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.
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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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