Rapidly-degraded Cas9-ODC422-461 fusion protein and application thereof

A fusion protein and rapid degradation technology, applied in the field of molecular biology, can solve problems such as off-target effects, achieve short half-life, small side effects, and reduce non-specific cleavage toxicity

Inactive Publication Date: 2015-04-22
SUZHOU KECHUANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this RNA-guided Cas9 technology has a big defect that it has serious off-target effects

Method used

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  • Rapidly-degraded Cas9-ODC422-461 fusion protein and application thereof
  • Rapidly-degraded Cas9-ODC422-461 fusion protein and application thereof
  • Rapidly-degraded Cas9-ODC422-461 fusion protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Cas9-ODC 422–461l and Cas9-ubiquitin G75A / G76V Preparation of fusion protein

[0037] Methods as below:

[0038] Part 1: Cas9-ODC 422-461 Construction of eukaryotic expression vectors:

[0039] PEST sequences rich in proline, glutamic acid, serine, and threonine are associated with rapid protein degradation. We used PESTFind (http: / / emboss.bioinformatics.nl / cgi-bin / emboss / pestfind) to detect Cas9 protein Analysis was performed and no potential PEST sequences were found. Cas9 has a long half-life. In order to reduce its half-life. We fused the PEST sequence ODC at the C-terminus of the Cas9 protein 422–461 and Ub-G75A / G76V, constructed into the following vector, pCas9-ODC 422–461 , pCas9-ubiquitin G75A / G76V , construct pCas9-ubiquitin G75A / G76V as a comparison.

[0040] The specific construction method is as follows:

[0041] (1) Using Cas9-ODC 422–461 DNA sequence as template (SEQ ID NO.2), using primer Primer_Cas9_F: CAGCCTCCGGACTCTAGA...

Embodiment 2

[0072] Example 2 Cas9-ODC 422–461l and Cas9-ubiquitin G75A / G76V Protein expression and degradation in eukaryotic cells

[0073] Wild-type pCas9, pCas9-ODC 422–461 and pCas9-ubiquitin G75A / G76V After transfection into 293T cells, 24 hours after transfection, they were treated with cycloheximide (50 mg / mL) for 6 hours to prevent the synthesis of new proteins. Cells were collected, lysed, and then used for Western blotting. Take 10ul of the fermentation broth supernatant and load the sample. After SDS-PAGE protein electrophoresis, the protein was transferred to PVDF membrane, combined with primary antibodies (Cas9 antibody, mouse antibody,) and HRP-labeled secondary antibody (rabbit anti-mouse,) was then developed onto film.

[0074] The result shows as figure 1 As shown, compared with wild-type Cas9, the expression of other fusion proteins is relatively low, indicating that Cas9-ODC 422–461l and Cas9-ubiquitin G75A / G76V The fusion protein is degraded and has a ...

Embodiment 3

[0076] pCas9, pCas9-ODC 422–461 and pCas9-ubiquitin G75A / G76V The promoter of the plasmid was replaced with TRE, and the vector pTRE-Cas9, pTRE-Cas9-ODC was constructed 422–461 and pTRE-Cas9-ubiquitin G75A / G76V2 , the three vectors were transfected into the HEK 293 Tet-On cell line, and then screened with G418. Find expression Cas9, Cas9-ODC 422–461 and Cas9-ubiquitin G75A / G76V2 expressing cells. Tetracycline was treated for 1 hour, and after tetracycline was removed, six hours later, western blot was used to detect the expression of the three proteins, as shown in figure 2 shown. The results indicated that Cas9-ODC 422–461 and Cas9-ubiquitin G75A / G76V2 Fusion proteins have a relatively short half-life.

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Abstract

The invention discloses a rapidly-degraded Cas9-ODC422-461 fusion protein and application thereof, and belongs to the technical field of molecular biology. According to the rapidly-degraded Cas9-ODC422-461 fusion protein and the application, a humanization Cas9 protein is transformed with a genetic engineering method, a PEST protein degradation sequence is added, and therefore the rapidly-degraded Cas9-ODC422-461l fusion protein is obtained. The Cas9-ODC422-461l fusion protein can conduct induced control over an expression of Cas9; the staying time of the Cas9-ODC422-461l fusion protein in cells is shortened; and nonspecific cutting toxicity of Cas9-ODC422-461l overexpression to the cells is accordingly reduced. The rapidly-degraded Cas9-ODC422-461l fusion protein is safer when being used for drug screening or gene modification; and the fusion protein can be further used for gene therapy.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a rapidly degradable Cas9-ODC 422-461 Fusion proteins and their applications. Background technique [0002] CRISPR-Cas is derived from the immune system of bacteria and archaea, and can use target-specific RNA to bring Cas9 nuclease to specific targets on the genome, thereby modifying specific genetic loci. RNA-guided Cas9 can function in a variety of cells and organisms, cleaving entire genomes at specific sites. The genome editing potential of Cas9 has made this technology widely used in human cell lines, mice, rats, gene knockout and knockin in multiple species. However, this RNA-guided Cas9 technology has a big defect that it has serious off-target effects. One aspect of this off-target effect is the relatively long half-life of Cas9. Contents of the invention [0003] The C-terminal amino acid of mouse ornithine decarboxylase (MODC) contains a deg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N9/88C12N15/55C12N15/60C12N5/10
CPCC12N9/22C12N9/88C12Y401/01017
Inventor 李云英
Owner SUZHOU KECHUANG BIOTECH
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