Nature research paper: RNA targeting unleashes indiscriminate nuclease activity of CRISPR–Cas12a2
-arabinose and antibiotics for both immune system and target or non-target plasmids. Colonies were counted in the highest countable spot in the dilution series and the relative transformation efficiency was calculated between the target and non-target plasmid.Binary complex of either WT Cas12a2 or Cas12a2 with crRNA was combined with various targets to final reaction conditions of 600 nM Cas12a2, 720 nM crRNA and 300 nM FAM-labelled target in 1× NEB 3.1 Buffer .
Reactions were quenched with phenol–chloroform pH 4.5 and mixed by flicking followed by spinning down for 30 s. Reaction products were run on a 12% fully denaturing formaldehyde polyacrylamide gel electrophoresis gel as described by previously with minor modifications. Loading dye was replaced with 30% glycerol, and gels were run at 50 V for 15 min before increasing the voltage to 150 V.Binary complex of Cas12a2 and crRNA was combined with FAM-labelled FL target RNA to a final reaction condition of 600 nM Cas12a2, 720 nM crRNA, 300 nM target RNA in 1× NEB 3.1 buffer . Binary complex was first formed as described in the activation assay. The 2× master mix was then combined with the target RNA and incubated at 37 °C.
To test the effect of pre-incubation of target with Cas12a2, 100 nM Cas12a2–crRNA complex was incubated for 2 h at room temperature with 200 nM target RNA in NEB 3.1 buffer . After incubation, Cas12a2/crRNA/target mixture was combined with 1 μM RNAse Alert or DNAse Alert , and the reactions were allowed to proceed for 60 min at room temperature. These reactions were compared to the same reaction condition with target RNA added simultaneously with RNAse Alert or DNAse Alert.
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