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.
Figure 1. Schematic of the T7EI assay, the basis of the Alt-R Genome Editing Detection Kit. Use PCR to amplify the targeted genomic region (note: reagents for PCR are not part of the kit). Denature and reanneal PCR products in a thermal cycler to allow potential heteroduplex formation between wild-type and CRISPR–mutated DNA. Digest reannealed PCR products with T7EI, which cleaves mismatched DNA heteroduplexes. Analyze results using gel or capillary electrophoresis.
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).
Figure 2. T7EI mismatch endonuclease assays provide a good estimate of genome editing efficiency, but underestimate efficiency when compared to NGS results. The Alt-R CRISPR-Cas9 RNA oligonucleotides (30 nM) were introduced by lipofection into HEK-293 cells that stably express Streptococcus pyogenes Cas9. 3 PAM sites were targeted in each of 8 genes. To estimate editing efficiency, samples of genomic DNA from the transfected cells were tested using the Alt-R Genome Editing Detection Kit (dark blue bars), which provides reagents needed to run T7EI assays. The same DNA samples were also analyzed using targeted next generation sequencing (NGS) (light blue bars). Amplicons were run on a MiSeq system (Illumina) and data were analyzed using a publicly available data processing program [Pinello L, Canver MC, et al. (2016) Nat Biotechnol 34:695–697.). Error bars represent standard deviation for triplicate lipofection experiments.
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.