Cell line engineering – tagged organelle markers
CRISPR/Cas9 methods were used for gene editing by homology directed repair (HDR) following established protocols. Ribonucleoprotein (RNP) complexes of S. pyogenes Cas9 and guide RNA were pre-assembled in vitro, mixed with double-stranded (dsDNA) HDR donors and delivered into HEK293T cells by electroporation in 96-well plates (see below). Each electroporation used 0.2x10^6 cells, 70 pmol RNP and 2 pmol HDR template. Electroporated cells were incubated for two days in the presence of 1 µM
nedisertib (M3814; Selleckchem # S8586) to enhance HDR efficiency.118 dsDNA HDR donors including tag sequence flanked by gene-specific homology arms were PCR-amplified from a corresponding plasmid template using 5’ biotinylated PCR primers as previously described.119 RNP complexes were freshly assembled prior to electroporation and combined with HDR template in a final volume of 10 µL. First, 0.7 µL gRNA (130 mM stock; source: Integrated DNA Technologies) was added to 2.2 µL high-salt RNP buffer {580 mM KCl, 40 mM Tris-HCl pH 7.5, 20% v/v glycerol, 2 mM TCEP-HCl pH 7.5, 2 mM MgCl2, RNAse-free} and incubated at 70°C for 5 min. 1.8 µL of purified Cas9 protein (40 mM stock in Cas9 buffer, ie. 70 pmol) was then added and RNP assembly was carried out at 37°C for 10 min. Finally, HDR template (2 pmol) and sterile RNAse-free H2O were added to 10 µL final volume. Electroporation was carried out in Amaxa 96-well shuttle Nucleofector device (Lonza) using SF solution (Lonza) following the manufacturer’s instructions. Cells were washed with PBS and resuspended to 10,000 cells/mL in SF solution (+ supplement) immediately prior to electroporation.
For each sample, 20 µL of cells (ie. 200,000 cells) were added to the 10 µL RNP/template mixture. Cells were immediately electroporated using the CM-130 program, after which 100 µL of pre-warmed media (containing 1 µM nedisertib) was added to each well of the electroporation plate to facilitate the transfer of 25,000 cells to a new 96-well culture plate containing 150 µL of pre-warmed media (containing 1 µM nedisertib). Electroporated cells were cultured for >5 days and transferred to 12-well plates prior to selection by fluorescence-activated cell sorting (FACS). For each target, 1,200 cells from the top 1% fluorescent cell pool were isolated on a SH800 instrument (Sony biotechnology) and collected in 96-well plates.
Cell line engineering – localization de-orphaning
HEK293T cell lines used in the analysis presented in Figure 4 were engineered using the mNeonGreen2(1-10/11) split fluorescent protein system using protocols previously described.14 In brief, CRISPR/Cas9 methods were used for gene editing by homology directed repair (HDR) using RNP electroporation methods as described in the previous section, with the exception that single-stranded deoxyoligonucleotide (ssODN) donors were used (Ultramer ssODN, Integrated DNA Technologies; 120 pmol donor per electroporation).