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PDA (at) CRISPR/Cas9/sgHMGA2 NPs complex, preparation method and application

Technology of a complex, sghmga2, applied in the biological field

Pending Publication Date: 2022-05-03
内蒙古自治区人民医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there has been no report on PDA as a carrier to deliver Cas9 RNP complexes

Method used

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  • PDA (at) CRISPR/Cas9/sgHMGA2 NPs complex, preparation method and application
  • PDA (at) CRISPR/Cas9/sgHMGA2 NPs complex, preparation method and application
  • PDA (at) CRISPR/Cas9/sgHMGA2 NPs complex, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] CRISPR / Cas9 -3NLS Preparation of endonuclease: In the Cas9 coding region of the backbone vector pET28a / Cas9-Cys (Addgene, Plasmid 53261) (before the nucleotide sequence encoding the SV40 nuclear localization signal sequence (NLS) (PKKKRKV)) additionally introduce two coding The nucleotide sequence of SV40 NLS, finally obtained the recombinant prokaryotic expression vector containing the nucleotide sequence of 3 SV40 NLS, named pET28a / Cas9-N3. The specific operation steps are as follows: according to the pET28a / Cas9-Cys vector sequence, Design specific primers N3-F and N3-R as shown in SEQ ID NO.:1, SEQ ID NO.:2: wherein the 5' end of N3-F contains a nucleotide sequence that can encode an NLS, N3-R The 5' end of contains nucleotide sequences encoding two NLSs. Use the above N3-F / -R primers and high-fidelity enzyme POD-plus-Neo (1U / ul) to perform PCR amplification on 100ng substrate pET28a / Cas9-Cys (conditions are as follows), purify and recover after agarose gel electro...

Embodiment 2

[0066]sgHMGA2, CRISPR sgRNA targeting HMGA2: CRISPR / Cas9 as shown in SEQ ID NO.:4, SEQ ID NO.:5 and SEQ ID NO.:6 was designed for the HMGA2 gene fragment shown in SEQ ID NO.:3 Target sequence, followed by synthesis of the linkers shown in SEQ ID NO.:7, SEQ ID NO.:8, SEQ ID NO.:9, SEQ ID NO.:10 and SEQ ID NO.:11, SEQ ID NO.:12 The target sequence and its complementary sequence were annealed to obtain three CRISPR sgRNA double-stranded DNA fragments as insert fragments, which were recombined with the DR274 vector treated with endonuclease (preferably BbsI endonuclease) to form three Recombinant prokaryotic expression plasmids targeting different sites of the HMGA2 gene: DR274-sgHMGA2-1, DR274-sgHMGA2-2 and DR274-sgHMGA2-3; the above three plasmids were digested with restriction endonuclease (DraI), The digestion product containing the T7 promoter and the CRISPR sgRNA targeting HMGA2 was recovered by electrophoresis and used as a template. After in vitro transcription and extract...

Embodiment 3

[0068] Preparation method of PDA@CRISPR / Cas9 / sgHMGA2 NPs complex:

[0069] S1, CRISPR / Cas9 -3NLS preparation of

[0070] S1.1. According to the pET28a / Cas9-Cys carrier sequence, design specific primers N3-F as shown in SEQ ID NO.:1, and design specific primers N3-R and N3-F as shown in SEQ ID NO.:2 The 5' end of N3-R contains a nucleotide sequence that can encode one NLS, and the 5' end of N3-R contains a nucleotide sequence that can encode two NLSs;

[0071] S1.2. Use N3-F and N3-R primers and high-fidelity enzyme POD-plus-Neo to perform PCR amplification on the substrate pET28a / Cas9-Cys;

[0072] S1.3. Purify and recover the target PCR product after agarose gel electrophoresis;

[0073] S1.4. After phosphorylation and self-ligation, the PCR product was subcloned into Escherichia coli DH5α, and then colony PCR and sequencing were performed to identify and screen the vector containing the nucleotide sequences encoding three NLSs;

[0074] S1.5. Using competent cell Rosetta...

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Abstract

The invention discloses a PDA (at) CRISPR / Cas9 / sgHMGA2 NPs complex as well as a preparation method and application thereof.The complex is a nano-particle formed by wrapping a CRISPR / Cas9 ribonucleoprotein complex with polydopamine, the complex efficiently and safely transports the CRISPR / Cas9 ribonucleoprotein complex into cells by utilizing the biomolecular transport function and good biocompatibility of the polydopamine, and the PDA (at) CRISPR / Cas9 / sgHMGA2 NPs complex has the advantages of being capable of effectively and safely transporting the CRISPR / Cas9 / sgHMGA2 NPs complex into cells. According to the present invention, the target gene HMGA2 can be efficiently edited, the complex can achieve the efficient delivery of the CRISPR / Cas9 gene editing system and the efficient targeting knockout of the cancer cell genome HMGA2, and the complex can be used for developing the accurate targeting treatment method of the CRISPR / Cas9 gene editing system, and can be researched as the novel targeting treatment means for the HMGA2 high expression type cancer; the PDA-coated CRISPR / Cas9 gene editing system is used for revealing the function of the CRISPR / Cas9 gene editing system in the HMGA2 high-expression cancer treatment process, and a preclinical data basis is provided for precise targeted biological treatment of HMGA2 high-expression cancer patients through the PDA-coated CRISPR / Cas9 gene editing system.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a PDA@CRISPR / Cas9 / sgHMGA2 NPs complex, a preparation method and an application. Background technique [0002] High mobility gene A2 (HMGA2) is a non-histone chromosomal protein encoded by HMGA2. Its role is to regulate transcription by affecting chromatin structure. Under physiological conditions, HMGA2 is highly expressed during embryogenesis, and is basically not expressed or expressed at a very low level in normal adult tissues; under pathological conditions, it is re-expressed in various types of cancer tissues, so it is speculated that HMGA2 plays an important role in the process of carcinogenesis. Indispensable. It has been reported that HMGA2 can regulate tumorigenesis, metastasis and recurrence by participating in apoptosis, cell cycle, angiogenesis, epithelial-mesenchymal transition (EMT) and drug resistance. The feature of HMGA2 re-expression in cancer tissue...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N15/113C12N11/089C12N15/70C12N1/21C12N15/10A61K48/00A61P35/00C12R1/19
CPCC12N9/22C12N15/113C12N11/089C12N15/70C12N15/10A61K48/005A61P35/00C12N2310/20C12Q2525/191C12Q2565/125Y02A50/30
Inventor 俞兰武洲英霍雪武婷冯宗琪王敏李凤
Owner 内蒙古自治区人民医院
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