System Biosciences

EF1-T7-hspCas9-T2A-RFP-H1-gRNA linearized SmartNuclease vector

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  • EF1-T7-hspCas9-T2A-RFP-H1-gRNA linearized SmartNuclease vector
  • EF1-T7-hspCas9-T2A-RFP-H1-gRNA linearized SmartNuclease vector
$1,069.20

Description

EF1-T7-hspCas9-T2A-RFP-H1-gRNA linearized SmartNuclease vector. Cat# CAS701R. Supplier: SBI System Biosciences

Overview

Conveniently check transfection efficiencies with this All-in-one Cas9 SmartNuclease Our All-in-one Cas9 and gRNA plasmids are an excellent way to simplify delivery of your CRISPR/Cas9 system by providing both Cas9 and gRNA from a single vector, and the addition of coordinate expression of RFP for monitoring transfection efficiencies helps make genome engineering projects more user-friendly. SBI’s CMV-T7-hspCas9-T2A-RFP-H1-gRNA All-in-one Cas9 SmartNuclease Plasmid includes a number of additional features that make it a great All-in-one choice for any genome engineering project involving transfectable cells:
  • Conveniently deliver Cas9 and gRNA with a single vector
  • Monitor transfection efficiencies with RFP, 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
As with all of our Cas9 delivery options, the CMV-T7-hspCas9-T2A-RFP-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:
 

How It Works

Using SBI’s All-in-one Cas9 SmartNuclease Plasmids

The workflow at-a-glance  

  1. 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’)
  2. Anneal the two oligonucleotides to generate a duplex
  3. Ligate the duplex into the pre-linearized All-in-one Cas9 SmartNuclease Plasmid
  4. Transform into competent cells and grow in LB/Kanamycin plate (50 µg/ml)
  5. Confirm positive clones by direct sequencing
  6. 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)
  7. 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.

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

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.

Figure 1. Schematic showing knock-out of a genomic luciferase gene by insertion of an RFP gene. Cas9 activity and gRNA were delivered using an All-in-one Cas9 SmartNuclease Plasmid.

To demonstrate the capabilities of our All-in-one Cas9 SmartNuclease Plasmids and also show how the choice of gRNA sequence can affect genome editing efficiencies, we designed a study to knock-out a genomic copy of a luciferase gene by inserting a copy of the RFP gene. This study used two different All-in-one constructs—EF1-hspCas9-H1-Luc-gRNA1 and EF1-hspCas9-H1-Luc-gRNA2—and an HR donor vector containing the RFP gene surrounded by sequences homologous to the DNA around the genomic luciferase gene (Figure 1). While both constructs enabled successful genome editing, as indicated by the recovery of RFP-positive clones, the construct with gRNA1 provided more efficient genome editing than the construct with gRNA2 (Figure 2).

Figure 2. The All-in-one Cas9 SmartNuclease Plasmid enabled successful genomic luciferase knock-out and replacement with RFP. (A) Insertion of the RFP gene into the genomically-integrated luciferase gene results in a decrease in luciferase activity, indicating successful genome editing. (B) A Surveyor Nuclease Assay reflects finding from (A) that the gRNA1 construct delivered slightly more cleavage than the gRNA2 construct. (C) Fluorescence imaging shows successful genome editing by the recovery of RFP-positive clones.

 
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