Chapter 4 —— 61 —— Figure 5. Investigating the role of AAV donor structures on genome editing. (A) Testing genome editing in hMSCs using single-stranded AAV HR and HMEJ donor constructs. Schematics of AAV donor structures are depicted. Horizontal gray bars, short tracts homologous to DNA flanking the AAVS1 target site of gRNA GS1 (‘short homology arms’); gray box, EGFP transgene; orange boxes, GS1 target sequence. hMSCs were transduced with AdVP.eC94NLSGS1 (4 GC cell-1), expressing high-specificity eCas94NLS:GS1 complexes, together with AAV-HRS1 or AAV-HMEJS1 donors (4 TU cell-1 each). The frequencies of transient and stable transduction levels are plotted in the top and bottom graphs, respectively. Datapoints correspond to mean ± SD values from three biological replicates. (B) Testing genome editing in HeLa cells using single-stranded versus doublestranded AAV donor constructs. Diagrams of regular and sc AAV HR and HMEJ donors, are shown. HeLa cells were transduced with AdVP.eC94NLSGS1 (20 GC cell−1) mixed with single-stranded AAV (2 TU cell-1) or scAAV donors (0.5 TU cell-1. The frequencies of transient and stable transduction levels are plotted in the top and bottom graphs, respectively. Datapoints correspond to mean ± SD values from three biological replicates. Numerals above the graph bars correspond to AAV stable transduction frequencies (mean ± SD) normalized to the initial transduction levels.
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