Cleavage-free human genome editing

Most gene editing technologies introduce breaks or nicks into DNA, leading to the generation of mutagenic insertions and deletions by non-homologous end-joining repair. Here, we report a new, cleavage-free gene editing approach based on replication interrupted template-driven DNA modification (RITDM). The RITDM system makes use of sequence-specific DLR fusion molecules that are specifically designed to enable localized, temporary blockage of DNA replication fork progression, thereby exposing single-stranded DNA that can be bound by DNA sequence modification templates for precise editing. We evaluate the use of zinc-finger arrays for sequence recognition. We demonstrate that RITDM can be used for gene editing at endogenous genomic loci in human cells and highlight its safety profile of low indel frequencies and undetectable off-target side effects in RITDM-edited clones and pools of cells.