Additionally, we explored the ITPN principle by demonstrating its compatibility with orthogonal and high-specificity CRISPR-Cas9 nickases and, importantly, report that in human induced pluripotent stem cells (iPSCs), in contrast to high-specificity CRISPR-Cas9 nucleases, neither regular nor high-specificity CRISPR-Cas9 nickases activate P53 signaling, a DNA damage-sensing response linked to the emergence of gene-edited cells with tumor-associated mutations. In the present study, we identified high-specificity CRISPR-Cas9 nucleases capable of outperforming parental CRISPR-Cas9 nucleases in directing genome editing through homologous recombination (HR) and homology-mediated end joining (HMEJ) with donor constructs having regular and ‘double-cut’ designs, respectively. Alternatively, in trans paired nicking (ITPN) involving tandem single-strand DNA breaks at target loci and exogenous donor constructs by CRISPR-Cas9 nickases, fosters seamless and scarless genome editing. Yet, next to precise gene knock-ins, programmable nucleases yield unintended genomic modifications resulting from non-homologous end-joining processes. Homology-directed recombination (HDR) between donor constructs and acceptor genomic sequences cleaved by programmable nucleases, permits installing large genomic edits in mammalian cells in a precise fashion.

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