CMV-hspCas9-T2A-Puro-H1-AAVS1 gRNA SmartNuclease Lentivector Plasmid
- Usually Shipped in 5 Working Days
- 10 ug
- Shipping Temperature:
- Blue Ice / Dry Ice
CMV-hspCas9-T2A-Puro-H1-AAVS1 gRNA SmartNuclease Lentivector Plasmid. Cat# CASLV601PA. Supplier: SBI System Biosciences
- Conveniently deliver Cas9 and gRNA with a single vector
- Monitor transfection efficiencies with GFP, which is coordinately expressed with the hspCas9 gene via the T2A element
- Drive Cas9 expression with the CMV promoter, which provides high expression levels in many common cell lines (HeLa, HEK293, HT1080)
- 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
How It Works
Using SBI’s All-in-one Cas9 SmartNuclease Plasmids
The workflow at-a-glance
- Design two DNA oligonucleotides that are sense and antisense sequences of the target DNA and are immediately upstream of a PAM sequence (5’ – NGG – 3’)
- Anneal the two oligonucleotides to generate a duplex
- Ligate the duplex into the pre-linearized All-in-one Cas9 SmartNuclease Plasmid
- 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) »
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.
Genome engineering with SBI’s All-in-one Cas9 SmartNuclease Plasmids
Note that this study uses a slightly different All-in-one Cas9 SmartNuclease Plasmid design, but the results are expected to be similar.