Fig. 1: The Cas9-AAV6 platform is an effective and versatile tool for genome editing in human MSCs.

a Schematics outline procedures for genome editing using the Cas9-AAV6 platform in human MSCs derived from the bone marrow (BM), adipose tissue (AD), and umbilical cord blood (UCB) for site-specific transgene integration. MS-modified guide RNAs and Cas9 nuclease are electroporated as All-RNA cocktail or RNP complex. Then, homologous repair templates containing GFP reporter cassette insert are delivered by electroporation-aided transduction (EAT) of AAV6 vectors for 15 min during recovery period. Integration of GFP-overexpression cassette can be detected ~7–10 days later. HA: homology arm b Representative single-channel and overlay images of a GFP⁺ hBM-MSC colony originated from low-density seeding (220 cells/cm²) one passage in culture after nuclease electroporation and EAT. Scale bars represent a distance of 400 µm. GFP⁺ colony formation is observed in targeting from all 12 biological hMSC donors described in this study. c Dots and lines represent average frequencies of GFP⁺ cells in hBM-MSCs (biological replicates: n = 6) at different timepoints after HBB locus targeting using All-RNA nuclease and AAV6 EAT or AAV EAT only. P_i signifies the end of the i^th passage post targeting. Error bars represent standard error of mean. d GFP⁺ cell frequencies at one passage post targeting of hBM-MSCs (biological replicates: n = 7), hAD-MSCs (n = 3), and hUCB-MSCs (n = 3), which were targeted with All-RNA cocktails or RNP complexes followed by AAV6 EAT are represented in dot plots for indicated gene loci (HBB, CCR5, and RANKL). Connected dots represent hMSCs from the same human donors. Mean indel frequencies were compared between two modes of targeting and two-tailed p-values from paired t-test are shown for significant differences of mean. Source data are available in the Source data file.