POCKET-seq method maps genome-wide off-target binding of dCas9 during CRISPR interference screens

CRISPR interference (CRISPRi) uses a catalytically inactive form of Cas9 (dCas9) fused to a transcriptional repressor domain — typically KRAB — to silence target genes without cutting DNA. The approach is widely used in functional genomic screens to study gene function at scale. A preprint posted to bioRxiv describes the development of POCKET-seq, a method that profiles dCas9-KRAB binding across the entire genome during CRISPRi experiments.

The researchers report that off-target binding of dCas9 occurs frequently. Critically, when this off-target binding occurs near the promoter of a gene relevant to the screen phenotype, it can produce false-positive associations — meaning a guide RNA appears to implicate a target gene when the observed effect is actually driven by inadvertent silencing elsewhere in the genome. The authors observed this problem during validation of a genetic screen, where non-overlapping guide RNAs targeting the same gene produced different phenotypic outcomes, prompting the development of the method.

The work has implications for the interpretation and design of CRISPRi screens broadly. As a preprint, the findings have not yet been peer-reviewed. Researchers using CRISPRi approaches for functional genomics may find the tool and the described quality-control considerations relevant to their experimental workflows.