Epidermal stem cell RAB23 gene knockout through CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) system

A technology of epidermal stem cell and gene editing, applied in the field of RAB23 gene editing, can solve the problem of siRNA not stable inheritance, etc., and achieve the effect of high knockout efficiency, stable passage, and strong knockout effect

Active Publication Date: 2018-11-27
浙江玉安康瑞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a kind of knockout RAB23 gene epidermal stem cells, which effectively overcomes the technical defect that the prior art uses siRNA for interference and cannot stably inherit

Method used

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  • Epidermal stem cell RAB23 gene knockout through CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, construction of CRISPR expression vector

[0023] gRNA design

[0024] According to the gene sequence of the target gene, the form of the specific sgRNA obtained through the applicant's optimization design and specific screening from dozens of gRNAs in the early stage is as follows:

[0025] RAB23-sgRNA1: 5'to 3'attacaaaggcctactatcg

[0026] RAB23-sgRNA2: 5'to 3'agtcactccggtcagaattc.

[0027] According to the above gRNA, add CACC to its 5' end to obtain the forward oligonucleotide sequence, add AAAC to the 5' end of its complementary strand to obtain the reverse oligonucleotide sequence, and synthesize forward and reverse oligonucleotides respectively Nucleotide sequence, then denature and anneal the synthesized sequence to obtain a double-stranded DNA fragment with BbsI sticky ends, as follows: Forward: 5'-CACCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

[0028] NNNNNNNNNNNNNNNNNNNNNCAAA-5', denaturation and annealing...

Embodiment 2

[0032] Example 2 Cloning of synergistic protein ESCS-higher and construction vector

[0033]Clone the synergistic protein ESCS-higher gene, and obtain the gene sequence described in SEQ ID NO: 1 through the method of whole gene synthesis. Using this sequence as a template, according to the sequences of the upstream and downstream primers are 5'-atgatatactttattagaat-3', 5 '-tcaagggatttccatttctc-3', primers and whole genome were synthesized by Shanghai Sangon Co., Ltd. The target gene fragment of ESCS-higher gene was amplified by PCR reaction. The amplification reaction system was as follows: 95°C, 40s, 57°C, 1min, 72°C, 1min, 72°C, 10min, cycled 35 times, and the PCR product was produced by Shanghai Shenggong Co., Ltd. Sequencing was performed, and by sequencing, the binding was a complete match to SEQ ID NO:1. Subsequently, the target gene amplified by PCR was connected to the empty vector lentiviral vector pHIV-CS-CDF-CG-PRE, and the recombinant lentiviral vector was identif...

Embodiment 3

[0034] Example 3 Application Analysis of CRISPR / Cas9 in Epidermal Stem Cells

[0035] The sgRNA expression plasmid prepared in Example 1, the known Cas9 expression plasmid co-transfected epidermal stem cells. Using liposome transfection method to construct transfection epidermal stem cell transfection system and reagents to make Lipofectamine TM 2000 (Invitrogen Company), the detailed steps of transfection refer to the transfection instructions. Stem cells not transfected with the synergistic gene of Example 2 were used as positive control.

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Abstract

The invention provides epidermal stem cell RAB 23 gene editing through a CRISPR-Cas system and particularly relates to establishment of an RAB23 gene knockout epidermal stem cell system for subsequentresearch on cutaneous squamous cell carcimona. According to the establishment of an RAB23 gene knockout epidermal stem cell system, a specific gRNA (guide ribonucleic acid) is structured and obtainedto significantly improve epidermal stem cell intracellular CRISPR/Cas9 RAB23 gene editing. A provided epidermal stem cell RAB23 knockout plasmid is high in hereditary stability and targeting efficiency.

Description

technical field [0001] The present invention provides a CRISPR-Cas system for RAB23 gene editing on epidermal stem cells, and in particular relates to the establishment of a RAB23 knockout epidermal stem cell line. Background technique [0002] Epidermal stem cells (Epidermal stem cells, EpiSCS) are stem cells with self-proliferation and multi-lineage differentiation potential. Its normal proliferation and differentiation are the basic requirements for maintaining the structural and functional integrity of the skin and its appendages (sweat glands, hair, sebaceous glands). Under physiological conditions, epidermal stem cells differentiate into a stem cell and a transit amplifying cell (TA cell) through asymmetric division, and the TA cell differentiates into post-mitotic cells (Post-mitotic cells) and terminal cells after multiple divisions. Differentiated cells (terminally-differentiated cells) to supplement the continuous renewal of epidermal cells. Studies have shown tha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N5/10
CPCC07K14/82C12N15/113C12N15/907C12N2310/10C12N2310/20
Inventor 杨骏朱成光
Owner 浙江玉安康瑞生物科技有限公司
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