Sh800 cytometer

eIF2Bβ-mNeonGreen-Flag-tagged cells were generated as described above with pMS074 used to PCR amplify the HDR template and sgMS001 used as the sgRNA. After recovery and expansion, the edited cells were sorted on a Sony SH800 cytometer, and the top 0.1% of mNeonGreen fluorescing cells were sorted into a polyclonal population. After expansion, recovery, and determining that the editing efficiency was over 90% in this population, the polyclonal cells were subjected to a second round of nucleofection using an HDR template amplified off of pMS075 to endogenously tag eIF2Bδ. sgMS004 was used to target the eIF2B2 locus. Nucleofection conditions were as described above. After ~1 week of recovery and expansion, cells were again sorted as described above to isolate the highest mScarlet-i fluorescing cells. After ~1 week of recovery, limiting dilutions were prepared as described above to isolate and validate editing in individual clones. A fully eIF2B2-edited and eIF2B4-edited clone was then subjected to a third round of nucleofection to introduce the eIF2Bα-FKBP12^F36V fusion. This was performed under identical conditions to those described above for the ISR reporter cell line.

Flow cytometry was performed using a Sony SH800 cytometer using a standard 408 nm laser configuration and a 100-μm sorting chip. Cells were gated on FSC-A and SSC-A to exclude debris followed by FSC-H and FSC-W to isolate single cells. For pure EcN culture experiments, cells were treated with calprotectin for 1 h prior to analysis. For human stool experiments, 1 mL of CS EcN-GFP grown to the log phase was cocultured with human stool samples for 1 to 3 h prior to analysis. Flow cytometry plots were generated with FlowJo (Becton, Dickinson & Company).