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Preparation method of CRISPR-Cas13a system based on near-infrared light control and application of CRISPR-Cas13a system

A near-infrared light and gene technology, applied in the biological field, can solve the problems of complex operation, low inhibition efficiency, easy to produce off-target effects, etc., and achieve the effects of controllable efficiency, inhibiting gene expression level and wide application range.

Pending Publication Date: 2020-04-21
JIANGSU UNIV
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Problems solved by technology

In response to this problem, RNA interference, CRISPR-Cas9 and other technologies are used to elucidate gene function by inhibiting gene expression, but these methods have some shortcomings, such as complex operation, easy off-target effects, and low inhibition efficiency. Therefore, new gene expression inhibitors have been developed. method is imminent

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  • Preparation method of CRISPR-Cas13a system based on near-infrared light control and application of CRISPR-Cas13a system
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  • Preparation method of CRISPR-Cas13a system based on near-infrared light control and application of CRISPR-Cas13a system

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Embodiment Construction

[0024] In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

[0025] The method for suppressing eukaryotic gene expression based on the near-infrared light-controlled CRISPR-Cas13a system proposed by the present invention is applicable to all eukaryotic organisms. In this embodiment, only Schizosaccharomycespombe is used as an example to illustrate in detail.

[0026] A method for preparing a CRISPR-Cas13a system based on near-infrared light control proposed by the present invention, the specific process is as follows:

[0027] Material preparation process:

[0028] 1. Obtaining of LwCas13a protein:

[0029] like image 3 , using the pC013-Twinstrep-SUMO-h...

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Abstract

The invention discloses a preparation method of a CRISPR-Cas13a system based on near-infrared light control and application of the CRISPR-Cas13a system. The preparation method comprises the followingsteps: designing and synthesizing CrRNA according to the sequence of the mRNA of a target gene, allowing the crRNA and purified Cas13a protein to form a Cas13a-crRNA compound, and connecting the Cas13a-crRNA compound with an up-conversion nanomaterial through photosensitive molecules to obtain UCNPs-Cas13a; and coating the UCNPs-Cas13a with PEI to obtain UCNPs-Cas13a@PEI. The prepared UCNPs-Cas13a@PEI is attached to the surface of an eukaryotic cell and enters the cell, and the PEI of the UCNPs-Cas13a@PEI is used for promoting the escape of an endosome; the Cas13a-crRNA is released under the irradiation of an NIR laser; a complementary sequence corresponding to a crRNA spacer sequence is searched for on the target gene mRNA; and then, the crRNA is matched with the target gene mRNA to activate the HEPN catalytic site of Cas13a to shear mRNA, and then the expression of an eukaryote target gene is inhibited.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for preparing a CRISPR-Cas13a system based on near-infrared light control and its application. Background technique [0002] With the completion of whole-genome sequencing of food microorganisms such as yeast and mold, rapid and efficient analysis of gene functions has become a problem to be solved by scientific researchers. In response to this problem, RNA interference, CRISPR-Cas9 and other technologies are used to elucidate gene function by inhibiting gene expression, but these methods have some shortcomings, such as complex operations, easy to produce off-target effects, and low inhibition efficiency. Therefore, new gene expression inhibitors have been developed. The method is imminent. [0003] The simple, fast and efficient near-infrared light-controlled CRISPR-Cas13a system can remotely control the release of Cas13a-crRNA complexes from upconversion nanomaterials thr...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22
CPCC12N9/22C12N15/902C12N2310/20
Inventor 刘蕊陈全胜吕鹏黄栋李欢欢欧阳琴贺培欢许婧
Owner JIANGSU UNIV
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