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MISSION esiRNA for RNAi Screening in Mammalian Cells
Published on: May 12, 2010
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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.
Plos One
|September 12, 2015
Summary
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.
