CN107245493B - Vector for expressing aptamer ribozyme modified sgRNA regulated and controlled by theophylline and application - Google Patents
Vector for expressing aptamer ribozyme modified sgRNA regulated and controlled by theophylline and application Download PDFInfo
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Abstract
The invention discloses a carrier for expressing aptamer ribozyme modified sgRNA regulated by theophylline, wherein aptamer ribozyme P1-F5 is inserted into the positions of a four-base ring (tetra loop) and a stem ring 2(stem loop2) of an expressed sgRNA-AZ2.0 framework; sequentially connecting a U6 promoter and a sgRNA-AZ2.0 framework into a vector pUC19/EKSHL through Kpn I and EcoRI sites and EcoRI and SpeI sites respectively to obtain a vector pU6-sgRNA-AZ 2.0; the vector pU6-sgRNA-AZ2.0 was digested with Bsa I, and the sgRNA sequence for the target gene was designed and ligated using sticky ends to obtain pU6-sgRNA-AZ 2.0-target site. The genome editing function of the Cas9 protein can be effectively mediated, and the editing activity of the Cas9 protein is regulated and controlled by theophylline; and can mediate dCas9-KRAB protein to inhibit the transcription of target gene and make its inhibiting activity be regulated by theophylline.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a vector, in particular to a vector for expressing an aptamer ribozyme modified sgRNA regulated and controlled by theophylline and application thereof.
Background
Aptamer ribozyme (Aptazyme) an aptamer ribozyme type riboswitch is an artificial gene regulation switch that has emerged in recent years. The most common aptamer ribozyme consists of hammerhead ribozyme and aptamer, has clear structure and is easy to design. As a cis-acting element, the aptamer ribozyme riboswitch can regulate the translation of mRNA by regulating the self-shearing reaction under the action of a specific ligand without the assistance of protein molecules, and can be applied to the gene regulation of various cells. One reported aptamer ribozyme P1-F5(
et al, 2010; ketzer, et al, 2014) can undergo self-shearing after being combined with theophylline, thereby causing the cutting and degradation of mRNA, thereby playing a role in regulating gene expression.
The Clustered Regulated Interstitial Short Palindromic Repeats (CRISPR)/CRISPR-associated (cas)9 system was successfully engineered into a third generation artificial endonuclease useful for the editing of various complex genomes as well as zinc finger endonucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). At present, the technology is successfully applied to the precise modification of human cells, zebra fish, mice and bacterial genomes, and the modification types comprise gene site-specific mutation, gene site-specific knock-in, simultaneous mutation of two sites and deletion of small fragments. Due to the characteristics of high mutation efficiency, simple preparation and low cost, the method is considered to be a genome site-specific modification molecular tool with wide application prospect. The Cas9 nuclease can bind to any DNA containing PAM sequence under the guidance of sgRNA, wherein 20 nucleic acid sequences in the sgRNA target specific DNA through base complementary pairing, and binding of Cas9 and the target DNA is stabilized. The sgRNA can be complementarily paired with the target DNA to ensure Cas9 action site specificity by altering the sequence of the sgRNA to bind to any site. However, subsequent studies found that Cas9 allowed different numbers of mismatches between the gRNA 20nt sequence and the target DNA, depending on the number, location, and nature of the base mismatches. The resulting off-target problem limits the application of Cas9 editing technology in the clinic. The existing research indicates that the off-target effect of excessive cutting can be avoided by controlling the action time of the CRISPR-Cas9 system and turning off the CRISPR-Cas9 system after the cutting and editing aim is achieved.
The modified CRISPR/Cas9 can also be used for activating or inhibiting the expression of a target gene, a mutant dCas9 without cleavage activity, which is obtained by mutating a Cas9 protein, has the characteristic of combining the target gene under the guidance of a sgRNA sequence but not cleaving, and a dCas9-VP64 molecule constructed by fusing dCas9 and a transcription activation domain VP64 becomes a transcription activation factor capable of targeting a specific gene. Similarly, the transcriptional repression domain KRAB is fused at the 3' end of dCas9, and the obtained dCas9-KRAB molecule becomes a transcriptional repressing factor capable of targeting a specific gene. However, in gene therapy and functional studies, more precise temporal control is often required for the regulation of expression of a particular gene. Therefore, effective control means are also required for dCas9-VP64 or dCas 9-KRAB-mediated gene expression.
The main control segment for CRISPR/Cas9 system-mediated genome editing and dCas9-VP64 or dCas 9-KRAB-mediated gene expression activation and inhibition is regulated at the transcription, post-translation, etc. level of Cas9 protein. For example, Davis et al have modified Cas9 protein, so that it can undergo post-translational protein editing in the presence of 4-hydroxyttamoxifen (4-Hydroxytamoxifen) as a drug to generate active Cas9 protein, thereby initiating CRISPR/Cas9 system for gene editing. There are also researchers using tetracycline regulation systems to regulate Cas9 protein or sgRNA expression to regulate CRISPR/Cas9 activity, but such regulation systems would require the introduction of additional cis-regulatory elements and trans-regulatory factors.
A method for controlling the activity of CRISPR/Cas9 by regulating sgRNA through a pathway without introducing additional cis-regulatory elements and trans-regulatory factors is a new strategy. The applicant researches and discovers that by constructing a vector for expressing the aptamer ribozyme modified sgRNA under theophylline control, when gene editing and transcription control are carried out, the aptamer ribozyme is caused to self-cut by adding the ligand theophylline of the aptamer ribozyme P1-F5 into a culture medium, so that the sgRNA is broken, and the sgRNA guided CRISPR/Cas9 system is inactivated. The regulation method avoids the introduction of additional cis-regulatory elements and trans-regulatory factors, and the vector plasmid occupies small volume and has the characteristic of being regulated by ligand drugs.
Disclosure of Invention
The invention aims to provide a vector for expressing an aptamer ribozyme modified sgRNA regulated by theophylline.
In order to realize the task, the invention adopts the following technical solution:
a carrier for expressing aptamer ribozyme modified sgRNA regulated by theophylline is characterized in that aptamer ribozyme P1-F5 is inserted into the positions of a four-base ring (tetra loop) and a stem loop 2(stem loop2) of an expressed sgRNA-AZ2.0 framework; sequentially connecting a U6 promoter and a sgRNA-AZ2.0 framework into a vector pUC19/EKSHL through Kpn I and EcoRI sites and EcoRI and SpeI sites respectively to obtain a vector pU6-sgRNA-AZ 2.0; the vector pU6-sgRNA-AZ2.0 was digested with Bsa I, and the sgRNA sequence for the target gene was ligated using a cohesive end to obtain pU6-sgRNA-AZ 2.0-target site.
According to the invention, the sequence of the inserted aptamer ribozyme is P1-F5:
GGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCC。
the complete sequence of the sgRNA-AZ2.0 framework is as follows:
GAATTCGGTCTCCGTTTTAGAGCTAGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAG GACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAG CCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCAAGTGGCACCGAGTCGGTGCTTTTTTACTAGT。
the sequence of the U6 promoter is as follows:
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGAGACCGAATTC
the complete sequence of the vector pU6-sgRNA-AZ2.0 is as follows:
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGAGACCGAATTCGGTCTCCGTTTTAGAGCTAGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGC CCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG GCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCA GGGTTCCTGGATTCCACTGCTATCCACGGCCAAGTGGCACCGAGTCGGTGCTTTTTTACTAGT。
according to the invention, the cohesive end is connected with 19 or 20 nucleotides in length aiming at a target gene editing target, and the sequence of a primer for annealing a sgRNA fragment is as follows:
GLRX3：ACCGTGAGGATAGGTAGGCCAAC andAAACGTTGGCCTACCTATCCTCA；
VEGFA：ACCGGGTGAGTGAGTGTGTGCGTG andAAACCACGCACACACTCACTCACC；
PGRN Pro：ACCGCGTCGGGACAGCCTCAGCA andAAACTGCTGAGGCTGTCCCGACG。
the sgRNA sequence aiming at the target gene aims at the target gene editing target and aims at any one of GLRX3 gene, VEGFA gene or PGRN gene promoters. But is not limited to these three.
According to research of an applicant, the HEK293 cell is co-transfected by the carrier pU6-sgRNA-AZ 2.0-target site for expressing the aptamer ribozyme modified sgRNA regulated by theophylline and the carrier hCas9, and an editing experiment on a target gene is performed, wherein an experiment result shows that after the carrier pU6-sgRNA-AZ 2.0-target site for expressing the aptamer ribozyme modified sgRNA regulated by theophylline and the hCas9 carrier are co-transfected into the cell, the target gene can be effectively edited, and the editing activity of the target gene is effectively regulated by theophylline; the experiment results of the regulation experiment of gene transcription inhibition carried out on the vector pU6-sgRNA-AZ 2.0-target site for expressing the aptamer ribozyme modified sgRNA regulated by theophylline, the vector pHAGE EF1 alpha dCas9-KRAB and the vector pGL3-PGRN Pro-Luc cotransformation HEK293 cell constructed by the construction method show that after the vector pU6-sgRNA-AZ 2.0-target site for expressing the aptamer ribozyme modified sgRNA regulated by theophylline, the vector pHAGE EF1 alpha dCas9-KRAB and the vector pGL3-PGRN Pro-Luc cotransfect the cell, the expression of luciferase started by a human PGRN promoter can be inhibited, and the inhibition activity of the vector is effectively regulated by theophylline.
The constructed vector for expressing the aptamer ribozyme modified sgRNA regulated by theophylline can effectively mediate the genome editing function of Cas9 protein, and the editing activity of the vector is regulated by theophylline; and can mediate dCas9-KRAB protein to inhibit the transcription of target gene, and the inhibiting activity is regulated by theophylline.
Drawings
Fig. 1 is a structural diagram of the sgRNA-AZ2.0 framework.
FIG. 2 is a diagram of the vector structure of pU6-sgRNA-AZ 2.0.
FIG. 3 is a diagram of the vector structure of pU6-sgRNA-AZ 2.0-target site.
FIG. 4 is a graph showing the efficiency of editing a target gene mediated by the pU6-sgRNA-AZ 2.0-GLRX3 vector constructed in example 1.
FIG. 5 is a map of the target gene editing efficiency mediated by the pU6-sgRNA-AZ2.0-VEGFA vector constructed in example 2.
FIG. 6 shows the construction of pGL3-PGRN Pro-Luc vector.
FIG. 7 is a graph showing the transcriptional repression activity of a target gene mediated by the pU6-sgRNA-AZ2.0-PGRN Pro vector constructed in example 3.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Detailed Description
The aptamer ribozyme P1-F5 is inserted into the four-base ring (tetra loop) and stem ring 2(stem loop2) positions of an expressed sgRNA-AZ2.0 framework of the vector for expressing the aptamer ribozyme modified sgRNA regulated by theophylline; sequentially connecting a U6 promoter and a sgRNA-AZ2.0 framework into a vector pUC19/EKSHL through Kpn I and EcoRI sites and EcoRI and SpeI sites respectively to obtain a vector pU6-sgRNA-AZ 2.0; the vector pU6-sgRNA-AZ2.0 was digested with Bsa I, and the sgRNA sequence for the target gene was ligated using a cohesive end to obtain the vector pU6-sgRNA-AZ 2.0-target site (FIG. 3).
The constructed vector for expressing the aptamer ribozyme modified sgRNA regulated by theophylline specifically comprises the following steps:
(1) synthesizing sgRNA-AZ2.0 skeletons with aptamer ribozymes P1-F5 inserted at the four-base ring (tetra loop) and stem loop 2(stem loop2) positions;
according to known sequences and the design of the inventors, a scaffold of sgRNA-AZ2.0 with an EcoRI site at the 5 'end and a SpeI site at the 3' end was synthesized, in which the aptamer ribozymes P1-F5 were inserted at the four base rings (tetra loop) and stem loop 2(stem loop2) of the original scaffold, respectively.
(2) Synthetic U6 promoter
Synthesizing a U6 promoter with KpnI at the 5 'end and EcoRI site at the 3' end according to a known sequence;
(3) construction of pU6-sgRNA-AZ2.0 vector
Cutting a U6 promoter fragment by Kpn I and EcoRI, cutting a sgRNA-AZ2.0 fragment by EcoRI and SpeI, carrying out 1% agarose gel electrophoresis, recovering two fragments, cutting a vector pUC19/EKSHL (Xiao, et al., 2016) by Kpn I and SpeI, carrying out 1% agarose gel electrophoresis, and recovering the vector; connecting a fragment U6 promoter, sgRNA-AZ2.0 and a vector pUC19/EKSHL by using T4 ligase, transforming a product into escherichia coli DH5 alpha, selecting a monoclonal strain, extracting plasmid DNA by an alkaline lysis method after culturing, and obtaining a positive clone which is a required vector through enzyme digestion and sequencing identification, wherein the vector is named as pU6-sgRNA-AZ 2.0.
(4) Construction of pU6-sgRNA-AZ 2.0-GLRX3 vector targeting human GLRX3 Gene
The designed sgRNA fragment targeting the human GLRX3 gene was formed by annealing primers synthesized by jingzhi, su, with the following sequences (underlined cohesive end sequences):
the forward primer sequence is:ACCGTGAGGATAGGTAGGCCAAC；
the reverse primer sequence is as follows:AAACGTTGGCCTACCTATCCTCA。
both primers were diluted with ultrapure water to 20. mu.M, and 20. mu.L of each primer was mixed and annealed at room temperature for 1 hour. The obtained fragment is sgRNA fragment targeting human GLRX3 gene, and the end of the sgRNA fragment has a specific cohesive end. The vector pU6-sgRNA-AZ2.0 is cut by Bsa I, the vector is recovered after agarose gel electrophoresis with the mass concentration of 1%, the sgRNA fragment targeting the human GLRX3 gene is connected with the vector pU6-sgRNA-AZ2.0 cut by Bsa I, the product is transformed into escherichia coli DH5 alpha, a monoclonal strain is selected, plasmid DNA is extracted by an alkaline lysis method after culture, the positive clone is obtained by enzyme digestion and sequencing identification and is named as pU6-sgRNA-AZ 2.0-GLRX 3.
(5) Construction of pU6-sgRNA-AZ2.0-VEGFA vector targeting human VEGFA Gene
The sgRNA fragment targeting the human VEGFA gene was formed by annealing of primers synthesized by jinzhi, suzhou (Fu, et al., 2013) of the following sequences (the viscous end sequences are underlined):
the forward primer sequence is:ACCGGGTGAGTGAGTGTGTGCGTG；
the reverse primer sequence is as follows:AAACCACGCACACACTCACTCACC。
both primers were diluted with ultrapure water to 20. mu.M, and 20. mu.L of each primer was mixed and annealed at room temperature for 1 hour. The obtained fragment is the sgRNA fragment targeting the human VEGFA gene, and the tail end of the sgRNA fragment is provided with a specific cohesive end. The vector pU6-sgRNA-AZ2.0 is cut by Bsa I, the vector is recovered after agarose gel electrophoresis with the mass concentration of 1%, the sgRNA fragment targeting the human VEGFA gene is connected with the vector pU6-sgRNA-AZ2.0 cut by Bsa I, the product is transformed into Escherichia coli DH5 alpha, a monoclonal strain is selected, plasmid DNA is extracted by an alkaline lysis method after culture, the positive clone is obtained by enzyme digestion and sequencing identification and is named as pU6-sgRNA-AZ 2.0-VEGFA.
(6) Construction of pU6-sgRNA-AZ2.0-PGRN Pro vector targeting human PGRN Gene promoter
The sgRNA fragment targeting the human PGRN gene promoter was formed by annealing primers synthesized by jingzhi, su, with the following sequences (underlined cohesive end sequences):
the forward primer sequence is:ACCGCGTCGGGACAGCCTCAGCA；
the reverse primer sequence is as follows:AAACTGCTGAGGCTGTCCCGACG。
both primers were diluted with ultrapure water to 20. mu.M, and 20. mu.L of each primer was mixed and annealed at room temperature for 1 hour. The obtained fragment is the sgRNA fragment of the targeted human PGRN gene promoter, and the tail end of the sgRNA fragment is provided with a specific cohesive tail end. The vector pU6-sgRNA-AZ2.0 is cut by Bsa I, the vector is recovered after agarose gel electrophoresis with the mass concentration of 1%, the sgRNA fragment of the target human PGRN gene promoter is connected with the vector pU6-sgRNA-AZ2.0 cut by Bsa I, the product is transformed into Escherichia coli DH5 alpha, a monoclonal strain is selected, plasmid DNA is extracted by an alkaline lysis method after culture, the positive clone is obtained by enzyme digestion and sequencing identification and is named as pU6-sgRNA-AZ2.0-PGRN Pro.
The following are specific examples given by the inventors, it should be noted that the following examples are preferred examples, and the present invention is not limited to these examples
Example 1:
the vector for expressing the theophylline-regulated aptamer ribozyme-modified sgRNA targeting the human GLRX3 gene constructed in this example is as follows:
in the expressed sgRNA-AZ2.0 framework (the framework structure is shown in figure 1), aptamer ribozyme P1-F5 is inserted at the positions of a four-base ring (tetra loop) and a stem ring 2(stem loop2), wherein the sequence of the aptamer ribozyme P1-F5 is as follows:
GGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCC。
the complete sequence of sgRNA-AZ2.0 framework is:
GAATTCGGTCTCCGTTTTAGAGCTAGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAG GACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAG CCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCAAGTGGCACCGAGTCGGTGCTTTTTTACTAGT。
the U6 promoter sequence is as follows:
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGAGACCGAATTC。
the complete sequence of the vector pU6-sgRNA-AZ2.0 is:
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGAGACCGAATTCGGTCTCCGTTTTAGAGCTAGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGC CCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTG GCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCA GGGTTCCTGGATTCCACTGCTATCCACGGCCAAGTGGCACCGAGTCGGTGCTTTTTTACTAGT。
the sgRNA sequences targeted to the human GLRX3 gene inserted in two Bsa i were as follows:
TGAGGATAGGTAGGCCAAC。
the construction method of the carrier of the aptamer ribozyme modified sgRNA which is controlled by theophylline and expresses the targeted human GLRX3 gene comprises the following steps:
1. synthesizing sgRNA-AZ2.0 skeletons with aptamer ribozymes P1-F5 inserted at the four-base ring (tetra loop) and stem loop 2(stem loop2) positions;
according to known sequences and the design of the inventor, a sgRNA-AZ2.0 framework with an EcoRI site at the 5 'end and a SpeI site at the 3' end is synthesized by Jiangsu Jinzhi company, and in the sgRNA-AZ2.0 framework, aptamer ribozymes P1-F5 are inserted at the positions of a four-base ring (tetraloop) and a stem-loop 2(stem loop2) of an original framework respectively.
2. Synthetic U6 promoter
The U6 promoter with KpnI at the 5 'end and EcoRI site at the 3' end was synthesized by Jiangsu Jinzhi.
3. Construction of pU6-sgRNA-AZ2.0 vector
Cutting a U6 promoter fragment by Kpn I and EcoRI, cutting a sgRNA-AZ2.0 fragment by EcoRI and SpeI, carrying out 1% agarose gel electrophoresis, recovering two fragments, cutting a vector pUC19/EKSHL (Xiao, et al., 2016) by Kpn I and SpeI, carrying out 1% agarose gel electrophoresis, and recovering the vector; the fragment U6 promoter, sgRNA-AZ2.0, was ligated to the vector pUC19/EKSHL using T4 ligase under the conditions: 0.5. mu.L of pUC19/EKSHL vector, 2. mu.L of the U6 promoter segment, 2. mu.L of sgRNA-AZ2.0 fragment, 5. mu.L of 2 XT 4DNA quick ligase buffer, 0.5. mu.L of T4DNA quick ligase, transformed competent cells DH 5. alpha. after 1 hour of reaction at 25 ℃ and plated on LB plates containing 100. mu.g/ml ampicillin. And selecting colonies, inoculating the colonies into LB culture solution containing 100 mu g/ml ampicillin, extracting plasmid DNA by an alkaline lysis method after 14-16 hours, and obtaining positive clones by enzyme digestion and sequencing identification. The obtained positive clone was named pU6-sgRNA-AZ2.0 vector, and the structure diagram of the pU6-sgRNA-AZ2.0 vector is shown in FIG. 2.
4. Construction of pU6-sgRNA-AZ 2.0-GLRX3 vector targeting human GLRX3 Gene
The designed sgRNA fragment targeting the human GLRX3 gene was formed by annealing primers synthesized by jingzhi, su, with the following sequences (underlined cohesive end sequences):
P1：ACCGTGAGGATAGGTAGGCCAAC；
P2：AAACGTTGGCCTACCTATCCTCA。
both primers were diluted with ultrapure water to 20. mu.M, and 20. mu.L of each primer was mixed and annealed at room temperature for 1 hour. The obtained fragment is sgRNA fragment targeting human GLRX3 gene, and the end of the sgRNA fragment has a specific cohesive end. After the vector pU6-sgRNA-AZ2.0 is cut by Bsa I, the vector is recovered after agarose gel electrophoresis with the mass concentration of 1%, the sgRNA fragment targeting the human GLRX3 gene is connected with the vector pU6-sgRNA-AZ2.0 cut by Bsa I, and the connection conditions are as follows: pU6-sgRNA-AZ2.0 vector 0.5. mu.L, sgRNA fragment targeting human GLRX3 gene 2. mu.L, 2 XT 4DNA quick ligase buffer 5. mu.L, T4DNA quick ligase 0.5. mu.L, deionized water 2. mu.L, transformed competent cell DH 5. alpha. after 1 hour of reaction at 25 ℃ and plated in LB plate containing 100. mu.g/ml ampicillin. And selecting colonies, inoculating the colonies into LB culture solution containing 100 mu g/ml ampicillin, extracting plasmid DNA by an alkaline lysis method after 14-16 hours, and obtaining positive clones by enzyme digestion and sequencing identification. The obtained positive clone was named pU6-sgRNA-AZ 2.0-GLRX 3.
Example 2:
the vector of the theophylline-regulated aptamer ribozyme-modified sgRNA expressing the targeted human VEGFA gene constructed in this example is as follows:
the sgRNA sequence targeting the human GLRX3 gene in example 1 was replaced by a sgRNA sequence targeting the human VEGFA gene. The sgRNA sequences targeted to the human VEGFA gene inserted in the two Bsa i were as follows:
GGTGAGTGAGTGTGTGCGTG。
the other structure of the carrier was the same as in example 1.
In the construction method step 4, the method for constructing the pU6-sgRNA-AZ2.0-VEGFA vector targeting the human VEGFA gene comprises the following steps:
the sgRNA fragment targeting the human VEGFA gene was formed by annealing primers synthesized by jingzhi, su, with the following sequences (underlined cohesive end sequences):
P3：ACCGGGTGAGTGAGTGTGTGCGTG；
P4：AAACCACGCACACACTCACTCACC。
the other construction methods are the same as example 1, and a vector pU6-sgRNA-AZ2.0-VEGFA of the theophylline-regulated aptamer ribozyme-modified sgRNA expressing the targeted human VEGFA gene is constructed.
Example 3:
the vector of the theophylline-regulated aptamer ribozyme-modified sgRNA expressing the human PGRN gene promoter constructed in this example is as follows:
the sgRNA sequence targeting the human GLRX3 gene in example 1 was replaced with a sgRNA sequence targeting the human PGRN gene promoter. The sgRNA sequences of the targeted human PGRN gene promoters inserted in the two Bsa i were as follows:
CGTCGGGACAGCCTCAGCA。
the other structure of the carrier was the same as in example 1.
In the step 4 of the construction method, the method for constructing the pU6-sgRNA-AZ2.0-PGRN Pro vector of the target human PGRN gene promoter comprises the following steps:
the sgRNA fragment targeting the human PGRN gene promoter was formed by annealing primers synthesized by jingzhi, su, with the following sequences (underlined cohesive end sequences):
P5：ACCGCGTCGGGACAGCCTCAGCA；
P6：AAACTGCTGAGGCTGTCCCGACG。
the other construction methods are the same as example 1, and a vector pU6-sgRNA-AZ2.0-PGRN Pro expressing the theophylline-regulated aptamer ribozyme-modified sgRNA targeting the human PGRN gene promoter is constructed.
In order to verify the beneficial effects of the constructed vector for expressing the theophylline-regulated aptamer ribozyme-modified sgRNA, the inventors carried out experiments using the theophylline-regulated aptamer ribozyme-modified sgRNA vector pU6-sgRNA-AZ 2.0-GLRX3 that was constructed in example 1 for expressing the human GLRX3 gene, the theophylline-regulated aptamer ribozyme-modified sgRNA vector pU6-sgRNA-AZ2.0-VEGFA that was constructed in example 2 for expressing the human VEGFA gene, and the theophylline-regulated aptamer ribozyme-modified sgRNA vector pU6-sgRNA-AZ2.0-PGRN Pro that was constructed in example 3 for expressing the human PGRN gene promoter, with the experimental conditions as follows:
1. editing efficiency and drug regulation effect of carrier expressing theophylline-regulated aptamer ribozyme modified sgRNA targeting human GLRX3 gene on target gene
The inventors cleaved the vector pU6-sgRNA-AZ2.0 constructed in example 1 with EcoRI and SpeI, recovered the vector after electrophoresis in 1% agarose gel, and inserted the sgRNA backbone fragment without aptamer ribozyme modification, which has the following sequence and is synthesized by Kingzhi corporation, Suzhou, with EcoRI site at the 5 'end and SpeI site at the 3' end:
GAATTCGGTCTCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTACTAGT。
the obtained vector is named as pU6-sgRNA, the vector is a vector for expressing sgRNA without aptamer ribozyme modification, and the expressed sgRNA is not regulated by theophylline drugs and is used as a control in the experiment. The sgRNA sequence targeting the human GLRX3 gene was also inserted into the vector pU6-sgRNA in the same manner as in step 4 of the construction method in example 1, and the obtained vector was named pU6-sgRNA-GLRX 3. HEK293 cells were seeded in 24-well plates at 1X 10 per well5Cells, co-transfected with the vector hCas9(Addgene, #41815) expressing the protein of Cas9 with pU6-sgRNA-AZ 2.0-GLRX3 into the cells; meanwhile, the vector hCas9 co-transfected with protein expressing Cas9 entered the cell with pU6-sgRNA-GLRX3 as a positive control. Theophylline was added to the cell culture medium with or without addition to a final concentration of 3 mM. Cell genomic DNA was extracted after 48 hours. Regions of the genome that contain editing sites are amplified using nested PCR.
Outer nest reaction:
primer:
P7:ACTCAAACTGGAGGCTGGAG；
P8:GCTGCACCCAGTAAAACACGA。
reaction system:
the conditions for the polymerase chain reaction are: 94 ℃, 30 seconds, 98 ℃, 10 seconds, 70 ℃, 30 seconds (1 ℃ per cycle), 72 ℃, 30 seconds, 10 cycles, 98 ℃, 10 seconds, 60 ℃, 30 seconds, 72 ℃, 30 seconds, 25 cycles.
Inner nest reaction:
primer:
P9:AGGTACCCCCCAGTCTCTGGGATTACA；
P10:AAGATCTAAAAGCAAAGCAGATCCTCCA。
reaction system:
the conditions for the polymerase chain reaction are: 94 ℃, 30 seconds, 98 ℃, 10 seconds, 60 ℃, 30 seconds, 72 ℃, 30 seconds, 30 cycles.
The procedure of denaturation annealing of the obtained PCR product by the PCR instrument is as follows:
5 minutes at 95 ℃; 95-85 deg.C, a reduction of 2 deg.C per second (-2 deg.C/s); 85-25 deg.C, 0.1 deg.C per second (-0.1 deg.C/s).
And (3) recovering the product after agarose gel electrophoresis with the mass concentration of 1%, measuring the content, and detecting the editing efficiency of the target genes in each group by using a classical T7Endonuclease I Endonuclease detection method. The specific method comprises the following steps: each group was prepared from 500ng PCR product DNA, 0.5. mu.L of T7Endonuclease I (NEB, M0302S) Endonuclease, 2. mu.L of 10 XBuffer, and supplemented with water to a total volume of 20. mu.L. The reaction was carried out at 37 ℃ for 20 minutes. After the end, the detection was carried out by 1% agarose gel electrophoresis. The T7Endonuclease I Endonuclease can cut off unpaired parts in the PCR product, if the PCR product is cut into two small bands, the target position is subjected to genome editing, and base mutation is generated, and the brighter the two small bands, the higher the genome editing efficiency is. The results are shown in fig. 4, in the absence of the addition of the drug theophylline, both pU6-sgRNA-AZ 2.0-GLRX3 and pU6-sgRNA-GLRX3 mediated the genome editing by Cas9, but after the addition of theophylline, the effect of vector pU6-sgRNA-AZ 2.0-GLRX3 mediated the genome editing by Cas9 was significantly attenuated, while the effect of pU6-sgRNA-GLRX3 mediated the genome editing by Cas9 was not significantly changed. This result suggests that the effect of vector-mediated Cas9 on genome editing at the human GLRX3 site on expression of theophylline-regulated aptamer ribozyme-modified sgRNA can be effectively controlled by theophylline.
2. Editing efficiency and drug regulation effect of theophylline-regulated aptamer ribozyme modified sgRNA vector for expressing targeted human VEGFA gene on target gene
The inventors also inserted sgRNA sequences targeting the human VEGFA gene into the vector pU6-sgRNA, the method of which is in step 4 of the construction method in example 1The obtained vector was designated pU 6-sgRNA-VEGFA. HEK293 cells were seeded in 24-well plates at 1X 10 per well5Co-transfecting a vector hCas9 expressing protein of Cas9 and pU6-sgRNA-AZ2.0-VEGFA into the cells; meanwhile, the vector hCas9 co-transfected with protein expressing Cas9 and pU6-sgRNA-VEGFA entered the cells as a positive control. Theophylline was added to the cell culture medium with or without addition to a final concentration of 3 mM. After 48 hours, the cells were harvested and genomic DNA was extracted from the cells. Touchdown PCR was used to amplify the portion of the genome that contained the editing site.
Primer:
P11:TCCAGATGGCACATTGTCAG；
P12:AGGGAGCAGGAAAGTGAGGT。
reaction system:
the conditions for the polymerase chain reaction are: 30 seconds at 94 ℃, 10 seconds at 98 ℃, 30 seconds at 70 ℃, 30 seconds at 72 ℃, 30 seconds at 10 cycles (1 ℃ drop per cycle), 30 seconds at 60 ℃, 30 seconds at 72 ℃, 30 seconds at 98 ℃, and 25 cycles.
The obtained PCR product is subjected to denaturation annealing by a PCR instrument, and the editing efficiency of the target genes in each group is detected by a T7E1 endonuclease detection method. The specific method was the same as in experiment 1. The results are shown in fig. 5, which shows that both pU6-sgRNA-AZ2.0-VEGFA and pU6-sgRNA-VEGFA mediated genome editing of Cas9 in the absence of the addition of the drug theophylline, but the effect of vector pU6-sgRNA-AZ2.0-VEGFA mediated genome editing of Cas9 was significantly attenuated after the addition of theophylline, while the effect of pU6-sgRNA-VEGFA mediated genome editing of Cas9 was not significantly changed. This result demonstrates that the effect of the theophylline-regulated aptamer ribozyme-modified sgRNA vector-mediated Cas9 on human VEGFA site genome editing can be effectively controlled by theophylline.
Experiments 1 and 2 show that the effect of the carrier-mediated Cas9 of the aptamer ribozyme modified sgRNA, the expression of which is regulated by theophylline, on the editing of the target site genome can be effectively controlled by theophylline.
3. Theine-regulated aptamer ribozyme-modified sgRNA vector-mediated dCas9-KRAB protein for expressing human PGRN gene promoter-targeted gene transcription inhibition efficiency and drug regulation effect
First, the inventors constructed a luciferase expression vector pGL3-PGRN Pro-Luc driven by the human PGRN promoter, as follows:
use of primers
P13：GGTACCAGGATACTTCTTTGTTG；
P14：AGATCTCTCCTCCCTGCTTCCTCT。
The human PGRN promoter is amplified by taking the genome of the human embryonic kidney HEK293 cell line as a template, and the sequence of the human PGRN promoter is as follows:
GGTACCAGGATACTTCTTTGTTGTGGGGGATTGTTCTGTGTGTCGTGTGATGTTTAGTGGGATTGCTGGCCCTTACCTACCAGATGCCAGTGTCCCTCCACCCTGAGTTGTGACAACCCAGATTGTCTCCAGACACTCCTAAATGTCCCTGGCCGGCAAAATTGCCGCTGCTCAAGAATCACGGCTTTGACGATTAGACTTTGTGATATTTGTTTCAGTCTGTTTAGGTTTTTTTTCTTCTACCTGTATTTTTTTCTGGTTCTGGGTGGTTGTAATTAGTAGGTTATTGATCGATTCACCTAACATTTCATGAAAGTTTCATGTGTGTGTGTGTTTCAATAGAAGCATAAACTATACTCCCTAGTCTCAAGATACACAGGAAGGAAAATAAGCACAAATGTGTCACCAGGGCACAGACTAGTACTAGGTCCTCAGCAGGCCAGGTGTCTTATCCGCTGTCTGGGTCTGCTCTAGCTCCAGGCTTAGAACCCCTGCCACACGACTCCACAGCTCGGTTGGCACCCTTTCCCTCCTCCGACTTCTGCTGCCTCGAGCTTGGTTAGCCATCCCCCTGCCCCTGCCTCATCCTCAGCTCCAGTTCCTTGCTCAGGCTGCAGCAGTCTCCATCCCCTGTGCAGACACTGCCGTTCCTCCACGGCCCAGTATCAGGCTTTCCCTGGGCCTCTCCTCTCTCCTGGCCCATCTCCCATCATCCATCTCTGCCTGGCCCAGGCCCTTTGGCACCAAGCAGGCTGACTCTTGTCACTGGCTAATCTGTTCTGTGGTACATTTTCTCTCCTCACCCTCCCATATCAATTCCTCGAAGGCAGGGCCGATCTGGAGACTAGGAAGCCACTTCTCTTTCGACAGCCCCCACCACAGCCCAGCCCGTGCCAGGCACCCAGCAGCTCCTGAAGCCCACTGGCATTGAACATGGCATTCAATCCCTGCCAAGCCTGCCCTTCCCATCTGGTTTCCCAGGGCTCTTCCCAACACCTCCTCCTCCACCTGCCAGTTAAAATCTTCCCAGACTCAGCTCAAGGAGATGCTCCTAAGGTGGAATGAAATCTCTTCTTCCCCACCTGGAGACAATCTACTTCCTCTCCCTACACCTGGCAACTGGCGCACAACCTTGTATCTTAAATTAGATTCAGCCTGAGACTGTCTCCCACCAATCCCTGCTCCCTGTCCTGCTGAGCACCTTGAGGAAAGGGCTTTGGGGCTGTTTATCTTTGTCCTGGAAACCATCCTTCAACTCACTCTGGGGCCTGCCTAGCATGTCAACCGAGTTTGGAGAATAGGGCAGAATAGGGCAGGACAGGACAGGACAAGACAGGGCAGGATAGGATAGGAGCGAGCCAGCTCAGTAGCTCACATTTGTAATCCCAGCGCCTTGGGGGGCTGCGGTAGGAGAATCGCTTTGGGAGCAGGAGTTGCAGGCCGCAGTGAGCTATGATCAGCTTGGGCGACTGAGCGAGACCCTGTCTCTAAAACAAACACACAAGTCCGGGCGCGGTGGCTCATGCCTGTAATCTTAGCACTTTGGGAGGCCGAGGTGGGCGGATCACGAGGTCAAGAAATCGAGACCATCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCTGGGCGTGGTGGTGCGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCAGAGGTTGCAGTGAGCCGAGATCGTGCCACTGCACTCCAGCCTGGCGACAGAGTGAGACTCCGTCTCAGAACAAACAAACAAAAGGATAGAAAGGCGAGCACAAATATTCCCAATTCATAACACTCCCTCGCACTGTCAATGCCCCAGACACGCGCTATCATCTCTAGCAAACTCCCCCAGGCGCCTGCAGGATGGGTTAAGGAAGGCGACGAGCACCAGCTGCCCTGCTGAGGCTGTCCCGACGTCACATGATTCTCCAATCACATGATCCCTAGAAATGGGGTGTGGGGCGAGAGGAAGCAGGGAGGAGAGATCT。
recovering the PCR product after agarose gel electrophoresis with the mass concentration of 1%, cloning to a pGEMT-easy vector, and obtaining a positive clone through sequencing identification, wherein the positive clone is the required vector and is named as pGEMT-easy-PGRN Pro; the vector pGEMT-easy-PGRN Pro is subjected to Kpn I and Bgl II double enzyme digestion, a PGRN Pro fragment is recovered after 1% agarose gel electrophoresis, the PGRN Pro fragment is connected to a pGL3-basic vector subjected to the same enzyme digestion, after transformation, cloning is selected, plasmid DNA is extracted through an alkaline lysis method after culture, and a positive clone is obtained through enzyme digestion and sequencing identification and is named as a required vector pGL3-PGRN Pro-Luc (the vector structure is shown in figure 6).
The inventors also inserted sgRNA sequences targeting the human PGRN gene promoter into the existing vector pU6-sgRNA, the method was the same as the construction method step 3 in example 1, and the obtained vector was named as pU6-sgRNA-PGRN Pro.
HEK293 cells were seeded in 24-well plates at 1X 10 per well5Cells co-transfected with a vector pHAGE EF1 alpha dCas9-KRAB (Addgene, #50919) expressing the protein of dCas9-KRAB, pGL3-PGRN Pro-Luc and pU6-sgRNA-AZ2.0-PGRN Pro; meanwhile, the co-transfection vectors pHAGE EF1 alpha dCas9-KRAB, pGL3-PGRN Pro-Luc, and pU6-sgRNA-PGRN Pro were introduced into cells as positive controls. Theophylline was added to the cell culture medium at various time points at a concentration of 3 mM. Cells were harvested after 24 hours, and the kit was used to test each group at different time points after theophylline was addedThe luciferase expression of the cells was varied and the results are presented as the ratio between the experimental and control values. The activity of PGRN promoter on pGL3-PGRN Pro-Luc is inhibited by sgRNA targeting the promoter and dCas-KRAB9 protein with transcription inhibition effect, and the aptamer ribozyme contained in the theophylline-controlled aptamer ribozyme modified sgRNA targeting the human PGRN gene promoter expressed by pU6-sgRNA-AZ2.0-PGRN Pro after theophylline is added can be self-sheared to relieve the inhibition activity of the whole system on the PGRN promoter, so the luciferase ratio in the experimental result is enlarged, which indicates that the activity of the PGRN promoter in the experimental group is improved, namely, indicates that the transcription inhibition effect in the system is eliminated by adding drugs. The results are shown in FIG. 7 and indicate that the activity of the luciferase reporter gene begins to increase rapidly 2 hours after theophylline addition.
The experiment 3 shows that the transcription inhibition effect of dCas9-KRAB mediated by the theophylline-regulated aptamer ribozyme-modified sgRNA vector for expressing the target human PGRN gene promoter can be effectively regulated and controlled by theophylline.
Nucleotide or amino acid sequence listing
<110> university of Shanxi university
<120> vector for expressing aptamer ribozyme modified sgRNA regulated and controlled by theophylline and application
<160>
<210> 1
<211> 108
<212> aptamer ribozyme P1-F5
<213> DNA
<220>
<400> 1
GGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCC
<210> 2
<211> 309
<212> sgRNA-AZ2.0 framework:
<213>
<220>
<400> 2
GAATTCGGTCTCCGTTTTAGAGCTAGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCAAGTGGCACCGAGTCGGTGCTTTTTTACTAGT
<210> 3
<211> 284
<212> U6 promoter
<213>
<220>
<400> 3
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGAGACCGAATTC
<210> 4
<211> 587
<212> pU6-sgRNA-AZ 2.0
<213>
<220>
<400> 4
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGAGACCGAATTCGGTCTCCGTTTTAGAGCTAGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCAAGTGGCACCGAGTCGGTGCTTTTTTACTAGT
<210> 5
<211> 23
<212> GLRX3 Forward primer
<213>
<220>
<400> 5
ACCGTGAGGATAGGTAGGCCAAC
<210> 6
<211> 23
<212> GLRX3 reverse primer
<213>
<220>
<400> 6
AAACGTTGGCCTACCTATCCTCA
<210> 7
<211> 24
<212> VEGFA Forward primer
<213>
<220>
<400> 7
ACCGGGTGAGTGAGTGTGTGCGTG
<210> 8
<211> 24
<212> VEGFA reverse primer
<213>
<220>
<400> 8
AAACCACGCACACACTCACTCACC
<210> 9
<211> 23
<212> PGRN Pro sgRNA forward primer
<213>
<220>
<400>9
ACCGCGTCGGGACAGCCTCAGCA
<210> 10
<211> 23
<212> PGRN Pro sgRNA reverse primer
<213>
<220>
<400>10
AAACTGCTGAGGCTGTCCCGACG
<210> 11
<211> 101
<212> sgRNA backbone fragments
<213>
<220>
<400>11
GAATTCGGTCTCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTACTAGT
<210> 12
<211> 20
<212> amplification of the outer nested primer P7 from the editing region of GLRX3
<213>
<220>
<400>12
ACTCAAACTGGAGGCTGGAG
<210> 13
<211> 21
<212> amplification of the outer nested primer P8 from the editing region of GLRX3
<213>
<220>
<400>13
GCTGCACCCAGTAAAACACGA
<210> 14
<211> 27
<212> amplification of the nested primer P9 in the editing region of GLRX3
<213>
<220>
<400>14
AGGTACCCCCCAGTCTCTGGGATTACA
<210> 15
<211> 28
<212> amplification of the nested primer P10 in the editing region of GLRX3
<213>
<220>
<400>15
AAGATCTAAAAGCAAAGCAGATCCTCCA
<210>16
<211>20
<212> VEGFA editing region amplification primer P11
<213>
<220>
<400>16
TCCAGATGGCACATTGTCAG
<210>17
<211>20
<212> VEGFA editing region amplification primer P12
<213>
<220>
<400>17
AGGGAGCAGGAAAGTGAGGT
<210>18
<211>23
<212> PGRN promoter amplification primer P13
<213>
<220>
<400>18
GGTACCAGGATACTTCTTTGTTG
<210>19
<211>24
<212> PGRN promoter amplification primer P14
<213>
<220>
<400>19
AGATCTCTCCTCCCTGCTTCCTCT
<210>20
<211>2033
<212> PGRN promoter
<213>
<220>
<400>20
GGTACCAGGATACTTCTTTGTTGTGGGGGATTGTTCTGTGTGTCGTGTGATGTTTAGTGGGATTGCTGGCCCTTACCTACCAGATGCCAGTGTCCCTCCACCCTGAGTTGTGACAACCCAGATTGTCTCCAGACACTCCTAAATGTCCCTGGCCGGCAAAATTGCCGCTGCTCAAGAATCACGGCTTTGACGATTAGACTTTGTGATATTTGTTTCAGTCTGTTTAGGTTTTTTTTCTTCTACCTGTATTTTTTTCTGGTTCTGGGTGGTTGTAATTAGTAGGTTATTGATCGATTCACCTAACATTTCATGAAAGTTTCATGTGTGTGTGTGTTTCAATAGAAGCATAAACTATACTCCCTAGTCTCAAGATACACAGGAAGGAAAATAAGCACAAATGTGTCACCAGGGCACAGACTAGTACTAGGTCCTCAGCAGGCCAGGTGTCTTATCCGCTGTCTGGGTCTGCTCTAGCTCCAGGCTTAGAACCCCTGCCACACGACTCCACAGCTCGGTTGGCACCCTTTCCCTCCTCCGACTTCTGCTGCCTCGAGCTTGGTTAGCCATCCCCCTGCCCCTGCCTCATCCTCAGCTCCAGTTCCTTGCTCAGGCTGCAGCAGTCTCCATCCCCTGTGCAGACACTGCCGTTCCTCCACGGCCCAGTATCAGGCTTTCCCTGGGCCTCTCCTCTCTCCTGGCCCATCTCCCATCATCCATCTCTGCCTGGCCCAGGCCCTTTGGCACCAAGCAGGCTGACTCTTGTCACTGGCTAATCTGTTCTGTGGTACATTTTCTCTCCTCACCCTCCCATATCAATTCCTCGAAGGCAGGGCCGATCTGGAGACTAGGAAGCCACTTCTCTTTCGACAGCCCCCACCACAGCCCAGCCCGTGCCAGGCACCCAGCAGCTCCTGAAGCCCACTGGCATTGAACATGGCATTCAATCCCTGCCAAGCCTGCCCTTCCCATCTGGTTTCCCAGGGCTCTTCCCAACACCTCCTCCTCCACCTGCCAGTTAAAATCTTCCCAGACTCAGCTCAAGGAGATGCTCCTAAGGTGGAATGAAATCTCTTCTTCCCCACCTGGAGACAATCTACTTCCTCTCCCTACACCTGGCAACTGGCGCACAACCTTGTATCTTAAATTAGATTCAGCCTGAGACTGTCTCCCACCAATCCCTGCTCCCTGTCCTGCTGAGCACCTTGAGGAAAGGGCTTTGGGGCTGTTTATCTTTGTCCTGGAAACCATCCTTCAACTCACTCTGGGGCCTGCCTAGCATGTCAACCGAGTTTGGAGAATAGGGCAGAATAGGGCAGGACAGGACAGGACAAGACAGGGCAGGATAGGATAGGAGCGAGCCAGCTCAGTAGCTCACATTTGTAATCCCAGCGCCTTGGGGGGCTGCGGTAGGAGAATCGCTTTGGGAGCAGGAGTTGCAGGCCGCAGTGAGCTATGATCAGCTTGGGCGACTGAGCGAGACCCTGTCTCTAAAACAAACACACAAGTCCGGGCGCGGTGGCTCATGCCTGTAATCTTAGCACTTTGGGAGGCCGAGGTGGGCGGATCACGAGGTCAAGAAATCGAGACCATCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCTGGGCGTGGTGGTGCGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGCAGAGGTTGCAGTGAGCCGAGATCGTGCCACTGCACTCCAGCCTGGCGACAGAGTGAGACTCCGTCTCAGAACAAACAAACAAAAGGATAGAAAGGCGAGCACAAATATTCCCAATTCATAACACTCCCTCGCACTGTCAATGCCCCAGACACGCGCTATCATCTCTAGCAAACTCCCCCAGGCGCCTGCAGGATGGGTTAAGGAAGGCGACGAGCACCAGCTGCCCTGCTGAGGCTGTCCCGACGTCACATGATTCTCCAATCACATGATCCCTAGAAATGGGGTGTGGGGCGAGAGGAAGCAGGGAGGAGAGATCT
Claims (9)
1. A carrier for expressing aptamer ribozyme modified sgRNA regulated by theophylline is characterized in that aptamer ribozyme P1-F5 is inserted into the positions of a four-base ring (tetra loop) and a stem ring 2(stem loop2) of an expressed sgRNA-AZ2.0 framework; sequentially connecting a U6 promoter and a sgRNA-AZ2.0 framework into a vector pUC19/EKSHL through Kpn I and EcoRI sites and EcoRI and SpeI sites respectively to obtain a vector pU6-sgRNA-AZ 2.0; the vector pU6-sgRNA-AZ2.0 was digested with Bsa I, and the sgRNA sequence for the target gene was ligated using a cohesive end to obtain pU6-sgRNA-AZ 2.0-target site.
2. The vector for expressing the theophylline-regulated aptamer ribozyme-modified sgRNA according to claim 1, wherein: the sequence of the inserted aptamer ribozyme P1-F5 is as follows:
GGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCC。
3. the vector for expressing the theophylline-regulated aptamer ribozyme-modified sgRNA of claim 1, wherein the complete sequence of the sgRNA-AZ2.0 backbone is:
GAATTCGGTCTCCGTTTTAGAGCTAGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACG AAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCAT ACCAGCCGAAAGGCCCTTGGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCAAGTGGCACCGAGTCGGTGCTTTTTTACTAGT。
4. the vector for expressing the theophylline regulated aptamer ribozyme modified sgRNA of claim 1, wherein the U6 promoter sequence is as follows:
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGAGACCGAATTC。
5. the vector for expressing the theophylline controlled aptamer ribozyme modified sgRNA of claim 1, wherein the complete sequence of the vector pU6-sgRNA-AZ2.0 is:
GGTACCAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGAGACCGAATTCGGTCTCCGTTTTAGAGCTAGGCCCTGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTT GGCAGGGTTCCTGGATTCCACTGCTATCCACGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGGCCC TGAGATGCAGGTACATCCAGCTGATGAGTCCCAAATAGGACGAAAGCCATACCAGCCGAAAGGCCCTTGGCAGGGT TCCTGGATTCCACTGCTATCCACGGCCAAGTGGCACCGAGTCGGTGCTTTTTTACTAGT。
6. the vector for expressing the theophylline regulated aptamer ribozyme modified sgRNA of claim 1, wherein the sgRNA sequence directed against the target gene is directed against the target gene editing target and is directed against any one of the promoters of GLRX3 gene, VEGFA gene or PGRN gene.
7. The vector for expressing the theophylline-regulated aptamer ribozyme-modified sgRNA according to claim 1, wherein the cohesive end of the sgRNA is ligated to a target gene editing site, which is 19 or 20 nucleotides in length, and the sequence of primers annealing to the sgRNA fragments of the sgRNA is as follows:
GLRX3：ACCGTGAGGATAGGTAGGCCAAC andAAACGTTGGCCTACCTATCCTCA；
VEGFA：ACCGGGTGAGTGAGTGTGTGCGTG andAAACCACGCACACACTCACTCACC；
PGRN Pro：ACCGCGTCGGGACAGCCTCAGCA andAAACTGCTGAGGCTGTCCCGACG。
8. use of a vector according to one of claims 1 to 7 for expressing a theophylline-regulated aptamer ribozyme-modified sgRNA in genome editing.
9. Use of a vector according to any one of claims 1 to 7 for expressing a theophylline-regulated aptamer ribozyme-modified sgRNA in gene transcription regulation.
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| CN108728441B (en) * | 2018-04-18 | 2022-07-22 | 深圳市第二人民医院 | Gene system for specifically recognizing P53 mutation |
| CN109852650B (en) * | 2018-12-18 | 2020-12-01 | 江南大学 | Artificial aptamer enzyme regulated and controlled by theophylline and application |
| EP3911334A4 (en) * | 2019-01-17 | 2023-07-26 | The Regents of the University of Colorado, a body corporate | Small-molecule regulation of crispr-cas9 using rna aptamers |
| CN112662674B (en) * | 2021-01-12 | 2023-04-11 | 广州瑞风生物科技有限公司 | gRNA for targeted editing of VEGFA gene exon region and application thereof |
| CN113564155B (en) * | 2021-07-22 | 2023-10-31 | 华侨大学 | Aptamer screening method and application thereof |
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