Chapter 2 —— 17 —— genetic switches [3,4]. Indeed, notwithstanding the growing mining for and adaption of CRISPR and CRISPR-like systems for genome editing purposes, engineered CRISPR-Cas9 nucleases based on the prototypic Streptococcus pyogenes CRISPR system and their molecularly evolved or structurallyguided designed variants (e.g., high-specificity and targeting range-expanded variants), remain leading tools for a wide variety of genome engineering applications [5,6]. Main attributes of CRISPR nuclease-assisted genome editing Chromosomal gene knock-in procedures often entail the delivery of CRISPRCas9 nucleases together with donor DNA constructs designed as substrates for either homology-independent or homology-dependent repair (HDR) pathways [7]. Generally, HDR-mediated transgene knock-ins are more precise than those resulting from homology-independent processes in that they are naturally inserted at the chromosomal target site in a predefined orientation and present neither multiple-copy insertions nor imprecise ‘foot- prints’ at the junctions between genomic and exogenous DNA sequences [8,9]. Importantly, as HDR takes place during the late G2 and S phases of the cell cycle, therapeutically relevant dividing cell types, such as induced pluripotent stem cells (iPSCs), natural killer (NK) cells and T lymphocytes, are amenable to precise HDR-mediated genome editing. For instance, in what valuable target cells is concerned, genetically engineered CAR-T cells, serving as personalized ‘living drugs’, are yielding impressive results in terms of treating CD19-positive hematological malignancies [3,10]. This is so despite their high costs that stem in part from the difficulties in generating the large amounts of the respective engineered cell products. Since 2017, a growing number of these CAR-T cell products have in fact started to be approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) [11]. Building on the resulting CD19-targeted cancer therapy datasets, over 500 CAR-T cell therapies directed at different antigens in liquid and solid tumors are currently undergoing clinical testing worldwide [11,12]. Significantly, CAR-NK cells are also entering the
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