title: Deciphering Seed Sequence Based Off-Target Effects in a Large-Scale RNAi Reporter Screen for E-Cadherin Expression.
abstract: Functional RNAi based screening is affected by large numbers of false positive and negative hits due to prevalent sequence based off-target effects. We performed a druggable genome targeting siRNA screen intended to identify novel regulators of E-cadherin (CDH1) expression, a known key player in epithelial mesenchymal transition (EMT). Analysis of primary screening results indicated a large number of false-positive hits. To address these crucial difficulties we developed an analysis method, SENSORS, which, similar to published methods, is a seed enrichment strategy for analyzing siRNA off-targets in RNAi screens. Using our approach, we were able to demonstrate that accounting for seed based off-target effects stratifies primary screening results and enables the discovery of additional screening hits. While traditional hit detection methods are prone to false positive results which are undetected, we were able to identify false positive hits robustly. Transcription factor MYBL1 was identified as a putative novel target required for CDH1 expression and verified experimentally. No siRNA pool targeting MYBL1 was present in the used siRNA library. Instead, MYBL1 was identified as a putative CDH1 regulating target solely based on the SENSORS off-target score, i.e. as a gene that is a cause for off-target effects down regulating E-cadherin expression.
authors.affiliation: Bayer Pharma AG, BPH-GDD-GTRG-CIPL, Muellerstr. 178, 13353, Berlin, Germany; Humboldt University of Berlin, Institute for Biology, Berlin, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Adams
authors.affiliation: Bayer Pharma AG, BPH-GDD-GTRG-CIPL, Muellerstr. 178, 13353, Berlin, Germany.
authors.forename: Barbara
authors.initials: B
authors.lastname: Nicke
authors.affiliation: Bayer Pharma AG, BPH-GDD-GTRG-CIPL, Muellerstr. 178, 13353, Berlin, Germany.
authors.forename: Hans-Dieter
authors.initials: HD
authors.lastname: Pohlenz
authors.affiliation: Bayer Pharma AG, BPH-GDD-GTRG-CIPL, Muellerstr. 178, 13353, Berlin, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Sohler
mesh_terms: D015703:Antigens, CD; D015820:Cadherins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D015894:Genome, Human; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D012680:Sensitivity and Specificity; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism
chemical_list: D015703:Antigens, CD; C513384:CDH1 protein, human; D015820:Cadherins; C088831:MYBL1 protein, human; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D015534:Trans-Activators