CRISPR-Cas9 Murine and Human AML Editing

sgRNA against murine Rosa26 (negative control) and Nmnat1 as well as human ENAM (negative control) and NMNAT1 were designed according to CRISPR DESIGN (http://crispr.mit.edu/). We cloned sgRNA with a T7 promoter sequence and transcribed using the HiScribe T7 In Vitro Transcription Kit (NEB, Ipswich, MA). We introduced the Cas9-sgRNA complex to murine AML cells as previously described (38 (link), 53 (link)). In brief, 1 μg of Cas9 protein (PNA Bio, Newbury Park, CA) was incubated with 1 μg of in vitro–transcribed sgRNA at room temperature for 5 min before being electroporated for murine AML cells (10⁵ cells per reaction) using the Neon Transfection System (MPK1096, Thermo Fisher Scientific) using the following parameters: 1700 V, 20 ms, and 1 pulse. Human AML cells were electroporated with the following parameters: 1350 V, 20 ms, and 1 pulse. Cells were then cultured for at least 12 hours before subsequent assays including transplantation, cell counting, and immunoblotting.