Lc6876

All oligonucleotides were purchased from IDT DNA (Coralville, IA) and were desalted for in vitro transcription experiments. The oligonucleotides used in the conditional activation experiments were purchased from IDT with PAGE purification. Oligo-annealing solutions were prepared by mixing complementary strands (10 μM final concentration) together in 1× Cas9 assay buffer (20 mM of Tris–HCl, pH = 7.5; 150 mM of KCl; 1 mM of EDTA; 50 mM of MgCl₂). The oligonucleotides were annealed by heating to 95 °C for five minutes followed by slow cooling to 25 °C at a rate of 0.1 °C s^–1 to produce a double-stranded oligonucleotide. The gRNA templates were generated by annealing a sequence-specific oligo with either a “universal” oligo containing the Cas-nuclease-specific tracrRNA backbone (SpCas9 and SaCas9) or a T7 oligo to yield a double-stranded T7 promoter (all Cpf1 nucleases). See Table S1† for a list of all sequences. RNA was transcribed using HiScribe T7 Quick High Yield RNA Synthesis Kits (NEB, E2050S). To assess gRNA quality, 200 ng of purified RNA was heated to 95 °C in loading buffer (ThermoFisher, LC6876) and 50 mM of EDTA for 10 minutes and run on a 15% TBE-Urea gel (ThermoFisher, EC68855) for 70 minutes at 200 V. The gels were stained with Sybr Green (ThermoFisher) and imaged by UV (Fig. S3†).