AAVS1 SparQ™ All-In-One Inducible System
Controllable Gene Modulation at Specific Site
Introducing the AAVS1 SparQTM All-In-One Inducible System, a groundbreaking solution that combines the power of the AAVS1 safe harbor site and CRISPR/Cas9 technology to revolutionize inducible gene expression. This innovative product provides researchers with a comprehensive tool for precise control and manipulation of gene expression, enabling advanced gene therapies, functional genomics studies, and molecular investigations.Discover the Advantages of SBI’s AAVS1 SparQTM All-In-One Inducible System:
- Targeted Integration at AAVS1 Safe Harbor Site
- Inducible Gene Expression
- High Specificity
- Effortless Cell Line Generation
- Easy Use with All-In-One Design
Products
| Catalog Number | Description | Size | Price | Quantity | Add to Cart | |||
|---|---|---|---|---|---|---|---|---|
| GEQM800A-1 | AAVS1 SparQTM All-In-One Inducible Donor Vector (AAVS1-SA-RFP-Puro-EF1-CymR-CuO-MCS) | 10 µg | $1400 |
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| GEQM820A-1 | AAVS1 SparQTM All-In-One Inducible Donor Vector (AAVS1-SA-RFP-Puro-EF1-CymR-CuO-MCS-P2A-GFP) | 10 µg | $1400 |
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| GEQM820A-Kit | AAVS1 SparQTM All-In-One Inducible System GEQM820A-1(AAVS1-SA-RFP-Puro-EF1-CymR-CuO-MCS-P2A-GFP) with CAS601A-1 (All-in-one Cas9 SmartNuclease AAVS1 Targeting Plasmid) and GEQM840PR-1 (Junction PCR Primer Mix to confirm AAVS1 site integration) | 1 Kit | $2333 |
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| GEQM800A-Kit | AAVS1 SparQTM All-In-One Inducible System GEQM800A-1 (AAVS1-SA-RFP-Puro-EF1-CymR-CuO-MCS) with CAS601A-1 (All-in-one Cas9 SmartNuclease AAVS1 Targeting Plasmid) and GEQM840PR-1 (Junction PCR Primer Mix to confirm AAVS1 site integration) | 1 Kit | $2333 |
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| GEQM840PR-1 | 5’ and 3’ Primer Mixes for Junction PCR Assays for GEQM-8xx (AAVS1 SparQTM All-In-One Inducible System, 10 µM) | 100 µL | $241 |
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Overview
The AAVS1 safe harbor site, located on chromosome 19 in humans, offers a reliable and stable genomic locus for targeted gene integration. The SparQTM All-In-One Inducible Expression Cassette utilizes a small molecule inducer Cumate to achieve precise control over gene expression in high efficiency. Combined with the versatility of CRISPR/Cas9 technology, the AAVS1 SparQTM All-In-One Inducible System provides a robust platform for controlled transgene expression and manipulation within AAVS1 safe harbor site.
To streamline your research journey, our AAVS1 SparQTM All-In-One Donor Vectors arrive with AAVS1 homology arms pre-cloned. Your task? Simply introduce your gene of interest and co-transfect with a Cas9/AAVS1 gRNA complex, such as our top-tier All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid
SBI’s AAVS1 SparQTM All-In-One Inducible System Offers:
- Targeted Integration at AAVS1 Safe Harbor Site: Harness the precision of CRISPR/Cas9 technology to achieve seamlessly integration at the AAVS1 safe harbor site for reliable gene control.
- Inducible Gene Expression: Achieve precise control over gene expression from AAVS1 safe harbor site, allowing you to turn genes on or off at will.
- High Specificity: Experience minimal off target integration
- Effortless Cell Line Generation: Streamline the construction of site-specific inducible isogenic cell lines.
- Easy Use with All-In-One Design: A unprecedented solution that simplifies the entire process of inducible gene expression, from targeted integration to control.
The AAVS1 safe harbor site stands out as a prime target for gene knock-ins, consistently delivering potent transgene expression. Research has demonstrated that insertions at this site are not only safe but also free from any reported phenotypic effects. Moreover, the neighboring DNA remains in an open conformation, ensuring dependable expression across a range of transgenes. With the integration of CRISPR/Cas9 technology, the AAVS1 SparQTM All-In-One Inducible System facilitates precise embedding of the Cumate inducible expression cassette right at the AAVS1 site. Such strategic placement ensures minimal interference with adjacent genes, presenting a holistic solution for meticulous gene manipulation and regulated expression.
Why Choose One of Our AAVS1 SparQTM All-In-One Donor Vectors?Our AAVS1 SparQTM All-In-One Donor Vectors are ingeniously crafted to ensure minimal off-target integration for highly-specific targeting at the AAVS1 site. Leveraging the AAVS1's strategic position within an intron, the puromycin marker is equipped solely with a splice acceptor site without promoter. This ensures that puromycin expression is activated only upon intron integration, significantly reducing the chances of off-target integrants during puromycin selection.
The All-In-One design incorporates all essential elements required for inducible gene expression into a single, compact construct. It includes the regulatory elements, promoter, cloning site, and the Cumate Switch for inducible control. This streamlined design simplifies experimental workflows, saves time, and enhances research efficiency.
By combining the power of the AAVS1 safe harbor site and CRISPR/Cas9 technology, the AAVS1 SparQTM All-In-One Inducible System empowers researchers to unlock the full potential of inducible gene expression and precise gene editing. Explore new frontiers in gene therapy, functional genomics, and molecular research with this comprehensive solution and drive scientific advancements with unparalleled control and precision.
How It Works
SparQ All-In-One Expression Cassette Knock-In at AAVS1 site
Figure 1. Knocking-in SparQTM All-In-One Expression Cassette at the AAVS1 Site. Step 1: Cas9 introduces a double-stranded break (DSB) at the AAVS1 site. The activity of Cas9 is directed to the AAVS1 site by an AAVS1-specific gRNA. Step 2: The DNA repair machinery is summoned to the DSB. When an HR Donor, which has homology to the region adjacent to the DSB (represented by the blue areas on both the genomic and plasmid DNA), is present, homologous recombination (HR) takes precedence over non-homologous end joining (NHEJ). Result: The HR event results in the insertion of the region from the HR Donor Vector between the two homology arms. As a consequence, the SparQTM All-In-One expression cassette becomes integrated into the AAVS1 site.
Figure 2. Cumate Switch System. The regulatory mechanisms of the bacterial operons cmt and cym have been engineered to regulate gene expression in mammalian cells. In the repressor configuration, regulation is mediated by strong binding of the CymR repressor to the Cumate operator site (CuO), which is downstream of the CMV5 promoter. Addition of cumate (depicted as yellow circle in the graphic scheme below), a non-toxic small molecule inducer, relieves the repression and drives gene activation; in this case, fluorescent proteins (RFP and GFP). Subsequent removal of cumate from the growth media reverts the activation process, rendering gene switches to off mode.
Workflow for Generation of Site Specifc Inducible Isogenic Cell Line
Figure 3. Generating an inducible expression cell line using the AAVS1 SparQTM All-In-One System. Step 1: Co-transfect your target cell line with the all-in-one vector, which expresses both Cas9 and AAVS1-specific gRNA, along with the AAVS1 SparQTM All-in-One donor vector GEQM800A-1 or GEQM820A-1. Step 2: Four to seven days after transfection, apply puromycin for stable cell line selection. Step 3: Perform single-cell sorting using FACS to isolate RFP positive (RFP+) and GFP negative (GFP-) cells. Step 4: For the clonal cell line, validate precise integration at the AAVS1 site using junction PCR. Step 5: Manipulate gene expression using cumate.
Supporting Data
AAVS1 SparQTM Inducible Cell Line is RFP+ and GFP- before induction
Figure 4. Selected clonal cell lines derived from AAVS1 SparQTM inducible system. The HEK 293 cells were co-transfected with GEQM820A-1 and CAS601A-1. After puromycin selection (1 μg/ml), single-cell sorting was performed using FACS to isolate RFP+ and GFP- cells.
Junction PCR Validation for Precise Integration of the SparQTM All-in-One Expression Cassette at the AAVS1 Site
Figure 5. The AAVS1 SparQTM All-in-One expression cell line is specifically integrated at the AAVS1 site. The HEK 293 cells were co-transfected with GEQM820A-1 and CAS601A-1. Following puromycin selection (1 μg/ml) and single-cell sorting using FACS for RFP+ and GFP- cells, individual clonal cell lines were generated. Junction PCR was used to verify the precise integration of the SparQTM All-in-One expression cassette at the AAVS1 site. A) Illustration of the junction PCR design. B) Junction PCR validation using pooled puromycin-resistant cells. C) Validation of individual clonal cell lines by junction PCR.
Gene Expression is Inducible and Reversible
Figure 6. Gene expression in the AAVS1 SparQTM All-in-One expression cell line is inducible and reversible. GFP expression in the GEQM820A-1 stable cell line was induced just one day after adding cumate (cumate (+)) at 30 μg/ml; images were taken on day 2 post-cumate addition. Upon removal of cumate (cumate (-)) from the culture medium, GFP expression gradually diminished. Reintroducing cumate to the culture medium led to further induction of GFP expression.
Precise Control Over Expression Dynamics
Figure 7. Achieve fine-tuned gene regulation in the AAVS1 SparQTM All-in-One expression cell line. The SparQTM inducible expression cassette allows for tight and dose-dependent control over transgene expressions. GFP expression in the GEQM820A-1 stable cell line was induced by adding cumate at various dosages. Images were taken on 1 day and 2 days after adding cumate. On day 3, cumate was removed from the culture medium, and GFP expression gradually diminished accordingly. Images were taken on 3 days after removing of cumate. Researchers can modulate gene expression levels simply by adjusting the concentration of cumate, enabling precise control over expression dynamics for their specific research needs.
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Products
| Catalog Number | Description | Size | Price | Quantity | Add to Cart | |||
|---|---|---|---|---|---|---|---|---|
| GEQM800A-1 | AAVS1 SparQTM All-In-One Inducible Donor Vector (AAVS1-SA-RFP-Puro-EF1-CymR-CuO-MCS) | 10 µg | $1400 |
|
||||
| GEQM820A-1 | AAVS1 SparQTM All-In-One Inducible Donor Vector (AAVS1-SA-RFP-Puro-EF1-CymR-CuO-MCS-P2A-GFP) | 10 µg | $1400 |
|
||||
| GEQM820A-Kit | AAVS1 SparQTM All-In-One Inducible System GEQM820A-1(AAVS1-SA-RFP-Puro-EF1-CymR-CuO-MCS-P2A-GFP) with CAS601A-1 (All-in-one Cas9 SmartNuclease AAVS1 Targeting Plasmid) and GEQM840PR-1 (Junction PCR Primer Mix to confirm AAVS1 site integration) | 1 Kit | $2333 |
|
||||
| GEQM800A-Kit | AAVS1 SparQTM All-In-One Inducible System GEQM800A-1 (AAVS1-SA-RFP-Puro-EF1-CymR-CuO-MCS) with CAS601A-1 (All-in-one Cas9 SmartNuclease AAVS1 Targeting Plasmid) and GEQM840PR-1 (Junction PCR Primer Mix to confirm AAVS1 site integration) | 1 Kit | $2333 |
|
||||
| GEQM840PR-1 | 5’ and 3’ Primer Mixes for Junction PCR Assays for GEQM-8xx (AAVS1 SparQTM All-In-One Inducible System, 10 µM) | 100 µL | $241 |
|
||||
Overview
The AAVS1 safe harbor site, located on chromosome 19 in humans, offers a reliable and stable genomic locus for targeted gene integration. The SparQTM All-In-One Inducible Expression Cassette utilizes a small molecule inducer Cumate to achieve precise control over gene expression in high efficiency. Combined with the versatility of CRISPR/Cas9 technology, the AAVS1 SparQTM All-In-One Inducible System provides a robust platform for controlled transgene expression and manipulation within AAVS1 safe harbor site.
To streamline your research journey, our AAVS1 SparQTM All-In-One Donor Vectors arrive with AAVS1 homology arms pre-cloned. Your task? Simply introduce your gene of interest and co-transfect with a Cas9/AAVS1 gRNA complex, such as our top-tier All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid
SBI’s AAVS1 SparQTM All-In-One Inducible System Offers:
- Targeted Integration at AAVS1 Safe Harbor Site: Harness the precision of CRISPR/Cas9 technology to achieve seamlessly integration at the AAVS1 safe harbor site for reliable gene control.
- Inducible Gene Expression: Achieve precise control over gene expression from AAVS1 safe harbor site, allowing you to turn genes on or off at will.
- High Specificity: Experience minimal off target integration
- Effortless Cell Line Generation: Streamline the construction of site-specific inducible isogenic cell lines.
- Easy Use with All-In-One Design: A unprecedented solution that simplifies the entire process of inducible gene expression, from targeted integration to control.
The AAVS1 safe harbor site stands out as a prime target for gene knock-ins, consistently delivering potent transgene expression. Research has demonstrated that insertions at this site are not only safe but also free from any reported phenotypic effects. Moreover, the neighboring DNA remains in an open conformation, ensuring dependable expression across a range of transgenes. With the integration of CRISPR/Cas9 technology, the AAVS1 SparQTM All-In-One Inducible System facilitates precise embedding of the Cumate inducible expression cassette right at the AAVS1 site. Such strategic placement ensures minimal interference with adjacent genes, presenting a holistic solution for meticulous gene manipulation and regulated expression.
Why Choose One of Our AAVS1 SparQTM All-In-One Donor Vectors?Our AAVS1 SparQTM All-In-One Donor Vectors are ingeniously crafted to ensure minimal off-target integration for highly-specific targeting at the AAVS1 site. Leveraging the AAVS1's strategic position within an intron, the puromycin marker is equipped solely with a splice acceptor site without promoter. This ensures that puromycin expression is activated only upon intron integration, significantly reducing the chances of off-target integrants during puromycin selection.
The All-In-One design incorporates all essential elements required for inducible gene expression into a single, compact construct. It includes the regulatory elements, promoter, cloning site, and the Cumate Switch for inducible control. This streamlined design simplifies experimental workflows, saves time, and enhances research efficiency.
By combining the power of the AAVS1 safe harbor site and CRISPR/Cas9 technology, the AAVS1 SparQTM All-In-One Inducible System empowers researchers to unlock the full potential of inducible gene expression and precise gene editing. Explore new frontiers in gene therapy, functional genomics, and molecular research with this comprehensive solution and drive scientific advancements with unparalleled control and precision.
How It Works
SparQ All-In-One Expression Cassette Knock-In at AAVS1 site
Figure 1. Knocking-in SparQTM All-In-One Expression Cassette at the AAVS1 Site. Step 1: Cas9 introduces a double-stranded break (DSB) at the AAVS1 site. The activity of Cas9 is directed to the AAVS1 site by an AAVS1-specific gRNA. Step 2: The DNA repair machinery is summoned to the DSB. When an HR Donor, which has homology to the region adjacent to the DSB (represented by the blue areas on both the genomic and plasmid DNA), is present, homologous recombination (HR) takes precedence over non-homologous end joining (NHEJ). Result: The HR event results in the insertion of the region from the HR Donor Vector between the two homology arms. As a consequence, the SparQTM All-In-One expression cassette becomes integrated into the AAVS1 site.
Figure 2. Cumate Switch System. The regulatory mechanisms of the bacterial operons cmt and cym have been engineered to regulate gene expression in mammalian cells. In the repressor configuration, regulation is mediated by strong binding of the CymR repressor to the Cumate operator site (CuO), which is downstream of the CMV5 promoter. Addition of cumate (depicted as yellow circle in the graphic scheme below), a non-toxic small molecule inducer, relieves the repression and drives gene activation; in this case, fluorescent proteins (RFP and GFP). Subsequent removal of cumate from the growth media reverts the activation process, rendering gene switches to off mode.
Workflow for Generation of Site Specifc Inducible Isogenic Cell Line
Figure 3. Generating an inducible expression cell line using the AAVS1 SparQTM All-In-One System. Step 1: Co-transfect your target cell line with the all-in-one vector, which expresses both Cas9 and AAVS1-specific gRNA, along with the AAVS1 SparQTM All-in-One donor vector GEQM800A-1 or GEQM820A-1. Step 2: Four to seven days after transfection, apply puromycin for stable cell line selection. Step 3: Perform single-cell sorting using FACS to isolate RFP positive (RFP+) and GFP negative (GFP-) cells. Step 4: For the clonal cell line, validate precise integration at the AAVS1 site using junction PCR. Step 5: Manipulate gene expression using cumate.
Supporting Data
AAVS1 SparQTM Inducible Cell Line is RFP+ and GFP- before induction
Figure 4. Selected clonal cell lines derived from AAVS1 SparQTM inducible system. The HEK 293 cells were co-transfected with GEQM820A-1 and CAS601A-1. After puromycin selection (1 μg/ml), single-cell sorting was performed using FACS to isolate RFP+ and GFP- cells.
Junction PCR Validation for Precise Integration of the SparQTM All-in-One Expression Cassette at the AAVS1 Site
Figure 5. The AAVS1 SparQTM All-in-One expression cell line is specifically integrated at the AAVS1 site. The HEK 293 cells were co-transfected with GEQM820A-1 and CAS601A-1. Following puromycin selection (1 μg/ml) and single-cell sorting using FACS for RFP+ and GFP- cells, individual clonal cell lines were generated. Junction PCR was used to verify the precise integration of the SparQTM All-in-One expression cassette at the AAVS1 site. A) Illustration of the junction PCR design. B) Junction PCR validation using pooled puromycin-resistant cells. C) Validation of individual clonal cell lines by junction PCR.
Gene Expression is Inducible and Reversible
Figure 6. Gene expression in the AAVS1 SparQTM All-in-One expression cell line is inducible and reversible. GFP expression in the GEQM820A-1 stable cell line was induced just one day after adding cumate (cumate (+)) at 30 μg/ml; images were taken on day 2 post-cumate addition. Upon removal of cumate (cumate (-)) from the culture medium, GFP expression gradually diminished. Reintroducing cumate to the culture medium led to further induction of GFP expression.
Precise Control Over Expression Dynamics
Figure 7. Achieve fine-tuned gene regulation in the AAVS1 SparQTM All-in-One expression cell line. The SparQTM inducible expression cassette allows for tight and dose-dependent control over transgene expressions. GFP expression in the GEQM820A-1 stable cell line was induced by adding cumate at various dosages. Images were taken on 1 day and 2 days after adding cumate. On day 3, cumate was removed from the culture medium, and GFP expression gradually diminished accordingly. Images were taken on 3 days after removing of cumate. Researchers can modulate gene expression levels simply by adjusting the concentration of cumate, enabling precise control over expression dynamics for their specific research needs.