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Method for activating gene expression by CRISPR-assisted trans-enhancer and application thereof

A gene expression and target gene technology, applied in the field of biomedicine, can solve problems such as low-efficiency gene activation activity

Active Publication Date: 2018-12-21
SOUTHEAST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current gene activators are still limited by their inefficient gene activation activity

Method used

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  • Method for activating gene expression by CRISPR-assisted trans-enhancer and application thereof
  • Method for activating gene expression by CRISPR-assisted trans-enhancer and application thereof
  • Method for activating gene expression by CRISPR-assisted trans-enhancer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] vector construction

[0059] experimental method:

[0060] To apply the CRISPR / dCas9 expression system for transfection in cells, a plasmid containing sgRNA driven by a U6 promoter was constructed. Using Pfu high-fidelity polymerase (Transgen, AS221-01), using primers Lac-px-F (Table 1) and Lac-px-R (Table 1) cloned from pEASY-Blunt-simple (Transgen, CB101-01) The lac operator sequence with BbsI and BsaI sites at both ends. This lac operator sequence was ligated into px458 (Addgene plasmid ID: 42230) to construct px458-lac. The ligation product was transformed into competent cells DH5α, and then blue colonies were screened. Verify px458-lac by sequencing. Three flanking sequences were then designed. Using the forward primer (U6-F; Table 1) and one of three reverse primers U6-1-R (Table 1), U6-2-R (Table 1), and U6-3- R (Table 1), flanking sequences were added to the 3'-end of the gRNA scaffold sequence by PCR amplification, where the gRNA scaffold sequence cloned ...

Embodiment 2

[0108] Effect of capture sequence on sgRNA function

[0109] experimental method:

[0110] DNA cleavage with Cas9 / csgRNA: Select sgRNA targeting the HNF4α promoter sequence. sgRNAs were prepared by in vitro transcription using T7 RNA polymerase (M0251S, NEB). The sgRNA transcription template was prepared by PCR amplification of the sgRNA coding sequence cloned in the sgRNA expression plasmid (pEASY-csgRNA) with the forward primer HNF4α-T7-F (Table 5); the forward primer contained the T7 promoter sequence (TAATACGACTCACTATAG, Transcription starts at 3'G), and one of four reverse primers U6-R, U6-1-R, U6-2-R and U6-3-R (Table 1). A normal sgRNA (HNF4α-sgRNA) and three csgRNAs (HNF4α-csgRNA) were prepared. A 732-bp HNF4α promoter fragment was amplified from pEZX-HP-ZsGreen-A by PCR using primers HNF4α-sP-F and HNF4α-sP-R (Table 5). The Cas9 digestion reaction system (30 μL) consists of 1×Cas9 nuclease reaction buffer, 1 μM Cas9 nuclease (NEB, M0386T) and 300 nM HNF4α-sgRNA or...

Embodiment 3

[0116] Activation of exogenous reporter genes through trans-enhancers

[0117] experimental method:

[0118] Cell culture and transfection:

[0119] 293T, HepG2, A549, SKOV3, HT29, PANC-1, and HeLa were obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and incubated at 37°C and 5% (v / v) CO 2 incubator culture. Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin. When cells are confluent >70% in each well of a 12-well plate, use 800ng total DNA, including 500ng plasmid (pcDNA-dCas9, pcDNA-dCas9-VP64 or pcDNA-dCas9-VPR), 150ng linear sgRNA expression template, ( U6-sgRNA or U6-csgRNA) and 150ng linear CMV, dissolved in Lipofectamine 2000 (ThermoFisher Scientific) liposomes were transfected using the following protocol. For each transfection, when cells grow to 4 x 10 5 At a density...

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PUM

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Abstract

The invention discloses a method for activating gene expression by a CRISPR-assisted trans-enhancer. The method is characterized in that an enhancer DNA is collected onto a target gene in a trans manner by virtue of a CRISPR system, and the target gene expression is activated by virtue of the trans enhancer DNA and the interaction of the trans enhancer DNA and CRISPR system. By improving the conventional guidance RNA(sgRNA), a capturing sgRNA(csgRNA) is developed; and the CMV enhancer is collected onto the target gene in a trans manner by virtue of the hybridization of csgRNA in a dCas9-AD / csgRNA complex and enhancer DNA with CMV, so that the expression of the target gene can be efficiently activated. The method has wide application value in the biological medicine, and can be applied to the preparation of biological detection and treatment reagents.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for CRISPR-assisted trans-enhancer activation of gene expression and its application. Background technique [0002] The expression of artificially activated genes plays an important role in basic biological research and biomedical applications. For example, the function of a gene is often explored by artificially activating its expression in cells or in vivo in basic research. In biomedicine, it is often necessary to reprogram cells into induced stem (iPS) cells or other differentiated cells by activating endogenous genes. In medicine, cancer can be treated by inhibiting various kinases and activating genes related to enhancing immunity, apoptosis and differentiation. Therefore, various artificial gene activators have been developed. For example, activation domain-fused zinc fingers (ZFs), transcriptional activator-like effectors (TALEs), and clustered...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/63
CPCC12N15/113C12N15/63C12N2830/00C12N2310/20
Inventor 王进科徐新慧
Owner SOUTHEAST UNIV
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