Str profiling

U2OS cells (a gift from Toni Cathomen, Freiburg) and U2OS.EGFP cells (containing a single integrated copy of an EGFP-PEST reporter gene)^{30 (link)} were cultured in Advanced DMEM supplemented with 10% HI FBS, 2 mM GlutaMax, and penicillin/streptomycin at 37°C with 5% CO₂. The growth media for U2OS.EGFP cells was additionally supplemented with 400 µg ml⁻¹ Geneticin. All cell culture reagents were obtained from Life Technologies. Cell line identity was validated by STR profiling (ATCC) and deep-sequencing, and cells were tested bi-weekly for mycoplasma contamination. Unless otherwise noted, cells were co-transfected with 750 ng of Cas9 plasmid and 250 ng of sgRNA plasmid. For negative control experiments, Cas9 plasmids were co-transfected with a U6-null plasmid. Nucleofections were performed using the DN-100 program on a Lonza 4-D Nucleofector with the SE Cell Line Kit according to the manufacturer’s protocol (Lonza). For T7E1 assays, GUIDE-seq experiments, and targeted deep sequencing, genomic DNA was extracted ~72 hours post-transfection using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter Genomics).

U2OS cells obtained from our collaborator Toni Cathomen (Freiburg) and U2OS.EGFP cells harboring a single integrated copy of a constitutively expressed EGFP-PEST reporter gene^{13 (link)} were cultured in Advanced DMEM media (Life Technologies) supplemented with 10% FBS, 2 mM GlutaMax (Life Technologies), penicillin/streptomycin, at 37 °C with 5% CO₂. Additionally, U2OS.EGFP cells were cultured in 400 µg/ml of G418. The identity of U2OS and U2OS.EGFP cell lines were validated by STR profiling (ATCC) and deep sequencing, and cells were tested bi-weekly for mycoplasma contamination. Cells were co-transfected with 750 ng of Cas9 plasmid and 250 ng of sgRNA plasmid (unless otherwise noted) using the DN-100 program of a Lonza 4D–nucleofector according to the manufacturer’s protocols. Cas9 plasmid transfected together with an empty U6 promoter plasmid was used as a negative control for spontaneous background EGFP loss for all human cell EGFP disruption experiments, and all engodenous gene disruption experiments (none of which showed detectable activity by T7E1). Target sites for endogenous gene experiments were selected within 200 bp of NGG sites cleavable by wild-type SpCas9 (see Extended Data Fig. 6a and Supplementary Table 2).

A549 cells were obtained from ATCC. DLD1-BRCA2KO cells were obtained from Horizon Discovery. EMT6 cells were provided by Dr. Sara Rockwell. Human cells were authenticated using STR profiling (ATCC). All cell lines were regularly tested for mycoplasma contamination (MycoAlert, Lonza). Cells were grown in F12K (A549), RPMI (DLD1-BRCA2KO), or DMEM (EMT6) media containing 10% fetal bovine serum without antibiotics. For pH-specificity experiments, Leibovitz L-15 media was titrated to pH=6.2 using HCl. Cells were treated with pHLIP-PNAs in pH-titrated media for 24 hours, followed by a series of several washes with phosphate buffered saline. Cells were then immediately processed for flow cytometry or collected 24 hours later for Western blot analysis.