Chapter 4 —— 60 —— design [7–9,13,26]. Of notice, incoming single-stranded AAV genomes normally undergo conversion to a double-stranded format via either the engagement of host cell DNA polymerases at the priming 3′ ITR or after the hybridization of genomes with plus and minus polarity whose packaging in AAV capsids seems to occur at similar rates [12,45,59,60]. Hence, the singleto-double strand AAV DNA conversion kinetics is likely to be contingent upon particular cell types and experimental conditions that, together, influence the build-up of double-stranded AAV HMEJ substrates susceptible to targeted DNA cleavage (Supplementary Figure S6). For instance, inhibitory cellular factors binding AAV ITR cis-acting elements, whose amounts vary in different cell types, can render second-strand AAV DNA synthesis or transgene expression rate-limiting to different extents [46–48]. Therefore, to readily present HMEJ-prone AAV donor substrates in transduced cells and, simultaneously, probe the performance of singlestranded versus double-stranded AAV donor structures, scAAV-HRS1 and scAAV-HMEJS1 were assembled and tested side-by-side with their respective AAV-HRS1 and AAV-HMEJS1 counterparts (Figure 5B). Similar to the previous experiments in hMSCs (Figure 5A), combining AdVP.eC94NLSGS1 with AAV-HRS1 or AAV-HMEJS1 yielded comparable CRISPR-Cas9dependent stable transduction frequencies in HeLa cells (Figure 5B, left graphs). Notably, comparable CRISPR-Cas9-dependent genome editing frequencies were also observed in HeLa cells initially co-transduced with AdVP.eC94NLSGS1 and each of the scAAV-HRS1 and scAAV-HMEJS1 vectors (Figure 5B, right graphs). Therefore, these data suggest that in contrast to HMEJ templates placed in the context of donor plasmids or adenovector genomes [7–9,13,26], HMEJ templates in AAV donor genomes do not necessarily outperform their HR counterparts during DSB-dependent genome editing.
RkJQdWJsaXNoZXIy MTk4NDMw