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A vector expressing aptamer ribozyme-modified sgRNA regulated by theophylline and its application

A technology of aptamers and vectors, applied in DNA/RNA fragments, recombinant DNA technology, using vectors to introduce foreign genetic material, etc.

Active Publication Date: 2021-02-19
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some researchers also use the tetracycline regulatory system to regulate the expression of Cas9 protein or sgRNA to regulate CRISPR / Cas9 activity, but such a regulatory system will require the introduction of additional cis-regulatory elements and trans-regulatory factors

Method used

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  • A vector expressing aptamer ribozyme-modified sgRNA regulated by theophylline and its application
  • A vector expressing aptamer ribozyme-modified sgRNA regulated by theophylline and its application
  • A vector expressing aptamer ribozyme-modified sgRNA regulated by theophylline and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The carrier constructed in this example to express the aptamer ribozyme-modified sgRNA targeting the human GLRX3 gene regulated by theophylline is as follows:

[0059] Expressed sgRNA-AZ 2.0 backbone (skeleton structure shown in figure 1 shown), the aptamer ribozyme P1-F5 is inserted at the tetraloop and stem loop 2 positions, wherein the sequence of the aptamer ribozyme P1-F5 is as follows:

[0060] GGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCC.

[0061] The complete sequence of the sgRNA-AZ 2.0 backbone is:

[0062] GAATTCGGTCTC CGTTTTAGGCTA GGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAG GACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCC TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAG CCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCC AAGTGGCACCGAGTCGGTGCTTTTTT ACTAGT .

[0063] The U6 promoter sequence is as follows:

[0064] GGTA...

Embodiment 2

[0082] The carrier constructed in this example to express the aptamer ribozyme-modified sgRNA targeting the human VEGFA gene and regulated by theophylline is as follows:

[0083] The sgRNA sequence targeting the human GLRX3 gene in Example 1 was replaced by the sgRNA sequence targeting the human VEGFA gene. The sgRNA sequence targeting the human VEGFA gene inserted in the two BsaIs is as follows:

[0084] GGTGAGTGAGTGTGTGCGTG.

[0085] Other structures of the carrier are the same as in Example 1.

[0086] In step 4 of the construction method, the method for constructing the pU6-sgRNA-AZ2.0-VEGFA vector targeting the human VEGFA gene is:

[0087] The sgRNA fragment targeting the human VEGFA gene is formed by annealing primers synthesized by Suzhou Jinweizhi Co., Ltd. The sequence is as follows (the underline indicates the cohesive end sequence):

[0088] P3: ACCG GGTGAGTGAGTGTGTGCGTG;

[0089] P4: AAAC CACGCACACACTCACTCACC.

[0090] The rest of the construction method ...

Embodiment 3

[0092] The carrier constructed in this example to express the aptamer ribozyme-modified sgRNA targeting the promoter of the human PGRN gene and regulated by theophylline is as follows:

[0093] The sgRNA sequence targeting the human GLRX3 gene in Example 1 was replaced by the sgRNA sequence targeting the promoter of the human PGRN gene. The sgRNA sequence targeting the promoter of the human PGRN gene inserted in two BsaIs is as follows:

[0094] CGTCGGGACAGCTCTCAGCA.

[0095] The other structures of the carrier are the same as in Example 1.

[0096] In step 4 of the construction method, the method for constructing the pU6-sgRNA-AZ2.0-PGRN Pro vector targeting the human PGRN gene promoter is:

[0097] The sgRNA fragment targeting the promoter of the human PGRN gene is formed by annealing primers synthesized by Suzhou Jinweizhi Co., Ltd. The sequence is as follows (the underline indicates the cohesive end sequence):

[0098] P5: ACCG CGTCGGGACAGCCTCAGCA;

[0099] P6: AAA...

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Abstract

The invention discloses a carrier for expressing an aptamer ribozyme-modified sgRNA regulated by theophylline, inserting an aptamer at the position of the four-base loop (tetraloop) and stem loop 2 (stem loop 2) of the expressed sgRNA-AZ 2.0 backbone. Ribozyme P1‑F5; the U6 promoter and the sgRNA‑AZ 2.0 backbone were sequentially connected into the vector pUC19 / EKSHL through the Kpn Ⅰ and EcoR Ⅰ sites and the EcoR Ⅰ and Spe Ⅰ sites, respectively, to obtain the vector pU6‑sgRNA‑AZ 2.0; the vector pU6‑sgRNA‑AZ 2.0 was digested with BsaⅠ, and the sgRNA sequence targeting the target gene was designed and ligated using sticky ends to obtain the pU6‑sgRNA‑AZ 2.0‑target site. It can effectively mediate the genome editing function of the Cas9 protein, and its editing activity is regulated by theophylline; it can also mediate the transcriptional inhibition of the target gene by the dCas9-KRAB protein, and its inhibitory activity is regulated by theophylline.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a carrier, in particular to a carrier expressing an aptamer ribozyme-modified sgRNA regulated by theophylline and an application thereof. Background technique [0002] Aptamer ribozyme (Aptazyme) aptamer ribozyme riboswitch is a kind of artificial gene regulation switch that appeared in recent years. The most common aptamer ribozyme consists of a hammerhead ribozyme and an aptamer, which has a clear structure and is easy to design. As a cis-acting element, under the action of a specific ligand, the aptamer ribozyme riboswitch can regulate the translation of mRNA by regulating its own shearing reaction without the assistance of protein molecules, and can be applied to the production of various cells. gene regulation. A reported aptamer ribozyme P1-F5 ( et al., 2010; Ketzer, et al., 2014) can undergo self-cleavage after binding with theophylline, resulting in cleavage and degradation ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/113C12N9/22
CPCC12N9/22C12N15/113C12N15/63C12N2320/50
Inventor 夏海滨陈皓郑晓晶代鑫李燕陈芳
Owner SHAANXI NORMAL UNIV
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