Dox-inducible CRISPR-Cas9 Lentivector System

Single-guide RNA (sgRNA) sequences used for the generation of Dox-inducible CRISPR-Cas9 lentivector systems are listed in table S1. sgRNA-targeting human NGFR was designed using the Vienna Bioactivity CRISPR score (82 (link)). A nontargeting sgRNA was used as a control (83 (link)). sgRNA oligonucleotides were cloned into the all-in-one Dox-inducible Cas9 (iCas9) LentiCRISPR v2 vector (TLCV2, Addgene no. 87360), which was a gift from A. Karpf (84 (link), 85 (link)). One Shot Stbl3 Chemically Competent Escherichia coli (Thermo Fisher Scientific) were transformed with plenti-iCas9-sgNGFR or plenti-iCas9-sgCtr plasmid. Plasmid isolation was performed using the NucleoBond Xtra Midi Plus EF Kit (Macherey-Nagel). Correct sequence insertion was confirmed by Sanger sequencing (Microsynth, Switzerland).