Fig 1: MFSD2A knockdown in in both HEK293T and MDA-MB-231 cells reduced intercellular transfer of Cas9.(A) Donor cells: HEK293T with stable overexpression of SBP-tagged Cas9-GFP/gRNA (SBP-Cas9/gRNA) (Ctrl), or MFSD2A knockdown. The recipient cell line is MDA-MB-231 with reporter plasmid. The donor cells and recipient cells were co-cultured for 3 days. Nluc/Fluc was measured and the data normalized to WT donor cells. Data represent mean ± SEM, n ≥ 3. ****p<0.0001, one-way ANOVA. (B) MFSD2A was knocked down in HEK293T with stable overexpression of SBP-tagged Cas9-GFP/gRNA (SBP-Cas9/gRNA). (C) Donor cells: HEK293T wild-type (WT) with stable overexpression of his-tagged Cas9-GFP/gRNA (His-Cas9/gRNA), with stable overexpression of SBP-tagged Cas9-GFP/gRNA (SBP-Cas9/gRNA) or with stable overexpression of SBP-Cas9-GFP/gRNA and myc-streptavidin-CD63-mCherry (Str-CD63/SBP-Cas9/gRNA). The recipient cells are MDA-MB-231 with reporter plasmid (siCtrl), or MFSD2A knockdown (siMFSD2A). Donor cells and recipient cells were co-cultured for 3 days. Nluc/Fluc assays were performed and normalized to an aliquot of co-cultured WT donor and reporter cells. Data represent mean ± SEM, n ≥ 3. (D) MFSD2A was knocked- own in MDA-MB-231 with reporter plasmid. (E) MFSD2A localization in HEK293T cells was detected by immunofluorescence using anti-MFSD2A antibody (green). The cells were stained by Hoechst 33342 and a CF640R-WGA conjugate. White arrows indicate the localization of MFSD2A at or near the plasma membrane. Scale bar is 20 μm. (F) MFSD2A-GFP was transfected in HEK293T cells. Cells were fixed and stained with a CF640R-WGA conjugate. White arrows indicate the localization of MFSD2A at or near the plasma membrane. Scale bar is 20 μm. (G) Model: donor and recipient cells form actin-based tubular protrusions projecting from donor cells (or both of donor and recipient cells) (left). Tubules adhere to recipient cell surface. The human endogenous fusogen, syncytin (especially syncytin-2), expressed on the acceptor cell surface then interacts with its receptor, MFSD2A, on the donor cell surface to facilitate plasma membrane fusion forming an open-ended tubular connection. Cargo, including endosomes and mitochondria, as well as Cas9 protein (free or bound to endosomes) and presumably other proteins and RNA, transfer from donor cells to recipient cells (right). Cytoplasmic Cas9 protein enters the nucleus inducing genome editing and Nluc expression (right). Of note, in cells such as MDA-MB-231, both syncytin-2 and MFSD2A are functional. Figure 9—source data 1.Uncropped Western blot images corresponding to Figure 9B. Figure 9—source data 2.Uncropped Western blot images corresponding to Figure 9D.