Uncovering the Dynamics of Precise Repair at CRISPR/Cas9-induced Double-Strand Breaks

Ben-Tov, D.; Mafessoni, F.; Cucuy, A.; Honig, A.; Bessudo, C.; Levy, A. A.

doi:10.1101/2023.01.10.523377

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bioRxiv · 10.1101/2023.01.10.523377

Uncovering the Dynamics of Precise Repair at CRISPR/Cas9-induced Double-Strand Breaks

Ben-Tov, D.·Mafessoni, F.·Cucuy, A.·Honig, A.·Bessudo, C.·Levy, A. A.

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Abstract

CRISPR/Cas9-mediated genome editing relies on error-prone repair of targeted DNA double-strand breaks (DSBs). Understanding CRISPR/Cas9-mediated DSB induction and subsequent repair dynamics requires measuring the rate of cutting and that of precise repair, a hidden-variable of the repair machinery. Here, we present a molecular and computational toolkit for multiplexed quantification of DSB intermediates and repairproducts by single-molecule sequencing. Using this approach, we characterized the dynamics of DSB induction, processing and repair at endogenous loci along a 72-hour time-course in tomato protoplasts. Combining this data with kinetic modeling reveals that indel accumulation is not an accurate reflection of DSB induction efficiency due to prominent precise re-ligation, accounting for 40-70% of all repair events. Altogether, this system exposes previously unseen flux in the DSB repair process, decoupling induction and repair dynamics, and suggesting an essential role of high-fidelity repair in limiting CRISPR editing efficiency in somatic cells.

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Ben-Tov, D., Mafessoni, F., Cucuy, A., Honig, A., Bessudo, C., Levy, A. A.. 2023-01-10. Uncovering the Dynamics of Precise Repair at CRISPR/Cas9-induced Double-Strand Breaks. https://doi.org/10.1101/2023.01.10.523377

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