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Near-infrared light controlled gene editing method

A technology of gene editing and infrared light, applied in the biological field, can solve problems such as poor penetration ability and canceration, and achieve the effect of improving targeting

Active Publication Date: 2019-07-05
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the penetration ability of ultraviolet light organisms is relatively poor and long-term exposure can easily cause cancer

Method used

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  • Near-infrared light controlled gene editing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] This embodiment provides the preparation method of UCNPs-Cas9@PEI:

[0036] UCNPs@SiO2 (100 mg) was carboxylated with 20 μL N-trimethoxysilylpropylethylenediaminetriacetic acid trisodium salt for 4.5 h. The carboxylated nanoparticles were then transferred to 10 mL (containing 100 μg ONA) of dry THF for esterification using dicyclohexylcarbodiimide and 4-dimethylaminopyridine as catalysts. After 12 hours, wash with THF 3 times, and then dissolve 20 μg of dicyclohexylcarbodiimide in THF to further activate the carboxyl group. After 8 hours, the particles were collected and transferred into PBS. At the same time, mix Cas9 protein and sgRNA (1:2) in PBS for 5 minutes to form a CRISPR / Cas9 system. Then, the CRISPR / Cas9 system was mixed with carboxylated nanoparticles-ONA (i.e. the aforementioned ligation product of carboxylated nanoparticles and ONA), and o C incubated overnight. After centrifugation, PEI was coated on UCNPS-Cas9 at a weight ratio of 5:1 (PEI:sgRNA), and...

Embodiment 2

[0038] This embodiment verifies the near-infrared response of the system, the method is as follows:

[0039] Using a collimator, fix the light intensity of the 980 nm near-infrared laser at 2 W / cm 2 . Put the UCNPs-Cas9 solution under the light intensity, centrifuge every 5 minutes to obtain the supernatant, and measure the UV absorption peak at 280 nm (common protein absorption peak), the results are as follows image 3 As shown in A, the absorption peak increases with time, which means that the protein is detached from the particle. In addition, in order to verify the role of upconversion nanoparticles in converting infrared light into ultraviolet light, the UCNPs-Cas9 solution can be exposed to ultraviolet light, centrifuged every 2.5 minutes to obtain the supernatant to measure the 280 nm absorption peak, and similar results were obtained Such as image 3 b.

Embodiment 3

[0041] The nanocomplex of test embodiment 1 (sgRNA targeting EGFP Genes whose targeting sequences are:

[0042] 5'-GGGCGAGGAGCTGTTCACCG-3' (SEQ ID NO.1)) cytotoxicity, the method is as follows:

[0043] Select lung cancer cells (A549) and irradiate them under different intensities of infrared light for 10 minutes. According to the results obtained by the CCK-8 detection method, 0.5~2.5 W / cm 2 Infrared light does not have much effect on cell activity ( Figure 4 A). Subsequently, at 2.0W / cm 2 Under the infrared light intensity, different concentrations of UCNPs-Cas9@PEI complexes were selected to incubate with cells, and it was found that the toxicity of nanoparticles to cells increased as the concentration increased ( Figure 4 B). Finally, the effect of different light durations on cell activity was investigated. In order to reduce the toxicity of the UCNPs-Cas9@PEI complex itself and infrared light conditions on cells, in subsequent cell experiments, 2.0 W / cm 2 The inf...

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Abstract

The invention discloses a near-infrared light controlled gene editing method. A UCNPs-Cas9 complex is obtained by covalent connection of carboxylated UCNPs@SiO2 with a CRISPR / Cas9 system under the action of 4-hydroxymethyl-3-nitrobenzoic acid, and after coating with PEI, nanoparticles are subjected to endocytosis; then, under infrared light irradiation, up-conversion nanoparticles emit ultravioletlight to disconnect Cas9 protein with the up-conversion nanoparticles, so that the protein is released to enter the cell nucleus to realize gene editing. Due to strong tissue penetration of infraredlight, the near-infrared light controlled gene editing method is evidently advantageous in in-vivo application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and discloses a method for regulating gene editing by near-infrared light. Background technique [0002] Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) technology is currently the most popular gene editing tool. ) or homology-directed repair (HDR) to correct gene mutations. However, the delivery of the CRISPR / Cas9 system has been problematic due to the simultaneous need to deliver two large molecules: the Cas9 protein (about 160 kd) and the sgRNA (over 100 bp), especially the precise control of the CRISPR / Cas9 system in space and time. in terms of delivery. In recent decades, on-demand delivery systems based on the design of stimuli-responsive nanomaterials have received extensive attention in nanomedicine. Among various control strategies, photomodulation has proven to be an ideal non-invasive way. By light, the release of bioactive molecules can b...

Claims

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Application Information

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IPC IPC(8): C12N15/90
CPCC12N15/907
Inventor 宋玉君潘永春
Owner NANJING UNIV
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