Poster for SEED 2019, New York City.

1. Synthetic Genetic Incompatibility:
dCas9-based programmable transcription activation for
invasive species biocontrol and transgene biocontainment
Synthetic Genetic Incompability (SGI) is a genetic engineering
technology with potential application in the control of invasive species
and the biocontainment of transgenes in ecosystems. SGI enables
the engineering of genetic barriers to sexual reproduction. This is
achieved with a dCas9-based transcriptional activator which drives
lethal overexpression of a target gene early in the development of an
embryo resulting from the mating of an SGI and wild type organism.
We are pursuing a specific application in preventing the spread of
invasive carp species in the freshwater ecosystems of the US
Midwest and Great Lakes regions. No reliable control method exists
which is cost-effective and does not adversely impact the surrounding
ecosystem. A regime of wild release of SGI carp combined with
existing methods could enable complete control or even eradication
of carp in lakes and rivers. The SGI system has been previously
demonstrated in Saccharomyces cerevisiae. We describe efforts to
demonstrate SGI in zebrafish Danio rerio and common carp Cyprinus
carpio.
Abstract
Background
The spread of common carp poses a threat to US
Midwest freshwater ecosystems
ABC System: Novel year-round carp
husbandry and spawning capability
Acknowledgements
Samuel E. Erickson1, Siba Das1, Przemyslaw G. Bajer2 and Michael Smanski1
1BioTechnology Institute, University of Minnesota Twin Cities, St. Paul, MN
2Department of Fisheries, Wildlife and Conservation Biology, University of Minnesota Twin Cities, St. Paul, MN
Fig. 3. Demonstration of SGI in Saccharomyces Cerevisiae (Maselko et al. 2017). A
dCas9-VP64 PTA construct was directed to bind to the S. cerevisiae WT ACT1
promoter. Mating of a WT strain with an engineered strain results in lethal
overexpression of actin, preventing growth on solid media.
This work was funded by Minnesota Aquatic Invasive
Species Research Center (MAISRC).
Common carp (Cyprinus carpio) is a benthic, omnivorous freshwater fish native to
Eurasia. Common carp was introduced to North America for aquaculture and have
since spread across the Mississippi River basin, vigorously bottom-feeding and
uprooting vegetation while creating a new, previously unoccupied ecological niche.
The invasive spread of common carp in the American Midwest poses a threat to
native freshwater ecosystems. A combination of field observation and
experimentation demonstrates that growing carp biomasses result in increased
turbidity, suspended solids, decreased macrophyte and macroinvertebrate
abundance. Bajer et al. observed a threshold biomass of 30 kg/ha, below which
native plant and bird species can thrive, above which vegetation and bird
populations are disturbed by large-scale habitat deterioration. Shallow lakes in the
Midwest frequently exceed this threshold 3-4 fold (Hydrobiologia, 2009).
Many control methods have been discussed to prevent further carp spread, such as
physical methods (trapping, electrofishing, netting), chemical methods (toxins,
mating pheromones), and biological methods (predators, pathogens, wild release of
sterile fish). All have significant limiting drawbacks. Synthetic genetic incompability
(SGI) technology (Maselko et. al, Nat. Commun, 2017) seeks to overcome these
limitations.
eric4204@umn.edu
Synthetic Genetic
Incompatibility (SGI) Concept
SGI in Zebrafish
SGI in Yeast
b
Fig 1. a) the common carp range extends across
Hydrologic Unit Code 8, the Mississippi River
basin. Adapted from USGS Nonindigenous
Aquatic Species (NAS) resource
(https://nas.er.usgs.gov). b) The author, holding a
common carp at Long Lake, Ramsey County, MN.
Genetic engineering of Synthetic Genetic
Incompatibility (SGI) via a dCas9-based
programmable transcription activator (PTA) opens
the possibility of invasive species biocontrol that is
consistently effective, economically feasible, and
avoids harming the surrounding environment
Fig. 4. a) Three gene targets (GATA5,
SHHA, and ERN) are lethal to embryos
when expressed on too strongly. All three
genes are central regulatory points
guiding early embryo development. b)
Lethal phenotypes are visually observable
within a day after fertilization.
a.
b.
c. d.
Fig 2. a) A single induced synthetic speciation event produces an SGI carp, which
can be stably propagated with conventional aquaculture methods. An SGI carp will
not productively mate with a wild-type carp. b) A regime consisting of a combination
of wild-release of SGI carp with existing physical methods could enable complete
control or eradication of carp and/or other sexually reproducing invasive species. c)
A programmable transcription activator (PTA) is composed of a dCas9 fused to a
transcription activating domain, enabling targeted upregulation of a gene of interest
by including an sgRNA complementary to the promoter. The PTA is directed to
upregulate an endogenous, tightly regulated gene, resulting in a synthetic lethal
phenotype. An SGI carp can be protected from the lethal effect of the PTA by
specific modifications to the promoter region, preventing premature sgRNA binding.
d) When an SGI carp mates with a WT carp, a WT copy of the gene of interest is
brought into contact with the PTA in the recently fertilized embryo. Binding of the
PTA to the gene of interest promoter results in lethal overexpression of the gene,
preventing development of the embryo. e) This concept could conceivably function
in any sexually reproducing organism, offering novel application for responding to
other invasive species threats, transgene biocontainment in agriculture, and even
disease vector control. Fig 5. Carp are determinate spawners – in the wild they only spawn once a year in
the late spring. Transgenesis experiments utilizing the Tol2 transposon system
require a steady supply of fresh eggs and sperm. We have designed and
constructed the ABC system – a novel approach to carp husbandry involving
temperature and photoperiod manipulation.
It has been long understood that temperature is a crucial regulator of fish
reproductive development. More recent results show that manipulation of
photoperiod allows precise control of annual gamete development. Rearing
conditions in the ABC system are directly inspired by results from Davies et al.
(Aquaculture, 1986).
Once carp have sufficiently developed gametes, spawning can be induced with a
hypophysation technique via injection of Ovaprim (Salmon Gonadotropin Releasing
Hormone Analog + Domperidone). Transgenic constructs can be introduced into
sperm by electroporation, followed by IVF to generate transgenic carp lines.
Current research efforts seek to identify strong programmable transcription activator
components, identify gene targets for lethal overexpression, and make specific edits
to gene of interest promoters such as to prevent PTA binding while minimizing off-
target impact. A range of promoters and activating regions are being tested in vivo.
Transgenesis is induced via the Tol2 transposon system. Transgenesis is induced in
embryos by microinjection of plasmids along with mRNA encoding transposase into
zebrafish embryos at the one-cell stage
a.
b.
Our unique approach to carp husbandry enables
year-round spawning and collection of eggs and
sperm needed for transgenesis experimentation
Target genes of interest have been identified in a
zebrafish model
a.
b.
e.
a.