EF1α-T7-hspCas9-H1-gRNA All-in-one Cas9 SmartNuclease™ Plasmid & Multiplex gRNA Cloning Kit
- Conveniently deliver Cas9 and gRNA with a single vector
- Drive Cas9 expression with the EF1α promoter, which provides medium expression levels in most cell types, including primary cells and stem cells
- Express gRNA from the H1 promoter for maximum specificity and choice of targets
- Ensure efficient import of Cas9 to the nucleus with N-term and C-term nuclear localization signals (NLSs)
- Boost Cas9 gene expression and stabilize the transcript via the WPRE regulatory element after the C-term NLS
Products
Catalog Number | Description | Size | Price | Quantity | Add to Cart | |||
---|---|---|---|---|---|---|---|---|
CAS700A-KIT | Multiplex gRNA Kit + All-in-one EF1α-T7-hspCas9-H1-gRNA linearized SmartNuclease vector | 10 Reactions | $1075 |
|
Overview
Overview
Enhance your All-in-one Cas9 SmartNuclease Plasmid with multiplexed gRNA delivery
Easily enhance the power of the EF1α-T7-hspCas9-H1-gRNA All-in-one Cas9 SmartNuclease™ Plasmid by combining it with the Multiplex gRNA Cloning Kit. Together, these two products can enable a wider range of sophisticated genome editing projects that require multiple gRNAs.
You get all the great features of the EF1α-T7-hspCas9-H1-gRNA All-in-one Cas9 SmartNuclease Plasmid:- Conveniently deliver Cas9 and gRNA with a single vector
- Drive Cas9 expression with the EF1α promoter, which provides medium expression levels in most cell types, including primary cells and stem cells
- Express gRNA from the H1 promoter for maximum specificity and choice of targets
- Ensure efficient import of Cas9 to the nucleus with N-term and C-term nuclear localization signals (NLSs)
- Boost Cas9 gene expression and stabilize the transcript via the WPRE regulatory element after the C-term NLS
- Easily detect and/or purify the Cas9 protein with the N-term myc-tag
- Produce Cas9 mRNA via in vitro transcription using the T7 promoter
- Edit multiple loci at once, save time and reagents
- Easily generate multi-cistronic gRNA expression constructs
- Ideal for Cas9 Nickase applications
- Great for studying signaling pathways
As with all of our Cas9 delivery options, the EF1α-T7-hspCas9-H1-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.
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 |
References
How It Works
How It Works
Using SBI’s All-in-one Cas9 SmartNuclease Plasmids with a Mulitplex gRNA Cloning Kit
The workflow at-a-glance
- Use the Multiplex gRNA Cloning Kit to prepare your multiplexed gRNA insert (Figure 1)
The PrecisionX Multiplex gRNA Cloning Kit provides H1 and U6 promoter blocks to easily build multiple gRNA cassettes (Figure 1). In Step 1, primers with overlapping ends containing the desired gRNA (designed by user) and the scaffold-promoter block (from the kit) are combined in a PCR reaction to generate a PCR amplicon containing both gRNAs. In Step 2, the PCR amplicon is combined with the fusion reaction mix along with the linearized expression vector for seamless construction.
- Transform into competent cells and grow in LB/Kanamycin plate (50 µg/ml)
- Confirm positive clones by direct sequencing
- Transfect sequence-verified All-in-one construct into mammalian cells using standard transfection protocols (co-transfect with an HR Targeting Vector, if required for your application)
- Perform a Surveyor Nuclease assay (or other suitable mismatch cleavage assays) to check for site-specific genome cleavage and select for desired clones
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.
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 | |||
---|---|---|---|---|---|---|---|---|
CAS700A-KIT | Multiplex gRNA Kit + All-in-one EF1α-T7-hspCas9-H1-gRNA linearized SmartNuclease vector | 10 Reactions | $1075 |
|
Overview
Overview
Enhance your All-in-one Cas9 SmartNuclease Plasmid with multiplexed gRNA delivery
Easily enhance the power of the EF1α-T7-hspCas9-H1-gRNA All-in-one Cas9 SmartNuclease™ Plasmid by combining it with the Multiplex gRNA Cloning Kit. Together, these two products can enable a wider range of sophisticated genome editing projects that require multiple gRNAs.
You get all the great features of the EF1α-T7-hspCas9-H1-gRNA All-in-one Cas9 SmartNuclease Plasmid:- Conveniently deliver Cas9 and gRNA with a single vector
- Drive Cas9 expression with the EF1α promoter, which provides medium expression levels in most cell types, including primary cells and stem cells
- Express gRNA from the H1 promoter for maximum specificity and choice of targets
- Ensure efficient import of Cas9 to the nucleus with N-term and C-term nuclear localization signals (NLSs)
- Boost Cas9 gene expression and stabilize the transcript via the WPRE regulatory element after the C-term NLS
- Easily detect and/or purify the Cas9 protein with the N-term myc-tag
- Produce Cas9 mRNA via in vitro transcription using the T7 promoter
- Edit multiple loci at once, save time and reagents
- Easily generate multi-cistronic gRNA expression constructs
- Ideal for Cas9 Nickase applications
- Great for studying signaling pathways
As with all of our Cas9 delivery options, the EF1α-T7-hspCas9-H1-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.
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 |
References
How It Works
How It Works
Using SBI’s All-in-one Cas9 SmartNuclease Plasmids with a Mulitplex gRNA Cloning Kit
The workflow at-a-glance
- Use the Multiplex gRNA Cloning Kit to prepare your multiplexed gRNA insert (Figure 1)
The PrecisionX Multiplex gRNA Cloning Kit provides H1 and U6 promoter blocks to easily build multiple gRNA cassettes (Figure 1). In Step 1, primers with overlapping ends containing the desired gRNA (designed by user) and the scaffold-promoter block (from the kit) are combined in a PCR reaction to generate a PCR amplicon containing both gRNAs. In Step 2, the PCR amplicon is combined with the fusion reaction mix along with the linearized expression vector for seamless construction.
- Transform into competent cells and grow in LB/Kanamycin plate (50 µg/ml)
- Confirm positive clones by direct sequencing
- Transfect sequence-verified All-in-one construct into mammalian cells using standard transfection protocols (co-transfect with an HR Targeting Vector, if required for your application)
- Perform a Surveyor Nuclease assay (or other suitable mismatch cleavage assays) to check for site-specific genome cleavage and select for desired clones
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.
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.