Chapter 6 —— 153 —— using nickases is dramatically biased towards precise HDR over imprecise NHEJ events. Of note, although genomic SSBs are per se weak HDR stimuli, earlier experiments demonstrated that concomitant SSB formation at acceptor genomic sequences and donor DNA substrates by CRISPR/Cas9 nickases fosters HDR-mediated gene KI [7-9]. The application of this generic in trans paired nicking principle yields genomic engineering of stem cells with minimal allelic indels, translocations, and P53-dependent activation of the DNA damage response (DDR) known to trigger apoptosis or, alternatively, halt the cell cycle progression needed for HDR [7,9]. Therefore, as pointed out in Chapter 2, cell therapy products derived from using RNAprogrammable nickases as such or coupled to heterologous effector domains, like prime editor reverse transcriptases or base editor deaminases, will start to increasingly offer a complementary set of ‘soft’ genome editing options whose performances and safety profiles are potentially higher than those resulting from exposing cells to programmable nucleases. In Chapter 3, the relevance of DSB-independent genome editing efforts is further elaborated through a commentary to a study by Chai and colleagues [10]. In this study, base editors based on Cas9 nickases fused to deaminase effector domains are tested for the purpose of restoring dystrophin synthesis in in vitro and in vivo models of Duchenne muscular dystrophy (DMD; MIM: 310200). In particular, the authors identify adenine base editors and cognate guide RNAs that, by installing A⋅T-to-G⋅C transitions at splicing motifs, lead to defective DMD reading frame repair via exon skipping and subsequent removal of premature stop codons that arose due to out-of-frame deletions. Indeed, by using dual AAV vectors for the trans-splicing assembly of complete base editor proteins, the authors demonstrate the rescue of dystrophic traits at the cellular and organismal levels in dystrophin-defective mice upon dual AAV vector administrations. Specifically, base editor constructs linked to N- and C-terminal intein domains were split and packaged in two distinct AAV vectors that, upon target cell co-transductions, led to intein-mediated protein trans-splicing and in situ assembly of full-
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