Non-canonical cleavage expands prime editing in 5' direction

Cleavage-dependent CRISPR-Cas gene editing relies on RNA-guided DNA cleavage that drives cellular machinery to incorporate intended edits. Beyond canonical cleavage, CRISPR nucleases can also execute non-canonical cleavage on the displaced DNA strand. Here, we report a modified design principle for prime editing that leverages the non-canonical cleavage activity, enabling editing in the 5' direction of a classic nick site. We elucidate the non-canonical-cleavage-driven reverse transcription pathway that initiates upstream of the classic nick site. We identify competition between the canonical and non-canonical routes, which can be modulated by rationally designing RNA templates carrying the intended edits. We demonstrate that cellular physiology enhances editing efficiency from individual reverse-transcription product yet exerts limited influence on pathway competitions. Our findings revise the design principle of prime editing and establish a new dimension for engineering CRISPR-Cas systems by harnessing its intrinsic, non-host-specific, non-canonical cleavage activity.