169Synthetic Biology: Parts, Devices and Applications, First Edition. Edited by Christina Smolke.
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2018 by Wiley-VCH Verlag GmbH & Co. KGaA.
8
8.1 Utility of the RNAi Pathway for Application
in Mammalian Cells
RNA interference (RNAi) is an efficient and convenient tool for transient gene
suppression (knockdown) in biomedical research. RNAi is beneficial for genetic
screening and basic studies involving loss‐of‐function phenotypes and as an
alternative protein inhibitor to small molecule drugs [1]. Since the first discovery
of the RNAi phenomena in Caenorhabditis elegans [2], intensive genetic and
biochemical research has uncovered the molecular mechanisms underlying
RNAi and identified analogous pathways and molecules to control RNAi in
eukaryotes [3–5].
In mammalian systems, RNAi is induced when microRNA (miRNA), short
hairpin RNA (shRNA), or small interfering RNA (siRNA) harness the endoge-
nous processing pathway and machinery (Figure 8.1). In the endogenous RNAi
pathway, primary miRNA (pri‐miRNA) embedded in coding or noncoding RNA
is transcribed from genetic or plasmid DNA by RNA polymerase II or III and is
cleaved at the base region of the stem‐loop structure (two black wedges) by the
RNase III nuclease Drosha. The cleaved stem‐loop precursor miRNA (pre‐
miRNA) is recognized by Expotin‐5b proteins, exported from the nucleus to the
cytoplasm and processed into mature miRNA (two black wedges) by another
RNase III nuclease, Dicer. Then, one strand of the mature miRNA is selected and
introduced into Ago2 to activate sequence‐specific mRNA degradation and
targeted gene repression. shRNA expressed from plasmids is exported to the
cytoplasm and processed only by Dicer in a similar manner to transfected shRNA
or siRNA molecules (Figure 8.1).
From a synthetic biology perspective, RNAi is a suitable and potent technology
for the development of genetic devices to rewire cell signaling. It is important to
generate RNAi‐modulated genetic devices that detect target input molecules
Design of Ligand‐Controlled Genetic Switches Based
on RNA Interference
Shunnichi Kashida1,2 and Hirohide Saito^1
(^1) Center for iPS Cell Research and Application, Kyoto University, Department of Life Science Frontiers,
53 Kawahara‐cho, Shogoin, Sakyo‐ku, Kyoto 606‐8507, Japan
(^2) Ecole Normale Supérieure, UMR 8640 CNRS‐ENS‐UPMC Pasteur, Department of Chemistry, 24 rue Lhomond Paris,
75005, France