Chapter 4 —— 41 —— of this generic approach results from its compatibility with large-scale genomic edits and the facile assembly of robust RNA-programmable CRISPR-Cas9 nucleases. Indeed, despite the discovery of a growing number of CRISPR systems, engineered CRISPR-Cas9 nucleases based on the prototypic Streptococcus pyogenes CRISPR system remain leading tools for genome editing purposes [1]. These designer nucleases consist of a single guide RNA (gRNA) and a Cas9 enzyme complex that binds to protospacer adjacent motif (PAM) sequences reading NGG. Targeted DSB formation, catalyzed by the HNH and RuvC nuclease domains of Cas9, follows whenever next to a PAM locates a circa 20-bp DNA stretch (protospacer) complementary to the 5′ end of the gRNA (spacer). Critically, directed evolution and structure-guided protein engineering efforts have yielded highspecificity Cas9 variants that, depending on the selected gRNA, have either undetectable or notably reduced off-target DNA cleaving activities [2]. Normally, gene targeting is achieved through the delivery of donor DNA substrates for ectopic homology-directed repair (HDR) [3] or nonhomologous end joining (NHEJ) [4,5] at site-specific DSBs created by RNAprogrammable nucleases. The former pathways engage donor DNA constructs whose designs favor homologous recombination (HR) [3], microhomology-mediated end joining (MMEJ) [6] or, more recently, homology-mediated end joining (HMEJ) [7-9]. MMEJ, HMEJ and HR donors contain sequences homologous to genomic sequences framing the targeted DSB (‘homology arms’) whilst NHEJ-prone donors lack ‘homology arms’ altogether [4,5]. As such, homology-bearing donors achieve directional single-step gene knock-ins in contrast to NHEJ donors including those with a homology-independent targeted integration (HITI) design [5]. Moreover, in contrast to HR donors, NHEJ, MMEJ and HMEJ donors have their targeting matrixes flanked by programmable nuclease cleaving sites. This ‘double-cut’ design assures the freeing of donor DNA substrates from delivery vehicle backbones which favors gene knock-ins via the processing
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