Chapter 2 —— 16 —— Abstract The integration of the gene and cell therapy fields through the application of genome editing principles permits generating ex vivo transplantable grafts from stem cells or from their differentiated progenies (e.g., T and NK cells) with novel genetically-engineered function(s). As such, these technologies are offering new therapeutic avenues to previously intractable inherited and acquired disorders (e.g., malignant and infectious diseases). In this article, we discuss the main characteristics, advantages and limitations of genome editing involving the targeted chromosomal insertion of transgenes upon site-specific double-stranded DNA break (DSB) formation by programmable nucleases, namely, RNA-programmable CRISPR nucleases. Subsequently, building on this information and recent findings, we put forward the view that targeted transgene insertion strategies based on CRISPR nickases, as opposed to nucleases, address important limitations of conventional DSB-dependent genome editing approaches. In particular, the cytotoxicity and high genotoxicity resulting from DSBs especially in cell types highly sensitive to DNA damage, including pluripotent and hematopoietic stem cells. Background Genome editing or genome engineering is a fast-evolving field with growing impact on basic science, biotechnology, and medicine [1]. Particularly versatile genome editing strategies consist of inserting exogenous donor DNA constructs into specific genomic loci (knock-in) subjected to double-stranded DNA breaks (DSBs) made by engineered nucleases derived from class 2 type II CRISPR systems consisting of single guide RNA (gRNA) and Cas9 ribonucleoprotein complexes (CRISPR-Cas9 nucleases) [2]. This versatility stems from the robust activity and straightforward designing of these RNAprogrammable nucleases and the amenability of gene knock-in strategies to genomic modifications spanning entire transgenes, including those encoding chimeric antigen receptors (CARs) and T-cell receptors (TCRs) alone or together with auxiliary factors, such as positive-selection markers and safety
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