Control the timing of gene editing with inducible lentiviral Cas9 expression



A lentiviral vector permitting temporal control of Cas9 expression helps reveal the function of essential genes, while avoiding constitutive expression of Cas9.

    temporal control image
  • Temporal control of Cas9 expression and gene editing
  • Facilitates enrichment of edited cells
  • High precision and improved experimental flexibility

 

 

The Edit-R™ Inducible Lentiviral Cas9 Expression Vector is based on one of two critical components of the CRISPR-Cas9 system required for gene editing in mammalian cells – the Cas9 nuclease enzyme. Used in conjunction with an Edit-R Lentiviral sgRNA designed to target the gene of interest, the Edit-R Inducible Lentiviral Cas9 Expression Vector provides researchers with temporal control of gene knockout using an optimized doxycycline inducible system.

Figure 1. Illustration  of CRISPR-Cas9 system. Cas9 nuclease (light blue), programmed by the targeting sequence (green) of the sgRNA, cutting both strands of genomic DNA 5' of the PAM (red).

Temporal control of Cas9 expression and gene editing

The Edit-R Inducible Lentiviral Cas9 Expression Vector contains a human codon-optimized version of the Cas9 gene under the control of the highly responsive Tet-On® 3G doxycycline inducible promoter. This allows gene knockouts to be obtained by treatment with an optimized concentration of doxycycline at a precisely defined time. As well as permitting tight regulation of Cas9 expression with minimal basal expression, the Edit-R Inducible Lentiviral Cas9 Expression Vector provides potent activation upon induction, even at very low doses of doxycycline.

 

inducible lentiviral cas9 nuclease reagent data

Figure 2. Dose response for doxycycline in inducible U2OS-Cas9 cells. Cells were transduced with the inducible (TRE3G-Cas9) Cas9 expression lentiviral particles at an MOI of 0.3, and selected with 10 µg/mL blasticidin in tetracycline-free medium for 10 days. Cas9-stable cells were then transduced with PPIB-sgRNA lentiviral particles at an MOI of 0.3. Cells were selected with 2 µg/mL puromycin tetracycline-free medium for 4 days, suspended with trypsin and seeded in a 96-well plate in medium containing increasing concentrations of doxycycline (0 to 1000 ng/mL). The cells were incubated for 72 hours, lysed and analyzed for indel formation using a DNA mismatch detection assay with T7EI. Upper panel, representative gel image of the DNA mismatch detection assay with T7EI for PPIB targeted amplicon; lower panel, mean ± standard deviation of the estimated percentage of gene editing from three independently treated wells.

Facilitates enrichment of edited cells

By constitutively expressing the blasticidin resistance gene (BlastR), the Edit-R Inducible Lentiviral Cas9 Expression Vector enables selection of Cas9-expressing cells prior to sgRNA transduction. In turn, this increases the percentage of gene edited cells available for downstream studies for identifying functional knockouts.

Figure 3. Gene knockout workflow using the Edit-R™ Inducible Lentiviral Cas9 Nuclease with Edit-R™ Lentiviral sgRNA.

High precision and improved experimental flexibility

The Edit-R Inducible Lentiviral Cas9 Expression Vector provides robust expression of the Cas9 nuclease when induced, and minimal promoter transcription in the off state for high precision in gene editing. It can be used in conjunction with an Edit-R Lentiviral sgRNA targeting any gene of interest, affording maximum flexibility.

 

 

Ryan Donnelly headshotWritten by Ryan Donnelly, Senior Product Manager

Ryan has led the gene editing reagents portfolio at Horizon for the past 2 years, concentrating on the needs of researchers using CRISPR methods and developing products to simplify their experiments and move their work forward. Previously, he worked as a Product Manager with Canon BioMedical where he focused on bringing inherited disease testing methods into the clinic. He holds a Professional Science Masters in Molecular Biotechnology from George Washington University and Bachelors in Chemistry from the University of Florida.

 

 

Ready to get started with inducible lentiviral Cas9 expression? See our product page.

 

 

Inducible lentiviral Cas9

 

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