10-12 April 2019: The OECD Conference on RNAi based pesticides provided an overview on the current status and future possibilities for the regulation of externally applied dsRNA-based products that are proposed for use as pesticides. The event facilitated exchanges between policy makers, academia, industry on their implications in health, environment, and regulation.

1. RNA Biology
Regensburg University
Universitätsstrasse 31
93053 Regensburg
Paris 10.04.2019
Potential for off-target effects in topically
applied dsRNA-based products used for
crop protection purposes

2. • Short introduction into Argonaute proteins
and their mechanisms of action
• different types of off-target effects in animals
• miRNA-like off-target effects
• reduction of off-target effects

3. miRNA-guided gene silencing
Treiber et al., 2019, Nature Reviews Molecular Cell Biology

4. Small RNA-guided
gene silencing
• Not all Ago proteins are catalytically
active
• Multiple Ago proteins exist in animal
species
• In nematodes and insects, Ago proteins
are specialized for binding to distinct
classes of small RNAs
• Nematodes and insects trigger RNAi
through long double stranded RNA

5. Argonaute domains
• N domain: required for small RNA loading
and duplex unwinding
• PAZ domain: binds the 3’ end of the small
RNA
• MID domain: anchors the 5’ end of the small
RNA
• PIWI domain: structurally similar to RNase H. Cleaves complementary
target RNAs
• N/PAZ and PAZ/MID are connected by flexible linkers
Schirle et al. Science, 2012

6. Elkyam et al. Cell, 2012; Schirle et al. Science, 2012, Pfaff et al. PNAS, 2013
Structure of human Ago2

7. - characterized by GW-rich Ago binding domain
- silencing domain that mediates miRNA-guided
gene silencing
GW proteins
Tritschler et al., 2010, Nature Reviews Molecular Cell Biology

8. 8
Model of the unstructured GW peptide binding to Ago2
TNRC6B
Ago2
Tryptophane
Cross-linked Lysine
Pfaff et al.
PNAS, 2013

9. microRNA-guided post-transcriptional gene silencing
Jonas and Izaurralde, 2015,
Nature Reviews Genetics

10. miRNA-guided gene silencing
Dueck and Meister, 2014, Biological Chemistry
Animals (rarely in plants)
Plants (rarely in animals)
• major differences between plant and animal miRNA systems

11. • Short introduction into Argonaute proteins
and their mechanisms of action
• different types of off-target effects in animals
• miRNA-like off-target effects
• reduction of off-target effects

12. „off-target effects” caused by siRNAs
• partially complementary siRNAs can
function as miRNAs. 10-14 nucleotides or less of a siRNA
is already enough for efficient knock down
• At least in nematodes, siRNAs or long dsRNAs are mobile and can be
exchanged between cells and tissues.
• off-target activity:
sequence-specific and -unspecific inhibition of unrelated genes
• sequence composition. Some sequences trigger immune responses
in higher animals
• high concentrations of exogenous siRNAs may compete
with systems for protein components (e.g. Ago proteins)
• both strands can trigger off-target effects.
Unintended loading of the passenger strand

13. Design and generation of siRNAs
Petri and Meister, 2013, Methods Mol Biol
• a siRNA ‘seed match’ (nt 2-8)
may efficiently inhibit gene
expression through the
miRNA system

14. • Short introduction into Argonaute proteins
and their mechanisms of action
• different types of off-target effects in animals
• miRNA-like off-target effects
• reduction of off-target effects

15. miRNA-guided gene silencing
Dueck and Meister, 2014, Biological Chemistry
Animals (rarely in plants)
Plants (rarely in animals)

16. miRNA-like off-target effects in RNAi screening
approaches
Data from Marine et al. J Biomol Screen 2012
replicate 1
replicate2
R = 0.94
siRNA 1
R = 0.073
siRNA2 High correlation
 Great assay reproducibility
 No correlation
 Non-interpretable results
*siRNA reagent used = single siRNA/pools of 3 siRNAs
Comparing two replicate values
for one siRNA reagent*
Comparing two siRNA reagents
targeting one gene
• Lessons from large scale and genome-wide RNAi screening approaches in
animals

17. • Short introduction into Argonaute proteins
and their mechanisms of action
• different types of off-target effects in animals
• miRNA-like off-target effects
• reduction of off-target effects

18. Design and generation of highly complex siRNA pools
Hannus et al., 2014, NAR

19. Efficient gene silencing (on target activity) by siPools
• reduced off target activity?

20. Analysis of siPool-off target activities
On target activity
Off target activity Sigoillot et al., 2012,
Nature Methods

21. Analysis of siPool-off target activities

22. siPools shown strongly reduced off target activity

23. siPools shown strongly reduced off target activity

24. •
siPOOLs – Proof of concept
viability siRNA 1
viabilitysiRNA2
viability siPOOL 1
siPOOLsiRNA
average gene expression average gene expression
ExpressionarrayPhenotypic
correlation
viabilitysiPOOL2
High
specificity
Robust phenotypesR2 = 0.19 R2 = 0.71
siPOOLsiRNA
 off-target gene
Expressionvscontrol
Expressionvscontrol
A single siRNA
deregulated many off-
target genes, while a
siPOOL containing the
same siRNA did not.
Two siPOOLs against the
same gene produced
better phenotypic
correlation than two
single siRNAs.
Hannus et al. Nucleic Acids Res 2014

25. Summary
• pooling of many different siRNAs (>30) reduces miRNA-guided off-target
effects below detection
• for crop protection purposes, long double stranded RNA is used and
taken up by herbivores
• In these animals, Dicer enzymes produce natural pools of siRNAs
• thus, based on our work, we conclude that miRNA-like off-target effects are
minimal and probably negligible in such a system
• differences between plant and animal miRNA systems. Accurate sequence
selection should not affect the host or other plant systems.

26. 26
Laboratory of RNA Biology
University of Regensburg
Julia Neumeier
Norbert Eichner
Gerhard Lehmann
Nora Treiber
Thomas Treiber
Johanns Danner
Eva Schöller
Alice Griselin
Tiana Hanelt
Acknowledgements
Collaborations
University of Würzburg
Utz Fischer
Balagopal Pai
Daniele Hasler
Robert Hett
Michaela Bauer
Claudia Latini
Kevin Heizler
Hung Ho
Daniela Zeitler
Franziska Weichmann
Simone Larivera
Helmholtz-Center Munich
Aloys Schepers, Regina Feederle
Technical University Darmstadt
Florian Hastert, Cristina Cardoso
JGU Mainz
Mark Helm