Chapter 4 —— 63 —— testing single-stranded AAV constructs as sources of HR donor templates. To this end, gene targeting experiments were carried out in HeLa cells with AdVP.eC94NLSGS1 and AAV-HRL.S1. The AAV-HRL.S1 donor construct has ‘homology arms’ larger than those in the previous single-stranded and sc AAV vectors (i.e. 600-bp instead of 200-bp per arm). Moreover, in these experiments, various doses of AAV-HRL.S1 were applied to assess the efficiency and precision of gene targeting as a function of AAV donor amounts (Figure 6C). As expected, the CRISPR-Cas9-dependent stable transduction frequencies determined at 14 days after transduction, were directly proportional to AAV donor amounts (Figure 6C). Interestingly, independently of AAV donor amounts, genome editing frequencies relative to the initial transduction levels were comparable (range: 84%–87%) (Figure 6C). Importantly, randomly isolated EGFP-expressing cell clones (n = 80) generated through AAV-HRL.S1 transductions at 2 and 0.1 TU per cell were all shown to be targeted (Figure 6D) with, respectively, 97.6% and 100% of these being precisely targeted (Figure 6D and E). Taken together, these data suggest that AAV HR donors engage target sequences more frequently through precise HDR than double-stranded AAV HMEJ donors and, reminiscent of double-stranded HR templates, extending the ‘homology arms’ in single-stranded AAV HR templates can improve the performance of gene targeting protocols.
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