Chapter 2 —— 21 —— endogenous human locus, namely, the prototypic safe harbour locus AAVS1 [34]. The application of this generic in trans paired nicking (ITPN) principle was subsequently expanded to other genomic sequences through the use of more versatile RNA-programmable CRISPR-Cas9 nickases [35,36] that are simply obtained through site-directed mutagenesis of one of the two nucleases domains of the parental Cas9 protein (i.e., HNH or RuvC) (Figure 1) [37]. Indeed, by stimulating otherwise inefficient SSB-dependent HDR, ITPN approaches based on the delivery of nicking CRISPR-Cas9 complexes and matched nickase-susceptible HDR donor constructs, are valuable for seamless and scarless chromosomal editing, including at multiple-copy or essential genomic tracts [19,38]. Additional examples regarding the application of ITPN methodologies in various mammalian cell types, e.g., iPSCs, keratinocytes and organoids featuring normal or cancer traits, encompass: (i) repairing or installing predefined gene mutations [35,38-41], (ii) maximizing the integrity of unmodified alleles during allele-specific gene editing [42,43], and (iii) streamlining one-step biallelic gene editing or onestep multiplexing gene knock-in or tagging [35,44,45]. It is equally worth mentioning that, in contrast to regular and high-specificity CRISPR-Cas9 nucleases, CRISPR-Cas9 nickases constitute poor P53-dependent signalling triggers in human cells, including in DNA damage-sensitive iPSCs [38,40]. Hence, it is expected that the aforementioned growing mining for CRISPR systems buried in large genomic and metagenomic databases, will start unearthing enzymes that, via either their intrinsic or engineered nicking activities, enlarge the toolset for DSB-free genome editing. Examples include
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