POCKET-seq enables genome-wide profiling of on- and off-target transcriptional regulation events by dCas9-KRAB during CRISPR interference experiments

CRISPR interference screens use catalytically inactive dCas9 fused to a repressor domain to enable genetic perturbations at the transcriptomic level. Interpretation of results involves identification of guide RNAs associated with the screen phenotype, followed by secondary analysis. During validation of a genetic screen, we observed different phenotypes from non-overlapping guide RNAs targeting one gene. Here, we developed POCKET-seq to map the binding of dCas9 genome-wide. We show that off-target binding occurs frequently and can generate false-positive interactions when it occurs near the promoter of genes associated with the screen phenotype. POCKET-seq classifies these false-positive and true-positive interactions using gene ontology.