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Updated: Apr 4, 2026
MISSION esiRNA for RNAi Screening in Mammalian Cells
Published on: May 12, 2010
Deciphering Seed Sequence Based Off-Target Effects in a Large-Scale RNAi Reporter Screen for E-Cadherin Expression
Robert Adams1, Barbara Nicke2, Hans-Dieter Pohlenz2
1Bayer Pharma AG, BPH-GDD-GTRG-CIPL, Muellerstr. 178, 13353, Berlin, Germany; Humboldt University of Berlin, Institute for Biology, Berlin, Germany.
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.
Insights
A new method, SENSORS, improves RNAi screening by accounting for off-target effects, enabling robust identification of true hits like MYBL1, a novel regulator of E-cadherin (CDH1) expression.
Area of Science:
- Molecular Biology
- Genetics
- Bioinformatics
Background:
- RNA interference (RNAi) screening is crucial for identifying gene function but is hampered by sequence-based off-target effects, leading to numerous false positives and negatives.
- E-cadherin (CDH1) is a key regulator of epithelial mesenchymal transition (EMT), and understanding its regulation is vital for cancer research.
Purpose of the Study:
- To develop and validate a novel computational method, SENSORS, to mitigate off-target effects in RNAi screening.
- To identify novel regulators of E-cadherin (CDH1) expression using a refined siRNA screen.
Main Methods:
- A druggable genome-targeting siRNA screen was conducted to identify regulators of CDH1 expression.
- The SENSORS (Seed ENrichment Strategy for Off-target analysis in RNAi Screens) method was developed to analyze and correct for siRNA off-target effects.
- Computational analysis of screening data using SENSORS to stratify results and identify true hits.
Main Results:
- The SENSORS method effectively stratified primary screening results by accounting for seed-based off-target effects.
- The SENSORS approach enabled the discovery of additional screening hits that were missed by traditional methods.
- MYBL1, a transcription factor, was identified as a novel, experimentally verified regulator of CDH1 expression, discovered solely through the SENSORS off-target score.
Conclusions:
- The SENSORS method significantly improves the accuracy and reliability of RNAi screening by robustly identifying and correcting for off-target effects.
- This approach facilitates the discovery of novel gene regulators, such as MYBL1, which are critical for understanding biological processes like EMT.
