The Edit-R guide RNA guarantee

We guarantee that these guides will provide successful editing at the target site when delivered as described in the Technical Manual.

The Edit-R guide RNA guarantee is valid when used with any wild type S. pyogenes Cas9 nuclease, including mRNA, expression plasmid, protein, or stable Cas9 expression.

Analysis of editing of the treated cell population must be shown using a T7EI or Surveyor mismatch detection assay. If successful editing is not observed for a predesigned Edit-R guide RNA while an appropriate side-by-side Edit-R positive control is successful, a one-time replacement of a different predesigned Edit-R guide RNA of the same format and quantity will be provided at no cost.

A replacement will only be approved upon discussion with our Scientific Support team.

Successful editing at the DNA level does not always lead to functional gene knockout; it is recommended to test multiple guide RNAs to determine the most effective guide RNA for knockout of your target gene.

This guarantee does not extend to any accompanying experimental costs, does not apply to guide RNAs ordered via the CRISPR Design Tool, and will not be extended to the replacement guide RNA.

Edit-R synthetic guide RNAs targeting T cell receptor constant regions are validated for high editing efficiency while maintaining high viability in primary T cells

Primary CD4+ T cells from two donors were nucleofected with Cas9-RNP complexes loaded with TRAC single sgRNAs (donor 1 n=3, donor 2 n=2, +/-SD). 72 hours after nucleofection, functional knockdown was assessed by flow cytometry for TCR α/β receptor expression using BioLegend APC anti-human TCR α/β antibody. Cells were co-stained with BioLegend Zombie Green Viability Dye to gate on the living cell population (Zombie Green Neg) of edited cells (displayed on the right y-axis, green diamonds). Overlaid histograms from the top performing sgRNAs are pictured to the right of the bar graph in blue (top), unedited (no sgRNA) and PPIB control sgRNA – both positive controls for TCR α/β gating in red and purple, respectively (below). For a full description of the methods, see our T cell electroporation protocol.

TCR knock-out is stable in CD8+ T cells

Primary CD8+ T cells were electroporated with Cas9-RNP loaded with Edit-RTM PPIB, NTC2, TRAC or TRBC sgRNAs. 72 hours post electroporation, edited cells were assessed for TCR α/β knock-out phenotype by flow cytometry. Displayed in overlapping histograms are ZombieGreenTM Dye-negative (living) unedited CD8+ T cells unstained (black), isotype stained (gray) or CD8+ T cells that were stained with PE anti-human TCR α/β antibody (blue, green, and orange traces in histograms). For a full description of the methods, see our T cell electroporation protocol and CD8+ CAR-T application note.

Workflow for RNP-based editing with synthetic sgRNAs in primary cells

Workflow demonstrating RNP-based knockout in primary cells using electroporation.