Chapter 5 —— 99 —— whole transgene knock-ins) and the straightforward programmability of RNA-guided CRISPR nucleases, such as those built on the prototypic CRISPR-Cas9 adaptive immune system from Streptococcus pyogenes (Pacesa et a. 2024). Engineered CRISPR-Cas9 nucleases, consisting of a sequence-tailored guide RNA (gRNA) and a Cas9 enzyme induce DSBs at DNA sequences that, next to a protospacer adjacent motif (i.e., NGG), have a circa 20-bp nucleotide tract (protospacer) complementary to the 5’ end of the gRNA (spacer). Subsequent DSB repair by donor DNA substrates tailored for homology-directed repair mechanisms, e.g., homologous recombination (HR) (Liao et al. 2024) or for alternative DNA end-joining processes (He et al. 2016; Suzuki et al. 2016), results in targeted genome editing. Critically, when compared to donor constructs tailored for DNA end-joining processes, HR donors yield directional and more accurate gene knock-ins by mitigating insertions and mutations at, respectively, off-target positions and endogenous-exogenous DNA junctions (Liao et al. 2024; He et al. 2016). Unfortunately, HR-mediated genome editing is an inefficient process that often requires auxiliary measurers, e.g., addition of inhibitors of competing and dominant error-prone DNA repair pathways, like non-homologous end joining (NHEJ) and microhomology end joining (MMEJ) or, more typically, incorporation of selectable marker expression units in donor constructs to enrich for gene-edited cell fractions (Chu et al. 2015; Wimberger et al. 2023; Schimmel et al. 2023). However, interfering with endogenous DNA repair processes raises genomic instability concerns (Bischoff et al. 2020); whilst chromosomal insertion of heterologous marker genes limits the applicability and increases the complexity of gene editing protocols (Mikkelsen and Bak, 2023). Interestingly, the co-transfection of donor HR plasmids with selectable markers and genes-of-interest targeting independent loci permits drugdependent enrichment for cells edited simultaneously at both loci, indicating the preferential isolation of HR-proficient cells amongst heterogeneous cell populations (Shy et al. 2016; Mitzelfelt et al. 2017). Building on this phenomenon, marker-free co-selection strategies have been devised where a
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