{Reference Type}: Journal Article
{Title}: Uncovering the dynamics of precise repair at CRISPR/Cas9-induced double-strand breaks.
{Author}: Ben-Tov D;Mafessoni F;Cucuy A;Honig A;Melamed-Bessudo C;Levy AA;
{Journal}: Nat Commun
{Volume}: 15
{Issue}: 1
{Year}: 2024 Jun 14
{Factor}: 17.694
{DOI}: 10.1038/s41467-024-49410-x
{Abstract}: CRISPR/Cas9 is widely used for precise mutagenesis through targeted DNA double-strand breaks (DSBs) induction followed by error-prone repair. A better understanding of this process requires measuring the rates of cutting, error-prone, and precise repair, which have remained elusive so far. Here, we present a molecular and computational toolkit for multiplexed quantification of DSB intermediates and repair products by single-molecule sequencing. Using this approach, we characterize the dynamics of DSB induction, processing and repair at endogenous loci along a 72 h time-course in tomato protoplasts. Combining this data with kinetic modeling reveals that indel accumulation is determined by the combined effect of the rates of DSB induction processing of broken ends, and precise versus error repair. In this study, 64-88% of the molecules were cleaved in the three targets analyzed, while indels ranged between 15-41%. Precise repair accounts for most of the gap between cleavage and error repair, representing up to 70% of all repair events. Altogether, this system exposes flux in the DSB repair process, decoupling induction and repair dynamics, and suggesting an essential role of high-fidelity repair in limiting the efficiency of CRISPR-mediated mutagenesis.