Surveyor nuclease assay

Surveyor assays were performed as previously descried [18]. Briefly, after electroporation of CRISPR/Cas9 plasmids and incubation for 3 days genomic DNA was extracted using GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific), following manufacturer’s instructions. PCR amplicons were generated spanning the Cas9 binding site using Accuprime™ Taq HF (Invitrogen) using the following PCR cycle: initial denaturation at 95°C for 5 min; 40x (95°C for 30 sec, 55°C or 60°C for 30 sec, 68°C for 40 sec); final extension at 68°C for 2 min. PCR amplicons were denatures and annealed as follows: 95°C for 5 minutes, 95–85°C at -2°C/s, 85–25°C at -0.1°C/s, 4°C hold. Primer sequences can be found in S1 Table. Three microliters of the annealed amplicon was then diluted with 6 μL of 1x Accuprime PCR buffer and treated with 1 μL of Surveyor nuclease with 1 μL of enhancer (Thermo Scientific) at 42°C for 20 min. The reaction was then stopped by the addition of 3 μL of 15% Ficol-400 and 0.05% Orange G solution containing 1 mM EDTA and subsequently run on a standard 10% TBE gel. Percent gene modification was calculated using Image J software as described [25,26].