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System Biosciences

Cas9 Nickase: CMV-hspCas9(D10A)-T2A-Puro SmartNickase Lentivector Plasmid

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SKU:
CASLV200PA-1
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  • Cas9 Nickase: CMV-hspCas9(D10A)-T2A-Puro SmartNickase Lentivector Plasmid
  • Cas9 Nickase: CMV-hspCas9(D10A)-T2A-Puro SmartNickase Lentivector Plasmid
€731.00

Description

Cas9 Nickase: CMV-hspCas9(D10A)-T2A-Puro SmartNickase Lentivector Plasmid. Cat# CASLV200PA. Supplier: SBI System Biosciences

When working with difficult-to-transfect-cells and you want to reduce off-target events, use our lentiviral Cas9 Nickase—CMV-hspCas9(D10A)-T2A-Puro.
  • 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

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)-EF1α-GFP Two Vector Cas9 SmartNickase Lentivector expresses human codon-optimized Cas9 nickase (the D10A mutant) from the strong CMV promoter. The vector also includes a GFP marker expressed from the EF1α promoter to verify transduction efficiencies. All pseudoviral particle preparations have been packaged to exacting QC standards and come with functional titer and in-house transduction data for each production lot of virus.
  • Conduct genome editing and engineering in difficult-to-transfect cell lines
  • Drive Cas9 expression from the CMV promoter, for high expression in many commonly-used cell lines (HeLa, HEK293, HT1080)
  • Check transduction efficiencies with the GFP marker
  • 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:
 

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 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.

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