Chapter 2 —— 22 —— Figure1. Standard versus in trans paired nicking genome editing. The relative weights of desired and undesired genome editing outcomes derived from, respectively, homologydirected repair (HDR) and imprecise events caused by competing end-joining DNA repair pathways, e.g., non-homologous end joining (NHEJ), are illustrated. DSB and SSB, doublestranded and single-stranded chromosomal breaks, respectively; ‘nickases’, sequence- and strand-specific nucleases. In contrast to standard donor constructs, modified donor constructs have nickase-susceptible target sites (TS) framing their targeting modules consisting of exogenous DNA (green bar) flanked by sequences homologous to the genomic target region (‘homology arms’). the HNH-negative IsrB nickase derived from the ancestral CRISPR-like system OMEGA and the RuvC-only CRISPR class 2 type V Cas12i nuclease that nick and preferentially nicks, respectively, double-stranded DNA substrates [46–48]. Moreover, often, newly discovered CRISPR systems also yield genome editing components whose small sizes renders them more fitting for delivery through commonly used adeno-associated viral (AAV) vectors [49].
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