Systematic identification of seed-driven off-target effects in Perturb-seq experiments
Hartman, A.; Blair, J. D.; Nguyen, T. P.; Dyson, K.; Bradu, A.; Takacsi-Nagy, O.; Santostefano, K.; Boade, T.; Bolanos, M.; Zhu, R.; Dann, E.; Marson, A.; Gitler, A.; Satija, R.; Satpathy, A. T.; Roth, T. L.
Show abstract
Genome-wide Perturb-seq (GWPS) has emerged as a powerful approach for unbiased mapping of gene regulatory networks. A key assumption underlying many Perturb-seq analyses is that each guide RNA exclusively perturbs a single target locus. Without methods to identify and filter off-target events, erroneous gene-pathway associations driven by off-target activity can propagate into downstream analyses. Here, we present a workflow for the systematic identification of candidate off-target events in CRISPRi Perturb-seq experiments. Our approach exploits the observation that cells harboring a guide which represses an off-target gene display transcriptional similarity to cells in which that gene is directly targeted by an on-target guide. We apply our workflow to multiple GWPS datasets and nominate off-target events in which a guide nominally targeting one gene also represses a distinct gene producing a phenotype likely attributable to the off-target perturbation. We use both off-target gene repression and guide seed sequence alignments at the off-target promoter locus as evidence for off-target effects and find independent evidence of putative off-target events in separate GWPS datasets. Together, these results establish a principled framework for the identification and filtering of off-target guide effects in Perturb-seq experiments.
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