Quantifying CasRx Editing Efficiency

CasRx-engineered cells were transduced with the pLenti-EGFPdestablized-Hygro (Addgene, cat. no. 138152 (ref. ^{43 (link)})) lentivirus and selected with hygromycin for 1 week. After selection, the cell culture was split into two different dishes. One of the dishes was transduced with the pLKO5-CasRx(DR1 30)–EFS-tRFP657 containing a NT gRNA and the other with a GPF-targeting gRNA. Four days after transduction, the cells were resuspended in FACS buffer (PBS, 3% FBS, 5 mM EDTA) and recorded using a Beckman Coulter Cytoflex LX system. FlowJo v.10.7.2 was used to analyze populations. CasRx activity was calculated as one minus the ratio between the GFP intensity (mean B525-FITC-A) of cells transduced with the gGFP gRNA and cells transduced with the gNT gRNA. The pLKO5-CasRx(DR1 30)–EFS-tRFP657 vector was cloned using the pLKO5.sgRNA.EFS.tRFP (Addgene cat. no. 57823 (ref. ^{83 (link)})).