Transfection details
Cells were seeded onto 48-well plates a day before transfection at a density of 3×104 cells/well. The next day, at around 40% confluence, cells were transfected with plasmid constructs using Turbofect reagent.), briefly as follows: 250 ng total plasmid DNA (2 ng GFxFP plasmid, 124 ng crRNA and nuclease expression plasmid, and 124 ng mCherry expression plasmid to monitor the transfection efficiency) and 1 μL Turbofect were mixed in 50 μL serum free DMEM and the mixture was incubated for 30 min at room temperature prior being added to cells. Three parallel transfections were made from each sample. Cells were analysed by flow cytometry two days post-transfection. The EGFP signal was analysed in the mCherry positive population. Background fluorescence was determined by using a crRNA-less, inactive LbCas12a nuclease expression vector as negative control and this background value was subtracted from each sample. Three parallel transfections were made for each case.

Addgene number #89052
Cloning site to accomodate the target sequence is between BamHI and EcoRI sites.