into the cells at the same time, collect the RNA of the GFP reporter gene after 48 hours and sequence it, and detect RNA editing events , proving that PARSE-d, PARSE1, and PARSE2 all have the ability to efficiently edit target RNA ( figure 2 A). By constructing a GFP mRNA containing an early stop codon, PARSE is used to realize the editing of A to G at a s...

Embodiment 2

[0230] The editing efficiency of ADAR on target RNA is improved by optimizing the ADAR catalytic domain, and the efficiency, precision and off-target rate of RNA editing enzymes are detected and analyzed by RNA seq high-throughput sequencing technology. A new point mutation was introduced in each of the catalytic domains of ADAR1 and ADAR2 to improve the editing efficiency of ADAR ( Figure 4 A), using RNA seq high-throughput sequencing technology to detect and analyze the efficiency and accuracy of RNA editing enzymes, and found that PARSE1, ePARSE1, PARSE2, and ePARSE2 can all edit target RNA, with efficiencies of 42%, 65%, and 67% respectively , 78% ( Figure 4 B). The analysis of RNA-seq results shows that there is a certain degree of off-target rate in PARSE editing, but compared with the editing efficiency of the target site, the off-target efficiency is lower, and the off-target rate can be reduced by reducing the transfection amount of PARSE in the later optimization ...

Embodiment 3

[0234] Optimize the RNA-binding protein PUF to improve the precision of PARSE editing RNA and reduce off-target efficiency. By optimizing PUF, PUF8, which recognizes 8 bases, is optimized to PUF10, which recognizes and binds to ten bases ( Figure 7 ), did not reduce the editing efficiency at the target site, but as the number of PUF recognition and binding bases increased, we believe that this strategy can effectively reduce off-target effects, and has the potential for further optimization, we can optimize PUF to recognition And combining 12 bases, 16 bases, or even longer, can greatly expand the scope of application of PARSE.

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Abstract

The invention provides RNA fixed-point editing by artificially constructing an RNA editing enzyme and related application. Specifically, the invention provides a new functional protein formed by fusing an RNA recognition domain for combining RNA and an effector domain for exerting functions, and the new functional protein specifically targets target RNA through the recognition domain and performsRNA editing by using the effector domain. The RNA fixed-point editing can be used for performing gene editing at an RNA level, thereby eliminating pathogenic RNA to correct pathogenic error point mutation; or the RNA fixed-point editing can be used for editing expressed RNA molecules to perform specific control on gene functions.

Description

technical field [0001] The invention belongs to the field of biology, in particular, the invention relates to the use of artificially constructed RNA editing enzymes for RNA fixed-point editing and related applications. Background technique [0002] DNA is the main genetic material in living organisms. At present, a large part of known human diseases are caused by genetic mutations, and single-base mutations are the largest category. Therefore, it is of great significance to develop a method to change the sequence of a single base in the genome and efficiently and accurately repair the disease-causing single base mutation for the research and treatment of genetic diseases. [0003] The current gene therapy strategies for single base mutation diseases are mainly strategies for treating diseases by directly repairing or replacing mutant genes at the DNA or RNA level. The main methods are the DNA-based base editing systems ABE and CBE based on CRISPR technology. These technol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N9/22C12N15/55C12N15/90
CPCC12N15/113C12N9/22C12N15/902C12N2310/10C12N2310/20C12N15/102C07K2319/00C07K2319/85C12N9/78A61K38/50A61K48/00C12N15/62C12N15/85C12Y305/04
Inventor 王泽峰韩文建
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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