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CRISPR (clustered regularly interspaced short palindromic repeats)-based multicolor orthogonal high-sensitivity RNA (ribonucleic acid) imaging method

An imaging, -AGUU|UUGA-3 technology, applied in the field of RNA imaging, can solve the problems of inability to perform RNA imaging, achieve excellent intracellular stability, reduce experimental costs, and improve efficiency

Pending Publication Date: 2022-04-15
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At the same time, dPspCas13b-3xEGFP-based methods have high background, making RNA imaging impossible without sufficient sequence numbers

Method used

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  • CRISPR (clustered regularly interspaced short palindromic repeats)-based multicolor orthogonal high-sensitivity RNA (ribonucleic acid) imaging method
  • CRISPR (clustered regularly interspaced short palindromic repeats)-based multicolor orthogonal high-sensitivity RNA (ribonucleic acid) imaging method
  • CRISPR (clustered regularly interspaced short palindromic repeats)-based multicolor orthogonal high-sensitivity RNA (ribonucleic acid) imaging method

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Experimental program
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Effect test

Embodiment 1

[0037] Construction of plasmids:

[0038] The plasmids used in other examples of the present invention were constructed, and the details of these plasmids and their applications in the examples are shown in Table 1. The sgRNA-aptamer structural sequence designed in the present invention is a whole gene synthesis (construction) on the PUC57 vector.

[0039] (1) The construction of pmRuby3-HSF1, pmRuby3-24xGCN4 and pHAGE-dPspCas13b-3xEGFP-2xNLS-IRES-puro (referred to as dPspCas13b-3xEGFP-2xNLS) is shown in the literature Yang, L. et al. Dynamic Imaging of RNA in Living Cells by CRISPR - Cas13 Systems. Mol. Cell 76, 981-997 (2019).

[0040] To construct pmRuby3-HSF1, the HSF1 sequence was amplified from HeLa cDNA and one-step cloned into the pmCherry-c1 plasmid (Yeasen) using EcoRI and BamHI restriction sites.

[0041] To construct pmRuby3-24xGCN4, 24xGCN4 was cloned into the pmCherry-cl plasmid (Yeasen) in one step using HindII and BamHI restriction sites.

[0042] To constru...

Embodiment 2

[0050] In vitro fluorescence characterization of sgRNA-aptamers:

[0051] (1) The sgRNA-aptamer was transcribed in vitro using T7 RNA polymerase. The primers corresponding to Table 5 were designed, and the forward primer contained a T7 promoter sequence located at the 5' end of the sgRNA, and the sgRNA-aptamer sequence on the PUC57 vector was PCR amplified to obtain an in vitro transcription template. The template was isolated by 0.8% agarose gel electrophoresis and purified using a gel cutting recovery kit (Axygen). Transcription reactions were performed in 1x buffer (pH=7.9) containing 100 mM HEPES-KOH containing 20 mM magnesium chloride, 30 mM DTT, 2 mM each NTP, 2 mM spermidine, 0.1 mg / mL T7 RNA polymerase and 300ng PCR fragments were reacted for a total of 4h. After the reaction was completed, DNA was degraded using DNaseI, and the transcribed RNA product was purified by the NaOAc / phenol / chloroform method. The purified sgRNA-aptamers were resuspended in RNase-free wate...

Embodiment 3

[0056] Cell Culture and Transfection:

[0057] Human renal epithelial cells 293T were cultured in a 20 mm glass-bottom petri dish (Nest), using high-glucose DMEM medium (Gibcol), supplemented with 10% (vol / vol) fetal bovine serum, and cultured at 37°C in 5% carbon dioxide. Cultivated in the box. During transfection, 200ng of dPspCas13b-RFP-2xNLS or dPspCas13b-3xEGFP-2xNLS or dPspCas13b-2xNLS, and 1 μg of sgRNA-aptamer-carrying vector were co-transfected with Liposome 3000 (ThermoFisher Science), and incubated for 24-48 Hour.

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Abstract

The invention discloses a multicolor, orthogonal and high-sensitivity RNA (Ribonucleic Acid) imaging method based on CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), and belongs to the technical field of RNA imaging. On the basis of Cas13 sgRNA and an aptamer, an sgRNA-aptamer into which an aptamer with a repeated structure is inserted is designed, and a multicolor, efficient and orthogonal RNA imaging method is established by utilizing the combination of the sgRNA-aptamer and dCas13b. According to the design, the stability of the sgRNA-aptamer is improved, the signal-to-noise ratio is improved on the premise of not influencing the targeting property, and imaging of low-abundance RNA and visualization of RNA interaction are realized. The method has universality, and spacer sequences can be designed for different target RNAs for fluorescence imaging; when multi-target multicolor imaging is carried out, only one dCas13b protein and several sgRNA-aptamers are needed, so that the protein design cost and related experimental operation are reduced.

Description

technical field [0001] The invention belongs to the technical field of RNA imaging, in particular to a CRISPR-based multicolor, orthogonal and high-sensitivity RNA imaging method. Background technique [0002] The spatiotemporal localization of RNA in cells is closely related to its biological function, so imaging methods in living cells are needed to reveal the dynamics of RNA. The MS2-MCP system and molecular tags are commonly used to visualize RNA, however they are difficult to track RNA interactions in living cells. The imaging technique of RNA aptamers greatly reduces background fluorescence and enables the design of a series of small molecule fluorophores providing a broad selection of emission wavelengths from cyan to red, however, although this method enables mRNA imaging, it requires exogenous Dozens of hairpin RNAs are inserted, which will inevitably affect the structure and function of the target RNA, making this system a barrier to studying the behavior of endog...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/115C12N15/85C12N15/55G01N21/84
CPCC12N15/113C12N15/115C12N15/85C12N9/22G01N21/84C12N2310/20C12N2310/16C12N2310/531C12N2800/107
Inventor 周翔翁小成唐恒彭俊然
Owner WUHAN UNIV
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