Alt-R™ Genome Editing Detection Kit for performing T7 endonuclease I (T7EI) mutation cleavage assay
How the T7EI assay works (Figure 1)
- Use PCR to amplify the targeted genomic region.
- 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.
Figure 1. Schematic of T7EI assay.
Why use T7EI, instead of Surveyor, for CRISPR genome editing detection?
We currently recommend using T7EI instead of the Surveyor® mismatch endonuclease for CRISPR mutation detection. The T7EI method is simple and provides clean electrophoresis results. T7EI is also compatible with a broader range of PCR buffers and does not usually require purification of the PCR product prior to digestion.
Note that T7EI activity is sensitive to the DNA:enzyme ratio, as well as incubation temperature and time . T7EI is able to recognize 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 underrepresents the total editing (see representative data).
Tips for PCR design for the T7EI assay
Design PCR primers that amplify your experimental target site and adjacent sequences. We recommend using PCR amplicons that are 600–1000 bp in length with at least 100 bp flanking each side of the CRISPR cut site. Position the CRISPR cut site off-center to allow for 2 distinctive digestion product sizes on the gel. Carefully optimize PCR conditions, and confirm by electrophoresis that a single PCR product is amplified from genomic DNA prior to editing. For PCR design assistance, use the PrimerQuest® Tool at www.idtdna.com/PrimerQuest.
For both Alt-R CRISPR-Cas9 System and Alt-R CRISPR-Cpf1 System, we offer recommended products and sequences for crRNA positive controls that target HPRT in human, mouse and rat cells. We have also designed and tested companion PCR primers for use with the Alt-R Genome Editing Detection Kit, which can be used to analyze editing efficiency in the positive control samples.
- Mean RJ, Pierides A, Pollet N. (2004) Modification of the enzyme mismatch cleavage method using T7 endonuclease I and silver staining. Biotechniques, 36(5):758–760.
- Vouillot L, Thélie A, et al. (2015) Comparison of T7EI and Surveyor mismatch cleavage assays to detect mutations triggered by engineered nucleases. G3: Genes|Genomes|Genetics, 5(3):407–415.