Fusion protein in Cpf1 and p300 core structural domain, corresponding DNA target activation system and application

A DNA targeting and fusion protein technology, applied in the fields of genetic engineering and biology, can solve the problems of numerous acting elements, complex acting systems, and low activation efficiency, and achieve the effects of fewer acting elements, low off-target rate and high activation efficiency.

Inactive Publication Date: 2017-12-19
SOUTHERN MEDICAL UNIVERSITY
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Problems solved by technology

[0007] The purpose of the present invention is to mutate the CRISPR/Cpf1 with enzymatic cleavage activity into a targeted gene with no enzymatic cleavage activity in view of the problems of low targeting, low activation efficiency, and complex action systems involving the

Method used

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  • Fusion protein in Cpf1 and p300 core structural domain, corresponding DNA target activation system and application
  • Fusion protein in Cpf1 and p300 core structural domain, corresponding DNA target activation system and application
  • Fusion protein in Cpf1 and p300 core structural domain, corresponding DNA target activation system and application

Examples

Experimental program
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Example Embodiment

[0051] Example

[0052] As / LbCpf1-3xHATag( Figure 3~4 ) Vector construction:

[0053] A commercial company synthesized a single-stranded DNA fragment Oligo-F and Oligo-R containing BamHI-3xHATag-XohI-AGC-(Ser)-AgeI, the base sequences of which are shown in SEQ ID NOs: 3-4.

[0054] Two partially complementary paired single-stranded DNA fragments synthesize a double-stranded DNA fragment. Specific steps are as follows:

[0055] 10ul 100uM Oligo-F and 10ul 100uM Oligo-R are premixed in a 1.5ml EP tube, boil 800ml of distilled water in a beaker, put the 1.5ml EP tube in boiling water for 5 minutes, and take out the 1.5ml EP tube and leave it at room temperature overnight.

[0056] The original plasmid AsCpf1 / LbCpf1 synthesized by a commercial company was cut with BamHI and NdeI (Figures 1-2), and the enzyme products were analyzed by 0.8% agarose gel electrophoresis. The gel was cut to recover 4015bp bands, and the concentration of recovered fragments was determined by NanoDrop. The line...

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Abstract

The invention discloses a fusion protein in a Cpf1 and p300 core structural domain, a corresponding DNA target activation system and application. CRISPR/Cpf1 with enzyme cutting activity is mutated to be free of enzyme cutting activity and of target gene identification characteristic, is fused with p300 protein transcriptional activation, reaches the purpose of target gene activation inside mammalian cells, and has the characteristics of being simple, efficient and high in specificity. The fusion protein has the advantages of low off-target rate, cost saving and the like. The system can be mutually complementary with an existing target gene activation method based on CRISPR/Cas9, the gene editing range of the CRISPR/Cas9 system is expanded, and the system has a good application prospect.

Description

technical field [0001] The invention relates to the fields of genetic engineering and biotechnology, in particular to the targeted gene activation of mammalian cells by using CRISPR / Cpf1 and its variants and p300 fusion protein. Background technique [0002] CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-Associated systems), the full name is the constant palindromic repeat sequence cluster / constant palindromic repeat sequence cluster-associated protein system, which is an acquisition of bacteria and archaea immune system. In 2010, Garneau et al. found that Cas9 is the only Cas nuclease that can mediate DNA cleavage in the CRISPR type II system of Streptococcus thermophilus. In 2013, Zhang Feng and other scientists first reported the application of CRISPR-Cas9 system in mammalian genome editing, which opened up the powerful gene editing function of CRISPR-Cas9 and became a new star in the field of life sciences, which has been widely studied and...

Claims

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Application Information

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IPC IPC(8): C12N9/22C12N9/10C12N15/113C12N15/85
CPCC12N9/1029C12N9/22C12N15/113C12N15/85C12N2310/10C12Y203/01048
Inventor 荣知立林瑛王为单琳马淑凤
Owner SOUTHERN MEDICAL UNIVERSITY
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