Kinetics and fidelity of the repair of Cas9-induced double-strand DNA breaks

The RNA-guided DNA endonuclease Cas9 is a powerful tool for genome editing. Little is known about the kinetics and fidelity of the double-strand break (DSB) repair process that follows a Cas9 cutting event in living cells. Here, we developed a strategy to measure the kinetics of DSB repair for single loci in human cells. Quantitative modeling of repaired DNA in time series after Cas9 activation reveals variable and often slow repair rates, with half-life times up to ~10 h. Furthermore, repair of the DSBs tends to be error-prone. Both classical and microhomology-mediated end-joining pathways contribute to the erroneous repair. Estimation of their individual rate constants indicates that the balance between these two pathways changes over time and can be altered by additional ionizing radiation. Our approach provides quantitative insights into DSB repair kinetics and fidelity in single loci, and indicates that Cas9-induced DSBs are repaired in an unusual manner. We transiently transfected K562#17 with a plasmid encoding a sgRNA. Twenty-four hours after transfection we stabilized Cas9 by adding Shield-1. We collected cells at various time points, isolated genomic DNA, amplified a ~300-bp region around the sgRNA target site by PCR and subjected the products to high-throughput sequencing to determine the intact and indel fractions. 8 replicates for sgRNA-LBR2, 6 replicates for sgRNA-AAVS1, 6 replicates sgRNA-LBR8, 5 replicates chr11, 2 replicates sgRNA LBR2/LBR8 combined. Double cut t=60 for sgRNA LBR2/LBR5, sgRNA LBR2/LBR6, sgRNA LBR2/LBR7, sgRNA LBR2/LBR8, sgRNA LBR2/LBR9