Drug-inducible CRISPR/Cas9 system for genome editing and transcription regulation

A genome editing 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: 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 genome editing or transcriptional activation, and the selected regulatory drugs or method...

Method used

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  • Drug-inducible CRISPR/Cas9 system for genome editing and transcription regulation
  • Drug-inducible CRISPR/Cas9 system for genome editing and transcription regulation
  • Drug-inducible CRISPR/Cas9 system for genome editing and transcription regulation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Drug-Inducible CRISPR / Cas9 System for Genome Editing

[0068] This example is used to illustrate the construction of the drug-inducible CRISPR / Cas9 system for genome editing described in the present invention.

[0069] Build method:

[0070] The drug-induced genome editing system constructed in the present invention includes the following two parts:

[0071] (1) Cas9 and two ERs in series with its C-terminus T2 Composition, namely Cas9-2ER T2 .

[0072] (2) in Cas9-2ER T2 Insert one / two tandem NES into the plasmid, i.e. Cas9-NES-2ER T2 / Cas9-2NES-2ER T2 .

[0073] The source of each part of the plasmid is as follows:

[0074] The Cas9 element was amplified from the plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (from Zhang Feng's laboratory, Addgene plasmid #42230 40). ER T2 Elements were amplified from plasmid pAd-CreER (a gift from T.C. He's laboratory, University of Chicago). NES sequence according to the report of Ding et al. (Ding, Y., Ai, H.W., Hoi,...

Embodiment 2

[0078] This embodiment is used to illustrate the application of the system described in Embodiment 1.

[0079] 1. Experimental materials

[0080] Cas9-2ER T2 , Cas9-NES-2ER T2 and Cas9-2NES-2ER T2 Plasmids, sgRNAs targeting different sites, donor plasmids for TLR systems and single-stranded DNA donors (ssDNA donors) for FCR experiments.

[0081] 2. Experimental method

[0082] HEK293T cells (ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS, 2mM GlutaMAX (Thermo Fisher), 100U / ml penicillin and 100μg / ml streptomycin, and placed at 37°C, 5% CO 2 cultured in an incubator. Monoclonal TLR and FCR stably transfected cell lines were obtained by lentiviral packaging and infection. The cell transfection reagent was the DNA transfection reagent of Biotool Company, and the transfection method was carried out according to the instructions. The total amount of DNA transfected per well was consistent in each experiment. Cell medium was changed 5 hou...

Embodiment 3

[0097] Example 3 A drug-inducible CRISPR / Cas9 system capable of simultaneous genome editing and transcriptional activation

[0098] In this example, genome editing of the BFP gene and transcriptional activation of the CD43 gene are taken as an example to illustrate the construction of a drug-inducible CRISPR / Cas9 system that simultaneously performs genome editing and transcriptional activation.

[0099] Build method:

[0100] The drug-inducible CRISPR / Cas9 system for simultaneous genome editing and transcriptional activation of the present invention includes Cas9-2NES-2ER T2 -GCN4 and scFv-2ER T2 -VPH, where Cas9-2NES-2ER T2 It is the plasmid constructed in Example 1, GCN4 is a short peptide of 10 copies of yeast transcriptional activator GCN4, V is VP64, P is P65, and H is HSF1. The sources and construction methods of each element of the plasmid are as follows:

[0101] VP64 was amplified using the pLenti-EF1a-SOX2 plasmid (Addgene plasmid #35388) from Feng Zhang’s labora...

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Abstract

The invention relates to the field of molecular biology, in particular to a drug inducible CRISPR/Cas9 system for genome editing, comprising 16-22nt of sgRNA and Cas9 fusion protein targeting a specific gene site, wherein the Cas9 fusion protein is composed of Cas9 and 2-5 ERT2 connected to the C end in series, and one or 2-10 NES in series are inserted between that Cas9 and the ERT2. Through a series of experiments and probes, a scheme is developed and optimized with the highest activity and the lowest background activity, and is applied to the editing of endogenous genes. In addition, the invention also provides that genome editing and transcription activation simultaneously carried out in a single system, so as to enrich and diversify the design and maximize the function of the drug-induced system in manipulating the genome. The establishment of such a drug induction system with multiple activities will provide a more powerful tool for clinical research and application in the fieldof precise genome engineering and gene therapy.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to various drug-inducible CRISPR / Cas9 systems for genome editing and transcription regulation. 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 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,...

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 王宇赵晨卢佳赵迎泽张竞方
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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