iGUIDE: an improved pipeline for analyzing CRISPR cleavage specificity

Genome engineering methods have advanced greatly with the development of programmable nucleases, but methods for quantifying on and off target cleavage sites and associated deletions remain nascent. Here we report an improvement of the GUIDE-seq method, named iGUIDE, which allows filtering of mispriming events to clarify the true cleavage signal. Using iGUIDE, we 1) specify the locations of Cas9- guided cleavage for four guide RNAs, 2) characterize associated deletions, and 3) show that naturally occurring background DNA double strand breaks are associated with open chromatin, gene dense regions, and chromosomal fragile sites. Protocols and software for running iGUIDE are provided.