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CRISPR/Cas9 gene vector as well as preparation method and application thereof

A gene carrier and construction method technology, applied in the biological field, can solve the problems of no genome integration ability, low transfection efficiency, complicated preparation process, etc., and achieve the effect of improving gene editing efficiency, high biological safety, and simple preparation method

Inactive Publication Date: 2021-01-22
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, lentiviral vectors tend to be inserted into high-expression regions of the host genome, and have strong carcinogenic and mutagenic properties, so clinical application has been discontinued.
At the same time, the targeting of viral vectors is not strong, the preparation process is complicated, and the defects of high cost also limit its clinical application.
Non-viral vectors have high biological safety and targeting, but have no genome integration ability, low transfection efficiency, and short expression time, which cannot meet the needs of CRISPR / Cas9 system delivery

Method used

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  • CRISPR/Cas9 gene vector as well as preparation method and application thereof
  • CRISPR/Cas9 gene vector as well as preparation method and application thereof
  • CRISPR/Cas9 gene vector as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Construction of "Sleeping Beauty" Transposon CRISPR / Cas9 Recombinant Plasmid

[0060] 1) Perform PCR amplification on the "Sleeping Beauty" transposon sequence in the plasmid pT2BH (purchased from addgene: #26556). The PCR reaction system was: 5 ng / μL pT2BH plasmid 6 μL, 5 μL 10×BeyoFusion Buffer (with Mg 2+ ), 10 μL of dNTPs (2.5 mM each), 2 μL of each primer (10 mM each), 1 μL of Fusion polymerase (2.5 U / μL), and 24 μL of sterile water.

[0061] The pT2BH primer sequence is as follows:

[0062] pT2BH-F: 5'-CCGGAATTCCGGGCTACCAAATACTAATTG-3' (SEQ ID NO.1)

[0063] pT2BH-R: 5'-TCCCCGCGGACAGTCAACTT-3' (SEQ ID NO.2)

[0064] The PCR reaction procedure is:

[0065] ①React at 92°C for 5 minutes; ②React at 92°C for 30 seconds; ③React at 56°C for 30 seconds; ④React at 68°C for 1 minute; ⑤Repeat steps ②~④ for 30 cycles;

[0066] After identification by agarose gel electrophoresis, the length of the "Sleeping Beauty" transposon sequence obtained by PCR amplificat...

Embodiment 2

[0090] Embodiment 2: Preparation of PDA / DEX-PEI@HA(PDPH) nanoparticles

[0091] Mix ammonia water (concentration 28%), absolute ethanol and deionized water evenly, the volume ratio of the three is 2:40:(90-180), and stir at 20°C-40°C for 30 minutes. Dissolve dopamine hydrochloride in deionized water, add dropwise to the above mixture solution (the final concentration of dopamine hydrochloride is 2-7mg / mL), stir and react at 20°C-40°C for 24 hours, and centrifuge the product at 13000rpm After 40-60 minutes, wash with water three times to obtain PDA nanoparticles. The PDA nanoparticles (3-6 mg) obtained above were dissolved in 10-20 ml of deionized water, and 2.5-5 mg of DEX powder was added, and stirred for 24 hours to obtain PDA / DEX. Add the resulting PDA / DEX dropwise to PEI 25K or PEI 10K In the aqueous solution (concentration 10mg / ml), the mass ratio of PDA / DEX to PEI is 1:(1~2), stirred for 24h, put the reaction mixture into a dialysis bag (MWCO=14000), and dialyzed in d...

Embodiment 3

[0093] Example 3: Preparation of PDPH / pT2SpCas9-Nanog transfection complex

[0094] Slowly add pT2SpCas9-Nanog into PDA / DEX-PEI@HA, the mass of the two is 1: (5-30), vortex, and incubate at room temperature for 30 minutes.

[0095] Figure 6(a) to Figure 6(e) It is the infrared spectrogram of the PDA / DEX-PEI@HA prepared in Example 2 of the present invention, Figure 6(a) is PDA, Figure 6(b) is DEX, Figure 6(c) is HA, Figure 6(d) for PDPH 10K , Figure 6(e) is PDPH 25K . The spectrum shows CH 2 The characteristic peak position of the skeleton is 2924cm -1 and 2890cm -1 , the characteristic peak position of the C-O-C group in hyaluronic acid is 1151cm -1 About, in the above two different molecular weights (PDPH 25K and PDPH 10K ) are reflected in, that is, the successful modification of DEX, PEI, and HA is verified.

[0096] Figure 7(a) to Figure 7(c) For the prepared PDPH in the embodiment of the present invention 3 25K and PDPH 10K TEM image of the complex with DNA...

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Abstract

The invention provides a CRISPR / Cas9 gene vector and a preparation method and application thereof. The CRISPR / Cas9 gene vector is surface-modified polydopamine nanoparticles and comprises polydopamine, polyethyleneimine, dexamethasone and hyaluronic acid, wherein the surface of polydopamine is modified with polyethyleneimine, dexamethasone and hyaluronic acid, dexamethasone is loaded on the surface of polydopamine through pi-pi conjugation, polyethyleneimine is modified on the surface of polydopamine, and hyaluronic acid acts on the surface of polydopamine through electrostatic adsorption. TheCRISPR / Cas9 gene vector is high in transmission efficiency, low in toxicity and good in biological safety, the preparation method is simple, conditions are mild, and the CRISPR / Cas9 gene vector has wide application prospects.

Description

technical field [0001] The invention belongs to the field of biotechnology, and provides a CRISPR / Cas9 gene carrier, its preparation method and application. [0002] technical background [0003] The occurrence of many major human diseases is related to gene defects, including most genetic diseases, sickle cell anemia, diabetes, tumors, etc. At present, traditional chemotherapy or surgical treatment still cannot effectively cure these major diseases, and gene therapy is considered to be an effective means to treat these major diseases. CRISPR / Cas9 gene editing technology has become a research hotspot for the treatment of genetic diseases due to its advantages of efficient targeting and easy operation. [0004] There are two types of gene vectors commonly used: viral vectors and non-viral vectors. At present, the delivery of CRISPR / Cas9 system mostly uses lentiviral vectors to improve the transfection efficiency of genes, so as to ensure that the CRISPR / Cas9 system can achie...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/55C12N15/87C08G81/00
CPCC12N15/113C12N9/22C12N15/87C08G81/00C12N2310/20
Inventor 马昆李文哲朱光池浩尹雅琳许建强郭兆明王黎崔昌浩
Owner DALIAN UNIV OF TECH
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