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Kit for editing or repairing HBB gene

A kit and gene technology, applied to other methods of inserting foreign genetic materials, DNA/RNA fragments, stably introducing foreign DNA into chromosomes, etc., can solve problems that affect the process of clinical transformation and application, and achieve the goal of improving gene editing tropism and target gene cleavage efficiency, strong targeting, and the effect of reducing gene mutations

Active Publication Date: 2018-01-26
SHENZHEN WINGOR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, as a gene editing technology, off-target effects are an inevitable shortcoming, which affects the process of its clinical translation application

Method used

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  • Kit for editing or repairing HBB gene
  • Kit for editing or repairing HBB gene
  • Kit for editing or repairing HBB gene

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0052] Verify the cutting efficiency of the kit for the HBB gene

[0053] 1. Modification of SpCas9 protein

[0054] The 848th lysine of SpCas9 protein (GI: 81533697) was mutated into alanine, the 1003rd lysine was mutated into alanine and the 1060th arginine was mutated into alanine, and the obtained The modified SpCas9 protein was named as SpCas9-SZ1 protein.

[0055] The 848th lysine of SpCas9 protein (GI:81533697) was mutated into alanine, the 925th arginine was mutated into proline, the 1003rd lysine was mutated into alanine and the 1060th Arginine was mutated into alanine, and the resulting transformed SpCas9 protein was named SpCas9-SZ2 protein.

[0056] 2. Construction of pSpCas9-SZ(BB) vector

[0057] Mutate the SpCas9 coding sequence in the pSpCas9(BB) (Addgene plasmaid ID: 42230) vector to encode the SpCas9-SZ1 enzyme described in step 1, and the new vector is named pSpCas9-SZ1 vector.

[0058] Mutate the SpCas9 coding sequence in the pSpCas9(BB) (Addgene plasmi...

experiment example 2

[0098] Verify the off-target efficiency of the kit

[0099] 1. Compare the sequence of HBB-sgRNA4-S with the human genome sequence, and select the closest target ( Figure 4 A), we design specific primers for these target regions for PCR amplification, and then sequence the amplified products. The sequencing results show that the psgRNA4-HBB-SpCas9-SZ1 vector has no cuts at these sites ( Figure 4 B). That is, the psgRNA4-HBB-SpCas9-SZ1 vector constructed using the sgRNA sequence provided by the kit of the present invention and the modified Cas9 protein has no off-target, strong specificity, and high safety.

experiment example 3

[0101] Verify the gene recombination efficiency of the kit

[0102] 1. Verify the driving efficiency of the EF1mini promoter

[0103] Adeno-associated virus is one of the most commonly used viral vectors in gene therapy, but its loading capacity is limited, which limits its packaging of CRISPR-Cas9 system for future applications. For this reason, we modified the promoter that starts Cas9, and adopted a new type of EF1mini promoter for our system. The size of the mini-promoter is only 500bp, and its nucleotide sequence is shown in Seq ID NO.16, which can well drive the expression of Cas9.

[0104] (1) Synthesize the EF1mini promoter by chemical synthesis method, and connect it into the psgRNA-HBB-SpCas9-SZ vector. In order to verify the activity of the EF1mini promoter, the promoter was first used to drive the expression of the reporter gene EGFP, and the pAAV-sgRNA4-HBB-EF1mini-EGFP vector was constructed. The vector structure diagram is shown in Figure 5 a.

[0105] (2) ...

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Abstract

The invention relates to a kit and particularly relates to a kit for editing or repairing human hemoglobin gene. The kit comprises sgRNA of specific target HBB gene or a carrier containing the nucleotide sequence of the sgRNA; and / or modified Cas9 enzyme or a sequence encoding the modified Cas9 enzyme or a carrier containing the sequence encoding the modified Cas9 enzyme; and / or a donor gene sequence for repairing the HBB gene of beta-thalassemia or a carrier containing the donor gene sequence. The invention also provides an application of the kit in cutting or repairing the HBB gene, especially an application in repairing the HBB gene of autologous hematopoietic stem cells of a patient with beta-thalassemia. The kit provided by the invention has the advantages that the targeting capability of the target HBB gene is strong, the cutting efficiency of the HBB gene is high, the off-target efficiency is low, the HBB gene can be effectively repaired and the prospect in clinical study and treatment application is wide.

Description

technical field [0001] The present invention relates to a kit, in particular to a kit for editing or repairing HBB gene. Background technique [0002] Thalassemia (Thalassemia, referred to as thalassemia) is also known as thalassemia. It is a common clinical hereditary hemolytic anemia. It is more common in Southeast Asia, the Mediterranean and other regions. Because it was first discovered in Italy, Greece, Malta and other regions along the Mediterranean Sea, it is called thalassemia. [0003] Thalassemia is an inherited hemoglobinopathy, which is a hemolytic anemia in which the synthesis ratio of α-chain and β-chain globin constituting hemoglobin is unbalanced and the life span of red blood cells is shortened due to the deletion or mutation of genes that regulate globin synthesis. [0004] According to the classification of gene defects, it is mainly divided into α-streptoglobin thalassemia and β-streptoglobin thalassemia clinically. β-thalassemia is a disorder of β-glo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N9/22
CPCC12N9/22C12N15/113C12N15/90
Inventor 张芸姜舒纪惜銮刘婕郭明罗朝霞杨顺
Owner SHENZHEN WINGOR BIO TECH
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