Fig. 1

CasKAS maps dCas9- and Cas9-mediated strand invasion and cleavage events genome-wide in vitro on purified DNA and in vivo in cell lines. a CasKAS is based on the KAS-seq assay for mapping ssDNA structures. N₃-kethoxal covalently modifies unpaired guanine bases (while having no activity for G bases paired within dsDNA). Strand invasion by Cas9/dCas9 carrying an sgRNA results in the formation of a ssDNA structure, which can be directly identified using N₃-kethoxal. b Outline of in vivo and in vitro CasKAS. For in in vitro CasKAS, gDNA is incubated with a dCas9/Cas9 RNP, then N₃-kethoxal is added to the reaction; for in vivo CasKAS, cells are transfected with an RNP, then treated with kethoxal. DNA is then purified, click chemistry is carried out, DNA is sheared, labeled fragments are pulled down with streptavidin beads, and sequenced. c, d Mapping of dCas9 targets in vitro. c Mouse gDNA was incubated with dCas9 RNPs carrying one of two sgRNAs targeting the mouse HOXA locus. Highly specific labeling is observed at the expected target location of each sgRNA. d Asymmetric strand distribution of in vitro dCas9 CasKAS reads around the sgRNA target site. e, f Mapping of Cas9 targets in vitro. e Mouse gDNA was incubated with Cas9 RNPs carrying one of the same two sgRNAs targeting the mouse HOXA locus. f The distribution of 5′ read ends around targets sites in in vitro CasKAS datasets shows direct capture of the intermediate cleavage state. g Reproducibility of in vivo dCas9 CasKAS datasets. Shown are RPM values for 500 bp windows centered on the top ∼7000 predicted target sites for the “sgRNA #1” in two in vitro CasKAS experiments. Off-target sites are color-coded by the number of mismatches relative to the sgRNA. h CasKAS requires a moderate sequencing depth of 10–20 × 10⁶ reads to accurately rank potential off-targets. A total of 10 different sets of subsamplings were generated, and the fraction of off-targets within 20% of their final quantification value was calculated for each. The mean ± SD is shown. i–k In vitro CasKAS maps Cas9 and dCas9 target sites. i Shown are CasKAS experiments with Cas9 and dCas9 and with the EMX1 sgRNA or with no sgRNA (negative control). j Assymmetric 5' end distribution around target sites in dCas9 in vivo CasKAS. k In vivo Cas9 CasKAS, a mixture distribution is observed between phased cleavage sites and broader ssDNA labeling