A drug-inducible CRISPR/Cas9 system for gene transcriptional activation

A gene transcription and inducible technology, applied in the field of molecular biology, can solve problems such as the inapplicability of regulatory drugs or methods

Active Publication Date: 2020-12-01
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of the existing successful programs are unilateral studies on gene editing or transcriptional activation, and the selected regulatory drugs or methods are not suitable for all fields, and the design of split Cas9 makes the results of drug induction irreversible, so , it is necessary to develop a new drug-induced system

Method used

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  • A drug-inducible CRISPR/Cas9 system for gene transcriptional activation
  • A drug-inducible CRISPR/Cas9 system for gene transcriptional activation
  • A drug-inducible CRISPR/Cas9 system for gene transcriptional activation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Direct fusion drug-inducible CRISPR / Cas9 system

[0063] Build method:

[0064] The drug-inducible gene editing system constructed by the present invention includes the following two parts:

[0065] (1) Design sgRNAs targeting specific gene transcriptional activation sites.

[0066] (2) Put one / two ERs T2 inserted between dCas9 and VPH or VPR (dCas9-E-VPH or dCas9-2E-VPH or dCas9-E-VPR or dCas9-2E-VPR) or fused to the C-terminus of dCas9-VPH / VPR (dCas9 -VPH-E or dCas9-VPH-2E or dCas9-VPR-E or dCas9-VPR-2E)

[0067] The sgRNA and expression fusion protein dCas9-E-VPH or dCas9-2E-VPH or dCas9-VPH-E or dCas9-VPH-2E or dCas9-E-VPR or dCas9-2E-VPR or dCas9-VPR-E or dCas9 -VPR-2E vectors are present in different plasmids or in the same plasmid.

[0068] The source of each part of the plasmid is as follows:

[0069] The dCas9 element was amplified from the plasmid pMSCV-LTR-dCas9-VP64-BFP (from Feng Zhang's laboratory, Addgene plasmid#42230). The ERT2 element ...

Embodiment 2

[0073] Example 2 HIT-SAM system

[0074] Build method:

[0075] The drug-inducible gene editing system constructed by the present invention includes the following two parts:

[0076] (1) Design sgRNAs targeting specific gene transcriptional activation sites.

[0077] (2) Fusing NLS to the C-terminus of dCas9 to obtain dCas9-NLS;

[0078] (3) Put one / two ERs T2 Insertion between MCP and ADs yields MCP-E-ADs or MCP-2E-ADs.

[0079] ADs include V, P, R, H and any combination thereof.

[0080] The sgRNA, the vector expressing the fusion protein dCas9-NLS and the vector expressing the fusion protein MCP-2E-VPH respectively exist in different plasmids or the same plasmid.

[0081] The source of each part of the plasmid is as follows:

[0082] Cas9, ER T2 Same as Example 1 with each transcription factor.

[0083] NLS was amplified from plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgeneplasmid#42230).

[0084] Construction of MCP Plasmid First, MCP was obtained by PCR amplific...

Embodiment 3

[0087] Example 3 HIT-SunTag System

[0088] Build method:

[0089] The drug-inducible gene editing system constructed by the present invention includes the following two parts:

[0090] (1) Design sgRNAs targeting specific gene transcriptional activation sites.

[0091] (2) Fusing NLS to the C-terminus of dCas9 to obtain dCas9-NLS, and fusing a 10×(GCN4)P peptide segment to its C-terminus to obtain dCas9-NLS-(GCN4)P;

[0092] put the two ERs T2 Inserted between scFv and VP64 to obtain scFv-2E-VP64;

[0093] put the two ERs T2 Insertion between scFv and PH yields scFv-2E-PH.

[0094] The sgRNA, the vector expressing the fusion protein dCas9-NLS-(GCN4)P, the vector expressing the fusion protein scFv-2E-VP64 and the vector expressing the fusion protein scFv-2E-PH respectively exist in different plasmids or the same plasmid.

[0095] The source of each part of the plasmid is as follows:

[0096] Cas9, NLS, ER T2 Same as Example 1 with V, P and H. Sequence references for s...

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Abstract

The invention relates to the field of molecular biology, in particular to a drug inducible CRISPR / Cas9 system for gene transcription activation. The drug inducible CRISPR / Cas9 system comprises 16-22ntsgRNA targeting a specific gene site and any one of vector / vector combination as (1)-(3) below: (1) a vector expressing fusion protein dCas9-(ERT2)n-ADs or dCas9-ADs-(ERT2)n; (2) a vector combinationexpressing fusion protein dCas9-(NLS) m or dCas9-(ERT2)n and MCP-(ERT2)n-ADs; (iii) a vector combination expressing fusion protein dCas9-(NLS)m-(GCN4)p and scFv-(ERT2)n-ADs. Compared with the prior art, the system of the invention can more effectively realize the regulation of drug-induced gene transcription activation, and has more flexible and convenient operation and lower background activity.It is helpful to control the dynamic biological process effectively and precisely, and has great value for the research and development of biomedicine.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a drug-inducible CRISPR / Cas9 system for gene transcription activation. Background technique [0002] CRISPR / Cas9 system, derived from the immune mechanism of bacteria to degrade invading viral DNA or other foreign DNA. Using RNA-mediated DNA-binding and endonuclease activities, this system can be regulated in the genome in a sequence-dependent manner. Cas9 protein binds and cleaves double-stranded DNA in a sequence-specific manner, and this sequence-specific binding is achieved through a guide RNA (gRNA) complementary to the target sequence and the adjacent protospacer-adjacent motif (PAM). The complex formed by Cas9 and gRNA mediates DNA double-strand breaks, and also completes targeted gene editing through two DNA repair mechanisms, NHEJ and HDR. The CRISPR / Cas9 system can also be combined with different effector molecules to expand its functions, enabling transcripti...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/90C07K19/00C12N9/22
CPCC07K2319/00C12N9/22C12N15/85C12N15/907C12N2810/10
Inventor 王宇卢佳赵晨张竞方
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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