Specificity of RNAi and genome editing
technologies in plants and animals
Brian D. Gregory
Department of Biology
Penn Genome Frontiers Institute
Genomics and Computational Biology Graduate Program
The University of Pennsylvania
RNAi in general
The active molecules in RNAi are small
RNAs (smRNAs)
Mechanisms for silencing by smRNAs in plants
and animals
Specificity of RNAi is conferred by
complementary base-pairing interactions
between a smRNA and its target RNA(s)
Very few complementary base pairing
interactions are required for functional
silencing of target RNAs by smRNAs in
animals
Witold Filipowicz, Suvendra N. Bhattacharyya & Nahum Sonenberg
Nature Reviews Genetics 9, 102-114 (February 2008)
Very few complementary base pairing interactions
are required for functional silencing of target
RNAs by smRNAs in animals
Front. Genet., 11 June 2013 | doi:
10.3389/fgene.2013.00107
Plant compared to animal smRNA-mediated
silencing and off-target effects
• In plants, near perfect complementarity is required for productive
silencing of target RNAs. This is likely responsible for the greater target
specificity that is observed in plant as compared to animal systems.
– However, in large plant genomes (which are common in horticulture and
agriculturally important plants) the likelihood of off-target effects increases as a
function of genome size.
• In plants, most smRNAs silence target RNAs by cleavage not
translation inhibition, so usual quite easy to identify off-target effects.
• In animals, only seed pairing (and maybe a few 3’ contacts) is required
for productive silencing of target RNAs. This results in the greater
expected and observed off-target effects observed for this technology
when trying to develop RNA-based silencing approaches.
• In animals, most smRNAs silence target RNAs through inhibition of
translation. Thus, even identifying off-target effects takes significantly
more time and effort than for similar experiments focused on
determining the answers to this same question in plant systems.
• In total, off-target effects are a serious concern with this pathway
Too many exogenous smRNAs can also
saturate the smRNA biogenesis and
function machinery
Too many exogenous smRNAs can also
saturate the smRNA biogenesis and
function machinery
• Increase in non-normal smRNAs can lead to exclusion of nomal,
endogenous smRNAs from AGO incorporation resulting in
developmental defects because of loss of developmentally important
smRNA-mediated silencing
Nuclease-mediated genome editing
Specificity determinants of
nuclease-mediated genome editing
Molecular Therapy Nucleic Acids (2012) 1, e3;
doi:10.1038/mtna.2011.5
CRISPR/CAS-mediated genome
editing
CRISPR/CAS-mediated genome
editing most likely to have off target
effects
Most important for
target cleavage
Method for detecting off-target effects of
nuclease-mediated genome editing
Method for detecting off-target effects of
nuclease-mediated genome editing
Summary of off-target effects as a result of
genome engineering by nucleases
• Detection of off target effects still in its infancy. Until recently, most
commonly used technique was whole genome sequencing to look for
new mutations induced by the genome engineering. This is time
consuming and labor intensive.
• New methods aimed at identifying double-strand breaks (DSBs) (i.e.
GUIDE-seq) are coming available and demonstrate that off target
effects more prevalent than thought for CRISPR/CAS system. These
approaches need to be used now on ZFN and TALEN-based
approaches as well, so that comprehensive comparisons can be
made.
• Early studies suggest that the specific CRISPR used and its targeting
region on the genome cause large-scale variation in off target effects.
Many more studies needed to truly determine how CRISPRs can be
made as specific as possible for all applications.
Some broad comparisons of the genomic effects
of RNAi/Genome Editing to normal plant breeding
techniques
• Breeding techniques such as introgression and mutagenesis are
likely to result in significantly more genomic changes than targeted
RNAi and genome editing approaches
– In fact, current breeding techniques (especially introgression and
mutagenesis) are currently known to effect more genomic loci and
resulting traits than the much more targeted RNAi and genome editing
technologies even when considering off-target effects of the latter
technologies.
• Stable transgene insertion not necessarily required for any of these
techniques. However, stable, multi-generation RNAi will require
transgenic technologies, and currently most genome editing
technologies do involve the use of transgenic approaches.
– However, genome editing approaches of desired crop traits is currently
being engineered to not rely on stable transgenic insertion.