Chapter 2 —— 20 —— Rationale for “soft” genome editing based on CRISPR nickases Although emerging high-specificity programmable nucleases can greatly minimize off-target DNA cleavage, e.g., eSpCas9(1.1) [32] and Cas9-HF1 [33], they are inherently incapable of eliminating the potentially deleterious effects resulting from on-target DSB formation. Therefore, the substantial genotoxicity and cytotoxicity profiles associated with conventional nucleaseassisted genome editing create a pressing need for the development of alternative genetic engineering systems that reliably generate safer and functionally robust cell products. Indeed, DSB-dependent genome editing is expected to be particularly risky in the context to cell therapies based on the transplantation of populations of genetically engineered pluripotent stem cells, T lymphocytes and NK cells. The reasons are twofold. Firstly, in the context of extensive ex vivo cell amplification protocols underpinning the generation of these cell transplantation products, DSB-derived mutations and/or chromosomal rearrangements can cooperate in cell transformation and clonal expansion. Secondly, in instances where targeting multiple genes is needed for achieving a robust anti-tumor effect, e.g., via combinatorial exogenous CAR transgene knock-ins and endogenous TCR or programmed cell death protein 1 (PD1) gene knockouts, simultaneous induction of the attendant DSBs at different genomic positions is expected to exacerbate the levels of undesirable genome editing by-products in the form of translocations and chromosomal rearrangements. In this context, investigations exploring alternative HDR-mediated gene knock-in approaches that rely on sequence- and strand-specific nucleases (‘nickases’) are valuable in that the resulting single-stranded DNA breaks (SSBs), or nicks, are substrates for neither NHEJ nor MMEJ. As a corollary, the balance between precise HDR to undesired end-joining events are dramatically biased towards the former. Moreover, although genomic SSBs are, per se, poor HDR stimuli, earlier experiments from our laboratory using the native adeno-associated virus Rep68/78 nickases demonstrated that concomitant SSB formation at acceptor sequences and donor DNA constructs fosters HDR-mediated gene knock-in at an
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