idtdna CRISPR/Cas9 editing: mutation detection.

Cas9-editing-mutation-detection-protocol From DiaGenode
T7 Endonuclease I cleavage assay

The method is pretty straightforward and consists of 4 parts: DNA isolation, PCR of desired locus, denaturation and re-annealing, and T7 endonuclease I cleavage.

* DNA isolation *

Use your favorite protocol, or the quick ‘n’ dirty method below

HotSHOT lysis

1. Trypsinize cells from a well of a 24-well or 12-well microfuge tube. Collect in an eppendorf, spin out the supernatant. You don’t need a lot of cells, eyeball maybe 10-20 uL cell pellet at most for efficient lysis

2. Add 75-100 µl Alkaline Lysis Reagent. Assure that the cell pellet is completely submerged.

3. Incubate at 95°C for 30-60 min.

4. Add 75-100 µl Neutralization Reagent using a new aerosol-barrier tip for each sample. Mix well, using tip to break up tissue. Some people like to centrifuge the tubes after this step and transfer the neutralized supernatant to a new tube, but this is not necessary.

5. Use 2-3 µl of neutralized supernatant per 20-25 µl PCR reaction, more and you might start to inhibit the PCR reaction (usually the case for mouse.

Alkaline Lysis Reagent

To 25 ml water, add:
62.5 µl of 10 N NaOH (final concentration is 25 mM.)
10.0 µl of 0.5 M disodium EDTA (final concentration is 0.2 mM, pH should be about 12 but should not have to be adjusted.)

Make fresh every one to two months. Keep solution at room temperature.
Neutralization Reagent

40 mM Tris-HCl pH should be about 5 but should not have to be adjusted.)

Keep solution at room temperature.

Make 1 M Tris-HCl with Tris hydrochloride salt (not the base).

* PCR reaction *

You preferably want to use a high-fidelity polymerase with hotstart characteristics. I used Q5 hotstart from NEB because it’s cheap and robust. Design primers of your targeted locus using Primer-BLAST; set the desired target size to be between 200-1000 bp, %GC-content 40-60%, melting temperature optimum at 60, oligo size 18-25 nt in length.

Protocol that I followed:

Component 25 µl Reaction
5X Q5 Reaction Buffer 5 µl
10 mM dNTPs 0.5 µl
10 µM Forward Primer 1.25 µl
10 µM Reverse Primer 1.25 µl
Template DNA 2 µl
Q5 Hot Start 0.25 µl
Nuclease-Free Water to 25 µl

You might need to add GC-enhancer if the region that you are PCRing up is especially GC-rich or has a GC-rich stretch of nucleotides.

Thermocycling Conditions for a Routine PCR

Initial Denaturation 98°C 30 seconds
25–35 Cycles 98°C 10 seconds
*50–72°C 30 seconds
72°C 30 seconds/kb
Final Extension 72°C 2 minutes
Hold 10°C

Use the NEB calculator to calculate the annealing temperature for your oligos. If it calculates your oligos for use at 72°C, do a 2-step reaction, but extend the 72°C to 60 seconds.

I do several reactions to achieve enough substrate for the assay — 3-4 reactions generally will yield a nice quantity. You will need at least ~200 ng per reaction. Load a couple of µl from a reaction on a gel to make sure that you have a single band (also load your negative reaction to make sure that you have no contamination).

I purify using a spin column and elute in 50 µl of warm (60°C) elution buffer. This will typically yield ~20-30 ng/µl of product.

* Re-annealing *

Add ~200-500 ng of product to a 20 µl reaction of H2O and (2 µl) 1X NEB Buffer 2. You will need 2 tubes per reaction, one for the nuclease and one for a negative (non-nuclease) control. Denature and re-anneal in a PCR machine using the parameters below:

Temperature Time
95 °C 10 min
95 °C to 85 °C (‐2.0 °C/s)
85 °C 1 min
85 °C to 75 °C (‐0.3 °C/s)
75 °C 1 min
75 °C to 65 °C (‐0.3 °C/s)
65 °C 1 min
65 °C to 55 °C (‐0.3 °C/s)
55 °C 1 min
55 °C to 45 °C (‐0.3 °C/s)
45 °C 1 min
45 °C to 35 °C (‐0.3 °C/s)
35 °C 1 min
35 °C to 25 °C (‐0.3 °C/s)
25 °C 1 min
10 °C Hold ∞

On our machine ‐2.0 °C/s is a 50% ramp rate and a ‐0.3 °C/s is a 2% ramp rate.

* T7 Endonuclease I cleavage assay *

To each tube add 1 µl of T7 endonuclease I (10 U). Incubate at 37°C for 15 min. Stop the reaction by the adding 2 µl of 0.5 M EDTA. Load half (or more) on a 2% TBE or 1.5% SB agarose gel. Staining afterword will give a cleaner signal-noise, in which case wait until the orange dye is near the bottom of the gel (and the cyan dye is about 1/3-1/2 the way down) before you stop and stain the gel (0.5 ug/ml of EtBr for 15 min, rinse in H2O, destain 15 min in H2O).