Chapter 2 —— 23 —— Finally, another recent ‘soft’ HDR-mediated genome editing concept that might be particularly suited for the repair of heterozygous or dominant mutations involves allele-specific chromosome nicking for the stimulation of interhomolog recombination (IHR) in somatic cells [50,51]. Through this process of allelic conversion, a pathogenic mutation in one allele can, in principle, be corrected using as donor template the endogenous ‘healthy’ allele (Figure 2). This elegant concept of using CRISPR-Cas9 nickases and endogenous homologous chromosomal DNA as repairing templates has been demonstrated in Drosophila models [51] and human cell lines [50,52]. Regarding the application of such exogenous donor DNA-free genome editing principles in human cells, recent investigations argue for multiplexing approaches in which primary allelic-specific gRNAs act in concert with secondary gRNAs to direct in trans paired nicking of homologous chromosomes and ensuing allelic conversion via IHR (Figure 2) [52]. Further research will be instrumental to advance CRISPR-Cas9 nickase-induced IHR from enticing proof-of-concept studies in cell lines to its application in human stem and progenitor cells. Translation Insight & Outlook There is a pressing need for investigating and validating alternative DSB-free and precise genome editing tools and strategies in various stem and progenitor cell types, e.g., bona fide T and NK cells as well as precursor iPSCs from which different effector cells can be differentiated, including immunotherapeutic T and NK cell candidates. Genome engineering strategies covering targeted and precise chromosomal incorporation of genetic payloads with varying sizes will become ever-more relevant. In this regard, CRISPR nickases per se and fused to reverse transcriptases offer a complementary
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