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SgRNA for beta-hemoglobinopathy gene editing and application

A technology for hemoglobinopathies and gene editing, which is applied to the sgRNA and application fields of gene editing for β-hemoglobinopathies, can solve the problems of high cost and low editing efficiency, and achieve the effects of cost reduction, improved gene editing efficiency, and improved gene editing efficiency

Pending Publication Date: 2021-05-04
GUANGZHOU REFORGENE MEDICINE CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] However, our study found that the editing efficiency of the CRISPR / Cas9 system targeting the GATA core element (target site BCL_01617) in the +58 enhancer region of the BCL11A gene provided by the Daniel Bauer group was relatively low, so the Daniel Bauer group used a higher dose of Cas9 protein + sgRNA complex achieves higher gene editing efficiency
Since the application of CRISPR / Cas9 system in therapy requires a large amount of high-purity Cas9 protein + sgRNA complex, the cost is very high

Method used

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  • SgRNA for beta-hemoglobinopathy gene editing and application
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  • SgRNA for beta-hemoglobinopathy gene editing and application

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Experimental program
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Effect test

Embodiment

[0038] The core sequence of BCL11A gene enhancer in 293FT cells was mutated by CRISPR-Sp Cas9 technology.

[0039] 1. Construct the sgRNA-spCas9 vector with optimized sgRNA structure.

[0040] On the basis of the sgRNA-spCas9 vector (PX459) we used, the original sgRNA expression cassette was replaced with a structurally optimized sgRNA expression cassette (such as figure 1 shown), to obtain the PX459-OP vector (attached figure 2 ), the base pairing number of the first complementary structural domain and the second complementary structural domain is increased to 16, and the sgRNA expressed by it contains SEQ ID NO: 1 (5'- UGCUG-3') or its antisense complementary sequence (5'-CAGCA-3').

[0041] The specific operation process is as follows:

[0042] 1) Using the PX459 carrier DNA as a template, using the sequences in the table below such as SEQ ID NO:7 and DNA of SEQ ID NO:10 as primers, PCR amplification to obtain product 1 (324bp), using the sequences in the table below su...

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Abstract

The invention relates to sgRNA for beta-hemoglobinopathy gene editing and application, and belongs to the technical field of gene editing. The sgRNA comprises a target domain, a first complementary domain, a connecting domain, a second complementary domain, a near-end domain and a tail domain from 5' to 3'; and the RNA sequence length of the target domain is 17-20 nt, and / or the number of base pairs between the first complementary domain and the second complementary domain is 14-18, wherein the number of base pairs close to the target domain is 6-8, and the number of base pairs close to the connecting domain is 8-10. The sgRNA disclosed by the invention has high gene editing efficiency, and the cost can be reduced on the premise of ensuring the high editing efficiency, so that the sgRNA is widely applied to preparation of medicines for treating beta-thalassemia or sickle cell anemia.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a sgRNA for gene editing of β-hemoglobinopathies and its application. Background technique [0002] Hemoglobin (Hb) is a special protein that carries and transports oxygen in red blood cells. Hemoglobin is composed of globin and heme. During embryonic development and shortly after birth, hemoglobin exists as fetal hemoglobin (HbF), a tetrameric protein consisting of two α-globin chains and two γ-globin chains. As infants develop, HbF is gradually replaced by adult hemoglobin (HbA), in which the gamma-globin chains of HbF are replaced by beta-globin chains through a process called globin turnover. HbF is more efficient than HbA at carrying oxygen, but the average adult's total hemoglobin contains less than 1% HbF. The α-globin gene is located on chromosome 16, while the β-globin gene (HBB), the γ(γA)-globin chain (HBG1, also known as γ-globin A) and the Gγ(γG)-globin chain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N5/10A61K48/00A61P7/06
CPCC12N15/113C12N5/0647A61K48/005A61P7/06C12N2310/20C12N2510/00
Inventor 梁峻彬古博徐辉陈昱
Owner GUANGZHOU REFORGENE MEDICINE CO LTD
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