1. The general conditions for high-throughput screening are as follows, which are described in detail in the subsequent points:
· Final concentrations in assay buffer: SpCas9 substrate dsDNA (0.5 nM), displacer ssDNA (2.5 nM), SpCas9 (5 nM), and gRNA (6 nM) in 1x assay buffer (20 mM Tris-HCl, pH 7.5, 0.1 M KCl, 5 mM MgCl2)
· A compound plate is tested in duplicate, such that compounds inhibiting SpCas9 in both tests are identified as hits.
2. Prepare SpCas9 substrate dsDNA solution by annealing two complementary SpCas9 substrate oligonucleotides.
· Mix 5 μL of SpCas9 substrate forward oligonucleotide solution (100 μM), 5 μL of SpCas9 substrate reverse oligonucleotide solution (100 μM), 5 μL of 10x assay buffer, and 35 μL of water in a PCR tube.
· Anneal the two complementary oligonucleotides with a thermocycler. Heat to 95°C for 5 min, then cool to 25°C at a rate of 0.1°C/s.
· Dilute the resulting 10 μM substrate dsDNA solution to 1 μM using 1x assay buffer and store at −20°C.
3. Prepare SpCas9 displacer ssDNA solution.
· Mix 5 μL of SpCas9 displacer ssDNA solution (100 μM) with 95 μL of 1x assay buffer. Store this 5 μM solution at −20°C.
4. Prepare 2x RNP solution and 2x apo SpCas9 solution.
· In a single tube, dilute SpCas9 stock solution to 1 μM and gRNA stock solution to 1.2 μM in 1x assay buffer to form SpCas9:gRNA RNP complex. Incubate for 5 min at 4°C, then dilute the RNP solution to 10 nM with 1x assay buffer and store at room temperature. Conical tubes (500 mL) can be used for storing large amounts of solution.
· For the apo SpCas9 positive control (no SpCas9 activity), dilute the SpCas9 stock solution to 10 nM in 1x assay buffer.
5. Add RNP solution or apo SpCas9 solution to 384-well assay plates using a Multidrop Combi Reagent Dispenser.
· Transfer 25 μL of 2x RNP solution to wells for test compounds and negative controls.
· Transfer 25 μL of 2x apo SpCas9 solution to wells for positive controls.
· Prepare two assay plates for each compound, as the screening is done in duplicate.
6. Transfer compound libraries into the assay plates via pinning and perform the 1st counter-screen.
· Note: For the pinning, we used an Epson Compound Transfer Robot with the assistance of the ICCB-Longwood Screening Facility, though the pinning process should be modified based on the setup of each laboratory.
· Pin compounds or vehicle (stocked in 384-well plates) into the 384-well assay plate. To each well, 100 nL of compound solution (usually 10 mM or 5 mg/mL stocks in DMSO) is added.
· Cover and incubate the pinned plates at room temperature for at least 30 minutes.
· Measure Alexa-Fluor 647 fluorescence to rule out compounds that auto fluorescence in the presence of RNP (1st counter-screen).
7. Add dsDNA substrate and displacer ssDNA solution to the assay plates and perform the 2nd counter-screen.
· Prepare the 2x solution of substrate dsDNA and displacer ssDNA by diluting 1 μM substrate dsDNA solution from step 2 to 1 nM and diluting 5 μM displacer ssDNA solution from step 3 to 5 nM in 1x assay buffer. Conical tubes (500 mL) can be used for storing large amounts of solution.
· Using a Multidrop Combi Reagent Dispenser, transfer 25 μL of the solution to every well.
· Immediately after addition, measure the Alexa-Fluor 647 fluorescence to identify compounds that are auto fluorescent in the presence of all the assay components (2nd counter-screen).
8. Perform the SpCas9 reaction and obtain the primary screen result.
· Incubate the covered plates at 37 °C for 2.5 h in a humidified incubator to prevent the vaporization of liquids. Minimize plate stacking to more rapidly reach the target temperature.
· Measure the Alexa-Fluor 647 fluorescence (primary screen).
9. Analyze the data.
· Export the data from Envision. Using Microsoft Excel, calculate the Z-score using [x − μ] / σ, where x is the signal from the sample, µ and σ are mean and standard deviation of the negative controls.
· Identify compounds with Z-score > 3 as potential hits for further testing in cell-based assays.
· Analyze counter-screen results to exclude auto fluorescent compounds.