CRISPR/CAS9 gene editing

YAP1 knock-outs were performed by CRISPR/CAS9 genome editing using the Alt-R CRISPR-CAS9 system (Integrated DNA Technologies, IDT) and Lonza 4D-Nucleofector, following previously described protocol (Richardson et al., 2016). Guide sequences for YAP1 were designed using Deskgen (deskgen.com), and the corresponding Alt-R CRISPR-Cas9 crRNAs (crRNA) were ordered from IDT. ThecrRNA was hybridized with Alt-R CRISPR-Cas9 tracrRNA (tracrRNA, IDT) by mixing 120 pmol of crRNA with 120 pmol of tracrRNA in 5 µl of CAS9 buffer (20 mM HEPES (pH 7.5), 150 mM KCl, 1 mM MgCl2, 10% glycerol and 1 mM TCEP), incubating the mixture at 95°C for 5 minutes and then letting the mixture cool to room temperature on benchtop (5–10 minutes). 100 pmol of Alt-R® S.p. Cas9 Nuclease V3 (IDT) in 5 µl of CAS9 buffer was slowly added to the crRNA:tracrRNA duplex and the subsequent solution was incubated for 20 minutes in room temperature to allow ribonucleoprotein complex (RNP) formation. The RNP complex was then added to 20 µl of cell suspension containing 300 000 cells suspended in Nucleofector SE Cell line solution, mixed and 20 µl of the cell/RNP mix was pipetted into one well of a Nucleocuvette Strip. The reaction mixtures were nucleofected using cell line -specific programs  in the 4D-Nucleofector, and finally transferred to 6-well plates.  The nucleofection conditions were optimized using the Cell Line Optimization 4D-Nucleofector X Kit following the kit protocol. The optimized programs used were: EN-138 for PC-9 and EBC-1 cells; CA-137 for H3122 cells; CM-137 for HCC4006 and DFCI243. All cell lines were nucleofected in SE Cell line solution.