Human cell culture assays for EGFP knockdown were performed essentially as described previously7, but with the following modifications. The X-001 program was used on a Lonza 2b nucleofector, and reaction mixtures were accordingly scaled up to 500,000 cells with 2000 ng Cas9 plasmid, 666 ng sgRNA plasmid, and 80 ng ptdTomato. Cells were harvested 52 h post transfection and then kept on ice, but were analyzed on a SONY SH800 cell cytometer with 20,000 events captured per sample. The percentage of EGFP-negative cells in each sample was determined from a subpopulation after gating for the top ~50% of tdTomato-expressing cells; the tdTomato-expressing population was identified by comparing control samples that were untransfected or transfected with ptdTomato alone. Each Cas9/sgRNA combination was tested in quadruplicate by performing two biological duplicates on separate days. Finally, a global average of 3.39% background EGFP loss (denoted with a black dashed line) was estimated from four biological duplicate experiments with a non-targeting sgRNA plasmid (BPK1520) performed on four separate days. The clonal U2OS.EGFP cell line used for these assays was obtained from the Joung lab, and harbors a constitutively expressed hEGFP cassette integrated in the genome49.