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CasRx preparation for silencing target gene and application thereof

Pending Publication Date: 2021-03-02
深圳瑞吉生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the AAV virus has a wide range of infections and lacks targeting, which makes the CasRx system function in normal tissues other than target organs and target cells, resulting in adverse reactions
At the same time, when the adenovirus vector binds to the target cells of the body, it can stimulate the body's antigen-presenting cells to produce a specific immune response to non-self antigens, or promote the body's own natural immunity to the adenovirus vector, which will affect drug tolerance. , thereby affecting gene silencing and therapeutic efficacy

Method used

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  • CasRx preparation for silencing target gene and application thereof
  • CasRx preparation for silencing target gene and application thereof
  • CasRx preparation for silencing target gene and application thereof

Examples

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preparation example Construction

[0073] In the present invention, the method for preparing CasRx mRNA preferably includes: reacting the system containing the DNA template at 37° C. for 6 h to obtain CasRx mRNA; each 1600 μL of the system containing the DNA template preferably includes: 440 μL RNA-free water, 160μL of ATP at a concentration of 7.5mM, 160μL of UTP at a concentration of 7.5mM, 160μL of CTP at a concentration of 7.5mM, 160μL of GTP at a concentration of 7.5mM, 160μL of Cap analogue at a concentration of 7.5mM, and a DNA template at a concentration of 150ng / μL, 160μL 10×Buffer and 160μL Enzyme Mix. In the present invention, when the DNA does not have polyA, polyA is added to the obtained CasRx mRNA. The method of adding polyA to CasRx mRNA in the present invention is not particularly limited, and those skilled in the art can add polyA according to conventional methods.

[0074] In the present invention, the sgRNA is set according to the target gene, and the sgRNA guides the CasRx protein to bind t...

Embodiment 1

[0087] Application of mRNA encoding CasRx in hyperlipidemia

[0088] Cell level: About 24 hours after inoculation of Huh-7 cells, observe the state of the cells in the 6-well plate, and the confluence is about 90%. In a biosafety cabinet, prepare a required volume of 90% DMEM+10% FBS medium. The medium in the well plate was discarded 30 minutes before transfection, and 1 ml of fresh medium (90% MSCBM+10% FBS) was added to each well. Prepare the transfection system: Take 200μl opti-MEM, add 2μg CasRx mRNA and 1μg sgRNA, add only 2μg CasRx mRNA to the negative control group, gently blow and mix with the tip of the pipette, then add 60μl PEI (concentration 1mg / ml), and place immediately Oscillate 10 times on a vortex oscillator, 1s each time, mix well, and let stand for 10min. Add the prepared transfection system directly and evenly into the cultured cells, and then shake it back and forth, so that the transfection system is evenly distributed on the cells. The medium was chan...

Embodiment 3

[0117] Application of mRNA encoding CasRx in maculopathy

[0118] Cell level: About 24 hours after inoculation of N2a cells, observe the state of the cells in the 6-well plate, and the confluence is about 90%. In a biosafety cabinet, prepare a required volume of 90% DMEM+10% FBS medium. The medium in the well plate was discarded 30 minutes before transfection, and 1 ml of fresh medium (90% MSCBM+10% FBS) was added to each well. Prepare the transfection system: take 200 μl opti-MEM, add 2 μg of CasRx mRNA (SEQ ID No.3~18) and 1 μg of sgRNA, and only add 2 μg of CasRx mRNA in the negative control group, gently blow and mix with the pipette tip, and then add 60 μl of PEI ( Concentration 1mg / ml), immediately place on a vortex shaker and vibrate 10 times, 1s each time, mix well, and let stand for 10min. Add the prepared transfection system directly and evenly into the cultured cells, and then shake it back and forth, so that the transfection system is evenly distributed on the ce...

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Abstract

The invention provides a CasRx preparation for silencing a target gene and application thereof, and belongs to the technical field of gene engineering. The CasRx preparation comprises CasRx mRNA and sgRNA; the CasRx mRNA is used for encoding CasRx protein, and the amino acid sequence of the CasRx protein is shown as SEQ ID No. 1; the sgRNA is set according to the target gene. According to the invention, CasRx mRNA and sgRNA aiming at the target gene are directly introduced into a target cell so that the blank of a CasRx-based efficient directional gene silencing system in the field of mRNA preparations is filled up, the defects of an AAV virus-carried CasRx system are overcome, and the CasRx preparation is simple, rapid and high in specificity, expression quantity knock-down and RNA interference can be quickly, efficiently and specifically carried out on the target gene.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a CasRx preparation for silencing a target gene and an application thereof. Background technique [0002] In recent years, CRISPR / Cas9 technology has attracted widespread attention due to its powerful and convenient DNA editing capabilities. In 2016, Zhang Feng's laboratory discovered a new Cas protein, Cas13a, which can target RNA for cutting. Later, Cas13b and Cas13c targeting RNA were successively discovered. Due to the characteristics of the Cas13 family protein targeting RNA, it has unique advantages in the detection and treatment of some specific diseases in theory, so it has become a research hotspot in recent years. In 2018, the Cas13d family was discovered by Patrick Hsu's laboratory at the University of California, Berkeley. They found that Cas13d-mediated gene silencing had higher specificity (no off-targets for Cas13d compared to hundreds of...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/85
CPCC12N15/113C12N9/22C12N15/85C12N2310/141C12N2310/20
Inventor 张苗苗洪丹胡迅
Owner 深圳瑞吉生物科技有限公司