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bioRxiv · 10.64898/2026.06.01.729390

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

Joyce, C. M.·Kramer, G. D.·Vu, J. T.·Tavasoli, K. U.·Gardner, B. M.·Richardson, C. D.
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Abstract

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.

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Joyce, C. M., Kramer, G. D., Vu, J. T., Tavasoli, K. U., Gardner, B. M., Richardson, C. D.. 2026-06-02. POCKET-seq enables genome-wide profiling of on- and off-target transcriptional regulation events by dCas9-KRAB during CRISPR interference experiments. https://doi.org/10.64898/2026.06.01.729390

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