UC Santa Barbara

Non-viral gene editing strategies have been developed for basic science and therapeutic applications. Major gains in efficiency have come from the optimization of nuclease activity and delivery, with comparatively little focus on the role of the homology-directed repair template (HDRT) in this process.

Here, we will describe a strategy to boost the efficiency of gene editing via homology-directed repair (HDR) by covalently modifying the template DNA molecule with interstrand crosslinks. Crosslinked templates (xHDRTs) increase Cas9-mediated editing efficiencies by up to fivefold in K562, HEK293T, U2OS, iPS, and primary T cells. Increased editing from xHDRTs is driven by events on the template molecule and requires ataxia telangiectasia and Rad3-related (ATR) kinase and components of the Fanconi anemia (FA) pathway.