In vitro cleavage sites were determined via mononoplex Digenome-seq with LbCpf1 (n = 8), AsCpf1 (n = 8), and SpCas9 (n = 2) in this study and by multiplex Digenome-seq with SpCas9 (n = 11) in our previous study. Using Digenome-seq to analyze genome-wide specificity of Cpf1 and SpCas9 RGENs. Genome-wide Circos plots showing DNA cleavage scores. Intact genomic DNA (gray) and genomic DNA digested by LbCpf1 (blue), AsCpf1 (red) or SpCas9 (yellow) were subjected to whole-genome sequencing and Digenome-seq analysis. The asterisk indicates one false-positive site found in the intact genomic DNA. The arrow indicates the on-target site. Sequence logos were obtained via WebLogo using DNA sequences at in vitro cleavage sites identified by Digenome-seq.