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Nucleic acid molecules and kit by combining to mutant RHO genes

A nucleic acid molecule and kit technology, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., to achieve the effect of improving targeting efficiency

Active Publication Date: 2020-11-13
CHIGENOVO CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the maximum loading capacity of AAV vectors is 4.7 kb. Usually, only sgRNA and Cas9 can be packaged separately

Method used

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  • Nucleic acid molecules and kit by combining to mutant RHO genes
  • Nucleic acid molecules and kit by combining to mutant RHO genes
  • Nucleic acid molecules and kit by combining to mutant RHO genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0129] Example 1. Collecting information of gene diagnosis of patients

[0130] In this study, in recent years, more than 1,000 RP patients who have been treated in Peking University Third Hospital have collected a number of genes with definite diagnosis. 希腊字母的第17字 Mutated RP family, found 希腊字母的第17字 See table 2 below for two mutation hotspots of the gene.

[0131] Table 2 希腊字母的第17字 Hot spot of mutation

[0132]

Embodiment 2

[0133] Example 2 design of sgrna and construction of pX601-SgRNA plasmid

[0134] According to the above two mutation sites, five sgRNA were designed for SaCas9 system using Benchling website, and the nucleotide sequences are shown in Table 3 below.

[0135] Table 3 SgRNA sequence

[0136]

[0137] The targeting vector used in this application is: PX601-AAV-CMV:: NLS-SACAS9-NLS-3xha-bGHPA; U6::BsaI-sgRNA, with the map as shown in 图一 Shown in (see https: / / www.addgene.org / 61591 / for carrier information).

[0138] aim at 希腊字母的第17字 Schematic diagram of sgRNA designed by p.Thr17Met is as follows 图2 Shown, for 希腊字母的第17字 Sequence diagram of coding gRNA designed by p.Arg135Trp is as follows 图3 Shown.

[0139] The specific steps of plasmid construction are as follows:

[0140] (1) annealing of 1)SgRNA

[0141] Melt T4 PNK and 10X T4 Ligation Buffer on ice for later use. The following reaction system was prepared:

[0142] Table 4 reaction system

[0143]

[0144] Put the prepared react...

Embodiment 3

[0187] Example 3 detection of sacas9-sgrna target efficiency detection kit 希腊字母的第17字 In vitro editing efficiency of each sgRNA

[0188] The specific experimental steps are as follows:

[0189] (1) in vitro transcription of 1)sgRNA:

[0190] 1) primer design of 1)SgRNA transcription in vitro

[0191] Table 9 Primer sequences of SGRNA transcription in vitro

[0192]

[0193] 2) Construction of in vitro transcription template of 2)sgRNA

[0194] PCR reaction system is as follows:

[0195] Table 10 Reaction system

[0196]

[0197]

[0198] 3) Use 2.0% DNA gel to run gel, and use OMEGA gel recovery kit to recover gRNA in vitro transcription template gel. The recovery steps are as follows:

[0199] A) adding equal volume of membrane binding solution into the PCR reaction product, adding 1l of membrane binding solution for every 1mg of gel cutting and recycling, heating at 50-60℃ for 7min until all gels are completely dissolved, vortexed and evenly mixed, and passing through the col...

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Abstract

The invention relates to a kit, which comprises 1) a vector, 2) a pharmaceutically acceptable carrier, wherein the vector comprises a nucleic acid molecule capable of encoding sgRNA that specificallybinds to an RHO gene comprising a mutation site selected from c.C50T and c.C403T. The invention also relates to a method for treating retinal pigment degeneration by using the kit.

Description

Technical field [0001] The application relates to the field of biomedicine, in particular to a pharmaceutical composition for China. 希腊字母的第17字 -Gene editing kit for -adRP patients based on CRISPR / Cas9 technology and AAV technology. technical background [0002] At present, Retinitis Pigmentosa (RP) is a group of hereditary blinding eye diseases whose main changes are the gradual loss of photoreceptor cells and / or retinal pigment epithelial cells. At present, there is no effective treatment (traditional medicine and surgical treatment are basically ineffective). In recent years, the rapid development of CRISPR / Cas9 technology has brought the dawn for gene therapy of RP. CRISPR / Cas9 technology is the most commonly used gene editing technology and one of the main tools for gene therapy of hereditary retinal degeneration. 希腊字母的第17字 It is the earliest found gene of RP, about 30%-40% of autosomal dominant RP (adRP) is caused by this gene, and it is the most important pathogenic gene of...

Claims

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Application Information

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IPC IPC(8): C12N15/864C12N15/113C12N15/55
CPCC12N9/22C12N15/113C12N15/86C12N2750/14143C12N2310/20
Inventor 杨丽萍柳小珍乔静张凡张天赋和赛超曾露颖裴红杰
Owner CHIGENOVO CO LTD
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