Generation of CRISPR/Cas9 Plasmids

CRISPR/Cas9 plasmids were generated as described in the Genome-Scale CRISPR Knock-out (GeCKO) protocol (Supplementary Table 2) [20 (link),24 (link)]. The plasmid pLentiCRISPR.v2 (Addgene #52961), which also contains a Puromycin-resistance cassette, was BsmBI digested to remove the filler fragment (1.89kb) and gel purified. Complementary DNA oligonucleotides containing selected CRISPR/Cas9 target sequences (Supplementary Table 3) were phosphorylated, annealed and ligated into the vector. Subsequently, plasmids were transformed into E. coli Stbl3 by heat-shock and colonies were screened by PCR (Supplemental Procedures). Positive clones were propagated followed by plasmid purification using the EndoFree Plasmid MaxiKit (Qiagen) or the GeneJET Endo-free Plasmid Maxiprep Kit (Thermo Scientific) according to the supplier´s protocol. Next, plasmids were confirmed by analytical digestions (BamHI: linearization of plasmid yielding one 12.98kb fragment; BamHI/EcoRV: yielding one 10.66kb and one 2.32kb fragment) and sequencing. Final plasmids are designated as pLentiCRISPR.v2-GFP, pLentiCRISPR.v2-SPRY1#1 and pLentiCRISPR.v2-SPRY1#2. pLentiCRISPR.v2-GFP and pLentiCRISPR.v2-SPRY1#1 were already employed in our previous study [13 ]. shRNA-encoding vectors required for SPRY1 gene knockdown were previously described [13 ]. A non-targeting shRNA was employed for comparison [10 (link)].