Generation of knockout cell lines

Gene knockout kits against BAX, BAK, and BID as well as Cas9 from Streptococcus pyogenes (SpCas9) were purchased from Synthego (Redwood City, CA). sgRNA sequences are detailed below. Lyophilized sgRNA was resuspended in TE (Tris-EDTA) buffer per the manufacturer’s instructions. OCI-AML2 cells were transfected per a ribonucleoprotein (RNP) strategy of SpCas9 combined with sgRNA against the gene of interest. For negative control cells, SpCas9 was paired with a non-targeting negative control sgRNA obtained from Synthego. RNP complexes were incubated for 15 min at 37 degrees Celsius prior to addition to OCI-AML2 cells. OCI-AML2 cells were washed once in PBS and then resuspended in nucleofection SF buffer obtained from Lonza (Basel, Switzerland) containing the RNP complex. The cell suspension was then immediately placed in a Lonza Amaxa 4D Nucleofector X Unit using a 96-well plate format, and nucleofection was performed using the FF-120 protocol. OCI-AML2 cells were placed back into A221 media and either underwent repeated rounds of nucleofection or were assessed for BAX/BAK or BID protein expression by western blot. In order to create near-complete knockouts in a bulk population of cells, we performed several rounds of RNP nucleofection because these mitochondrial proteins are intracellular, precluding cell sorting for live knockout (KO) cells. We noted that protein expression by western blot would return by 4–5 days post-RNP nucleofection, suggesting that wild-type cells remained, which could outgrow the knockout population, and thus experiments were performed within 4 days of the terminal nucleofection. The control sample underwent the equivalent number of Cas9/nucleofection treatments with a control scramble gRNA as detailed above.