Fig. 5. Kinetics of DSB formation and mutation induction following EMX1 genome editing.

a INDUCE-seq reveals the kinetics of EMX1-induced DSB formation in a cell population during the editing process. Quantification of the number of nuclease-induced breaks detected per million reads for each sample revealed high Cas9 activity at both on- and off-targets immediately following cell nucleofection. b The comparison between off-targets identified by INDUCE-seq with established in vitro methods CIRCLE-seq and Digenome-seq, as well as cell-based methods GUIDE-seq, BLISS, and HTGTS. INDUCE-seq detects many off-targets that were previously only identifiable by in vitro methods. Substantially more off-target sites were identified than by any of the current cell-based methods. INDUCE-seq also identifies multiple off-targets not detected by any other method. c Amplicon-sequencing to measure the indel frequency at INDUCE-seq-identified off-targets. Four of the 60 off-targets discovered using INDUCE-seq were mutated with an indel frequency above the background false-discovery rate of 0.1% for amplicon-seq. (c, far-right) Indel frequency reported previously for EMX1 48 h post RNP nucleofection. Source data are provided as a Source Data file.