System Biosciences

Transfection-ready hspCas9-T2A-RFP SmartNuclease mRNA (wildtype Cas9 with RFP marker)

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CAS531R-1
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  • Transfection-ready hspCas9-T2A-RFP SmartNuclease mRNA (wildtype Cas9 with RFP marker)
  • Transfection-ready hspCas9-T2A-RFP SmartNuclease mRNA (wildtype Cas9 with RFP marker)
£345.72

Description

Transfection-ready hspCas9-T2A-RFP SmartNuclease mRNA (wildtype Cas9 with RFP marker). Cat# CAS531R. Supplier: SBI System Biosciences

Overview

Be more confident with your in vivo genome editing

When you want a quick way to verify the efficiency of your Cas9 mRNA transfections, SBI offers the hspCas9-T2A-RFP SmartNuclease™ mRNA (an GFP version is also available). This construct delivers coordinated translation of Cas9 and RFP from the same mRNA, enabling the use of the RFP signal to assess transfection efficiency.

Use with an appropriate gRNA and, if your application needs it, a homologous recombination (HR) targeting vector for gene knock-ins, knock-outs, editing, and tagging. The Cas9 SmartNuclease will generate a double-strand break (DSB) at the site specified by the gRNA, and this DSB can be efficiently and precisely repaired in a manner that includes your desired mutation through the use of an HR targeting vector.

As with all of our Cas9 delivery options, the hspCas9-T2A-RFP SmartNuclease mRNA 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.
  • Ready to use for in vivo genome editing applications
  • Enables assessment of Cas9 mRNA transfection efficiency through the RFP signal
  • Delivers functionally validated Cas9 SmartNuclease
  • Backed by expert, easy-to-reach technical support
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.

Efficiently generate gRNA with the T7 gRNA SmartNuclease Synthesis Kit

Pair your hspCas9-T2A-RFP SmartNuclease mRNA with our T7 gRNA SmartNuclease Synthesis Kit, which comes with a pre-linearized, ready-for-cloning gRNA expression vector and all the reagents you need for T7-driven in vitro transcription of your cloned gRNA.

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 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

Assess transfection efficiency with the hspCas9-T2A-RFP SmartNuclease mRNA

Figure 1. Assess transfection efficiency with the hspCas9-T2A-RFP SmartNuclease mRNA. HEK2932 cells were transfected with either hspCas9-T2A-GFP SmartNuclease mRNA (top panels) or the hspCas9-T2A-RFP SmartNuclease mRNA (bottom panels). Cells were imaged for GFP and RFP fluorescence one day post-transfection.

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