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Novel CRISPR Cas13a-gRNA expression vector and application thereof

An expression vector, a new type of technology, applied in the biological field of cancer gene therapy, can solve the problem of knocking down the expression of tumor-promoting genes in cancer cells

Active Publication Date: 2020-06-19
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, it is still impossible to artificially control the expression of Cas13a-gRNA only in cancer cells, and this system has not been reported to be used to specifically knock down the expression of cancer-promoting genes in cancer cells

Method used

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  • Novel CRISPR Cas13a-gRNA expression vector and application thereof
  • Novel CRISPR Cas13a-gRNA expression vector and application thereof
  • Novel CRISPR Cas13a-gRNA expression vector and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment

[0055] pDCUg knocks down target gene expression in cancer cells in vivo and in vitro and inhibits tumor growth in vivo

[0056] 1. Materials and methods

[0057] 1.1, carrier structure

[0058] The chemically synthesized NF-κB specific promoter (DMP) was cloned into pMD19-T simple (TAKARA) to obtain pMD19-T-DMP. The human codon-optimized Cas13a coding sequence was amplified from pC013-Twinstrep-SUMO-huLwCas13a (Addgene) by PCR, and cloned into pMD19-T-DMP to obtain pMD19-T-DMP-Cas13a. The chemically synthesized U6 promoter sequence and the direct repeat sequence of the guide RNA (gRNA) separated by the BbsI restriction site were cloned into pMD19-T-DMP-Cas13a to generate the pDMP-Cas13a-U6-gRNA vector ( Referred to as pDCUg). Table 1 lists the genes targeted by gRNA and their target sequences.

[0059] Table 1. Target sequence of guide RNA in Cas13a-gRNA interference test

[0060]

[0061]

[0062] Note: PFS is the protospacer flanking site (the flanking site of the original spacer)...

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Abstract

The invention discloses a novel CRISPR Cas13a-gRNA expression vector and application thereof. The novel CRISPR Cas13a-gRNA expression vector is formed by respectively controlling expression of Cas13aand gRNA by virtue of an NF-kB specific promoter DMP and a U6 promoter; a gRNA 5' terminal of the Cas13a-gRNA expression vector is a 28nt sequence capable of targeting mRNA of a specific gene, and generated gRNA is capable of guiding binding of Cas13a proteins and cutting the mRNA of the specific gene, thereby causing expression inhibition of the specific gene. The constructed novel CRISPR Cas13a-gRNA expression vector is capable of specifically knocking down expression of cancer-promoting genes in cancer cells so as to cause growth inhibition and apoptosis of the cancer cells, but expressionand growth of target genes of normal cells are not influenced; and the expression vector can be applied to preparation of novel gene therapy reagents for cancers.

Description

Technical field [0001] The invention relates to the field of cancer gene therapy biotechnology, in particular to a novel CRISPR Cas13a-gRNA expression vector and its application in cancer cell growth inhibition. Background technique [0002] Cancer is an important disease that bothers human health and threatens human life. In the long-term struggle against cancer, humans have developed various cancer treatment methods such as surgery, chemotherapy, targeted therapy, and immunotherapy, and have also made significant progress, and the cancer survival rate has been significantly improved. However, the current level of cancer treatment is still far away from the expectations of patients for health and life. Therefore, actively developing new cancer treatment technologies is still the goal of continuous efforts in the medical field. Gene therapy is the cutting-edge field and technological highland of future medicine. The progress made by gene therapy in the field of genetic disease ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90A61K48/00A61P35/00
CPCC12N15/85C12N15/907C12N5/0693A61K48/005A61P35/00C12N2800/107C12N2830/34
Inventor 王进科高金良
Owner SOUTHEAST UNIV
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