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Drug-inducible CRISPR/Cas9 system for gene transcription activation

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

Active Publication Date: 2019-01-15
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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  • Drug-inducible CRISPR/Cas9 system for gene transcription activation
  • Drug-inducible CRISPR/Cas9 system for gene transcription activation
  • Drug-inducible CRISPR/Cas9 system for gene transcription 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 in the present invention includes the following two parts:

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

[0066] (2) Put one / two ER 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] Said 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 Zhang Feng's laboratory, Addgene plasmid #42230). The ERT2 element w...

Embodiment 2

[0073] Embodiment 2 HIT-SAM system

[0074] Build method:

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

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

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

[0078] (3) Put one / two ER 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 exist in different plasmids or the same plasmid respectively.

[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 the plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgeneplasmid #42230).

[0084] The construction of the MCP plasmid first uses the MS2-P65-H...

Embodiment 3

[0087] Embodiment 3 HIT-SunTag system

[0088] Build method:

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

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

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

[0092] combine two ER T2 Inserted between scFv and VP64 to obtain scFv-2E-VP64;

[0093] combine two ER T2 Insertion between scFv and PH yielded 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 exist in different plasmids or the same plasmid respectively.

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

[0096] Cas9, NLS, ER T2 And V, P, H are with embodiment 1. Sequence references for scFv an...

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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 invention relates to the field of molecular biology, in particular to a drug-inducible CRISPR / Cas9 system for gene transcription activation. Background technique [0002] The CRISPR / Cas9 system is derived from the immune mechanism of bacteria to degrade invading viral DNA or other foreign DNA. Utilizing RNA-mediated DNA-binding activity and endonuclease activity, the 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 by guide RNA (gRNA) complementary to the target sequence and adjacent protospacer-adjacent motif (PAM). The complex formed by Cas9 and gRNA mediates DNA double-strand breaks, and the targeted gene editing is also completed through the two DNA repair mechanisms of NHEJ and HDR. The CRISPR / Cas9 system can also be combined with different effector molecules to expand its functions, enabling i...

Claims

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Application Information

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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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