RT Journal Article
SR Electronic
T1 Quantitative analysis shows that repair of Cas9-induced double-strand DNA breaks is slow and error-prone
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 142802
DO 10.1101/142802
A1 Eva K. Brinkman
A1 Tao Chen
A1 Marcel de Haas
A1 Hanna A. Holland
A1 Waseem Akhtar
A1 Bas van Steensel
YR 2017
UL http://biorxiv.org/content/early/2017/05/26/142802.abstract
AB 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 a relatively slow repair rate (~6h). Furthermore, the double strand break is predominantly repaired in an error-prone fashion (at least 70%). Both classical and microhomology-mediated end-joining pathways are active and contribute to the repair in a stochastic manner. However, the balance between these two pathways changes over time and can be altered by chemical inhibition of DNAPKcs or additional ionizing radiation. Our strategy is generally applicable to study DSB repair kinetics and fidelity in single loci, and demonstrates that Cas9-induced DSBs are repaired in an unusual manner.