gRNA sequence of targeted editing bcr-abl fusion gene and application thereof

A technology of bcr-abl and fusion genes, which is applied in the field of targeted editing of gRNA sequences of bcr-abl fusion genes, which can solve problems such as limited application and poor specificity

Pending Publication Date: 2019-07-02
CHONGQING MEDICAL UNIVERSITY
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  • Claims
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AI Technical Summary

Problems solved by technology

Compared with ZFNs and TALENs, CRISPR / Cas9 has the advantages of low requirement for target gene sequence characteristics, simple assembly, high modification rate, and convenient switching for different sites. However, the relatively poor specificity of CRISPR / Cas9 limits its use in Applications in the field of gene therapy

Method used

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  • gRNA sequence of targeted editing bcr-abl fusion gene and application thereof
  • gRNA sequence of targeted editing bcr-abl fusion gene and application thereof
  • gRNA sequence of targeted editing bcr-abl fusion gene and application thereof

Examples

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Embodiment 1

[0038] Example 1 Design and Construction of gRNA Expression Plasmid for Specific Target Site Sequence of Human bcr-abl Gene (1) According to the gene sequence of human bcr-abl gene, we designed 3 pairs of gRNA and 1 pair of gRNA- At half-site, corresponding oligonucleotides were synthesized and corresponding gRNA expression plasmids and donors were constructed. The sequences are shown in Table 1, and the corresponding vector plasmid is PSQT1313 (Addgene #53370). The expression plasmid of FokI-dCas9 is PSQT1601 (Addgene #53369).

[0039] Table 1 oligo synthesis sequence of gRNA expression plasmid

[0040]

[0041]

[0042] (2) The 3 long fragments of Sangong Synthesis were cloned into the PCDH-CMV-MCS-EF1-copGFP vector, and the 338 bp near the target sequence of the K562 genomic DNA was used as a template to insert the 8-base NotI restriction site (GCGGCCGC) for a total of 346bp. The double-stranded PCR product of the target fragment was amplified with the upstream primer...

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Abstract

The invention provides a gRNA sequence of a targeted editing bcr-abl fusion gene and an application thereof. By location of three specific target sequence site DNA fragments in a bcr-abl gene, a gRNAexpression plasmid is constructed for a specific site, the plasmid and a FokI-dCas9 expression plasmid and a donor nuclear are transfected into a K562 cell, so that the bcr-abl gene is subjected to fixed-point fracture, homologous repair is performed by using a donor DNA as a template, and 8 basic groups are inserted to ultimately lead to disruption of the bcr-abl gene. Significant advantages of the sequence and the application thereof are that RFNs constructed for the pecific target sequence site in a bcr-abl sequence can efficiently target the bcr-abl gene to cause occurrence of DNA double-strand break, repair is performed in a HDR manner to enable bcr-abl to undergo mutation such as frame shift and to be destroyed, thereby losing potential for malignant transformation such as proliferation and apoptosis suppression.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gRNA sequence for targeted editing of a bcr-abl fusion gene and an application thereof. Background technique [0002] Chronic myeloid leukemia (CML) is a type of malignant clonal proliferative disease originating from hematopoietic stem cells. The root cause of the disease is the bcr-abl fusion gene formed by the balanced translocation of chromosome 9 and chromosome 22. The fusion gene can encode a BCR-ABL fusion protein with strong tyrosine kinase activity, which continuously activates downstream JAK-STAT, MEK-ERK, CRKL and other signaling pathways, promotes malignant cell proliferation and inhibits apoptosis. The clinical use of tyrosine kinase inhibitors (tyrosine kinase inhibitors, TKIs) in the treatment of CML has achieved good results, but more than 25% of patients are still resistant to TKIs, and the insensitivity of CML stem cells to TKIs is one ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/85C12N15/90A61K48/00A61P35/02
CPCC12N15/113C12N15/102C12N15/85C12N15/907A61K48/005A61P35/02C12N2310/10C12N2310/20C12N2800/107C12N2810/10
Inventor 黄峥兰骆贞红高淼冯文莉
Owner CHONGQING MEDICAL UNIVERSITY
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