Biolux cypridinia

Media containing secreted luciferase was harvested at 48 hours post transfection, unless otherwise noted. Media was diluted 1:5 in PBS and then luciferase activity was measured using the BioLux Cypridinia and Biolux Gaussia luciferase assay kits (New England Biolabs) on a Biotek Synergy 4 plate reader with an injection protocol. All replicates were performed as biological replicates.

To assess REPAIR activity in mammalian cells, we transfected 150 ng of REPAIR vector, 300 ng of guide expression plasmid, and 45 ng of the RNA editing reporter. We then harvested media with the secreted luciferase after 48 hours and diluted the media 1:10 in Dulbecco’s phosphate buffered saline (PBS) (10 μl of media into 90 μl PBS). We measured luciferase activity with BioLux Cypridinia and Biolux Gaussia luciferase assay kits (New England Biolabs) on a plate reader (Biotek Synergy Neo2) with an injection protocol. All replicates performed are biological replicates.

To assess RNA targeting in mammalian cells with reporter constructs, 150 ng of Cas13 construct was co-transfected with 300 ng of guide expression plasmid and 45 ng of the dual luciferase reporter construct. Forty-eight hours post-transfection, media containing secreted luciferase was harvested and measured for activity with BioLux Cypridinia and Biolux Gaussia luciferase assay kits (New England Biolabs) on a plate reader (Biotek Synergy H4) with an injection protocol. Signal from the targeted Gluc was normalized to signal from un-targeted Cluc, and subsequently, experiments with PbCas13b mutant luciferase signal were normalized to experiments with guide–only luciferase signal (the average of three bioreplicates). All replicates performed are biological replicates.