Gene editing or autophagic delivery of mutant Mpl to the cell surface rescue receptor function in vitro. (A) Transient overexpression of GRASP55 tagged with a V5 epitope results in accumulation of the lower-molecular-weight core-glycosylated form of Mpl regardless of WT or mutant status. Receptors are shown to be signaling competent on the basis of phosphorylation of key signaling proteins in the Jak/STAT and PI3K pathways in response to Tpo. (B-C) XTT-II proliferation assays performed on UT-7 or Ba/F3 cell lines expressing WT or mutant Mpl^mNG and selected for growth in the presence of Tpo (panel C, solid lines) or eltrombopag (Elt) (panel C, dotted lines). CRISPR-Cas9–edited cells that were reverse-engineered to restore the WT sequences in MPL exon 5 from the mutated W272R sequence (labeled Mpl W272R>WT) are represented by blue open circles. UT-7 cells were edited by using the D10A Cas9 mutant and 2 single gRNAs in a double nickase approach. A classical WT Cas9 approach (ie, coupled to a unique single gRNA) was used to edit Ba/F3 cells. **P < .005; ***P < .0001.