Platinum cas9

We generated HEK-293A cells endogenously tagged with the split NG2 protein at the Pfn1 and SHIP2 (INPPL1) loci, following a previously published general protocol (57 (link)). Briefly, we electroporated a Platinum Cas9 (Thermo Fisher Scientific#B25640)–gRNA complex with a single-stranded HDRT (IDT) into our stably expressing HEK293^NG2-1-10 cells. The HDRT is composed of 70 bp homology arms to align with the gene of interest, the NG2 11th β-strand insertion sequence, and a flexible linker. HDRT and gRNA sequences for INPPL1 are provided in Table S2. HDRT and gRNA sequences for Pfn1 are from OpenCell database. Following electroporation, we screened cells for fluorescence using our confocal microscope and subsequently used fluorescence-activated cell sorting (University of Pittsburgh Flow Cytometry Core) to isolate and expand NG2-positive cells.

HeLa cells are endogenously tagged with split GFP (Leonetti et al., 2016 (link)). HeLa cells stably expressing sfGFP-1-10 (Zewe et al., 2018 (link)) were electroporated with 200 pmol single-stranded homologous-directed repair (HDR) template (IDT) and 5 pmol precomplexed gRNA and Platinum Cas9 (Thermo Fisher Scientific) in a 10 μl reaction volume using a Neon Electroporation system (Thermo Fisher Scientific) with a single, 20 ms, 1,500 V pulse according to the manufacturer’s instructions. Sequences are provided in Table 5. All HDR templates contained 70-bp homology arms, the GFP-11 sequence, and a flexible linker in frame with the gene to be labeled (5′-CGT​GAC​CAC​ATG​GTC​CTT​CAT​GAG​TAT​GTA​AAT​GCT​GCT​GGG​ATT​ACA​GGT​GGC​GGC-3′). 48 h after electroporation, cells were sorted by FACS for GFP-positive cells.