Competition-based cell proliferation assay

Cas9-expressing cell lines were lentivirally transduced with LRG2.1T sgRNA linked with GFP or mCherry reporter. Percentage of GFP positive cell population was measured at day 3 or day 4 as initial time point using a Guava Easycyte HT (Millipore) or a MACSQuant (Miltenyl Biotec) instrument. GFP% (mCherry%) was then measured every two days (for leukemia cell lines) or every three days (for non-leukemia cell lines) over a time course. The relative change in the GFP% (or mCherry%) percentage at each time point was then normalized to initial time point GFP% (or mCherry%). This relative change was used to assess the impact of individual sgRNAs on cellular proliferation, which reflects cells with a genetic knockout being outcompeted by non-transduced cells in the cell culture.