CMV-hspCas9(D10A)-T2A-Puro Two Vector Cas9 SmartNickase™ Lentivector
- Conduct genome editing and engineering in difficult-to-transfect cell lines
- Stay more on-target with Cas9 nickase activity
- Drive Cas9 expression from the CMV promoter, for high expression in many commonly-used cell lines (HeLa, HEK293, HT1080)
- Perform in vivo engineering of model organisms
- Supports synthetic biology applications, gene- and cell-based therapy development, and genome-wide functional screening
Products
| Catalog Number | Description | Size | Price | Quantity | Add to Cart | |||
|---|---|---|---|---|---|---|---|---|
| CASLV200PA-1 | Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro Lentivector Plasmid) | 10 µg | $643 |
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| CASLV200PA-KIT | KIT: Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro Lentivector Plasmid) plus LentiStarter Packaging Kit | 1 Kit | $1075 |
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| CASLV200VA-1 | Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro LentivectorPre-Packaged Lentiviral Particles (>10^6 IFUs)) | 2 x 25 µL | $643 |
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Overview
Overview
On-target genome editing in transfection-resistant cells
When you’re genome editing in a transfection-resistant cell line and need to keep off-target events to a minimum, turn to one of SBI’s Cas9 SmartNickase™ Lentivector Systems. Unlike the wildtype Cas9 protein which introduces double-strand breaks (DSBs), the CMV-hspCas9(D10A)-T2A-Puro Two Vector Cas9 SmartNickase introduces paired nicks at the gRNA-directed site. Creating nicks favors the higher-fidelity homologous recombination process over non-homologous end joining (NHEJ), with paired nicking shown to reduce off-target activity by 50- to 1,500-fold in cell lines, and to facilitate gene knockout in mice without losing on-target cleavage efficiency1.
Available as lentivector plasmids, ready-to-transduce pre-packaged pseudovirus, and in a package-your-own-lentivector kit with the lentivector plasmid and the LentiStarter 3.0 Packaging Kit, the CMV-hspCas9(D10A)-T2A-Puro Two Vector Cas9 SmartNickase Lentivector expresses human codon-optimized Cas9 nickase (the D10A mutant) from the strong CMV promoter and includes the puromycin selection marker.
- Conduct genome editing and engineering in difficult-to-transfect cell lines
- Stay more on-target with Cas9 nickase activity
- Drive Cas9 expression from the CMV promoter, for high expression in many commonly-used cell lines (HeLa, HEK293, HT1080)
- Perform in vivo engineering of model organisms
- Supports synthetic biology applications, gene- and cell-based therapy development, and genome-wide functional screening
Why an HR targeting vector is a recommended
Even though gene knock-outs can result from DSBs caused by Cas9 alone, SBI recommends the use of HR targeting vectors (also called HR donor vectors) for more efficient and precise mutation. HR donors can supply elements for positive or negative selection ensuring easier identification of successful mutation events. In addition, HR donors can include up to 6-8 kb of open reading frame for gene knock-ins or tagging, and, when small mutations are included in either 5’ or 3’ homology arms, can make specific, targeted gene edits.
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 |
- Ran, FA. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013 Oct 24; 8:2281-2308. PMCID: PMC3969860.
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 | |||
|---|---|---|---|---|---|---|---|---|
| CASLV200PA-1 | Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro Lentivector Plasmid) | 10 µg | $643 |
|
||||
| CASLV200PA-KIT | KIT: Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro Lentivector Plasmid) plus LentiStarter Packaging Kit | 1 Kit | $1075 |
|
||||
| CASLV200VA-1 | Cas9 expression vector for the Two Vector SmartNickase System (CMV-hspCas9(D10A)-T2A-Puro LentivectorPre-Packaged Lentiviral Particles (>10^6 IFUs)) | 2 x 25 µL | $643 |
|
||||
Overview
Overview
On-target genome editing in transfection-resistant cells
When you’re genome editing in a transfection-resistant cell line and need to keep off-target events to a minimum, turn to one of SBI’s Cas9 SmartNickase™ Lentivector Systems. Unlike the wildtype Cas9 protein which introduces double-strand breaks (DSBs), the CMV-hspCas9(D10A)-T2A-Puro Two Vector Cas9 SmartNickase introduces paired nicks at the gRNA-directed site. Creating nicks favors the higher-fidelity homologous recombination process over non-homologous end joining (NHEJ), with paired nicking shown to reduce off-target activity by 50- to 1,500-fold in cell lines, and to facilitate gene knockout in mice without losing on-target cleavage efficiency1.
Available as lentivector plasmids, ready-to-transduce pre-packaged pseudovirus, and in a package-your-own-lentivector kit with the lentivector plasmid and the LentiStarter 3.0 Packaging Kit, the CMV-hspCas9(D10A)-T2A-Puro Two Vector Cas9 SmartNickase Lentivector expresses human codon-optimized Cas9 nickase (the D10A mutant) from the strong CMV promoter and includes the puromycin selection marker.
- Conduct genome editing and engineering in difficult-to-transfect cell lines
- Stay more on-target with Cas9 nickase activity
- Drive Cas9 expression from the CMV promoter, for high expression in many commonly-used cell lines (HeLa, HEK293, HT1080)
- Perform in vivo engineering of model organisms
- Supports synthetic biology applications, gene- and cell-based therapy development, and genome-wide functional screening
Why an HR targeting vector is a recommended
Even though gene knock-outs can result from DSBs caused by Cas9 alone, SBI recommends the use of HR targeting vectors (also called HR donor vectors) for more efficient and precise mutation. HR donors can supply elements for positive or negative selection ensuring easier identification of successful mutation events. In addition, HR donors can include up to 6-8 kb of open reading frame for gene knock-ins or tagging, and, when small mutations are included in either 5’ or 3’ homology arms, can make specific, targeted gene edits.
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 |
- Ran, FA. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013 Oct 24; 8:2281-2308. PMCID: PMC3969860.
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.