Journal: Genome Biology

Article Title: CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping

doi: 10.1186/s13059-023-02930-z

Figure Lengend Snippet: 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. N 3 -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 N 3 -kethoxal. b Outline of in vivo and in vitro CasKAS. For in in vitro CasKAS, gDNA is incubated with a dCas9/Cas9 RNP, then N 3 -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 × 10 6 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

Article Snippet: For each well, 6250 ng of Cas9 (MCLAB CAS9-200) or dCas9 (MCLAB dCAS9B-200) and 1200 ng sgRNA was complexed with CRISPRMAX (Thermo Fisher CMAX00008) reagent in Opti-MEM (Thermo Fisher 51,985,091) following manufacturer’s protocol.

Techniques: Genome Wide, In Vitro, Purification, In Vivo, Activity Assay, Incubation, Transfection, Labeling, Sequencing, Generated, Negative Control

Journal: Genome Biology

Article Title: CasKAS: direct profiling of genome-wide dCas9 and Cas9 specificity using ssDNA mapping

doi: 10.1186/s13059-023-02930-z

Figure Lengend Snippet: CasKAS profiles sgRNA specificity genome-wide. a Summary of de novo peak calls for sgRNA #1 (using MACS2). b CasKAS signal is stronger over predicted off-target sites, but legitimate interactions are also found elsewhere in the genome. c CasKAS profile over predicted (by Cas-OFFinder) off-target sites for sgRNA #1 with dCas9 (all such sites and focusing only on the top 100 ranked by dCas9 CasKAS signal). d CasKAS profile over peak calls outside predicted (by Cas-OFFinder) off-target sites for sgRNA #1 with dCas9. e Determinants of sequence specificity as measured by dCas9 CasKAS (for sgRNA #1). PAM-distal regions of the sgRNA are less constrained than its PAM-proximal parts. The on-target sgRNA is highlighted in yellow. f Active Cas9 signal read profiles can be used to distinguish off-targets associated with cutting from those where only binding occurs. Shown are the same off-target sites as in e and the plus- and minus-strand active Cas9 5′ end profiles around the sgRNA. In this case (sgRNA #1), only the on-target site shows a Cas9 CasKAS pattern indicating cleavage; at the other sites even active Cas9 likely only binds but does not cut. A simple cutting score metric (“ C -score”) based on multiplying the 5′ end forward- and reverse-strand profiles can be used to quantify cutting vs. binding. (g and h) Comparison between in vitro and in vivo CasKAS signal over predicted off-target sites for the EMX1 sgRNA. In vivo CasKAS is quantified as the difference in read per million (± 500 bp of the sgRNA site) between the sgRNA KAS-seq and the no-guide control KAS-seq (“RPM diff ”). The on-target site is shown in blue

Article Snippet: For each well, 6250 ng of Cas9 (MCLAB CAS9-200) or dCas9 (MCLAB dCAS9B-200) and 1200 ng sgRNA was complexed with CRISPRMAX (Thermo Fisher CMAX00008) reagent in Opti-MEM (Thermo Fisher 51,985,091) following manufacturer’s protocol.

Techniques: Genome Wide, Sequencing, Binding Assay, In Vitro, In Vivo