同源重组（HDR）是指将可编程核酸酶切割的接受基因组序列与供体构建物进行重组，可以精确地在哺乳动物细胞中进行大规模基因组编辑。然而，除了精确的基因敲入外，可编程核酸酶还会产生非同源末端联接过程中的意外基因组修改。相反，利用CRISPR-Cas9单链切割酶在目标位点和外源供体构建物间形成串联的单链DNA断裂，实现了无缝和无瘢痕的基因组编辑。在本研究中，我们鉴定了高特异性的CRISPR-Cas9核酸酶，可以通过同源重组（HR）和同源介导的末端联接（HMEJ）与具有常规和“双切割”设计的供体构建物一起指导基因组编辑。此外，我们探讨了ITPN原理，通过展示与正交和高特异性的CRISPR-Cas9单链切割酶的兼容性，并且重要的是，我们在人类诱导多能干细胞（iPSC）中报告了与高特异性CRISPR-Cas9核酸酶不同的现象，即常规或高特异性的CRISPR-Cas9单链切割酶不会激活P53信号传导，该信号传导与出现与肿瘤相关的基因编辑细胞有关。最后，在人类iPSC中的实验揭示，与基于高特异性CRISPR-Cas9核酸酶的HR和HMEJ基因组编辑不同，使用高特异性CRISPR-Cas9单链切割酶的ITPN可编辑与基因组中的基本性和复发性相关的等位序列。 ©2023年作者（们）。由牛津大学出版社代表核酸研究出版。

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. Yet, next to precise gene knock-ins, programmable nucleases yield unintended genomic modifications resulting from non-homologous end-joining processes. 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. 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. 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. Finally, experiments in human iPSCs revealed that differently from HR and HMEJ genome editing based on high-specificity CRISPR-Cas9 nucleases, ITPN involving high-specificity CRISPR-Cas9 nickases permits editing allelic sequences associated with essentiality and recurrence in the genome.© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.