Applications and reagents for CRISPR genome editing are developing quickly. Whether you are using CRISPR-Cas9, CRISPR-Cpf1, or another CRISPR system, identifying on-target editing events is vital for interpreting your experimental results. We currently recommend the Alt-R Genome Editing Detection Kit to perform T7EI assays for CRISPR mutation detection (Figure 1). This method provides clean electrophoretic results, requires only standard molecular biology equipment, and does not usually require purification of PCR products prior to T7EI digestion.
The Alt-R Genome Editing Detection Kit supplies the T7EI enzyme and buffer, as well as template-primer mixes for T7EI positive control reactions. You must design the target-specific PCR primers (see sidebar, Tips for using the Alt-R Genome Editing Detection Kit). Use them with your preferred PCR reagents.
Specificity of T7EI for insertions or ≥2 base deletions
T7EI recognizes insertions and deletions (indels) of ≥2 bases that are generated by non-homologous end joining (NHEJ) activity in CRISPR experiments. Because T7EI does not recognize 1 bp indels, T7EI assays can underrepresent total editing. The variance depends on the target and is affected by the type of NHEJ-mediated repair events that follow DNA cleavage (Figure 2). To enrich for successfully transfected cells, we offer a fluorescently labeled tracrRNA that enables fluorescence-activated cell sorting (FACS) (see sidebars, Tips for using the Alt-R™ Genome Editing Detection Kit and Additional resources).
Note that in Alt-R CRISPR-Cpf1 experiments, our preliminary results suggest that comparable editing efficiencies are obtained using T7EI assays and NGS. Cpf1 endonuclease generates 5′ staggered cuts, and presumably, NHEJ repair results in fewer single base indels compared to NHEJ repair of the blunt-ended cuts created by Cas9 endonuclease.
For more information and protocols, visit the Alt-R Genome Editing Detection Kit web page.