gRNAs were designed using either the optimized CRISPR (http://crispr.mit.edu/) or the CRISPR-ERA (http://crispr-era.stanford.edu) web tools. gRNAs were selected based on their specific locations at decreasing distances from the TSS as well as their lack of predicted off targets and E scores (http://crispr-era.stanford.edu). For lentiviral cloning: synthesized oligonucleotides (Thermo Fisher Scientific; Table S2) were annealed (37°C for 30 min, 95°C for 5 min, ramp-down to 25°C at 5°C per min), diluted 1:100 and then ligated into BsmB1-digested lentiGuide-dTomato or lentiGuide-mTagBFP2-Puro (described below). For IVT production: PCR assembly of gRNA DNA template using synthetic forward and reverse oligonucleotides for SNAP91 (Table S2) with the Tracr Fragment + T7 Primer Mix was performed as per GeneArt Precision gRNA Synthesis Kit (Thermo Fisher Scientific, A29377) instructions. The SNAP91 gRNA was generated by in vitro transcription and purified as per the GeneArt Precision gRNA Synthesis Kit instructions.
