T7 endonuclease I assay to detect Cas9 induced mutations
The following protocol has been adopted from a protocol developed by Keith Joung’s lab for T7 endo I assay in zebrafish. This assay detects heteroduplex DNA that results from annealing DNA stands that have been modified after a sgRNA/Cas9 mediated cut to DNA strands without modifications. We use this assay to get a first estimate of whether our targeting was successful or not. There are multiple other assays available that do essentially the same thing (e.g. Surveyor assay). We use T7 endo I mostly because it is easy and fast and does not need any specialized equipment. The assay seems to be pretty sensitive to different DNA/enzyme ratios as well as incubation time and as a result we only use it to compare samples within the same assay.
Materials you will need:
– buffer to prepare genomic DNA
– specific primers
– PCR kit
– gel extraction kit
– T7 endonuclease I (NEB)
– 0.25M EDTA
Step1: Prepare genomic DNA
Any method that gives DNA suitable for a 1kb PCR is fine. A good protocol from the VDRC can be found here. We use microLYSIS-PLUS (Cambio), mostly because it is fast and easy.
Step2: Amplify your target site by PCR
Choose primers either side of your target sequence. The final PCR product should be somewhere in the range of 600bp – 1kb. Run the PCR, load it on a gel and cut out the right band. Extract the DNA from the gel.
Step3: T7 endo I digest
Add 2ul NEBuffer 2, 200ng of purified PCR product and dH2O to a total of 19ul in a PCR tube. Run a hybridisation reaction in a PCR cycler: 5min, 95C; ramp down to 85C at -2C/s; ramp down to 25C at -0.1C/s; hold at 4C. Add 1ul (10U) T7 endo I and incubate at 37C for 15min. Stop the reaction by adding 2ul of 0.25M EDTA. Load immediately on a 1.5% agarose gel.
Here is an example of one of our assays: