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bioRxiv · 10.1101/2025.05.20.655067

Unveiling the hidden window of prime editing

Chen, P.-R.·Wei, Y.·Yuan, X.-Z.·Wang, S.-G.·Xia, P.-F.
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Abstract

Cleavage-dependent CRISPR-Cas gene editing relies on RNA-guided DNA cleavages that push cellular machinery to incorporate intended edits. Yet, a second cleavage on the already cut DNA strand can also be executed by CRISPR nucleases. This feature, however, has never been repurposed for gene editing. Here, we report the first integration of the second cleavage activity of Cas9 for precision gene editing, allowing previously impossible prime editing in the 5 direction of a nick. We elucidate the second-cleavage-driven pathway that primes non-canonical reverse transcription events upstream of the nick. We identify the competition between the non-canonical and canonical routes, which can be modulated by rationally designing RNA templates with intended edits. We demonstrate that cellular physiologies elevate editing efficiency from individual reverse-transcripts yet exert limited influence on the pathway competitions. Our findings reshape the design principle of prime editing and open an entirely new dimension for engineering CRISPR-Cas systems with the intrinsic, non-host-specific second cleavage activity.

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Chen, P.-R., Wei, Y., Yuan, X.-Z., Wang, S.-G., Xia, P.-F.. 2025-05-21. Unveiling the hidden window of prime editing. https://doi.org/10.1101/2025.05.20.655067

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