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 overco

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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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Example Embodiment

[0027] Example 1. Cloning of the enhanced protein ESCS-higher and construction of the vector

[0028] The ESCS-higher gene was cloned, and the gene sequence described in SEQ ID NO:1 was obtained through the method of full gene synthesis. Using this sequence as a template, the sequence of the upstream and downstream primers was 5'-atgatatactttattagaat-3', 5 '-tcaagggatttccatttctc-3', primers and whole genome were synthesized by Shanghai Shenggong Co., Ltd. The PCR reaction amplifies the target gene fragment of the ESCS-higher gene. The amplification reaction system is as follows: 95°C, 40s, 57°C, 1min, 72°C, 1min, 72°C, 10min, cycle 35 times, PCR products are produced by Shanghai Shenggong Co., Ltd. Sequencing was performed, and the binding completely matched SEQ ID NO:1 through 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 identified by methods such as P...

Example Embodiment

[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 well-known BSD gene, 5'-PCR primer with HindIII site, 3'-PCR primer to introduce I-SceI and EcoRI site. Insert the PCR product (BSD) into the EGFP plasmid (the EGFP nucleotide sequence is a well-known sequence in the art, such as the sequence 1 and sequence 2 in CN105647968A) HindIII and EcoRI between the CMV driver and the EGFP coding region Click to generate a 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] The 5’-PCR primers are

[0033] CTCAAGCTTAACTAAACCATGGCCAA...

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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...

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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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