Chapter 3 —— 34 —— robust expression of Becker-like dystrophins was detected in cardiomyocytes differentiated from base-edited DMD iPSCs. Interestingly, it was found that DMD exon 51 targeting serendipitously established an in-frame 11-nucleotide deletion instead of the intended exon skipping, presumably due to internal cryptic splice site usage. This finding per se stresses the importance of carefully assessing gene-edited products even when using subtle DSBindependent systems. Moreover, various amounts of bystander A·T-to-G·C transitions were also detected (range: 3.3%–91%). These bystander changes might have limited consequences as they map to either spliced-out intron or, if accompanied with the intended SA edits, to skipped exon sequences. Further investigations will be, nonetheless, necessary to probe for slight (or otherwise) splicing alterations in different cell types or contexts. Regardless, base editors with narrower “editing windows” should facilitate more favorable target-to-bystander ratios. AAV vectors are attractive in vivo gene-editing tool delivery vehicles owing to their lack of viral genes and serotype diversity of their parental viruses. Indeed, packaging vector genomes in AAV serotype capsids with a strong tropism for certain cell types (pseudotyping) facilitates tissue-directed transductions. However, the limited AAV packaging capacity (<4.7 kb) permits delivering neither base editing nor other large constructs. To obviate this limitation, researchers are developing base editors with compact architectures [7], testing alternative delivery systems or applying dual AAV strategies in which split constructs linked to N- and C-terminal intein domains are packaged in different AAV vectors (Figure 2). Upon target cell cotransductions, intein-mediated protein trans-splicing results in the in situ assembly of full-length proteins. Indeed, dual AAV-vectored base editor trans-splicing is currently undergoing intense investigation for tackling various disease-causing mutations, including DMD mutations [8,9]. In Chai et al. [5], a dual AAV ABE trans-splicing system is assembled to address frequent DMD deletions through exon skipping modulation. By exploiting high human-murine conservation over intron 44 to exon 45 junctions and
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