CRISPR Genotyping by Sanger Sequencing

For genotype analysis, cells were pelleted and resuspended in lysis buffer (0.45% NP-40 (NP40S, Sigma-Aldrich), 0.45% Tween-20 (P9416, Sigma-Aldrich), 0.2 mg mL⁻¹ Proteinase K, 1× DreamTaq PCR buffer in water). Volume was adapted to cell amount, for one-quarter of a 96-well plate, 30–50 µL were used. For PCR, 1 µL of DNA containing lysate, 1× DreamTaq Green buffer, 0.2 mM dNTPs, 1 µM primer mix (Ex12F2 5′-CAGCATACTGCCTTGCAAATAA-3′, Ex12R2 5′-TGATTCCACAAAAATAATCCCAG-3′) and Taq polymerase were mixed and brought to 15 µL with H₂O. Clones were screened for Tasor-null alleles using Sanger sequencing. Results were analyzed with TIDE (Brinkman et al. 2014 (link)).