In vitro cleavage assay and Electrophoretic Mobility Shift Assay (EMSA)

1.     Anneal crRNA with tracrRNA

a.     Add 1 µl of 100 µM caged crRNA with 1 µl of 100 µM tracrRNA in a PCR tube, both dissolved in duplex buffer (Integrated DNA Technologies, IDT)

b.     Heat to 95°C in a thermocycler for 3 minutes, cool on benchtop for 5 minutes

c.      Add 8 µl of duplex buffer (IDT) to make 10 µM cr/trRNA solution

2.     Mix to form reaction solution containing Cas9 in complex with cr/trRNA

a.     In a PCR tube, add 8 µl nuclease free water (NFW), add 1 µl of NEBuffer 3.1 (New England Biolabs, NEB), mix thoroughly by pipetting up and down 10 times

b.     Add 0.5 µl of caged 10 µM cr/trRNA solution

c.      Add 0.3 µl of 10 µM Cas9, thoroughly mix by pipetting up and down 10 times

d.     Incubate at room temperature for 30 minutes to form Cas9-gRNA complex (RNP)

3.     Cas9 cleavage reaction

a.     Add 2 µl (~60 fmol) of purified target DNA generated from PCR, mix thoroughly

b.     Transfer to thermocycler heated at 37°C

c.      To activate vfCRISPR caged gRNA, keeping the PCR tube lid open, hold the illumination LED flashlight 10 cm above the tube for 30 seconds

d.     Incubate at 37°C for variable amount of time (30 minutes reaches saturation)

e.     To stop the reaction at a given time point, transfer to a 95°C heated thermocycler for 5 minutes

4.     EMSA Readout

a.     Mix 4 µl of reaction solution with 16 µl of NFW. Mix thoroughly then load all into a E-Gel™ EX Agarose Gel, 4% (Invitrogen)

b.     Run gel for 10 minutes, visualize with SYBR® Gold channel in gel imager

5.     Nucleic acid-only Readout

a.     Add 1 µl of 10 mg/ml Proteinase K, mix thoroughly, incubate for 15 min at 55°C

b.     Mix 4 µl of reaction solution with 16 µl of NFW. Mix thoroughly then load all into a E-Gel™ EX Agarose Gel, 4% (Invitrogen)

c.      Run gel for 10 minutes, visualize with SYBR® Gold channel in gel imager

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