Application of virus-mediated Cpf1 protein in CRISPR/Cpf1 gene editing system

A gene editing and virus genome technology, applied in the biological field, can solve the problems of difficult gene editing and low transduction efficiency

Inactive Publication Date: 2017-03-08
SHANGHAI GENEPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for some difficult-to-transfect cells (such as ATDC5 cells derived from mice and C6 cells derived from rats), the transduction efficiency of Cpf1 protein is low, making it difficult to achieve gene editing

Method used

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  • Application of virus-mediated Cpf1 protein in CRISPR/Cpf1 gene editing system
  • Application of virus-mediated Cpf1 protein in CRISPR/Cpf1 gene editing system
  • Application of virus-mediated Cpf1 protein in CRISPR/Cpf1 gene editing system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1, Application of virus-mediated AsCpf1 protein in CRISPR / Cpf1 gene editing system

[0080] 1. Selection of target gene and target sequence

[0081] Select hRb1 gene (Gene ID: 5925), mRb1 gene (Gene ID: 19645), rRb1 gene (Gene ID: 24708), hP53 gene (Gene ID: 7157), mP53 gene (Gene ID: 22059), rP53 gene (Gene ID: ID: 24842), hDicer1 gene (Gene ID: 23405), mDicer1 gene (Gene ID: 192119) and rDicer1 gene (Gene ID: 299284) were used as target genes to detect the gene editing ability of the CRISPR / Cpf1 system.

[0082] Select target sequence 1 according to the nucleotide sequence of hRb1 gene, select target sequence 2 according to the nucleotide sequence of mRb1 gene, select target sequence 3 according to the nucleotide sequence of rRb1 gene, and select target sequence according to the nucleotide sequence of hP53 gene 4. Select target sequence 5 based on the nucleotide sequence of the mP53 gene, select target sequence 6 based on the nucleotide sequence of the rP53 g...

Embodiment 2

[0205] Example 2, Application of virus-mediated FnCpf1 protein in CRISPR / Cpf1 gene editing system

[0206] 1. Selection of target gene and target sequence

[0207] Same as Step 1 of Example 1.

[0208] 2. Preparation of chemically synthesized crRNA

[0209] Synthesize the crRNAs shown in Table 2. In Table 2, the single underline is the Fn crRNA backbone sequence, the double underline is the 1st to 20th position from the 5' end of the target sequence 1, the thick underline is the 1st to 20th position from the 5' end of the target sequence 2, and the dotted underline is the target Positions 1 to 20 from the 5' end of sequence 3, dotted underlines represent positions 1 to 20 from the 5' end of target sequence 4, dot-dash underlines represent positions 1 to 20 from the 5' end of target sequence 5, and waves The line represents the 1st to 20th position from the 5' end of the target sequence 6, and the dot-dot-dash underline represents the 1st to 20th position from the 5' end of ...

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Abstract

The invention discloses an application of virus-mediated Cpf1 protein in a CRISPR / Cpf1 gene editing system. The CRISPR / Cpf1 gene editing system provided by the invention consists of a recombinant virus or a recombinant vector. The recombinant virus is capable of expressing a Cpf1 gene; and the Cpf1 gene is capable of encoding the Cpf1 protein. The recombinant vector comprises a virus genome and the Cpf1 gene. The virus is either lentivirus or adenovirus. Experiments prove that virus-mediated FnCpf1 protein or AsCpf1 protein causes mutation of an hRb1 gene, an hP53 gene, an hDicer1 gene, an mRb1 gene, an mP53 gene, an mDicer1 gene, an rRb1 gene, an rP53 gene and an rDicer1 gene in the CRISPR / Cpf1 gene editing system. Therefore, the virus-mediated Cpf1 protein is applicable to gene editing in the CRISPR / Cpf1 gene editing system; and the Cpf1 protein has an important application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of virus-mediated Cpf1 protein in CRISPR / Cpf1 gene editing system. Background technique [0002] Gene editing is a technology for precise modification at the genome level, which can complete gene-directed deletion (InDel) mutation, gene-directed insertion mutation, simultaneous mutation of multiple sites, and deletion of small fragments. Gene editing technology can be used in the study of gene function and disease pathogenesis, the construction of disease animal models, biological therapy, research on genetic and tumor-related diseases, research on integrated viral diseases, and improvement of agricultural and livestock species. Gene editing technology is a tool for fundamentally changing the genetic material DNA of a species, and has extremely wide application value and development prospects. [0003] Zinc finger nuclease (ZFN), transcription activator-like effector...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/867C12N15/861
CPCC12N7/00C12N9/22C12N15/86C12N2710/10021C12N2710/10043C12N2740/15021C12N2740/15043C12N2800/107C12N2810/10
Inventor 董长贵杜永华张佩琢王德华李琴
Owner SHANGHAI GENEPHARMA CO LTD
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