Method for specifically improving gene editing efficiency of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-CAS system in epidermal stem cell

An epidermal stem cell and gene editing technology, applied in the field of improving the gene editing efficiency of CRISPR-cas system in epidermal stem cells, can solve the problem of low homologous recombination probability, and achieve the effect of improving gene editing efficiency and efficiency

Active Publication Date: 2018-10-02
河北万玛生物医药有限公司
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Problems solved by technology

[0011] The purpose of the present invention is to provide a method for improving the gene editing efficiency of the CRISPR-cas system in epidermal stem cells, which effectively overcomes the technical defects of the prior art that the low probability of homologous recombination in animals affects precise editing, especially for stem cells Drawbacks of gene editing

Method used

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  • Method for specifically improving gene editing efficiency of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-CAS system in epidermal stem cell

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Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1, cloning synergistic protein ESCS-higher and construction vector

[0028]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 the binding was a complete match to SEQ ID NO:1 by sequencing. 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 2

[0029] Example 2 Application Analysis of CRISPR / Cas9 in Epidermal Stem Cells and Bone Marrow Mesenchymal Stem Cells

[0030] CRISPR / Cas9 editing vector based on pBGN plasmid containing BSD-fsEGFP fusion gene

[0031] (1) BSD-fsEGFP fusion gene: use conventional PCR to amplify the known BSD gene, 5'-PCR primers with HindIII sites, and 3'-PCR primers to introduce I-SceI and EcoRI sites. Insert the PCR product (BSD) into the HindIII and EcoRI positions between the CMV driver and the EGFP coding region in the EGFP plasmid (the EGFP nucleotide sequence is a sequence known in the art, such as shown in sequence 1 and sequence 2 in CN105647968A) Point, generate the plasmid pBGN containing the BSD-fsEGFP fusion gene, the nucleotide sequence of the BSD-fsEGFP fusion gene is as shown in sequence 3 and sequence 4 in CN105647968A). The fusion gene is driven by CMV driver or PGK driver, but EGFP is inactive due to frameshift, so it is called fsEGFP.

[0032] 5'-PCR primers are

[0033] C...

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Abstract

The invention provides some new enhancins ESCS-higher, which can obviously improve the gene editing efficiency of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas9 in an epidermal stem cell. The invention provides a new way to improve the gene editing efficiency in the cell, and further provides a more efficient genome editing system. When the enhancin is used with the CRISPR / Cas9, the genome editing efficiency of the CRISPR / Cas9 in the epidermal stem cell can be obviously improved.

Description

technical field [0001] The present invention provides a method for improving the gene editing efficiency of the CRISPR-cas system in epidermal stem cells, and in particular relates to a new synergistic factor that can significantly increase the probability of homologous recombination, combining the synergistic factor with the CRISPR-cas system Can significantly improve the efficiency of genome editing. 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-mito...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N15/90C12N9/22
Inventor 杨骏朱成光
Owner 河北万玛生物医药有限公司
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