Chapter 2 —— 25 —— foreign DNA despite the need for substantial optimization of extended primeediting gRNAs (pegRNAs) [53]. Moreover, in contrast to HDR-based genome editing, prime editing can take place in post-mitotic cells albeit to lower efficiencies than in cycling cells [54]. Recent prime editing developments include the combinatorial use of dual pegRNAs and sitespecific recombinases designed for replacing genomic sequences with up 250-bp of foreign DNA and inserting entire transgenes at prime editordefined recombinase target sites, respectively [53]. Despite powerful and versatile, such combinatorial strategies require the delivery of large and multicomponent reagents into target cells. An aspect warranting attention when considering multiplexing approaches concerns the importance of introducing balanced amounts of the attendant individual components to maximize the performance and precision of genome editing interventions [55]. In addition, prime editing involving the delivery of dual pegRNAs is not compatible with large edits whereas sequential prime editing and site-specific recombination is not amenable to subtle genomic edits underlying endogenous gene repair due to discontinuous ‘footprint’ installation in the form of recombinase target sites. In conclusion, considering the herein discussed findings and matters, one can submit that cell therapy products derived from the use of RNA-programmable nickases as such or with heterologous domains, will offer a complementary set of ‘soft’ genome engineering options whose safety profiles are potentially higher than those associated with the exposure of cells to programmable nucleases.
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