Download rNAi Biotechnology: Pros and Cons for Crop Improvement

Public Policy Update
rNAi BIOTEChNOLOGY DISCuSSIONS ENCOurAGED
Jan Leach, Public Policy Board Chair, [email protected], and Elizabeth Grabau, Public Policy Board Member, [email protected]
Biotechnology for improved management of plant
diseases has the potential to reduce dependency
of commercial and noncommercial growers
on pesticides and to enhance food security for
an increasing global population . APS has long
opposed regulating food, feed, and fiber products
based solely on the particular technology that was used to create
the varieties/cultivars (www .apsnet .org/members/outreach/ppb/
positionstatements/Pages/BiotechnologyPositionStatement .aspx) .
However, regulation of newly emerging approaches to crop
modification, such as RNAi technology to silence expression of
plant, pathogen, or insect genes, is now being discussed . The APS
Public Policy Board (PPB) will hold a hot topics discussion session
on RNAi use for biotechnology at the upcoming APS-CPS Joint
Meeting . As a prelude to the meeting, and to encourage discussions
on the topic among APS members now, we asked Vicki Vance, a
virologist in the Biological Sciences Department of the University of
South Carolina, to provide the following article as an introduction
to RNAi and to summarize the pros and cons of using the approach
for biotechnology purposes . Please feel free to add to the scientific
discussion of the process by e-mailing any PPB member (www .
apsnet .org/members/directories/Pages/PPB .aspx) with your
comments or plan to attend the session during the joint meeting .
rNAi Biotechnology: Pros and Cons for Crop Improvement
Vicki Vance, University of South Carolina, [email protected]
risk Assessment
Genetically modified (GM) crops are
subject to an environmental risk-assessment
process designed to evaluate their safety, and
approval by the relevant regulatory agency is
required before commercial release of GM
crops in all countries where these crops are
currently grown, including the United States .
The majority of approved GM crops have
been transformed to produce one or more
novel proteins that confer useful agronomic
traits such as pathogen or insect resistance .
However, a number of genetic engineering
approaches use RNAi technology to confer
useful traits to plants, and the measures
needed to assess the safety of these approaches
are currently under investigation .
What exactly is RNAi? It stands for “RNA
interference” and it refers to a set of related
processes in which small regulatory RNAs
direct sequence-specific repression of gene
expression . RNAi pathways are evolutionarily
ancient and various versions of these
processes are found in virtually all eukaryotic
organisms . All RNAi processes begin with
an RNA precursor that is completely or
partially double-stranded (dsRNA) . This
precursor is then processed to produce the
active small RNA by an enzyme called Dicer
(or Dicer-like) . There are two major kinds
of small RNAs that are currently used in
biotechnological applications in plants: 1)
short interfering RNAs (siRNAs), and 2)
40 March 2014
microRNAs (miRNAs) . The siRNA pathway
is induced by invasive nucleic acids, such as
viruses, transposons, or transgenes, and serves
to defend the host plant against such invaders
(Alvarado and Scholthof, 2009) . In contrast,
miRNAs are produced by an endogenous
pathway that is used to control an organism’s
own gene expression (Vazquez et al ., 2010) .
Once produced, the small RNA (either
siRNA or miRNA) incorporates into a protein
complex called RISC, where it serves as a
guide to find target messenger RNAs using
the base-pairing rules . The targeted messages
are either degraded or their translation is
blocked—either way, the encoded protein
is not produced and the gene is said to be
silenced .
engineered to produce a transgene in which
the endogenous miRNA in the precursor is
replaced with one that is complementary to
the targeted messenger RNA . The modified
precursor is then processed to produce a novel
miRNA called an artificial miRNA (Ossowski,
et al ., 2008) . This approach produces a single
small RNA rather than a population of small
RNAs . The miRNA approach may be less
effective in silencing the target gene (because
only one small RNA is produced rather than
a population), but it is more selective and less
likely to result in off-target silencing . Artificial
miRNAs thus provide an elegant method to
silence genes in a very specific manner (Sablok
et al ., 2011)
sirNAs versus mirNAs
Applications of rNAi
Biotechnology (PrOs)
RNAi technology differs depending on
whether the approach relies on siRNAs or
miRNAs . In a siRNA-based approach, a
transgene is designed to produce dsRNA
directly—these are called hairpin transgenes
or RNAi constructs (Waterhouse et al ., 1998) .
They produce a population of siRNAs that
represents the entire dsRNA region and has
complementarity to a chosen target messenger
RNA . The siRNA population is generally
very effective at silencing the target gene
because there are many different small RNAs
all targeting the same transcript . A miRNAbased approach is more precise . A natural
endogenous miRNA gene is genetically
RNAi has proven to be a powerful approach
for silencing genes to improve agronomic
traits in crop plants . The approach has
been used to target specific plant genes for
improved traits such as modified oil content
in soybeans, increased lysine content in corn,
and reduced caffeine content in coffee and to
provide resistance to numerous viral diseases
by targeting viral transcripts produced in
planta (reviewed in Frizzi and Huang, 2010) .
Remarkably, this same technology can be used
to target genes in pest organisms that feed
on plants, such as root-knot nematodes, corn
rootworm, and bollworm (Baum et al ., 2007;
Huang et al., 2006; Mao et al., 2007; Nowara
et al., 2010). Thus, this technology provides a
novel and potent new kind of pesticide.
Limitations of RNAi
Biotechnology (CONs)
Because siRNA-based RNAi is an antiviral
defense mechanism, many plant viruses
encode proteins that suppress silencing
(Burgyan, 2008; Alvarado and Scholthof,
2009). Furthermore, plant viral infections
are very common. As a result, it is possible
that viral infection in the field might suppress
transgene-induced RNAi. In addition,
suppressors of silencing from unrelated
viruses are structurally unrelated and employ
a variety of mechanisms to block silencing,
making it difficult to engineer broad-spectrum
protection against this problem. A second
potential limitation arises from the fact that
siRNAs comprise a population of molecules
representing the entire sequence of the dsRNA
trigger. Although this sequence heterogeneity
could make it easy to silence a family of
related genes with only one construct, it
also opens the door to off-target effects, in
which genes with regions of homology to the
intended target get silenced unintentionally.
A third potential limitation stems from the
fact that post-transcriptional silencing in
plants is mobile. It can be induced locally
and will then spread throughout the plant.
Thus, siRNA-based RNAi strategies might not
be suitable for some applications requiring
tissue-specific silencing of genes. The use of
artificial miRNAs may alleviate some of these
concerns, but they also bring their own set of
limitations. On one hand, artificial miRNAs
provide a much more specific way to silence
genes than do siRNAs because miRNAs
use only a single 21- or 22-nt sequence to
identify the target, whereas siRNAs comprise
a population of sequences. Thus, there is
a reduced chance of off-target effects with
miRNA-based RNAi, making it easier to
target individual genes, even in a closely
related gene family. Artificial miRNAs also
provide a better option for tissue-specific
silencing because miRNA-directed silencing
tends not to move throughout the plant.
However, a potential limitation of artificial
miRNA-based RNAi is that the silencing
might not be very durable because only a
single 21- or 22-nt specificity determinant
is involved. Escape from miRNA-directed
silencing via mutation of the target, therefore,
would be easier than in siRNA-directed
silencing, in which a much larger sequence
is targeted. Using two (or more) different
GM Crop Strategies
GM Crop Strategies
A. PROTEIN PRODUCT
Sense Transgene
B. RNA INTERFERENCE
Hairpin or miRNA Transgene
transcription
transcription
mRNA
dsRNA
translation
dicer
Protein
siRNA or miRNA
Protein function
confers desired
trait
Silence
plant genes
Silence genes
in organism
that eats plant
Figure 1. Strategies for genetically modified (GM) crops. A) Most approved GM crops are
based on producing a protein product. The plant is engineered with a sense transgene that
is transcribed to produce a messenger RNA (mRNA) and then translated to produce the
encoded protein. The protein is the functional element in this approach as well as the novel
element from a risk assessment standpoint. B) In RNA interference (RNAi) strategies, the
plant is engineered with either a hairpin transgene or with a transgene that that encodes a
microRNA (miRNA) precursor. These transgenes produce RNA that is either completely
double stranded (hairpin transgene) or partially double stranded (miRNA precursor). The
small RNAs (either siRNAs or miRNAs) are the functional elements in this approach and
the novel elements from the standpoint of safety assessment.
artificial miRNAs against the target is a
strategy that has been used to overcome this
problem.
Safety concerns for RNAi biotechnology
(CONs). The current risk assessment
procedures were designed to address potential
risks posed by genetically engineered plants
that encode and produce novel proteins.
In the case of RNAi strategies, however,
the genetically engineered plants encode
and produce novel siRNAs or miRNAs, as
well as their dsRNA precursors, rather than
proteins. A new safety concern that is unique
to RNAi approaches has recently been raised
by a report that miRNAs made in plants are
taken up by humans and other mammals
when they eat plants (Zhang et al., 2012). The
paper presented evidence that endogenous
plant miRNAs from plant-derived foods
are absorbed by cells of the mammalian GI
tract and packaged into microvesicles, which
protects them from degradation. The miRNAs
are then trafficked via the bloodstream to a
variety of tissues, where they are capable of
regulating mammalian genes. The report has
generated a good deal of excitement because
of the implications with regard to uptake
of therapeutic small RNAs to treat human
disease. However, it also has implications for
the safety of RNAi crop plants for human
consumption. Many of these GM crops
will harbor populations of small RNAs that
don’t exist in nature and may have potential
to suppress mammalian gene expression in
unexpected ways. This is perhaps more of a
concern for RNAi-based pesticides because
that approach produces small RNAs designed
to kill the targeted organism when it feeds
on the plant. Subsequent publications have
challenged whether uptake of ingested plant
small RNAs occurs to any significant extent
(Snow et al., 2013; Witwer et al., 2013).
However, in view of the novel potential risks
of RNAi-based GM crops, it seems that a
careful re-evaluation of current risk assessment
strategies is warranted in order to ensure the
safety of RNAi crop plants.
Biotechnology continued on page 42
Phytopathology News 41