Chapter 4 —— 62 —— Knowledge on the relative contribution of different AAV donor designs (i.e. regular versus ‘double-cut’) and structures (i.e. single-stranded versus double-stranded) to genome editing endpoints is scant. To fill this knowledge gap, we capitalized on the isogenic set of structurally diverse AAV donors to systematically investigate the role of such donor substrates on the precision of genome editing. In particular, AAV donors with HR or HMEJ templates in single-stranded AAV or double-stranded scAAV genomes with targeting modules matching the commonly used AAVS1 safe harbor locus. After cotransducing HeLa cells with AdVP.eC94NLSGS1 and each of the AAV donors, we performed junction PCR screens on randomly selected EGFP-expressing cell clones, each of which corresponding to individual genome-modifying events (Figure 6A). In the set of clones modified through the transfer of AdVP.eC94NLSGS1 and AAV-HRS1, the AAVS1-targeted fraction was 97.7%, with 88.6% of the total DNA-modifying events representing precise genome editing (i.e. jT+/jC+) (Figure 6B and Supplementary Figure S13). The scAAV vectors scAAV-HRS1 and scAAV-HMEJS1 yielded similar and lower numbers of precise genome-editing events, respectively, when compared to those obtained with AAV-HRS1 (Figure 6B and Supplementary Figure S13). Indeed, although the AAVS1-targeted cell fraction resulting from using scAAV-HMEJS1 was 97.7%, only 71.7% of the total DNA-modifying events were precisely targeted (Figure 6B and Supplementary Figure S13). These data are consistent with previous studies showing that ‘double-cut’ donors placed in circular plasmids or protein-capped linear adenovector genomes normally yield less accurate genome-editing products than their respective HR donor counterparts [6,7,9,13,26,36]. Hence, it is also feasible that in the context of scAAV genomes with closed palindromic structures, generation of free-ended HMEJ donor products created by targeted DNA cleavage exacerbates the engagement of less precise NHEJ and/or MMEJ processes linking exogenous to endogenous DNA. Consequently, as single-stranded AAV-HRS1 and scAAV-HMEJS1 yielded similar genome editing frequencies but the former presented higher HDR precision, we next focused on further
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