Chapter 6 —— 152 —— Genome editing (aliases, genomic engineering and gene editing) is a fastpaced field with increasing impact on fundamental science, biotechnology, and medicine [1]. With the aid of engineered RNA-guided nucleases (RGNs) derived from clustered regularly interspaced short palindromic repeat (CRISPR) systems initially discovered as prokaryotic antiviral machineries in bacteria and archaea [2], genome editing has become more versatile and customizable with regards to the options for achieving gene knock-out (KO), gene knock-in (KI), targeted DNA replacement and tagging, amongst other chromosomal DNA modification endpoints [1,2]. Despite the increasing discovery and adaption of new CRISPR and CRISPR-like systems for genome editing purposes [3], engineered CRISPR-derived RGNs based on the prototypic Streptococcus pyogenes CRISPR-Cas9 system, and their variants created by directed evolution or rational design (e.g., high-specificity and targeting range-expanded variants), remain commonly used reagents for a broad range of genome engineering applications [4]. This stems in large part from their relative robustness in engaging eukaryotic chromatin [5]. Yet, although nuclease-induced double-stranded DNA break (DSB) formation yields robust and cell cycle-independent gene KO endpoints upon the installation of small insertions and deletions (indels) by canonical and alternative non-homologous end joining (NHEJ) pathways, these DNA repair pathways are disruptive to cell genotypes in the context of gene KI procedures based on homology-directed repair (HDR) [6]. Moreover, the possibility for RGN off-target and, more pervasively, on-target activities and ensuing mutagenic effects, which intrinsically stem from NHEJ processes, demands the exploration of more precise and less mutagenic (‘soft’) genome editing strategies. An emerging class of such DSB-independent strategies is reviewed in Chapter 2 where sequence- and site-specific nucleases ('nickases’) derived from CRISPR systems are exploited for precise HDR-mediated gene KI using tailored donor DNA substrates. Indeed, in contrast to DSBs, single-stranded DNA breaks (SSBs), or nicks, are not substrates for mutagenic NHEJ DNA repair pathways, canonical or otherwise. As corollary, genomic engineering
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