title: Morphological Profiles of RNAi-Induced Gene Knockdown Are Highly Reproducible but Dominated by Seed Effects.
abstract: RNA interference and morphological profiling-the measurement of thousands of phenotypes from individual cells by microscopy and image analysis-are a potentially powerful combination. We show that morphological profiles of RNAi-induced knockdown using the Cell Painting assay are in fact highly sensitive and reproducible. However, we find that the magnitude and prevalence of off-target effects via the RNAi seed-based mechanism make morphological profiles of RNAi reagents targeting the same gene look no more similar than reagents targeting different genes. Pairs of RNAi reagents that share the same seed sequence produce image-based profiles that are much more similar to each other than profiles from pairs designed to target the same gene, a phenomenon previously observed in small-scale gene-expression profiling experiments. Various strategies have been used to enrich on-target versus off-target effects in the context of RNAi screening where a narrow set of phenotypes are measured, mostly based on comparing multiple sequences targeting the same gene; however, new approaches will be needed to make RNAi morphological profiling (that is, comparing multi-dimensional phenotypes) viable. We have shared our raw data and computational pipelines to facilitate research.
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America.
authors.forename: Shantanu
authors.initials: S
authors.lastname: Singh
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: Wu
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America.
authors.forename: Vebjorn
authors.initials: V
authors.lastname: Ljosa
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America.
authors.forename: Mark-Anthony
authors.initials: MA
authors.lastname: Bray
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America.
authors.forename: Federica
authors.initials: F
authors.lastname: Piccioni
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America.
authors.forename: David E
authors.initials: DE
authors.lastname: Root
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America.
authors.forename: John G
authors.initials: JG
authors.lastname: Doench
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America.
authors.forename: Jesse S
authors.initials: JS
authors.lastname: Boehm
authors.affiliation: Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America.
authors.forename: Anne E
authors.initials: AE
authors.lastname: Carpenter
mesh_terms: D002460:Cell Line; D020869:Gene Expression Profiling / Q000379:methods; D055785:Gene Knockdown Techniques / Q000379:methods*; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism
chemical_list: D034741:RNA, Small Interfering