CMV-hspCas9-T2A-Puro-H1-AAVS1_gRNA All-in-one Cas9 SmartNuclease™ & AAVS1 gRNA Lentivector
- Easy, precise knock-in of any gene
- Consistent, robust transgene expression from the AAVS1 Safe Harbor Site
- Simplified construction of isogenic cell lines
- Minimal off-target integration when using our AAVS1 HR Targeting Vectors
- Streamlined CRISPR/Cas9 library screening
Products
| Catalog Number | Description | Size | Price | Quantity | Add to Cart | |||
|---|---|---|---|---|---|---|---|---|
| CASLV601PA-1 | All-in-one Cas9 SmartNuclease AAVS1 Targeting Lentivector (CMV-hspCas9-T2A-Puro-H1-AAVS1_gRNA) | 10 µg | $968 |
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| CASLV601VA-1 | All-in-one Cas9 SmartNuclease AAVS1 Targeting Lentivector (CMV-hspCas9-T2A-Puro-H1-AAVS1_gRNA) pre-packaged lentiviral particles (>10^6 IFUs) | 2 x 25 µL | $643 |
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Overview
Overview
Target the AAVS1 Safe Harbor Site for genome editing in hard-to-transfect cells
When you have hard-to-transfect cells and want to take advantage of the robust and reliable expression that’s possible from the AAVS1 Safe Harbor Site, SBI offers a streamlined plasmid system that’s ready to go. The CMV-hspCas9-T2A-Puro-H1-AAVS1_gRNA All-in-one Cas9 SmartNuclease™ & AAVS1 gRNA Lentivector expresses both Cas9 and an AAVS1-targeting gRNA—just co-infect with a homologous recombination (HR) Targeting Vector (if necessary) and select for your desired colonies.
Our Cas9 SmartNuclease Lentivectors are available as both ready-to-package plasmids and ready-to-transduce lentiviral particles—just choose the format that works best for your studies.
The power of the AAVS1 Safe Harbor Site
Delivering consistent, robust transgene expression, the AAVS1 safe harbor site is a preferred target for gene knock-ins. Insertion at the site has been shown to be safe with no phenotypic effects reported, and the surrounding DNA appears to be kept in an open confirmation, enabling stable expression of a variety of transgenes.
SBI’s AAVS1 Safe Harbor Targeting products deliver:
- Easy, precise knock-in of any gene
- Consistent, robust transgene expression from the AAVS1 Safe Harbor Site
- Simplified construction of isogenic cell lines
- Minimal off-target integration when using our AAVS1 HR Targeting Vectors
- Streamlined CRISPR/Cas9 library screening
Note that we’ve specially designed a series of AAVS1 HR Targeting Vectors that greatly reduce off-target integration events (Cat.# GE620A-1, GE622A-1, and GE624A-1). Taking advantage of the AAVS1's location within an intron, these vectors come with a puromycin marker that has no promoter, only a splice acceptor site—expression of puromycin can only occur when the construct integrates within an intron, reducing the probability of recovering off-target integrants in the presence of puromycin selection.
As with all of our Cas9 delivery options, the All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid is functionally validated and comes backed by our expert technical support team—if you’ve got a genome engineering question just ask by emailing tech@systembio.com.
Not sure whether you need a CRISPR/Cas9 plasmid, purified protein, or mRNA?
Use this table to choose the CRISPR/Cas9 product that’s right for you:
For This Application | In these types of cells | Use These Products |
|---|---|---|
MODIFYING ORGANISMS
| Embryos—to create transgenic animals | Injectable Cas9 mRNA & gRNA Synthesis Kits Cas9 Protein EGFP-labeled Cas9 Protein |
| Animals models—in vivo genome editing | AAV-Cas9 Vectors Cas9 Protein EGFP-labeled Cas9 Protein |
|
MODIFYING CELL LINES
| Cells that are transfectable | Cas9 Plasmids Cas9 Protein EGFP-labeled Cas9 Protein |
Difficult-to-transfect cell lines:
| AAV-Cas9 Vectors Lenti Cas9 Systems |
|
SCREENING
| All cell types requiring stable Cas9 overexpression | Lenti Cas9 Systems AAVS1 Safe Harbor Site Cas9 Gene Knock-in System Cas9 Protein EGFP-labeled Cas9 Protein |
PRE-CLINICAL APPLICATIONS
| All cell types and applications | Cas9 Nickase, available in all delivery formats Cas9 Protein EGFP-labeled Cas9 Protein |
| SIMULTANEOUS ENGINEERING OF MULTIPLE MUTATIONS | All cell types and applications | Multiplex gRNA cloning kit, compatible with all Cas9 delivery options |
References
How It Works
How It Works
Genome engineering with CRISPR/Cas9
For general guidance on using CRISPR/Cas9 technology for genome engineering, take a look at our CRISPR/Cas9 tutorials as well as the following application notes:
CRISPR/Cas9 Gene Knock-Out Application Note (PDF) »
CRISPR/Cas9 Gene Editing Application Note (PDF) »
CRISPR/Cas9 Gene Tagging Application Note (PDF) »
CRISPR/Cas9 Basics
Through careful selection of the target sequence and design of a donor plasmid for homologous recombination, you can achieve efficient and highly targeted genomic modification with CRISPR/Cas9.
The system
Cas9 protein—uses guide RNA (gRNA) to direct site-specific, double-strand DNA cleavage adjacent to a protospacer adapter motif (PAM) in the target DNA.
gRNA—RNA sequence that guides Cas9 to cleave a homologous region in the target genome. Efficient cleavage only where the gRNA homology is adjacent to a PAM.
PAM—protospacer adapter motif, NGG, is a target DNA sequence that spCas9 will cut upstream from if directed to by the gRNA.
The workflow at-a-glance
DESIGN: Select gRNA and HR donor plasmids. Choice of gRNA site and design of donor plasmid determines whether the homologous recombination event results in a knock-out, knock-in, edit, or tagging.
CONSTRUCT: Clone gRNA into all-in-one Cas9 vector. Clone 5’ and 3’ homology arms into HR donor plasmid. If creating a knock-in, clone desired gene into HR donor.
CO-TRANSFECT or CO-INJECT: Introduce Cas9, gRNA, and HR Donors into the target cells using co-transfection for plasmids, co-transduction for lentivirus, or co-injection for mRNAs.
SELECT/SCREEN: Select or screen for mutants and verify.
VALIDATE: Genotype or sequence putative mutants to verify single or biallelic conversion.
Supporting Data
FAQs
Resources
Citations
Related Products
Products
| Catalog Number | Description | Size | Price | Quantity | Add to Cart | |||
|---|---|---|---|---|---|---|---|---|
| CASLV601PA-1 | All-in-one Cas9 SmartNuclease AAVS1 Targeting Lentivector (CMV-hspCas9-T2A-Puro-H1-AAVS1_gRNA) | 10 µg | $968 |
|
||||
| CASLV601VA-1 | All-in-one Cas9 SmartNuclease AAVS1 Targeting Lentivector (CMV-hspCas9-T2A-Puro-H1-AAVS1_gRNA) pre-packaged lentiviral particles (>10^6 IFUs) | 2 x 25 µL | $643 |
|
||||
Overview
Overview
Target the AAVS1 Safe Harbor Site for genome editing in hard-to-transfect cells
When you have hard-to-transfect cells and want to take advantage of the robust and reliable expression that’s possible from the AAVS1 Safe Harbor Site, SBI offers a streamlined plasmid system that’s ready to go. The CMV-hspCas9-T2A-Puro-H1-AAVS1_gRNA All-in-one Cas9 SmartNuclease™ & AAVS1 gRNA Lentivector expresses both Cas9 and an AAVS1-targeting gRNA—just co-infect with a homologous recombination (HR) Targeting Vector (if necessary) and select for your desired colonies.
Our Cas9 SmartNuclease Lentivectors are available as both ready-to-package plasmids and ready-to-transduce lentiviral particles—just choose the format that works best for your studies.
The power of the AAVS1 Safe Harbor Site
Delivering consistent, robust transgene expression, the AAVS1 safe harbor site is a preferred target for gene knock-ins. Insertion at the site has been shown to be safe with no phenotypic effects reported, and the surrounding DNA appears to be kept in an open confirmation, enabling stable expression of a variety of transgenes.
SBI’s AAVS1 Safe Harbor Targeting products deliver:
- Easy, precise knock-in of any gene
- Consistent, robust transgene expression from the AAVS1 Safe Harbor Site
- Simplified construction of isogenic cell lines
- Minimal off-target integration when using our AAVS1 HR Targeting Vectors
- Streamlined CRISPR/Cas9 library screening
Note that we’ve specially designed a series of AAVS1 HR Targeting Vectors that greatly reduce off-target integration events (Cat.# GE620A-1, GE622A-1, and GE624A-1). Taking advantage of the AAVS1's location within an intron, these vectors come with a puromycin marker that has no promoter, only a splice acceptor site—expression of puromycin can only occur when the construct integrates within an intron, reducing the probability of recovering off-target integrants in the presence of puromycin selection.
As with all of our Cas9 delivery options, the All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid is functionally validated and comes backed by our expert technical support team—if you’ve got a genome engineering question just ask by emailing tech@systembio.com.
Not sure whether you need a CRISPR/Cas9 plasmid, purified protein, or mRNA?
Use this table to choose the CRISPR/Cas9 product that’s right for you:
For This Application | In these types of cells | Use These Products |
|---|---|---|
MODIFYING ORGANISMS
| Embryos—to create transgenic animals | Injectable Cas9 mRNA & gRNA Synthesis Kits Cas9 Protein EGFP-labeled Cas9 Protein |
| Animals models—in vivo genome editing | AAV-Cas9 Vectors Cas9 Protein EGFP-labeled Cas9 Protein |
|
MODIFYING CELL LINES
| Cells that are transfectable | Cas9 Plasmids Cas9 Protein EGFP-labeled Cas9 Protein |
Difficult-to-transfect cell lines:
| AAV-Cas9 Vectors Lenti Cas9 Systems |
|
SCREENING
| All cell types requiring stable Cas9 overexpression | Lenti Cas9 Systems AAVS1 Safe Harbor Site Cas9 Gene Knock-in System Cas9 Protein EGFP-labeled Cas9 Protein |
PRE-CLINICAL APPLICATIONS
| All cell types and applications | Cas9 Nickase, available in all delivery formats Cas9 Protein EGFP-labeled Cas9 Protein |
| SIMULTANEOUS ENGINEERING OF MULTIPLE MUTATIONS | All cell types and applications | Multiplex gRNA cloning kit, compatible with all Cas9 delivery options |
References
How It Works
How It Works
Genome engineering with CRISPR/Cas9
For general guidance on using CRISPR/Cas9 technology for genome engineering, take a look at our CRISPR/Cas9 tutorials as well as the following application notes:
CRISPR/Cas9 Gene Knock-Out Application Note (PDF) »
CRISPR/Cas9 Gene Editing Application Note (PDF) »
CRISPR/Cas9 Gene Tagging Application Note (PDF) »
CRISPR/Cas9 Basics
Through careful selection of the target sequence and design of a donor plasmid for homologous recombination, you can achieve efficient and highly targeted genomic modification with CRISPR/Cas9.
The system
Cas9 protein—uses guide RNA (gRNA) to direct site-specific, double-strand DNA cleavage adjacent to a protospacer adapter motif (PAM) in the target DNA.
gRNA—RNA sequence that guides Cas9 to cleave a homologous region in the target genome. Efficient cleavage only where the gRNA homology is adjacent to a PAM.
PAM—protospacer adapter motif, NGG, is a target DNA sequence that spCas9 will cut upstream from if directed to by the gRNA.
The workflow at-a-glance
DESIGN: Select gRNA and HR donor plasmids. Choice of gRNA site and design of donor plasmid determines whether the homologous recombination event results in a knock-out, knock-in, edit, or tagging.
CONSTRUCT: Clone gRNA into all-in-one Cas9 vector. Clone 5’ and 3’ homology arms into HR donor plasmid. If creating a knock-in, clone desired gene into HR donor.
CO-TRANSFECT or CO-INJECT: Introduce Cas9, gRNA, and HR Donors into the target cells using co-transfection for plasmids, co-transduction for lentivirus, or co-injection for mRNAs.
SELECT/SCREEN: Select or screen for mutants and verify.
VALIDATE: Genotype or sequence putative mutants to verify single or biallelic conversion.