TITLE: CasX Cleavage Assay
AUTHORS: Liz O'Brien, Connor Tsuchida

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes a CasX Cleavage Assay.</div></div>

[STEPS]
?. [Annealing Duplex DNA]
Prepare stock of labelled target strand DNA to be  of .
100 µl
?. [Annealing Duplex DNA]
Mix  of  labelled target strand with  of  unlabelled nontarget strand to make a  stock of duplex substrate.
50 µl
50 µl
100 µl
Reverse concentrations for labelled nontarget, unlabelled target.
?. [Annealing Duplex DNA]
Mix 1:1.2 molar ratio of 32P labelled target strand (TS) to unlabelled nontarget strand (NTS) of duplex substrate:Stock of unlabelled TS at , : dilute  TS stock in  DEPC.Stock of unlabelled NTS at , : dilute  TS stock in  DEPC.
50 µl
4.48 µl
45.42 µl
50 µl
3.56 µl
46.44 µl
?. [Annealing Duplex DNA]
Place on  heat block for  - , then slow cool for about . This is the Duplex DNA stock for cleavage assays.
95 °C
DNA stock was diluted to 120nM with CasX reaction buffer.
?. [Prepare Buffers and Solutions]
Prepare CasX reaction buffer.
?. [Prepare Buffers and Solutions]
Mix together , , , , and .
[HEPES (pH 7.5)]
[magnesium chloride]
[potassium chloride]
[glycerol]
[TCEP]
?. [Prepare Buffers and Solutions]
Prepare CasX dilution buffer.
?. [Prepare Buffers and Solutions]
Mix together , , , , and .
[NaCl]
[glycerol]
[Tris-HCl (pH 7.5)]
[magnesium chloride]
[TCEP]
?. [Prepare Buffers and Solutions]
Prepare quencher.
?. [Prepare Buffers and Solutions]
Mix together  and .
[heparin]
[EDTA]
?. [CasX Activity Assays]
Dilute CasX to  in CasX dilution buffer (components shown above in ).
?. [CasX Activity Assays]
Dilute sgRNA to  in CasX reaction buffer (components shown above in ).
?. [CasX Activity Assays]
Mix an equimolar ratio of CasX and sgRNA ( sgRNA stock, CasX stock,  CasX reaction buffer).
2 µl
3 µl
1 µl
?. [CasX Activity Assays]
Allow samples to mix for  at .
0 Room temperature
?. [CasX Activity Assays]
Dilute annealed, labelled duplex DNA into CasX reaction buffer ( Duplex DNA stock,  reaction buffer).
2 µl
43 µl
?. [CasX Activity Assays]
Add RNP stock (,  RNP) to the DNA:buffer mixture. Total volume should now be 50 μl.
5 µl
?. [CasX Activity Assays]
Incubate at . For each timepoint (0, 1, 5, 10, 30, 60, 120 minutes), remove  and add to a tube filled with  quencher (CasX mixture and quencher solution should have equal volumes).
37 °C
5 µl
5 µl
?. [CasX Activity Assays]
Incubate with quencher solution at  for .
0 Room temperature
?. [CasX Activity Assays]
Add  formamide loading dye and incubate at  for .
5 µl
95 °C
?. [CasX Activity Assays]
After quenching, load samples (4 μl/well, i.e. approximately 200 cpm/well was loaded per sample) on a 12 % PAGE gel and run at 40 – 45 W for .
Large gel was pre-warmed at 25W for ~45minutes.
Loading less (50-100cpm/well) sample works fine. You may need to expose overnight.
?. [Prepare Buffers and Solutions]
Prepare Formamide Loading Dye (2x concentrated).
?. [Prepare Buffers and Solutions]
Add .
[formamide]
?. [Prepare Buffers and Solutions]
Add EDTA to a final concentration of  ( of  stock).
2.2 ml
?. [Prepare Buffers and Solutions]
Add a spatula tip-ful of powdered xylene cyanol.
?. [Prepare Buffers and Solutions]
Add a spatula tip-ful of powdered bromophenol blue.