Chapter 1 —— 10 —— General introduction and outline of this thesis Nowadays, advanced tools deployed for genome editing (GE) mainly derive from the CRISPR/Cas9 system found to be in 2012 as a prokaryotic RNAguide antiviral system. Typically, Cas9 proteins or engineered Cas9 proteins, e.g., nucleases, can make endogenously site-specific double-stranded DNA breaks (DSBs) which are predominantly repaired via the non-homologous end joining (NHEJ) DNA repair pathway leading to the introduction of mutagenic insertions/deletions (indels). Hence, NHEJ-mediated GE can lead to the removal of pre-existing genetic information, i.e., knock-out (KO). Alternatively, in the presence of exogenous donor DNA templates, e.g., a transgene cassette flanked by DNA sequences identical to those present around the targeted DSB made by engineered RNA-guided nucleases, can result in gene knock-in (KI) through the homology-directed repair (HDR) DNA repair pathway. Differently from DSBs made by RNA-guided Cas9 nucleases, there are Cas9 protein variants that induce instead single-strand DNA breaks (SSBs), or nicks, by virtue of having the D10A or H840A mutations that result in the catalytic inactivation of their RuvC or HNH nuclease domains, respectively. Significantly, in contrast to DSBs, nicks are not substrates for error-prone NHEJ processes. Furthermore, nicks caused by nicking Cas9 proteins, i.e., nickases, can also serve as stimuli for HDR in the presence of exogenous donor DNA templates for the purpose of achieving KI at target chromosomal sites. Ideally, SSB-based KI strategies can alleviate or even avoid the buildup of indels caused by DSBs, and hence it can be regarded as a preferable GE strategy choice in the regards to therapeutical applications. In Chapter 2, the pros and cons of GE involving DSB-based homologydirected repair are elaborated. We discuss the pressing need for the development and application of less mutagenic GE procedures, namely, via using DSB-independent nickases which, as aforementioned, lead only to residual amounts of mutagenic indels and can be tailored together with matched donor DNA templates for precise SSB-mediated HDR. As such, SSB-mediated HDR can serve as a valuable approach for editing
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